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New Topoisomerase Inhibitors in Clinical Trials

Posted in Cancer and Current Therapeutics, CANCER BIOLOGY & Innovations in Cancer Therapy, Cell Biology, Drug Delivery Platform Technology, Pharmaceutical Analytics, Pharmaceutical Discovery, Pharmacologic toxicities, Small Molecules in Development of Therapeutic Drugs, tagged Cancer - General, Cell Biology, Clinical trial, DNA, DNA repair, FDA, Food and Drug Administration, medicine, research, topoisomerase on October 14, 2015| Leave a Comment »

New Topoisomerase Inhibitors in Clinical Trials

Curator: Stephen J. Williams, Ph.D.

Below is a great review of topoisomerase in cancer, approved inhibitors as well as some in clinical trials.

Biomolecules 2015, 5, 1652-1670; doi:10.3390/biom5031652

OPEN ACCESS

biomolecules

ISSN 2218-273X

Review

Inhibition of Topoisomerase (DNA) I (TOP1): DNA Damage Repair and Anticancer Therapy

Yang Xu and Chengtao Her *

School of Molecular Biosciences, College of Veterinary Medicine, Washington State University, Mail Drop 64-7520, Pullman, WA 99164, USA; E-Mail: davidxy22@vetmed.wsu.edu

* Author to whom correspondence should be addressed; E-Mail: cher@wsu.edu; Tel.: +1-509-335-7537; Fax: +1-509-335-4159.

Academic Editors: Wolf-Dietrich Heyer, Thomas Helleday and Fumio Hanaoka Received: 22 May 2015 / Accepted: 14 July 2015 / Published: 22 July 2015

Abstract: Most chemotherapy regimens contain at least one DNA-damaging agent that preferentially affects the growth of cancer cells. This strategy takes advantage of the differences in cell proliferation between normal and cancer cells. Chemotherapeutic drugs are usually designed to target rapid-dividing cells because sustained proliferation is a common feature of cancer [1,2]. Rapid DNA replication is essential for highly proliferative cells, thus blocking of DNA replication will create numerous mutations and/or chromosome rearrangements—ultimately triggering cell death [3]. Along these lines, DNA topoisomerase inhibitors are of great interest because they help to maintain strand breaks generated by topoisomerases during replication. In this article, we discuss the characteristics of topoisomerase (DNA) I (TOP1) and its inhibitors, as well as the underlying DNA repair pathways and the use of TOP1 inhibitors in cancer therapy.

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Type IB Topoisomerases and Inhibitors
1.1. TOP1

DNA topoisomerases resolve topological constraints that may arise from DNA strand separation and are therefore important for transcription and replication [4]. There are six topoisomerases in humans, classified as Type IA, IB and IIA. Type IA topoisomerases TOP3a and TOP3b cleave one DNA strand to relax only negative supercoiling. In addition, TOP3a forms the BTR complex with BLM and RMI1/2, which plays a role in the dissolution of double-Holliday junctions [5]. Type IIA topoisomerases TOP2a and TOP2b generate double-strand breaks on one DNA molecule to allow the passing of other DNA strands [6]. Topoisomerases are attractive drug targets in cancer therapy. For example, the commonly used anticancer agents doxorubicin and etoposide (VP-16) are TOP2 inhibitors [7]. Type IB topoisomerases include the nuclear TOP1 and mitochondrial TOP1mt [4]. TOP1 initiates the DNA relaxation by nicking one DNA strand. It then forms a TOP1-DNA cleavage complex (TOP1cc) by covalently linked to the 3′-phosphate end via its tyrosine residue Y723 (3′-P-Y). Following the resolution of topological entanglements and the removal of TOP1, the 5′-hydroxyl end is realigned with the 3′-end for religation. Each nicking-closing cycle enables the relaxation of one DNA supercoiling (Figure 1).

Figure 1. A schematic representation of strand passages catalyzed by three types of topoisomerases (adapted from ref. [8]).

TOP1 is essential for embryonic development in mammals [9]. Although TOP1 plays an important role in the deconvolution of supercoils arising amid DNA replication, the precise steps involved with

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the recruitment of TOP1 to topological constraints remains to be revealed. It appears that in yeast TOP1 travels at a distance of 600 bp ahead of the replication fork [10] and remains associated with the GINS-MCM complex [11]. However, the yeast TOP1 is distinct from its human counterpart in that it has little effect on fork progression or the firing of replication origin [12]. In humans, TOP1 binds to the regions of the pre-replicative complex in cells during the M, early G1, and G1/S phases of the cell cycle to control the firing of replication origins [12]. This difference may explain why yeast cells are viable in the absence of TOP1. In addition, TOP1 also has functions in transcription that are independent of its role in resolving DNA topological entanglements. First, TOP1 is known to repress transcription by binding to TFIID [13]. Second, inhibition of TOP1 can cause the induction of c-Jun in leukemia cells, suggesting its additional role in the control of transcription [14]. Furthermore, TOP1 interacts with the splicing factor ASF/SF2 by which it promotes the maturation of RNA—through suppressing the formation of R-loops (RNA-DNA hybrids)—and prevents collision between transcription bubble and replication fork [15,16]. It appears that the levels of TOP1 have to be dynamically regulated. In B cells, TOP1 is reduced by activation-induced cytidine deaminase (AID) to facilitate class-switch recombination (CSR) and somatic hypermutation (SHM) [17,18]. Although TOP1mt is important for mitochondrial integrity and metabolism, mice lacking mitochondrial TOP1mt are viable and fertile but they are associated with increased negative supercoiling of mtDNA [19,20].

1.2. TOP1 Inhibitors

Stabilization of TOP1cc by topoisomerase poison is detrimental to cells due to the disruption of DNA uncoiling, increased strand breaks, and unstable RNA transcripts as well as incomplete DNA replication [21]. The TOP1 inhibitor camptothecin (CPT), first isolated from the Chinese tree Camptotheca acuminate, was clinically used for cancer treatment long before it was identified as a TOP1 inhibitor [22]. Due to side effects, CPT is no longer used clinically and it has been replaced by more effective and safer TOP1 inhibitors [23]. Currently, CPT derivatives topotecan (trade name: Hycamtin) and irinotecan (CPT-11, trade name: Camptosar) are routinely used to treat colorectal, ovarian and lung cancers, while a few other TOP1 inhibitors are being tested in clinical trials.

CPT is a 5-ring alkaloid that is active in its closed E-ring (lactone) form but it is inactive with an open E-ring (carboxylate) at physiological and alkaline pH [24]. Therefore, CPT is not effective for inhibiting TOP1mt due to a higher pH mitochondrial environment. The inactive form of CPT tends to bind to serum albumin, which might be a reason for its side effects. CPT is highly specific for TOP1 and the binding is of relatively low affinity and can be reversed after drug removal. These features make the action of CPT controllable [24], and in fact CPT is widely used in studies of replication-associated DNA damage response. There are a few CPT derivatives and non-CPT TOP1 inhibitors [4,8,24]. For example, CPT derivatives Diflomotecan and S39625 were designed to stabilize the E-ring. Irinotecan has the bis-piperidine side chain to increase its water solubility, but it also contributes to some side effects. Non-CPTs—such as indolocarbazoles, phenanthrolines (e.g., ARC-111) and indenoisoquinolines—refer to drugs that have no typical CPT E-ring structures but they can still specifically target TOP1 and bind irreversibly to TOP1cc. Some of the CPT derivatives (i.e., Gimatecan and Belotecan) and non-CPTs (i.e., NSC 725776 and NSC 724998) are presently tested in clinical trials [23].

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How does CPT trap TOP1cc? Analysis of the crystal structure and modeling suggest that CPT-TOP1-DNA forms a ternary complex to prevent the two DNA ends from religation [25–27]. Although it is still controversial on how CPT is intercalated into DNA, it seems that CPT traps TOP1cc with a thymine (T) at the -1 position and a guanine (G) at the +1 position on the scissile strand, and it is therefore sequence-specific [28]. Three amino acid residues of the TOP1 enzyme, R364, D533 and N722, combined with DNA bases, contribute to the stabilization of the ternary complex by forming hydrogen bonds and hydrophobic interactions. It is of note that several point mutations, including N722S, in Camptotheca acuminata TOP1 confer resistance to CPT [29]. Interestingly, the same amino acids also contribute to the inhibition of TOP1 by non-CPT drugs [24].

Repair of TOP1 Poison-Induced DNA Lesions

As aforementioned, CPT-induced trapping of TOP1cc creates a single strand break with a free 5′-hydroxyl group, whereas the 3′-phosphate is connected to Y723 of TOP1 (3′-P-Y). At least two pathways contribute to the repair of DNA lesions created by TOP1 poison [30]. The tyrosyl-DNA-phosphodiesterase (TDP1) pathway starts with the ubiquitination and proteasome-mediated degradation of TOP1 in the CPT-TOP1-DNA complex to generate a 3′-P end linked to a short peptide [31]. TDP1 then cleaves the P-Y bond to release the 3′-P end; however, the 3′-P end cannot be directly ligated to the 5′-OH end because of the requirements of DNA ligases. The human polynucleotide kinase (PNKP) can process the DNA ends by functioning as both a 3′-phosphatase and a kinase to generate the required 3′-OH and 5′-P termini for direct ligation. The rest of the repair events can be best described by the single-strand break (SSB) repair pathway, which will be discussed below. Indeed, TDP1 and PNKP are tightly associated with the SSB repair machinery [32,33].

The endonuclease pathway requires multiple endonucleases to excise the DNA—usually at a few nucleotides away from the 3′-P-TOP1 end – on the scissile strand to release the DNA-TOP1 complex [30]. Initial studies were carried out to identify genes that functioned in CPT repair in the absence of TDP1 in yeast [34,35]. These studies led to the identification of RAD1-RAD10, SLX1-SLX4, MUS81-MMS4, MRE11-SAE2 as well as genes involved in recombination. The RAD1-RAD10 (human XPF/ERCC4-ERCC1) complex is a DNA structure-specific endonuclease that can act on 5′ overhang structures [36]. Interestingly, the cleavage site of XPF-ERCC1 is in the non-protruding DNA strand, about 3–4 nucleotides away from the 3′ end [36]. Therefore, trapped TOP1ccs can be removed by this endonuclease activity. Likewise, MUS81-MMS4 (human MUS81-EME1) can also cleave nicked duplex at the 5′ of the nick [37]. The SLX1-SLX4 endonuclease, although not tested on nicked duplexes, is able to process 3′ flap and other DNA structures [38,39]. In human cells, SLX4 also associates with XPF-ERCC1 and MUS81-EME1 endonucleases to process specific DNA intermediates [39,40]. Moreover, MRE11-RAD50 cleaves the 3′-P-Y bond and resects DNA to produce a 3′-OH end [41]. A direct role of SAE2 (human CtIP) in processing 3′-P-TOP1 is unknown, and its endonuclease activity appears to be limited to the 5′ flap or DNA “hairpin” structures [42,43]. Nonetheless, the endonuclease activity of CtIP is essential for processing CPT adducts [42]. In addition, like CtIP, the 5′ flap endonuclease RAD27 (human FEN1) seems to be unable to directly process 3′-P-TOP1 ends [44]. However, the gap endonuclease activity of FEN1 is important for processing stalled replication forks and CPT-induced adducts [45]. The role of FEN1 in SSB repair will be discussed further in the next section.

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During DNA replication, SSBs created by CPT are most likely converted to double-strand breaks (DSBs) by replication fork runoff. This conversion appears to be dependent on the proteolysis of TOP1 [46]. The repair of one-ended DSBs, as will be discussed in the next section, is largely dependent on homologous recombination (HR). However, low doses of CPT may also induce PARP1 and/or RAD51 dependent replication fork regression—generating no or few DSBs [47,48]. The regressed fork leads to the formation of a “chicken foot” DNA structure by newly synthesized strands [3,49,50]. The formation of regressed fork can be largely suppressed by ATR, EXO1, and DNA2 [51–53]. However, fork reversal can also be beneficial as it provides time for the repair of TOP1-induced DNA lesions by TDP1, thereby preventing DSB formation and the activation of error-prone non-homologous end-joining (NHEJ) [30].

Pathways Involved in the Repair of CPT-Induced DNA Lesions

Normal cells use DNA damage response (DDR) pathways to maintain genomic stability [54]. As aforementioned, SSB and DSB repair mechanisms are the two major DDR pathways that repair TOP1-induced DNA lesions. Paradoxically, cancer cells exploit DDR pathways to accumulate necessary genomic alterations for promoting proliferation. Furthermore, altered DDR and apoptotic responses in cancer cells are the major obstacles to successful chemotherapy. Thus, the delineation of TOP1-related SSB and DSB repair mechanisms is of great importance for identifying drug targets that can selectively affect cancer cell survival.

3.1. Single-Strand Break (SSB) Repair

Trapping of TOP1cc results in a 3′-P-TOP1 end and a 5′-OH terminus. Because the two ends cannot be directly religated, the persisting SSB is likely to be detected by PARP1 in which activated PARP1 catalyzes the synthesis of poly(ADP-ribose) (PAR) chains for recruiting repair proteins [55]. This reaction can be rapidly reversed by PARG, which hydrolyzes the PAR chains. The PAR chains at the SSB sites are important for the recruitment of XRCC1 that functions as a loading dock for other SSB repair proteins including TDP1 and PNKP. TDP1 generates 3′-P and PNKP converts 3′-P to 3′-OH, and PNKP also converts 5′-OH to 5′-P, making ends compatible for religation with no base loss. The rejoining of the 3′-OH and 5′-P ends is mainly mediated by LIG3, in which XRCC1 mediates the recruitment of LIG3.

If the trapped TOP1cc intermediates are processed by endonucleases, the initial SSBs will be converted to 3′-OH and 5′-OH ends with a gap over a few nucleotides (in the case of XPF-ERCC1, the loss is in the range of 3–4 nt), leading to the activation of PARP1 and XRCC1 recruitment. Consequentially, Pol3 recruited by XRCC1 can catalyze the gap filling, and PCNA-Polö/E also plays a role in this process [55]. If the 5′-OH is not processed by PNKP, the 5′-flap resulted from gap filling is likely to be removed by FEN1, which explains why FEN1 deficiency also leads to an increased CPT sensitivity. The final ligation is catalyzed by LIG1 because of the presence of PCNA.

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3.2. Double-Strand Break (DSB) Repair

Successful DSB repair requires concerted actions of proteins involved in DNA damage signaling and repair [54]. To repair TOP1 poison-induced DNA lesions, ATR signaling is required due to the runoff of replication fork and the presence of long single-strand DNA (ssDNA) [56]. The full activation of ATR follows a “two-man” rule—the ssDNA-ATRIP-dependent recruitment of ATR kinase and the RAD17 clamp loader/9-1-1/TOPBP1 mediator loading at the ssDNA-dsDNA junction. ATR phosphorylates CHEK1 to harness cell cycle arrest. If one-ended DSB is formed, ATM will be activated through the action of the MRE11-RAD50-NBS1 (MRN) complex. ATM mainly phosphorylates CHEK2 to mediate cell cycle arrest. Both ATM and ATR are able to phosphorylate hundreds of proteins in response to DSB formation [57]. One remarkable substrate is the histone H2AX, which can be phosphorylated by both kinases to yield g-H2AX. It is conceived that the propagation of g-H2AX signaling along the chromatin facilitates MDC1 recruitment and BRCA1 signaling via the MDC1-RNF8-RNF168-RAP80 ubiquitin cascade—events that are essential for HR [58].

The repair of TOP1 poison-induced DNA lesions is in essence the repair of one-ended DSBs, which facilitates the restoration of replication forks to restart DNA replication. It is important to note that one-ended DSB repair occurs in the S phase and relies on HR rather than NHEJ [59]. The first step in HR is end resection to generate a 3′-overhang for homology searching. A TOP1 cleavage in the leading strand may require end resection by the MRN-CtIP-BRCA1 and BLM-EXO1-DNA2 complexes [60], whereas a cleavage in the lagging strand automatically forms a 3′-overhang. Rad51 then associates with the 3′-ssDNA to form a nucleofilament for strand invasion, which leads to the formation of a D-loop structure [61]. This process continues with DNA synthesis, branch migration and the resolution of Holliday junction structures to reconstitute a functional replication fork [62]. TOP1 poisons can also lead to the formation of two-ended DSB if two replication forks collide into each other at the site of SSB. The repair of this type of DSBs is not aimed for fork restoration and can be accomplished by the classical DSB repair mechanisms [61].

3.3. Genes Involved in CPT-Induced Damage Repair

A long list of genes, in which mutations confer sensitivity to CPT in yeast, chicken or mammalian cells, has been compiled [24,30,63]. With no surprise, many genes involved in SSB and DSB repair are on the list, such as PARP1, XRCC1, PNKP, TDP1 for SSB repair; MRN, ATM-CHK2, ATR-CHK1 for DSB signaling; BRCA1/2, XRCC2, XRCC3 for HR. Most recently, the hMSH5-FANCJ complex has also been implicated to play a role in CPT-induced DNA damage response and repair [64]. Mutations in the binding partners of these repair factors are also likely to sensitize cells to CPT treatment. For example, depletion of the MRN-binding partner hnRNPUL increases the sensitivity to CPT [65]; and deficiencies in ZRANB3 and SPIDR, binding partners of PCNA and RAD51, cause CPT hypersensitivity in cancer cells [66–68]. In addition, the two DNA helicases BLM and WRN have also been implicated in the repair of CPT-induced DNA lesions [69,70]. Early studies revealed that chicken BLM knockout cells and human BLM-deficient fibroblasts showed increased sensitivity to CPT [71,72]. On the contrary, mouse BLM knockout embryonic stem cells showed mild resistance to

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CPT [73]. This discrepancy is likely attributable to the complexity of CPT-induced DNA lesion repair as well as different treatment conditions and experimental systems.

Interstrand crosslinks (ICLs) resemble CPT-induced lesions in that they block both replication and transcription [74]. They may induce replication fork reversal and fork collapse, which require DNA incision for lesion processing and HR for repair. ICL repair is accomplished by the coordinated actions of 17 Fanconi anemia (FA) genes whose mutations contribute to FA in patients [75]. Depletion of FANCP/SLX4 or FANCQ/XPF causes cellular sensitivity to CPT because they form an endonuclease complex involved in the repair of trapped TOP1cc [38]. Likewise, depletion of FANCS/BRCA1, FANCD1/BRCA2, FANCN/PALB2 or FANCO/RAD51C sensitizes cells to CPT because of their involvement in HR [76]. Accordingly, depletion of the FA core complex except FANCM—involved in fork reversal—is not expected to increase CPT sensitivity because they are unable to recognize the trapped TOP1cc [76]. However, the roles of FANCI, D2, J and FAN1 in the process are elusive due to conflicting reports presumably reflecting different experimental systems [76–78]. For example, in a multicolor competition assay, loss of FANCI or FAN1 rendered cells sensitive to CPT treatment [77]. However, this observation could not be recapitulated in studies performed with FANCI-deficient lymphoblasts and FAN1-depleted HEK293 cells [76,79], indicating that the involvement of these two genes in CTP sensitivity might be cell type specific.

It is interesting to note that the MMS22L-TONSL complex plays a prominent role in mediating CPT sensitivity [80–83]. Depletion of this complex impairs RAD51 foci formation and triggers G2/M arrest, indicating that the MMS22L-TONSL complex participates in HR repair. Furthermore, this complex associates with MCM, FACT, ASF1 and histones. FACT and ASF1 are histone chaperones that function in H2A/H2B and H3/H4 chromatin assembly and disassembly, respectively [84]. They recycle parental histones from old DNA strands unwound by MCM and incorporate them into newly synthesized DNA strands. FACT and ASF1 also function in checkpoint signaling; therefore the involvement of MMS22L-TONSL in CPT response implies the existence of a close association between HR, DNA damage signaling and replication restart.

TOP1 Inhibition in Cancer Treatment

The understanding of the function of TOP1 and the cellular effects of TOP1 inhibition has been a stepping-stone for the development of effective CPT derivatives in cancer therapy. Since TOP1 functions in normal and cancer cells, the use of low doses of TOP1 inhibitors are actively sought to treat cancers that heavily rely on the function of TOP1 for survival (e.g., highly malignant, rapid-dividing tumor cells). In fact, the FDA-approved CPT derivatives topotecan and irinotecan are currently used to treat ovarian and colorectal cancers, respectively [24].

Furthermore, the promising results from a Phase I trial have warranted further evaluation of the CPT derivative Diflomotecan in Phase II trials [85]. Other derivatives like Gimatecan, Lurtotecan and Exatecan are also being tested in clinical trials (Table 1). The non-CPT indolocarbazole BMS-250749 showed great anti-tumor activity against preclinical xenograft models [86], but no further evaluation beyond Phase I trials is presently available (Table 2). Another indolocarbazole compound Edotecarin has shown promising anti-tumor activity in xenograft models and it is now advanced to Phase II studies of patients with advanced solid tumors [87]. By contrast, Phenanthroline ARC-111 (topovale)

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was potently against human tumor xenografts and displayed anti-cancer activity in colon and Wilms’ tumors [88]; however, no result from Phase I clinical trials is available owing to profound bone marrow toxicity [89]. To date, indenoisoquinolines are the most promising non-CPT inhibitors in clinical trials. LMP400 (NSC 743400, indotecan) and LMP776 (NSC 725776, indimitecan) show significant anti-tumor activities in animal models and both are being evaluated in Phase I clinical trials for relapsed solid tumors and lymphomas [8,90].

Table 1. CPT derivatives in clinical trials [91].

Name Structure Clinical Trial Malignancy Reference

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Given the observation that CPT-mediated TOP1 inhibition provokes DNA repair activities, a synergistic effect is then anticipated on cancer cells by inhibition of TOP1 and downregulation of DNA repair activities. The rationale for this approach is to accelerate the accumulation of DNA breaks and trigger cellular apoptosis, probably through mitotic catastrophe [92]. Which DNA repair pathways can we exploit? Currently, the major interests are in SSB and DSB repair mechanisms. Indeed, PARP inhibitors can enhance the cytotoxicity of TOP1 inhibitors in cancer cell lines as well as in mouse models [93–96]. Phase I studies of combination therapy using PARP inhibitors veliparib or olaparib (FDA-approved) together with topotecan were carried out in patients with advanced solid tumors but showed some dose-dependent side effects [97,98]. TDP1 can be another potential target because it functions directly downstream of PARP1 in the repair of TOP1 poison-induced DNA lesions [99]. TDP1 inhibitors sensitize cells to CPT treatment in vitro [100,101], however in vivo evaluation is presently unavailable due to unsuitable properties of the compounds [102].

Table 2. Non-CPT derivatives in preclinical and clinical trials [91].

Name Structure Clinical Trial Malignancy Reference

Indolocarbazoles
(Edotecarin,
BMS-250749)

Phase II

(Edotecarin, Pfizer)

Stomach, breast
neoplasms

Preclinical
(BMS-250749)

Anti-tumor activity
in preclinical
xenograft models

[86,87,103]

Phenanthridines
(ARC-111/topovale)

Anti-tumor activity

Preclinical in preclinical [88,89,103]
xenograft models

Indenoisoquinolines
(LMP400, LMP776)

Phase I Lymphomas [8,90,103]

DSB repair can be targeted by either inhibition of DSB signaling or inhibition of HR. ATM and ATR inhibitors can largely increase the sensitivity to CPT in cancer cells [104,105]. This can be explained by the fact that abrogation of the cell cycle arrest will allow cells with unreplicated or unrepaired chromosomes to enter mitosis thereby triggering mitotic catastrophe and cell death. Similarly, CHEK1 and CHEK2 inhibitors are tested in Phase I studies in combination with irinotecan [106,107]. Inhibitors that can directly block HR proteins are very limited [108]. This is partially attributed to the fact that HR genes are often mutated in cancer cells, thus diminishing the enthusiasm for developing HR inhibitors. One diterpenoid compound, however, was found to be able to inhibit the function of BRCA1 and render cytotoxicity in human prostate cancer cells [109]. Several RAD51 inhibitors have also been

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identified but have not been tested in cell lines [110]. Inhibition of BRCA1 and RAD51 can be also achieved indirectly by harnessing corresponding kinases [106]. Clearly, defective hMRE11 sensitizes colon cancer cells to CPT treatment [111]. Although MRE11-deficeint tumor xenografts failed to display significant growth inhibition by irinotecan alone, combining thymidine with irinotecan caused a dramatic growth delay [112].

TOP1 inhibitors might be also useful for treating cancers with BRCA1/2 mutations. The successful use of PARP inhibitors in treating BRCA1/2-deficient tumors has ignited a broad interest in searching for synthetic lethality among DNA damage response and repair genes [113,114]. In the PARP-BRCA1/2 example, the accumulation of SSBs by PARP inhibition would lead to the formation of DSBs during replication. In HR-deficient cells, DSBs can only be repaired by illegitimate (toxic) NHEJ—joining one-ended DSBs from different locations—leading to cell death [115,116]. However, resistance to PARP inhibitors can arise in BRCA1-deficient tumors during treatment from either genetic reversion of BRCA1 mutations or the loss of NHEJ [117–122]. Therefore, it would be beneficial to explore the possibility of developing a similar synthetic lethal strategy to use TOP1 inhibitors in the treatment of BRCA1/2-deficient tumors.

Figure 2. An overview of the effects of TOP1 inhibition is provided. Inhibitors and key DNA repair factors are highlighted.

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Conclusions

Trapping of TOP1 by inhibitors generates SSBs and DSBs that are repaired by their corresponding repair pathways (Figure 2). Therefore, developing effective TOP1 inhibitors not only provides powerful tools to study DNA replication and repair but also establishes a foundation to devise new synthetic lethal strategies for efficient cancer treatments. The accumulation of DNA strand breaks (SSBs and DSBs) by TOP1 inhibition in HR-deficient tumor cells is expected to enhance cytotoxicity. However, increased DNA repair activities in cancer cells can make TOP1 inhibitors less effective, so silencing of repair pathways in conjunction with the use of TOP1 inhibitors offers an attractive new means for cancer control. Since each tumor is unique, it would be advantageous to identify the individualities of DNA repair pathways or biomarkers reflecting the changes of DNA repair activities in tumor cells [92,123]. This will make it possible to achieve better and predictable prognosis through tailored therapeutic regimens. Given that TOP1 is essential for transcription and DNA replication, future design of novel TOP1 inhibitors and combinational therapy strategies should aim to increase therapeutic efficacy of the inhibitors, thus reducing side effects.

Acknowledgments

The work in the Her laboratory is supported by the NIH grant GM084353.

Author Contributions

Yang Xu and Chengtao Her wrote and revised the article.

Conflicts of Interest

The authors declare that they have no conflicts of interest with the contents of this article.

Please see the following file for the referencesReferences for top paper

From a 2015 Clinical Cancer Research paper:

Phase 1 clinical pharmacology study of F14512, a new polyamine-vectorized anti-cancer drug, in naturally occurring canine lymphoma

Dominique Tierny 1, Francois Serres 1, Zacharie Segaoula 1, Ingrid Bemelmans 1, Emmanuel Bouchaert 1,

Aurelie Petain 2, Viviane Brel 3, Stephane Couffin 4, Thierry Marchal 5, Laurent Nguyen 6, Xavier Thuru 7,

Pierre Ferre 2, Nicolas Guilbaud 8, and Bruno Gomes 9,*

Abstract

Purpose: F14512 is a new topoisomerase II inhibitor containing a spermine moiety that facilitates selective uptake by tumor cells and increases topoisomerase II poisoning. F14512 is currently in Phase I/II clinical trial in patients with acute myeloid leukemia. The aim of this study was to investigate F14512 potential in a new clinical indication. Because of the many similarities between human and dog lymphomas, we sought to determine the tolerance, efficacy, PK/PD relationship of F14512 in this indication, and potential biomarkers that could be translated into human trials. Experimental design: Twenty-three dogs with stage III-IV naturally occurring lymphomas were enrolled in the Phase 1 dose-escalation trial which consisted of three cycles of F14512 intravenous injections. Endpoints included safety and therapeutic efficacy. Serial blood samples and tumor biopsies were obtained for PK/PD and biomarker studies. Results: Five dose levels were evaluated in order to determine the recommended dose. F14512 was well tolerated, with the expected dose-dependent hematological toxicity. F14512 induced an early decrease of tumoral lymph node cells, and a high response rate of 91% (21/23) with 10 complete responses, 11 partial responses, 1 stable disease and 1 progressive disease. Phosphorylation of histone H2AX was studied as a potential pharmacodynamic biomarker of F14512. Conclusions: This trial demonstrated that F14512 can be safely administered to dogs with lymphoma resulting in strong therapeutic efficacy. Additional evaluation of F14512 is needed to compare its efficacy with standards of care in dogs, and to translate biomarker and efficacy findings into clinical trials in humans.

AND From ASCO 2015 Annual Meeting

Survival impact of switching to different topoisomerase I or II inhibitors-based regimens (topo-I or topo-II) in extensive-disease small cell lung cancer (ED-SCLC): supplemental analysis from JCOG0509.

Abstract:

Background: The J0509 (phase III study for chemotherapy-naive ED-SCLC) demonstrated amrubicin plus cisplatin (AP) was inferior to irinotecan plus cisplatin (IP). However, median overall survival (OS) of both AP and IP (15 and 17 mo) was more favorable than those of previous trials (9-12 mo), probably because switching to different topo-I or topo-II in the second-line therapy, especially the use of topo-II in IP arm, was frequent. This analysis aimed to investigate whether observed survival benefit of IP arm can be explained by the treatment switching, and how post-protocol chemotherapy affected the result of J0509. Methods: Two analysis sets from J0509 were used: all randomized 283 pts and 250 pts who received post-protocol chemotherapy. One pt without initiation date of second-line therapy was excluded. A rank-preserving structural failure time (RPSFT) model was used to estimate “causal survival benefit” that would have been observed if all pts had been followed with the same type of regimen as randomized throughout the follow-up period. Additionally, to assess the survival impact of second-line use of topo-II, OS after initiating second-line therapy (OS2) was analyzed by multivariate Cox models. Results: %treatment switching in IP arm and AP arm was 65.2% (92/141) and 43.7% (62/142). By RPSFT model, estimated OS excluding the effect of the treatment switching was 2.7-fold longer in IP (topo-I) arm than AP (topo-II) arm. This causal survival benefit was stronger than the original report of J0509 (nearly 1.4-fold extension by Cox model), indicating that re-challenging topo-I in IP arm appeared beneficial. The multivariate Cox analysis for OS2 (n = 250) revealed second-line use of topo-II was detrimental (hazard ratio, 1.5; 95%CI, 1.1-2.1). Among sensitive relapsed pts in IP arm, OS2 was favorable in the following order: irinotecan-based regimen > the other topo-I > topo-II. Conclusions: IP remains the standard therapy. Re-challenging topo-I, especially irinotecan-based topo-I, seemed beneficial for IP-sensitive pts. This result should be confirmed in further investigations with large sample size. Clinical trial information: 000000720.

Below is actively recruiting clinical trials evaluating topoisomerase inhibitors. Shown are only a few trials for a complete list from CancerTrials.gov please see this link:

https://clinicaltrials.gov/ct2/results?term=topoisomerase+inhibitor&recr=Open#wrapper

A service of the U.S. National Institutes of Health

897 studies found for: topoisomerase inhibitor | Open Studies

Include only open studies Exclude studies with Unknown status

Status

Study

Recruiting

A Study of Standard Treatment +/- Enoxaparin in Small Cell Lung Cancer

Condition:	Small Cell Lung Cancer
Interventions:	Drug: cisplatinum or carboplatin and e.g.etoposide.; Drug: cisplatinum or carboplatin and e.g.etoposide+enoxaparin

Recruiting

A Phase I Study of Indenoisoquinolines LMP400 and LMP776 in Adults With Relapsed Solid Tumors and Lymphomas

Conditions:	Neoplasms; Lymphoma
Interventions:	Drug: LMP 400; Drug: LMP 776

Recruiting

A Dose-Ranging Study Evaluating the Efficacy, Safety, and Tolerability of GSK2140944 in the Treatment of Uncomplicated Urogenital Gonorrhea Caused by Neisseria Gonorrhoeae

Condition:	Gonorrhea
Intervention:	Drug: GSK2140944

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Conditions:	Esophageal Cancer; Gastric Cancer
Interventions:	Drug: Selinexor; Drug: Irinotecan

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Condition:	Neuroblastoma Recurrent
Interventions:	Drug: Dasatinib; Drug: Rapamycin; Drug: Irinotecan; Drug: Temozolomide

Unknown ^†

Study of the Farnesyl Transferase Inhibitor, R115777, in Combination With Topotecan (NYU 99-32)

Condition:	Cancer
Interventions:	Drug: R115777 (farnesyl transferase inhibitor); Drug: Topotecan

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Pegylated Irinotecan NKTR 102 in Treating Patients With Relapsed Small Cell Lung Cancer

Condition:	Recurrent Small Cell Lung Carcinoma
Interventions:	Other: Laboratory Biomarker Analysis; Drug: Pegylated Irinotecan; Other: Pharmacological Study

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Interventions:	Drug: mitoxantrone hydrochloride; Drug: etoposide; Drug: cytarabine; Drug: selinexor; Other: laboratory biomarker analysis; Other: pharmacological study

Recruiting

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Conditions:	Childhood Solid Neoplasm; Recurrent Childhood Medulloblastoma; Recurrent Childhood Supratentorial Primitive Neuroectodermal Tumor; Recurrent Neuroblastoma
Interventions:	Drug: Irinotecan Hydrochloride; Other: Laboratory Biomarker Analysis; Other: Pharmacological Study; Drug: WEE1 Inhibitor AZD1775

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PARP Inhibitor BMN-673 and Temozolomide or Irinotecan Hydrochloride in Treating Patients With Locally Advanced or Metastatic Solid Tumors

Conditions:	Metastatic Cancer; Unspecified Adult Solid Tumor
Interventions:	Drug: PARP inhibitor BMN-673; Drug: temozolomide; Drug: irinotecan hydrochloride; Other: pharmacological study; Other: laboratory biomarker analysis

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A Phase II Multicenter, Randomized, Placebo Controlled, Double Blinded Clinical Study of KD018 as a Modulator of Irinotecan Chemotherapy in Patients With Metastatic Colorectal Cancer

Condition:	Colorectal Neoplasms
Interventions:	Drug: KD018; Drug: Irinotecan; Drug: Placebo

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The Efficacy of the 7 Days Tailored Therapy as 2nd Rescue Therapy for Eradication of H. Pylori Infection

Condition:	Helicobacter Infection
Interventions:	Procedure: H. pylori culture and antimicrobial susceptibility testing; Drug: 14 days empirical bismuth quadruple therapy (Proton pump inhibitor); Drug: Metronidazole; Drug: Tetracycline; Drug: tripotassium dicitrate bismuthate; Drug: 7 days tailored therapy Proton Pump Inhibitor; Drug: Moxifloxacin; Drug: Amoxicillin

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Interventions:	Drug: G1T28 + carboplatin/ etoposide; Drug: Placebo + carboplatin/ etoposide

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Trial of Topotecan With VX-970, an ATR Kinase Inhibitor, in Small Cell Lung Cancer

Conditions:	Carcinoma, Non-Small -Cell Lung; Ovarian Neoplasms; Small Cell Lung Carcinoma; Uterine Cervical Neoplasms; Carcinoma, Neuroendocrine
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Condition:	Metastatic Colorectal Cancer
Interventions:	Genetic: UGT1A1 genotyping (6,6); Genetic: UGTIA1 genotyping (6,7); Genetic: UGTIA1 genotyping (7,7); Genetic: UGT1A1 non-genotyping; Drug: bevacizumab (Avastin); Drug: irinotecan; Drug: Leucovorin; Drug: 5-FU

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Condition:	Lymphoma
Interventions:	Drug: Ibrutinib; Drug: Placebo; Drug: Rituximab; Drug: Cyclophosphamide; Drug: Doxorubicin; Drug: Vincristine; Drug: Prednisone (or equivalent)

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Irinotecan Combination Chemotherapy for Refractory or Relapsed Brain Tumor in Children and Adolescents

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A Study To Evaluate PF-04449913 With Chemotherapy In Patients With Acute Myeloid Leukemia or Myelodysplastic Syndrome

Condition:	Acute Myeloid Leukemia
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Recruiting

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Condition:	Acute Myeloid Leukemia (Relapsed/Refractory)
Interventions:	Drug: Selinexor; Drug: Idarubcin; Drug: Cytarabine

ANNE FASSL HTTPS://ORCID.ORG/0000-0003-2777-0585YAN GENGAND PIOTR SICINSKI HTTPS://ORCID.ORG/0000-0002-8859-5234 Authors Info & Affiliations

Are Cyclin D and cdk Inhibitors A Good Target for Chemotherapy?

Posted in Biological Networks, Cancer and Current Therapeutics, CANCER BIOLOGY & Innovations in Cancer Therapy, Cell Biology, Clinical & Translational, Disease Biology, Disease Biology, Small Molecules in Development of Therapeutic Drugs, Drug Toxicity, FDA, FDA Regulatory Affairs, Pharmaceutical Discovery, Pharmaceutical Drug Discovery, tagged anti-cancer therapy, Biology, breast cancer, Cancer - General, CDER, CDK4, CDK6, cell cycle, cell cycle protein, Center for Drug Evaluation and Research, chemotherapeutic toxicity, Clinical trial, cyclin D, DNA, FDA approval, Food and Drug Administration, health, letrazole, Pfizer, research, science, serious adverse event on October 14, 2015| Leave a Comment »

Are Cyclin D and cdk Inhibitors A Good Target for Chemotherapy?

Curator: Stephen J. Williams, Ph.D.

UPDATED 7/12/2022

see below for great review

CDK4 and CDK6 kinases: From basic science to cancer therapy

SCIENCE

14 Jan 2022

Vol 375, Issue 6577

DOI: 10.1126/science.abc1495

Targeting cyclin-dependent kinases

Cyclin-dependent kinases (CDKs), in complex with their cyclin partners, modulate the transition through phases of the cell division cycle. Cyclin D–CDK complexes are important in cancer progression, especially for certain types of breast cancer. Fassl et al. discuss advances in understanding the biology of cyclin D–CDK complexes that have led to new concepts about how drugs that target these complexes induce cancer cell cytostasis and suggest possible combinations to widen the types of cancer that can be treated. They also discuss progress in overcoming resistance to cyclin D–CDK inhibitors and their possible application to diseases beyond cancer. —GKA

Structured Abstract

BACKGROUND

Cyclins and cyclin-dependent kinases (CDKs) drive cell division. Of particular importance to the cancer field are D-cyclins, which activate CDK4 and CDK6. In normal cells, the activity of cyclin D–CDK4/6 is controlled by the extracellular pro-proliferative or inhibitory signals. By contrast, in many cancers, cyclin D–CDK4/6 kinases are hyperactivated and become independent of mitogenic stimulation, thereby driving uncontrolled tumor cell proliferation. Mouse genetic experiments established that cyclin D–CDK4/6 kinases are essential for growth of many tumor types, and they represent potential therapeutic targets. Genetic and cell culture studies documented the dependence of breast cancer cells on CDK4/6. Chemical CDK4/6 inhibitors were synthesized and tested in preclinical studies. Introduction of these compounds to the clinic represented a breakthrough in breast cancer treatment and will likely have a major impact on the treatment of many other tumor types.

ADVANCES

Small-molecule CDK4/6 inhibitors (palbociclib, ribociclib, abemaciclib) showed impressive results in clinical trials for patients with hormone receptor–positive breast cancers. Addition of CDK4/6 inhibitors to standard endocrine therapy substantially extended median progression-free survival and prolonged median overall survival. Consequently, all three CDK4/6 inhibitors have been approved for treatment of women with advanced or metastatic hormone receptor–positive breast cancers. In the past few years, the renewed interest in CDK4/6 biology has yielded several surprising discoveries. The emerging concept is that CDK4/6 kinases regulate a much wider set of cellular functions than anticipated. Consequently, CDK4/6 inhibitors, beyond inhibiting tumor cell proliferation, affect tumor cells and the tumor environment through mechanisms that are only beginning to be elucidated. For example, inhibition of CDK4/6 affects antitumor immunity acting both on tumor cells and on the host immune system. CDK4/6 inhibitors were shown to enhance the efficacy of immune checkpoint blockade in preclinical mouse cancer models. These new concepts are now being tested in clinical trials.

OUTLOOK

Palbociclib, ribociclib, and abemaciclib are being tested in more than 300 clinical trials for more than 50 tumor types. These trials evaluate CDK4/6 inhibitors in combination with a wide range of therapeutic compounds that target other cancer-relevant pathways. Several other combination treatments were shown to be efficacious in preclinical studies and will enter clinical trials soon. Another CDK4/6 inhibitor, trilaciclib, is being tested for its ability to shield normal cells of the host from cytotoxic effects of chemotherapy. New CDK4/6 inhibitors have been developed and are being assessed in preclinical and clinical trials. The major impediment in the therapeutic use of CDK4/6 inhibitors is that patients who initially respond to treatment often develop resistance and eventually succumb to the disease. Moreover, a substantial fraction of tumors show preexisting, intrinsic resistance to CDK4/6 inhibitors. One of the main challenges will be to elucidate the full range of resistance mechanisms. Even with the current, limited knowledge, one can envisage the principles of new, improved approaches to overcome known resistance mechanisms. Another largely unexplored area for future study is the possible involvement of CDK4/6 in other pathologic states beyond cancer. This will be the subject of intense studies, and it may extend the utility of CDK4/6 inhibitors to the treatment of other diseases.

Targeting cyclin D–CDK4/6 for cancer treatment.

D-cyclins (CycD) activate CDK4 and CDK6 in G₁ phase of the cell cycle and promote cell cycle progression by phosphorylating the retinoblastoma protein RB1. RB1 inhibits E2F transcription factors; phosphorylation of RB1 activates E2F-driven transcription. In many cancers, CycD-CDK4/6 is constitutively activated and drives uncontrolled cell proliferation. The development of small-molecule CDK4/6 inhibitors provided a therapeutic tool to repress constitutive CycD-CDK4/6 activity and to inhibit cancer cell proliferation. As with several targeted therapies, tumors eventually develop resistance and resume cell proliferation despite CDK4/6 inhibition. New combination treatments, involving CDK4/6 inhibitors plus inhibition of other pathways, are being tested in the clinic to delay or overcome the resistance.

Abstract

Cyclin-dependent kinases 4 and 6 (CDK4 and CDK6) and their activating partners, D-type cyclins, link the extracellular environment with the core cell cycle machinery. Constitutive activation of cyclin D–CDK4/6 represents the driving force of tumorigenesis in several cancer types. Small-molecule inhibitors of CDK4/6 have been used with great success in the treatment of hormone receptor–positive breast cancers and are in clinical trials for many other tumor types. Unexpectedly, recent work indicates that inhibition of CDK4/6 affects a wide range of cellular functions such as tumor cell metabolism and antitumor immunity. We discuss how recent advances in understanding CDK4/6 biology are opening new avenues for the future use of cyclin D–CDK4/6 inhibitors in cancer treatment.

Cyclin D1, the activator of CDK4 and CDK6, was discovered in the early 1990s (1, 2). The role of cyclin D1 in oncogenesis was already evident at the time of its cloning, as it was also identified as the protein product of the PRAD1 oncogene, which is rearranged and overexpressed in parathyroid adenomas (3), and of the BCL1 oncogene, which is rearranged in B-lymphocytic malignancies (4). Subsequently, the remaining two D-type cyclins, D2 and D3, were discovered on the basis of their homology to cyclin D1 (1).

Cyclins serve as regulatory subunits of cyclin-dependent kinases (CDKs) (5). Shortly after the discovery of D-cyclins, CDK4 and CDK6 were identified as their kinase partners (6). Mouse gene knockout studies revealed that CDK4 and CDK6 play redundant roles in development, and combined ablation of CDK4 and CDK6 was found to result in embryonic lethality (7). The essentially identical phenotype was seen in cyclin D–knockout mice, thereby confirming the role of D-cyclins as CDK4/6 activators in vivo (8). Surprisingly, these analyses revealed that many normal nontransformed mammalian cell types can proliferate without any cyclin D–CDK4/6 activity (7, 8).

CDK4 and CDK6 are expressed at constant levels throughout the cell cycle. By contrast, D-cyclins are labile proteins that are transcriptionally induced upon stimulation of cells with growth factors. For this reason, D-cyclins are regarded as links between the cellular environment and the cell cycle machinery (6).

Cell cycle inhibitors play an important role in regulating the activity of cyclin D–CDK4/6 (Fig. 1). The INK inhibitors (p16^INK4A, p15^INK4B, p18^INK4C, p19^INK4D) bind to CDK4 or CDK6 and prevent their interaction with D-type cyclins, thereby inhibiting cyclin D–CDK4/6 kinase activity. By contrast, KIP/CIP inhibitors (p27^KIP1, p57^KIP2, p21^CIP1), which inhibit the activity of CDK2-containing complexes, serve as assembly factors for cyclin D–CDK4/6 (6, 9). This was demonstrated by the observation that mouse fibroblasts devoid of p27^KIP1 and p21^CIP1 fail to assemble cyclin D–CDK4/6 complexes (10).

Fig. 1. Molecular events governing progression through the G₁ phase of the cell cycle.

The mammalian cell cycle can be divided into G₁, S (DNA synthesis), G₂, and M (mitosis) phases. During G₁ phase, cyclin D (CycD)–CDK4/6 kinases together with cyclin E (CycE)–CDK2 phosphorylate the retinoblastoma protein RB1. This activates the E2F transcriptional program and allows entry of cells into S phase. Members of the INK family of inhibitors (p16^INK4A, p15^INK4B, p18^INK4C, and p19^INK4D) inhibit cyclin D–CDK4/6; KIP/CIP proteins (p21^CIP1, p27^KIP1, and p57^KIP2) inhibit cyclin E–CDK2. Cyclin D–CDK4/6 complexes use p27^KIP1 and p21^CIP1 as “assembly factors” and sequester them away from cyclin E–CDK2, thereby activating CDK2. Proteins that are frequently lost or down-regulated in cancers are marked with green arrows, overexpressed proteins with red arrows.

p27^KIP1 can bind cyclin D–CDK4/6 in an inhibitory or noninhibitory mode, depending on p27^KIP1 phosphorylation status. Cyclin D–p27^KIP1-CDK4/6 complexes are catalytically inactive unless p27^KIP1 is phosphorylated on Tyr⁸⁸ and Tyr⁸⁹ (11). Two molecular mechanisms may explain this switch. First, Tyr⁸⁸/Tyr⁸⁹ phosphorylation may dislodge the helix of p27^KIP1 from the CDK active site and allow adenosine triphosphate (ATP) binding (12). Second, the presence of tyrosine-unphosphorylated p27^KIP1 within the cyclin D–CDK4 complex prevents the activating phosphorylation of CDK4’s T-loop by the CDK-activating kinase (CAK) (12). Brk has been identified as a physiological kinase of p27^KIP1 (13); Abl and Lyn can phosphorylate p27^KIP1 in vitro, but their in vivo importance remains unclear (11, 14).

The activity of cyclin D–CDK4/6 is also regulated by proteolysis. Cyclin D1 is an unstable protein with a half-life of less than 30 min. At the end of G₁ phase, cyclin D1 is phosphorylated at Thr²⁸⁶ by GSK3β (15). This facilitates association of cyclin D1 with the nuclear exportin CRM1 and promotes export of cyclin D1 from the nucleus to the cytoplasm (16). Subsequently, phosphorylated cyclin D1 becomes polyubiquitinated by E3 ubiquitin ligases, thereby targeting it for proteasomal degradation. Several substrate receptors of E3 ubiquitin ligases have been implicated in recognizing phosphorylated cyclin D1, including F-box proteins FBXO4 (along with αB crystallin), FBXO31, FBXW8, β-TrCP1/2, and SKP2 (17). The anaphase-promoting complex/cyclosome (APC/C) was also proposed to target cyclin D1 while F-box proteins FBXL2 and FBXL8 target cyclins D2 and D3 (17, 18). Surprisingly, the level and stability of cyclin D1 was unaffected by depletion of several of these proteins, indicating that some other E3 plays a rate-limiting role in cyclin D1 degradation (19). Indeed, recent studies reported that D-cyclins are ubiquitinated and targeted for proteasomal degradation by the E3 ubiquitin ligase CRL4, which uses AMBRA1 protein as its substrate receptor (20–22).

Cyclin D–CDK4/6 in cancer

Genomic aberrations of the cyclin D1 gene (CCND1) represent frequent events in different tumor types. The t(11;14)(q13;q32) translocation juxtaposing CCND1 with the immunoglobulin heavy-chain (IGH) locus represents the characteristic feature of mantle-cell lymphoma and is frequently observed in multiple myeloma or plasma cell leukemia (23, 24). Amplification of CCND1 is seen in many other malignancies—for example, in 13 to 20% of breast cancers (23, 24), more than 40% of head and neck squamous cell carcinomas, and more than 30% of esophageal squamous cell carcinomas (23). A higher proportion of cancers (e.g., up to 50% of mammary carcinomas) overexpress cyclin D1 protein (24). Also, cyclins D2 and D3, CDK4, and CDK6 are overexpressed in various tumor types (5, 9). Cyclin D–CDK4/6 can also be hyperactivated through other mechanisms such as deletion or inactivation of INK inhibitors, most frequently p16^INK4A (5, 9, 23). Altogether, a very large number of human tumors contain lesions that hyperactivate cyclin D–CDK4/6 (5).

An oncogenic role for cyclin D–CDK4/6 has been supported by mouse cancer models. For example, targeted overexpression of cyclin D1 in mammary glands of transgenic mice led to the development of mammary carcinomas (25). Also, overexpression of cyclin D2, D3, or CDK4, or loss of p16^INK4a resulted in tumor formation (9).

Conversely, genetic ablation of D-cyclins, CDK4, or CDK6 decreased tumor sensitivity (9). For instance, Ccnd1– or Cdk4-null mice, or knock-in mice expressing kinase-inactive cyclin D1–CDK4/6, were resistant to develop human epidermal growth factor receptor 2 (HER2)–driven mammary carcinomas (26–29). An acute, global shutdown of cyclin D1 in mice bearing HER2-driven tumors arrested tumor growth and triggered tumor-specific senescence while having no obvious impact on normal tissues (30). Likewise, an acute ablation of CDK4 arrested tumor cell proliferation and triggered tumor cell senescence in a KRAS-driven non–small-cell lung cancer (NSCLC) mouse model (31). These observations indicated that CDK4 and CDK6 might represent excellent therapeutic targets in cancer treatment.

CDK4/6 functions in cell proliferation and oncogenesis

The best-documented function of cyclin D–CDK4/6 in driving cell proliferation is phosphorylation of the retinoblastoma protein, RB1, and RB-like proteins, RBL1 and RBL2 (5, 6) (Fig. 1). Unphosphorylated RB1 binds and inactivates or represses E2F transcription factors. According to the prevailing model, phosphorylation of RB1 by cyclin D–CDK4/6 partially inactivates RB1, leading to release of E2Fs and up-regulation of E2F-transcriptional targets, including cyclin E. Cyclin E forms a complex with its kinase partner, CDK2, and completes full RB1 phosphorylation, leading to activation of the E2F transcriptional program and facilitating S-phase entry (5, 6). In normal, nontransformed cells, the activity of cyclin D–CDK4/6 is tightly regulated by the extracellular mitogenic milieu. This links inactivation of RB1 with mitogenic signals. In cancer cells carrying activating lesions in cyclin D–CDK4/6, the kinase is constitutively active, thereby decoupling cell division from proliferative and inhibitory signals (5).

This model has been questioned by the demonstration that RB1 exists in a monophosphorylated state throughout G₁ phase and becomes inactivated in late G₁ by cyclin E–CDK2, which “hyperphosphorylates” RB1 on multiple residues (32). However, recent single-cell analyses revealed that cyclin D–CDK4/6 activity is required for the hyperphosphorylation of RB1 throughout G₁, whereas cyclin E/A–CDK maintains RB1 hyperphosphorylation in S phase (33). Moreover, phosphorylation of RB1 by cyclin D–CDK4/6 was shown to be required for normal cell cycle progression (34).

In addition to this kinase-dependent mechanism, up-regulation of D-cyclin expression and formation of cyclin D–CDK4/6 complexes lead to redistribution of KIP/CIP inhibitors from cyclin E–CDK2 complexes (which are inhibited by these proteins) to cyclin D–CDK4/6 (which use them as assembly factors), thereby activating the kinase activity of cyclin E–CDK2 (6). Cyclin E–CDK2 in turn phosphorylates RB1 and other cellular proteins and promotes cell cycle progression.

Cyclin D1–CDK4/6 directly phosphorylates, stabilizes, and activates the transcription factor FOXM1. This promotes cell cycle progression and protects cancer cells from entering senescence (35). Cyclin D–CDK4 also phosphorylates and inactivates SMAD3, which mediates transforming growth factor–β (TGF-β) antiproliferative response. CDK4/6-dependent phosphorylation of SMAD3 inhibits its transcriptional activity and disables the ability of TGF-β to induce cell cycle arrest (36). FZR1/CDH1, an adaptor protein of the APC complex, is another phosphorylation substrate of CDK4. Depletion of CDH1 in human cancer cells partially rescued the proliferative block upon CDK4/6 inhibition, and it cooperated with RB1 depletion in restoring full proliferation (37).

Cyclin D–CDK4/6 also phosphorylates and inactivates TSC2, a negative regulator of mTORC1, thereby resulting in mTORC1 activation. Conversely, inhibition of CDK4/6 led to decreased mTORC1 activity and reduced protein synthesis in cells representing different human tumor types. It was proposed that through TSC2 phosphorylation, activation of cyclin D–CDK4/6 couples cell growth with cell division (38). Consistent with this, the antiproliferative effect of CDK4/6 inhibition was reduced in cells lacking TSC2 (38).

MEP50, a co-regulatory factor of protein arginine-methyltransferase 5 (PRMT5), is phosphorylated by cyclin D1–CDK4. Through this mechanism, cyclin D1–CDK4/6 increases the catalytic activity of PRMT5/MEP50 (39). It was proposed that deregulation of cyclin D1–CDK4 kinase in tumor cells, by increasing PRMT5/MEP50 activity, reduces the expression of CUL4, a component of the E3 ubiquitin-ligase complex, and stabilizes CUL4 targets such as CDT1 (39). In addition, by stimulating PRMT5/MEP50-dependent arginine methylation of p53, cyclin D–CDK4/6 suppresses the expression of key antiproliferative and pro-apoptotic p53 target genes (40). Another study proposed that PRMT5 regulates splicing of the transcript encoding MDM4, a negative regulator of p53. CDK4/6 inhibition reduced PRMT5 activity and altered the pre-mRNA splicing of MDM4, leading to decreased levels of MDM4 protein and resulting in p53 activation. This, in turn, up-regulated the expression of a p53 target, p21^CIP1, that blocks cell cycle progression (41).

During oncogenic transformation of hematopoietic cells, chromatin-bound CDK6 phosphorylates the transcription factors NFY and SP1 and induces the expression of p53 antagonists such as PRMT5, PPM1D, and MDM4 (42). Also, in acute myeloid leukemia cells expressing constitutively activated FLT3, CDK6 binds the promoter region of the FLT3 gene as well as the promoter of PIM1 pro-oncogenic kinase and stimulates their expression. Treatment of FLT3-mutant leukemic cells with a CDK4/6 inhibitor decreased FLT3 and PIM1 expression and triggered cell cycle arrest and apoptosis (43). The relevance of these various mechanisms in the context of human tumors is unclear and requires further study.

Mechanism of action of CDK4/6 inhibitors

Three small-molecule CDK4/6 inhibitors have been extensively characterized in preclinical studies: palbociclib and ribociclib, which are highly specific CDK4/6 inhibitors, and abemaciclib, which inhibits CDK4/6 and other kinases (Table 1). It has been assumed that these compounds act in vivo by directly inhibiting cyclin D–CDK4/6 (9). This simple model has been recently questioned by observations that palbociclib inhibits only cyclin D–CDK4/6 dimers, but not trimeric cyclin D–CDK4/6-p27^KIP1 (44). However, it is unlikely that substantial amounts of cyclin D–CDK4 dimers ever exist in cells, because nearly all cyclin D–CDK4 in vivo is thought to be complexed with KIP/CIP proteins (11, 14, 44). Palbociclib also binds monomeric CDK4 (44). Surprisingly, treatment of cancer cells with palbociclib for 48 hours failed to inhibit CDK4 kinase, despite cell cycle arrest, but it inhibited CDK2 (44). Hence, palbociclib might prevent the formation of active CDK4-containing complexes (through binding to CDK4) and indirectly inhibit CDK2 by liberating KIP/CIP inhibitors. This model needs to be reconciled with several observations. First, treatment of cells with CDK4/6 inhibitors results in a rapid decrease of RB1 phosphorylation on cyclin D–CDK4/6-dependent sites, indicating an acute inhibition of CDK4/6 (45–47). Moreover, CDK4/6 immunoprecipitated from cells can be inhibited by palbociclib (48) and p21^CIP-associated cyclin CDK4/6 kinase is also inhibited by treatment of cells with palbociclib (49). Lastly, CDK2 is dispensable for proliferation of several cancer cell lines (50, 51), hence the indirect inhibition of CDK2 alone is unlikely to be responsible for cell cycle arrest.

Name of compound	IC₅₀	Other known targets	Stage of clinical development
Palbociclib (PD-0332991)	D1-CDK4, 11 nM; D2-CDK6, 15 nM; D3-CDK4, 9 nM		FDA-approved for HR⁺/HER2^– advanced breast cancer in combination with endocrine therapy; phase 2/3 trials for several other tumor types
Ribociclib (LEE011)	D1-CDK4, 10 nM; D3-CDK6, 39 nM		FDA-approved for HR⁺/HER2^– advanced breast cancer in combination with endocrine therapy; phase 2/3 trials for several other tumor types
Abemaciclib (LY2835219)	D1-CDK4, 0.6 to 2 nM; D3-CDK6, 8 nM	Cyclin T1–CDK9, PIM1, HIPK2, CDKL5, CAMK2A, CAMK2D, CAMK2G, GSK3α/β, and (at higher doses) cyclin E/A–CDK2 and cyclin B–CDK1	FDA-approved for early (adjuvant) and advanced HR⁺/HER2^– breast cancer in combination with endocrine therapy; FDA-approved as monotherapy in advanced HR⁺/HER2^– breast cancer; phase 2/3 trials for several other tumor types
Trilaciclib (G1T28)	D1-CDK4, 1 nM; D3-CDK6, 4 nM		FDA-approved for small-cell lung cancer to reduce chemotherapy-induced bone marrow suppression; phase 2/3 trials for other solid tumors
Lerociclib (G1T38)	D1-CDK4, 1 nM; D3-CDK6, 2 nM		Phase 1/2 trials for HR⁺/HER2^– advanced breast cancer and EGFR-mutant non–small-cell lung cancer
SHR6390	CDK4, 12 nM; CDK6, 10 nM		Phase 1/2/3 trials for HR⁺/HER2^– advanced breast cancer and other solid tumors
PF-06873600	CDK4, 0.13 nM (K_i), CDK6, 0.16 nM (K_i)	CDK2, 0.09 nM (K_i)	Phase 2 trials for HR⁺/HER2^– advanced breast cancer and other solid tumors
FCN-437	D1-CDK4, 3.3 nM; D3-CDK6, 13.7 nM		Phase 1/2 trials for HR⁺/HER2^– advanced breast cancer and other solid tumors
Birociclib (XZP-3287)	Not reported		Phase 1/2 trials for HR⁺/HER2^– advanced breast cancer and other solid tumors
HS-10342	Not reported		Phase 1/2 trials for HR⁺/HER2^– advanced breast cancer and other solid tumors
CS3002	Not reported		Phase 1 trial for solid tumors

Table 1. Currently available CDK4/6 inhibitors.

This table lists major inhibitors of CDK4 and CDK6, half-maximal inhibitory concentration (IC₅₀) for different cyclin D–CDK4/6 complexes (if known), other known targets, and the stage of clinical development. K_i, inhibitory constant.

Palbociclib, ribociclib, and abemaciclib were shown to block binding of CDK4 and CDK6 to CDC37, the kinase-targeting subunit of HSP90, thereby preventing access of CDK4/6 to the HSP90-chaperone system (52). Because the HSP90-CDC37 complex stabilizes several kinases (53), these observations suggest that CDK4/6 inhibitors, by disrupting the interaction between CDC37 and CDK4 or CDK6, might promote degradation of CDK4 and CDK6. However, depletion of CDK4/6 is typically not observed upon treatment with CDK4/6 inhibitors (54). More studies are needed to resolve these conflicting reports and to establish how CDK4/6 inhibitors affect the cell cycle machinery in cancer cells.

Validation of CDK4/6 inhibitors as anticancer agents

Consistent with the notion that RB1 represents the major rate-limiting substrate of cyclin D–CDK4/6 in cell cycle progression (55–57), palbociclib, ribociclib, and abemaciclib were shown to block proliferation of several RB1-positive cancer cell lines, but not cell lines that have lost RB1 expression (46, 58, 59). Breast cancer cell lines representing the luminal, estrogen receptor–positive (ER⁺) subtype were shown to be most susceptible to cell proliferation arrest upon palbociclib treatment (45). Palbociclib, ribociclib, abemaciclib, and another CDK4/6 inhibitor, lerociclib, were demonstrated to display potent antitumor activity in xenografts of several tumor types, including breast cancers (46, 60–62). Palbociclib and abemaciclib cross the blood-brain barrier and inhibit growth of intracranial glioblastoma (GBM) xenografts, with abemaciclib being more efficient in reaching the brain (63, 64). Recently, additional CDK4/6 inhibitors were shown to exert therapeutic effects in mouse xenograft models of various cancer types, including SHR6390 (65), FCN-437 (66), and compound 11 (67); the latter two were reported to cross the blood-brain barrier. In most in vivo studies, the therapeutic effect was dependent on expression of intact RB1 protein in tumor cells (46, 63). However, antitumor effects of palbociclib were also reported in bladder cancer xenografts independently of RB1 status; this was attributed to decreased phosphorylation of FOXM1 (68).

Tumor cell senescence upon CDK4/6 inhibition

In addition to blocking cell proliferation, inhibition of CDK4/6 can also trigger tumor cell senescence (63), which depends on RB1 and FOXM1 (35, 54). The role of RB1 in enforcing cellular senescence is well established (69). In addition, cyclin D–CDK4/6 phosphorylates and activates FOXM1, which has anti-senescence activity (35, 70). Senescence represents a preferred therapeutic outcome to cell cycle arrest, as it may lead to a durable inhibition of tumor growth.

It is not clear what determines the extent of senescence upon treatment of cancer cells with CDK4/6 inhibitors. A recent study showed that inhibition of CDK4/6 leads to an RB1-dependent increase in reactive oxygen species (ROS) levels, resulting in activation of autophagy, which mitigates the senescence of breast cancer cells in vitro and in vivo (71). Co-treatment with palbociclib plus autophagy inhibitors strongly augmented the ability of CDK4/6 inhibitors to induce tumor cell senescence and led to sustained inhibition of cancer cell proliferation in vitro and of xenograft growth in vivo (71). Decreased mTOR signaling after long-term CDK4/6 inhibition was shown to be essential for the induction of senescence in melanoma cells, and activation of mTORC1 overrode palbociclib-induced senescence (72). Others postulated that expression of the chromatin-remodeling enzyme ATRX and degradation of MDM2 determines the choice between quiescence and senescence upon CDK4/6 inhibition (73). Inhibition of CDK4 causes dissociation of the deubiquitinase HAUSP/USP7 from MDM2, thereby driving autoubiquitination and proteolytic degradation of MDM2, which in turn promotes senescence. This mechanism requires ATRX, which suggests that expression of ATRX can be used to predict the senescence response (73). Two additional proteins that play a role in this process are PDLIM7 and type II cadherin CDH18. Expression of CDH18 correlated with a sustained response to palbociclib in a phase 2 trial for patients with liposarcoma (74).

Markers predicting response to CDK4/6 inhibition

Only tumors with intact RB1 respond to CDK4/6 inhibitor treatment by undergoing cell cycle arrest or senescence (9, 58). In addition, “D-cyclin activating features” (CCND1 translocation, CCND2 or CCND3 amplification, loss of the CCND1-3 3′-untranslated region, and deletion of FBXO31 encoding an F-box protein implicated in cyclin D1 degradation) were shown to confer a strong response to abemaciclib in cancer cell lines (58). Moreover, co-deletion of CDKN2A and CDKN2C (encoding p16^INK4A/p19^ARF and p18^INK4C, respectively) confers palbociclib sensitivity in glioblastoma (75). Thr¹⁷² phosphorylation of CDK4 and Tyr⁸⁸ phosphorylation of p27^KIP1 (both associated with active cyclin D–CDK4) correlate with sensitivity of breast cancer cell lines or tumor explants to palbociclib (76, 77). Surprisingly, in PALOMA-1, PALOMA-2, and PALOMA-3 trials (78–80), and in another independent large-scale study (81), CCND1 gene amplification or elevated levels of cyclin D1 mRNA or protein were not predictive of palbociclib efficacy. Conversely, overexpression of CDK4, CDK6, or cyclin E1 is associated with resistance of tumors to CDK4/6 inhibitors (see below).

Synergy of CDK4/6 inhibitors with other compounds

Several preclinical studies have documented the additive or synergistic effects of combining CDK4/6 inhibitors with inhibitors of the receptor tyrosine kinases as well as phosphoinositide 3-kinase (PI3K), RAF, or MEK (Table 2). This synergism might be because these pathways impinge on the cell cycle machinery through cyclin D–CDK4/6 (82–86). In some cases, the effect was seen in the presence of specific genetic lesions, such as EGFR, BRAF^V600E, KRAS, and PIK3CA mutations (59, 87–89) (Table 2). When comparing different dosing regimens, continuous treatment with a MEK inhibitor with intermittent palbociclib resulted in more complete tumor responses than other combination schedules (90). Treatment with CDK4/6 inhibitors sensitized cancer cells to ionizing radiation (63) or cisplatin (68). The synergism with platinum-based chemotherapy was attributed to the observation that upon this treatment, CDK6 phosphorylates and stabilizes the FOXO3 transcription factor, thereby promoting tumor cell survival. Consequently, inhibition of CDK6 increases platinum sensitivity by enhancing tumor cell death (91).

CDK4/6 inhibitor	Synergistic target	Inhibitor	Disease
Palbociclib	PI3K	Taselisib, pictilisib	PIK3CA mutant TNBC
	AR	Enzalutamide	Androgen receptor–positive TNBC
	EGFR	Erlotinib	TNBC, esophageal squamous cell carcinoma
	RAF	PLX4720	BRAF-V600E mutant melanoma
	MEK	Trametinib	KRAS mutant colorectal cancer
	MEK	PD0325901 (mirdametinib)	KRAS or BRAFV600E mutant colorectal cancer
	MEK	MEK162 (binimetinib)	KRAS mutant colorectal cancer
	MEK	AZD6244 (selumetinib)	Pancreatic ductal adenocarcinoma
	PI3K/mTOR	BEZ235 (dactolisib), AZD0855, GDC0980 (apitolisib)	Pancreatic ductal adenocarcinoma
	IGF1R/InsR	BMS-754807	Pancreatic ductal adenocarcinoma
	mTOR	Temsirolimus	Pancreatic ductal adenocarcinoma
	mTOR	AZD2014 (vistusertib)	ER⁺ breast cancer
	mTOR	MLN0128 (sapanisertib)	Intrahepatic cholangiocarcinoma
	mTOR	Everolimus	Melanoma, glioblastoma
Ribociclib	PI3K	GDC-0941 (pictilisib), BYL719 (alpelisib)	PIK3CA mutant breast cancer
	PDK1	GSK2334470	ER⁺ breast cancer
	EGFR	Nazartinib	EGFR-mutant lung cancer
	RAF	Encorafenib	BRAF-V600E mutant melanoma
	mTOR	Everolimus	T-ALL
	Inflammation	Glucocorticoid dexamethasone	T-ALL
	γ-Secretase	Compound E	T-ALL
Abemaciclib	HER2	Trastuzumab	HER2⁺ breast cancer
	EGFR and HER2	Lapatinib	HER2⁺ breast cancer
	RAF	LY3009120, vemurafenib	KRAS mutant lung or colorectal cancer, NRAS or BRAF-V600E mutant melanoma
		Temozolomide (alkylating agent)	Glioblastoma

Table 2. Combination treatments that demonstrated synergy with CDK4/6 inhibitors in preclinical studies.

TNBC, triple-negative breast cancer; AR, androgen receptor; ER⁺, estrogen receptor–positive; T-ALL, T cell acute lymphoblastic leukemia; HER2⁺, human epidermal growth factor receptor 2–positive; PI3K, phosphoinositide 3-kinase; EGFR, epidermal growth factor receptor; IGF1R, insulin-like growth factor 1 receptor, InsR, insulin receptor.

In several instances, co-treatment with CDK4/6 inhibitors prevented the development of resistance to other compounds or inhibited the proliferation of resistant tumor cells. Co-treatment of melanoma patient-derived xenografts (PDXs) with ribociclib plus the RAF inhibitor encorafenib delayed or prevented development of encorafenib resistance (92). PDXs that acquired encorafenib resistance remained sensitive to the combination of encorafenib plus ribociclib (59). Treatment of BRAF^V600E-mutant melanoma xenografts with palbociclib plus the BRAF^V600E inhibitor PLX4720 prevented development of resistance (89). BRAF^V600E-mutant melanoma cell lines that acquired resistance to the BRAF^V600E inhibitor vemurafenib remained sensitive to palbociclib or abemaciclib, and xenografts underwent senescence and tumor regression upon CDK4/6 inhibition (72, 93). Treatment of ALK-mutant, ALK kinase inhibitor–resistant neuroblastoma xenografts with palbociclib restored the sensitivity to these compounds (94). A combination of PI3K and CDK4/6 inhibitors overcame the intrinsic and acquired resistance of breast cancers to PI3K inhibitors and resulted in regression of PIK3CA-mutant xenografts (88).

Up-regulation of cyclin D1 expression was shown to mediate acquired resistance of HER2⁺ tumors to anti-HER2 therapies in a mouse breast cancer model (95). Treatment of mice bearing trastuzumab-resistant tumors or PDXs of resistant HER2⁺ mammary carcinomas with abemaciclib restored the sensitivity of tumors to HER2 inhibitors and inhibited tumor cell proliferation. Moreover, in the case of treatment-naïve tumors, co-administration of abemaciclib significantly delayed the development of resistance to anti-HER2 therapies (95).

Several anticancer treatments, such as chemotherapy, target dividing cells. Because CDK4/6 inhibitors block tumor cell proliferation, they might impede the effects of chemotherapy. Indeed, several reports have documented that co-administration of CDK4/6 inhibitors antagonized the antitumor effects of compounds that act during S phase (doxorubicin, gemcitabine, methotrexate, mercaptopurine) or mitosis (taxanes) (96, 97). However, some authors reported synergistic effects (98, 99), although the molecular underpinnings are unclear.

A recent report documented that administration of CDK4/6 inhibitors prior to taxanes inhibited tumor cell proliferation and impeded the effect of taxanes (100). By contrast, administration of taxanes first (or other chemotherapeutic compounds that act on mitotic cells or cells undergoing DNA synthesis), followed by CDK4/6 inhibitors, had a strong synergistic effect. The authors showed that by repressing the E2F-dependent transcriptional program, CDK4/6 inhibitors impaired the expression of genes required for DNA-damage repair via homologous recombination. Because treatment of cancer cells with chemotherapy triggers DNA damage, the impairment of DNA-damage repair induced cytotoxicity, thereby explaining the synergistic effect (100).

Cells with impaired homologous recombination rely on poly-(ADP-ribose) polymerase (PARP) for double-stranded DNA-damage repair, which renders them sensitive to PARP inhibition. Indeed, a strong synergistic effect has been demonstrated between CDK4/6 inhibitors and PARP inhibitors in PDX-derived cell lines (100). Such synergy was also reported for ovarian cancer cells (101). Another study found that inhibition of CDK4/6 resulted in down-regulation of PARP levels (102).

Protection against chemotherapy-induced toxicity

Administration of palbociclib to mice induced reversible quiescence in hematopoietic stem/progenitor cells (HSPCs). This effect protected mice from myelosuppression after total-body irradiation. Moreover, treatment of tumor-bearing mice with CDK4/6 inhibitors together with irradiation mitigated radiation-induced toxicity without compromising the therapeutic effect (103). Co-administration of a CDK4/6 inhibitor, trilaciclib, with cytotoxic chemotherapy (5-FU, etoposide) protected animals from chemotherapy-induced exhaustion of HSPCs, myelosuppression, and apoptosis of bone marrow (60, 104). These observations led to phase 2 clinical trial, which evaluated the effects of trilaciclib administered prior to etoposide and carboplatin for treatment of small-cell lung cancer. Trilaciclib improved myelopreservation while having no adverse effect on antitumor efficacy (105). A similar phase 2 clinical trial investigating trilaciclib in combination with gemcitabine and carboplatin chemotherapy in patients with metastatic triple-negative breast cancer (TNBC) did not observe a significant difference in myelosuppression. However, this study demonstrated an overall survival benefit of the combination therapy (106, 107).

Metabolic function of CDK4/6 in cancer cells

The role of CDK4/6 in tumor metabolism is only starting to be appreciated (Fig. 2A). Treatment of pancreatic cancer cells with CDK4/6 inhibitors was shown to induce tumor cell metabolic reprogramming (108). CDK4/6 inhibition increased the numbers of mitochondria and lysosomes, activated mTOR, and increased the rate of oxidative phosphorylation, likely through an RB1-dependent mechanism (108). Combined inhibition of CDK4/6 and mTOR strongly suppressed tumor cell proliferation (108). Moreover, CDK4/6 can phosphorylate and inactivate TFEB, the master regulator of lysosomogenesis, and through this mechanism reduce lysosomal numbers. Conversely, CDK4/6 inhibition activated TFEB and increased the number of lysosomes (109). Another mechanism linking CDK4/6 and lysosomes was provided by the observation that treatment of TNBC cells with CDK4/6 inhibitors decreased mTORC1 activity and impaired the recruitment of mTORC1 to lysosomes (110). Consistent with the idea that mTORC1 inhibits lysosomal biogenesis, CDK4/6 inhibition increased the number of lysosomes in tumor cells. Because an increased lysosomal biomass underlies some cases of CDK4/6 inhibitor resistance (see below) (111), stimulation of lysosomogenesis by CDK4/6 inhibitors might limit their clinical efficacy by inducing resistance.

Fig. 2. CDK4 and CDK6: More than cell cycle kinases.

Although the role of CDK4 and CDK6 in cell cycle progression has been well documented, both kinases regulate several other functions that are only now starting to be unraveled. (A) Inhibition of CDK4/6 (CDK4/6i) affects lysosome and mitochondrial numbers as well as oxidative phosphorylation. Cyclin D3–CDK6 phosphorylates glycolytic enzymes 6-phosphofructokinase (PFKP) and pyruvate kinase M2 (PKM2), thereby controlling ROS levels via the pentose phosphate (PPP) and serine synthesis pathways. (B) Inhibition of CDK4/6 affects antitumor immunity, acting both within cancer cells and on the immune system of the host. In tumor cells, inhibition of CDK4/6 impedes expression of an E2F target, DNA methyltransferase (DNMT). DNMT inhibition reduces methylation of endogenous retroviral genes (ERV) and increases intracellular levels of double-stranded RNA (dsRNA) (*114*). In effector T cells, inhibition of CDK4/6 stimulates NFAT transcriptional activity and enhances secretion of IFN-γ and interleukin 2 (IL-2) (*115*).

Lastly, CDK4/6 inhibition impaired lysosomal function and the autophagic flux in cancer cells. It was argued that this lysosomal dysfunction was responsible for the senescent phenotype in CDK4/6 inhibitor–treated cells (110). Because lysosomes are essential for autophagy, the authors co-treated TNBC xenografts with abemaciclib plus an AMPK activator, A769662 (which induces autophagy), and found that this led to cancer cell death and subsequent regression of tumors (110).

Cyclin D3–CDK6 phosphorylates and inhibits two rate-limiting glycolytic enzymes, 6-phosphofructokinase and pyruvate kinase M2. This redirects glycolytic intermediates into the pentose phosphate pathway (PPP) and serine synthesis pathway. Through this mechanism, cyclin D3–CDK6 promotes the production of reduced nicotinamide adenine dinucleotide phosphate (NADPH) and reduced glutathione (GSH) and helps to neutralize ROS (112). Treatment of tumors expressing high levels of cyclin D3–CDK6 (such as leukemias) with CDK4/6 inhibitors reduced the PPP- and serine-synthesis pathway flow, thereby depleting the antioxidants NADPH and GSH. This increased ROS levels and triggered tumor cell apoptosis (112).

Another link between cyclin D–CDK4/6 in metabolism and cancer was provided by the observation that livers of obese/diabetic mice up-regulate cyclin D1 expression (113). Treatment of these mice with an antidiabetic compound, metformin, reduced liver cyclin D1 levels and largely protected mice against development of hepatocellular carcinoma. Also, genetic ablation of cyclin D1 protected obese/diabetic mice from liver cancer, and administration of palbociclib inhibited liver cancer progression. These treatments had no effect on tumors in nonobese animals (113). These observations raise the possibility of using antidiabetic compounds with CDK4/6 inhibitors for treatment of liver cancers in obese patients.

CDK4/6 inhibitors and antitumor immune responses

Several recent reports have started to unravel how inhibition of CDK4/6 influences antitumor immune responses, acting both on tumor cells as well as on the tumor immune environment (Fig. 2B). Treatment of breast cancer–bearing mice or breast cancer cells with abemaciclib activated expression of endogenous retroviral elements in tumor cells, thereby increasing the levels of double-stranded RNA. This, in turn, stimulated production of type III interferons and increased presentation of tumor antigens. Hence, CDK4/6 inhibitors, by inducing viral gene expression, trigger antiviral immune responses that help to eliminate the tumor (114).

Inhibition of CDK4/6 also affects the immune system by impeding the proliferation of CD4⁺FOXP3⁺ regulatory T cells (T_regs), which normally inhibit the antitumor response. Because cytotoxic CD8⁺ T cells are less affected by CDK4/6 inhibition, abemaciclib treatment decreases the T_reg/CD8⁺ ratio of intratumoral T cells and facilitates tumor cell killing by cytotoxic CD8⁺ T cells (114).

Inhibition of CDK4/6 also resulted in activation of T cells through derepression of NFAT signaling. NFAT4 (and possibly other NFATs) are phosphorylated by cyclin D3–CDK6 (115). Inhibition of CDK4/6 decreased phosphorylation of NFATs, resulting in their nuclear translocation and enhanced transcriptional activity. This caused up-regulation of NFAT targets, resulting in T cell activation, which enhanced the antitumor immune response. In addition, CDK4/6 inhibitors increased the infiltration of effector T cells into tumors, likely because of elevated levels of chemokines CXCL9 and CXCL10 after CDK4/6 inhibitor treatment (115). Abemaciclib treatment also induced inflammatory and activated T cell phenotypes in tumors and up-regulated the expression of immune checkpoint proteins CD137, PD-L1, and TIM-3 on CD4⁺ and CD8⁺ cells (116).

CDK4/6 inhibition also caused up-regulation of PD-L1 protein expression in tumor cells (117, 118). This effect was shown to be independent of RB1 status in the tumor. Mechanistically, CDK4/6 phosphorylates and stabilizes SPOP, which promotes PD-L1 polyubiquitination and degradation (118). Cyclin D–CDK4 also represses expression of PD-L1 through RB1. Specifically, cyclin D–CDK4/6-mediated phosphorylation of RB1 on S249/T252 promotes binding of RB1 to NF-κB protein p65, and this represses the expression of a subset NF-κB–regulated genes, including PD-L1 (119).

These observations prompted tests of the efficacy of combining CDK4/6 inhibitors with antibodies that elicit immune checkpoint blockade. Indeed, treatment of mice bearing autochthonous breast cancers, or cancer allografts, with CDK4/6 inhibitors together with anti-PD-1/PD-L1 antibodies enhanced the efficacy of immune checkpoint blockade and led to complete tumor regression in a high proportion of animals (114, 115, 118). Conversely, activation of the cyclin D–CDK4 pathway by genomic lesions in human melanomas correlated with resistance to anti–PD-1 therapy (117).

Some authors did not observe synergy when abemaciclib was administered concurrently with immune checkpoint inhibitors in allograft tumor models (116, 120). However, a strong synergistic antitumor effect was detected when abemaciclib was administered first (and continued) and anti–PD-L1 antibody was administered later. The combined treatment induced immunological memory, as mice that underwent tumor regression were resistant to rechallenge with the same tumor (116). Abemaciclib plus anti–PD-L1 treatment increased infiltration of CD4⁺ and CD8⁺ T cells into tumors, and increased the expression of major histocompatibility complex class I (MHC-I) and MHC-II on tumor cells and on macrophages and MHC-I on dendritic cells (116). In the case of anti–CTLA-4 plus anti–PD-1 treatment in melanoma allograft model, the synergistic effect was observed when immune checkpoint inhibitor treatment was started first, followed by abemaciclib (120).

The synergistic antitumor effect of PI3K and CDK4/6 inhibitors in TNBC is mediated, in part, by enhancement of tumor immunogenicity (121). Combined treatment of TNBC cells with ribociclib plus the PI3K inhibitor apelisib synergistically up-regulated the expression of immune-related pathways in tumor cells, including proteins involved in antigen presentation. Co-treatment of tumor-bearing mice also decreased proliferation of CD4⁺FOXP3⁺ T_reg cells, increased activation of intratumoral CD4⁺ and CD8⁺ T cells, increased the frequency of tumor-infiltrating NKT cells, and decreased the numbers of intratumoral immunosuppressive myeloid-derived suppressor cells. Moreover, combined treatment strongly augmented the response to immune checkpoint therapy with PD-1 and CTLA-4 antibodies (121).

Single-cell RNA sequencing of human melanomas identified an immune resistance program expressed by tumor cells that correlates with T cell exclusion from the tumor mass and immune evasion by tumor cells. The program can predict the response of tumors to immune checkpoint inhibitors. Treatment of human melanoma cells with abemaciclib repressed this program in an RB1-dependent fashion (120).

Together, these findings indicate that CDK4/6 inhibitors may convert immunologically “cold” tumors into “hot” ones. The most pressing issue is to validate these findings in a clinical setting. The utility of combining CDK4/6 inhibitors with PD-1 or PD-L1 antibodies is currently being evaluated in several clinical trials. Note that the effects of CDK4/6 inhibition on the immune system of the host are independent of tumor cell RB1 status, raising the possibility of using CDK4/6 inhibitors to also boost the immune response against RB1-negative tumors.

CDK4/6 inhibitors in clinical trials

Table 3 summarizes major clinical trials with CDK4/6 inhibitors. Given early preclinical data indicating that breast cancers—in particular, the hormone receptor–positive ones—are very sensitive to CDK4/6 inhibition (as discussed above), many clinical trials have focused on this cancer type. Most studies have evaluated CDK4/6 inhibitors administered together with anti-estrogens (the aromatase inhibitors letrozole or anastrozole, or the estrogen receptor antagonist fulvestrant) for treatment of advanced/metastatic HR⁺/HER2^– breast cancers in postmenopausal women. Addition of CDK4/6 inhibitors significantly extended median progression-free survival (78, 122–130) and prolonged median overall survival (131–134). Moreover, abemaciclib has shown clinical activity when administered as a single agent (135). Consequently, palbociclib, ribociclib, and abemaciclib have been approved by the US Food and Drug Administration (FDA) for treatment of patients with advanced/metastatic HR⁺/HER2^– breast cancer (Box 1). A recent phase 3 clinical trial, MonarchE, evaluated abemaciclib plus standard endocrine therapy in treatment of patients with early-stage, high-risk, lymph node–positive HR⁺/HER2^– breast cancer. Addition of abemaciclib reduced the risk of breast cancer recurrence (136). This is in contrast to the similar PALLAS study reported this year, which found no benefit of adding palbociclib to endocrine therapy for women with early-stage breast cancer (137). Analysis of patient populations in these two trials may help to explain the different outcomes. It is also possible that the favorable outcome of the MonarchE study reflects a broader spectrum of kinases inhibited by abemaciclib. The utility of CDK4/6 inhibitors in early-stage breast cancer remains unclear and is being addressed in ongoing clinical trials (PALLAS, PENELOPE-B, EarLEE-1, MonarchE) (138).

CDK4/6 inhibitor	Trial name	Trial details	Treatment	Patients	Outcome	Ref.	Other outcomes
Palbociclib	PALOMA-1	Randomized phase 2	Aromatase inhibitor letrozole alone (standard of care) versus letrozole plus palbociclib	Postmenopausal women with advanced ER⁺/HER2^– breast cancer who had not received any systemic treatment for their advanced disease	Addition of palbociclib markedly increased median PFS from 10.2 months in the letrozole group to 20.2 months in the palbociclib plus letrozole group	(78)	On the basis of this result, palbociclib received a “Breakthrough Therapy” designation status from FDA and was granted accelerated approval, in combination with letrozole, for the treatment of ER⁺/HER2^– metastatic breast cancer
Palbociclib	PALOMA-2	Double-blind phase 3	Palbociclib plus letrozole as first- line therapy	Postmenopausal women with ER⁺/HER2^– breast cancer	Addition of palbociclib strongly increased median PFS: 14.5 months in the placebo- letrozole group versus 24.8 months in the palbociclib-letrozole group	(123)	Palbociclib was equally efficacious in patients with luminal A and B breast cancers, and there was no single biomarker associated with the lack of clinical benefit, except for RB1 loss; CDK4 amplification was associated with endocrine resistance, but this was mitigated by addition of palbociclib; tumors with high levels of FGFR2 and ERBB3 mRNA displayed greater PFS gain after addition of palbociclib (79)
Palbociclib	PALOMA-3	Randomized phase 3	Estrogen receptor antagonist fulvestrant plus placebo versus fulvestrant plus palbociclib	Women with HR⁺/HER2^– metastatic breast cancer that had progressed on previous endocrine therapy	The study demonstrated a substantial prolongation of median PFS in the palbociclib- treated group: 4.6 months in the placebo plus fulvestrant group versus 9.5 months in the palbociclib plus fulvestrant group; addition of palbociclib also extended median overall survival from 28.0 months (placebo-fulvestrant) to 34.9 months (palbociclib- fulvestrant); estimated rate of survival at 3 years was 41% versus 50%, respectively	(124, 125, 135)
Palbociclib	NeoPalAna	Palbociclib in an neoadjuvant setting (i.e., prior to surgery)	Compared the effects of an aromatase inhibitor anastrozole versus palbociclib plus anastrozole on tumor cell proliferation	Women with newly diagnosed clinical stage II/III ER⁺/HER2^– breast cancer	Addition of palbociclib enhanced the antiproliferative effect of anastrozole	(161)
Palbociclib	PALLAS	Randomized phase 3	Palbociclib plus standard endocrine therapy versus endocrine therapy alone	Patients with early (stage 2 or 3), HR⁺/HER2^– breast cancer	Preliminary results indicate that the trial is unlikely to show a statistically significant improvement of invasive disease-free survival	(138)
Palbociclib	PENELOPE-B		Palbociclib in patients with early breast cancer at high risk of recurrence		Ongoing
Ribociclib	MONA LEESA-2	Randomized phase 3	Ribociclib plus letrozole versus placebo plus letrozole	First-line treatment for postmenopausal women with HR⁺/HER2^– recurrent or metastatic breast cancer who had not received previous systemic therapy for advanced disease	At 18 months, PFS was 42.2% in the placebo-letrozole group and 63.0% in the ribociclib- letrozole group	(126)
Ribociclib	MONA LEESA-3	Phase 3	Ribociclib plus fulvestrant	Patients with advanced (metastatic or recurrent) HR⁺/HER2^– breast cancer who have either received no treatment for the advanced disease or previously received a single line of endocrine therapy for the advanced disease	Addition of ribociclib significantly extended median PFS, from 12.8 months (placebo-fulvestrant) to 20.5 months (ribociclib- fulvestrant); overall survival at 42 months was also extended from 45.9% (placebo-fulvestrant) to 57.8% (ribociclib-fulvestrant)	(127, 133)
Ribociclib	MONA LEESA-7	Phase 3 randomized, double-blind	Ribociclib versus placebo together with an anti- estrogen tamoxifen or an aromatase inhibitor (letrozole or anastrozole)	Premenopausal and perimenopausal women with HR⁺/HER2^– advanced breast cancer who had not received previous treatment with CDK4/6 inhibitors	Ribociclib significantly increased median PFS from 13.0 months in the placebo-endocrine therapy group to 23.8 months in the ribociclib-endocrine therapy group; overall survival was also strongly prolonged in the ribociclib group (estimated overall survival at 42 months was 46.0% for the placebo group and 70.2% in the ribociclib group)	(128, 132)
Ribociclib	EarLEE-1	Phase 3 trial	Ribociclib in the treatment of early- stage, high-risk HR⁺/HER2^– breast cancers		Ongoing
Abemaciclib	MONARCH 1	Phase 2 trial	Abemaciclib as a single agent	Women with HR⁺/HER2^– metastatic breast cancer who had progressed on or after prior endocrine therapy and had 1 or 2 chemotherapy regimens in the metastatic setting	Abemaciclib exhibited promising activity in these heavily pretreated patients with poor prognosis; median PFS was 6.0 months and overall survival 17.7 months	(136)	The most common adverse events were diarrhea, fatigue, and nausea (136)
Abemaciclib	MONARCH 2	Double-blind phase 3	Abemaciclib in combination with fulvestrant	Women with HR⁺/HER2^– breast cancer who had progressed while receiving endocrine therapy, or while receiving first-line endocrine therapy for metastatic disease	Addition of abemaciclib significantly increased PFS from 9.3 months in the placebo-fulvestrant to 16.4 in the abemaciclib-fulvestrant group; median overall survival was also extended from 37.3 months to 46.7 months	(129, 134)
Abemaciclib	MONARCH 3	Randomized phase 3 double-blind	Abemaciclib plus an aromatase inhibitor (anastrozole or letrozole)	Postmenopausal women with advanced HR⁺/HER2^– breast cancer who had no prior systemic therapy in the advanced setting	Addition of abemaciclib prolonged PFS from 14.8 months (in the placebo-aromatase inhibitor group) to 28.2 months (abemaciclib-aromatase inhibitor group)	(130, 131)
Abemaciclib	MonarchE	Phase 3 study	Endocrine with or without abemaciclib	Patients with HR⁺/HER2^– lymph node–positive, high-risk early breast cancer	Preliminary analysis indicates that addition of abemaciclib resulted in a significant improvement of invasive disease-free survival and of distant relapse- free survival	(137)
Trilaciclib		Randomized phase 2 study	Chemotherapy alone (gemcitabine and carboplatin), versus concurrent administration of trilaciclib plus chemotherapy, versus administration of trilaciclib prior to chemotherapy (to mitigate the cytotoxic effect of chemotherapy on bone marrow)	Patients with recurrent or metastatic triple-negative breast cancer who had no more than two previous lines of chemotherapy	Addition of trilaciclib did not offer detectable myeloprotection, but resulted in increased overall survival (from 12.8 months in the chemotherapy-only group to 20.1 months in the concurrent trilaciclib and chemotherapy group and 17.8 months in trilaciclib before chemotherapy group)	(162)	The most common adverse events were neutropenia, thrombocytopenia, and anemia (162)

Table 3. Major past clinical trials with CDK4/6 inhibitors.

ER⁺, estrogen receptor–positive; HER2^–, human epidermal growth factor receptor 2–negative; HR⁺, hormone receptor–positive; PFS, progression-free survival. FGFR2, fibroblast growth factor receptor 2; ERBB3, receptor tyrosine-protein kinase erbB-3.

Palbociclib

Approved by FDA in 2016, in combination with fulvestrant for the treatment of hormone receptor–positive, HER2-negative (HR⁺/HER2^–) advanced or metastatic breast cancer in women with disease progression following endocrine therapy. Approved in 2017 for the treatment of HR⁺/HER2^– advanced or metastatic breast cancer in combination with an aromatase inhibitor as initial endocrine-based therapy in postmenopausal women.

Palbociclib is administered at a dose of 125 mg (given orally) daily for 3 weeks followed by 1 week off, or 200 mg daily for 2 weeks followed by 1 week off. The rate-limiting toxicities are neutropenia, thrombocytopenia, and anemia.

Ribociclib

Approved by FDA in 2017, in combination with an aromatase inhibitor as initial endocrine-based therapy for the treatment of postmenopausal women with HR⁺/HER2^– advanced or metastatic breast cancer. In 2018, the FDA expanded the indication for ribociclib in combination with an aromatase inhibitor for pre/perimenopausal women with HR⁺/HER2^– advanced or metastatic breast cancer, as initial endocrine-based therapy. FDA also approved ribociclib in combination with fulvestrant for postmenopausal women with HR⁺/HER2^– advanced or metastatic breast cancer, as initial endocrine-based therapy or following disease progression on endocrine therapy.

Ribociclib is administered at a dose of 600 mg (given orally) daily for 3 weeks followed by 1 week off. The main toxicities are neutropenia and thrombocytopenia.

Abemaciclib

Approved by FDA in 2017, in combination with fulvestrant for women with HR⁺/HER2^– advanced or metastatic breast cancer with disease progression following endocrine therapy. In addition, abemaciclib was approved as monotherapy for women and men with HR⁺/HER2^– advanced or metastatic breast cancer with disease progression following endocrine therapy and prior chemotherapy in the metastatic setting. Approved by FDA in 2018 in combination with an aromatase inhibitor as initial endocrine-based therapy for postmenopausal women with HR⁺/HER2^– advanced or metastatic breast cancer. Approved by FDA in 2021 for adjuvant treatment of early-stage HR⁺/HER2^– breast cancer in combination with endocrine therapy.

Abemaciclib is administered at a dose of 200 mg (given orally) every 12 hours. The dose-limiting toxicity is fatigue. Neutropenia is also observed but is not rate-limiting. Other severe side effects include diarrhea and nausea.

Currently, palbociclib is being used in 164 active or recruiting clinical trials, ribociclib in 69 trials, and abemaciclib in 98 trials for more than 50 tumor types (139). These trials evaluate combinations of CDK4/6 inhibitors with a wide range of compounds (Table 4). Trials with trilaciclib test the benefit of this compound in preserving bone marrow and the immune system.

Additional target	Inhibitor	Immune checkpoint inhibitor	Tumor type	Trial identifier
*Palbociclib*
Aromatase	Letrozole, anastrozole, exemestane		HR⁺ breast cancer, HR⁺ ovarian cancer, metastatic breast cancer, metastatic endometrial cancer	NCT04130152, NCT03054363, NCT03936270, NCT04047758, NCT02692755, NCT02806050, NCT03870919, NCT02040857, NCT04176354, NCT02028507, NCT03220178, NCT02592083, NCT02603679, NCT04256941, NCT03425838, NCT02894398, NCT02297438, NCT02730429, NCT02142868, NCT02942355
LHRH	LHRH agonists: goserelin, leuprolide		HR⁺ breast cancer	NCT03969121, NCT03423199, NCT01723774, NCT02917005, NCT02592746, NCT03628066
ER	ER antagonists: fulvestrant, tamoxifen		HR⁺ breast cancer, metastatic breast cancer	NCT02668666, NCT02738866, NCT03184090, NCT04526028, NCT02513394, NCT03560856, NCT02760030, NCT03079011, NCT03227328, NCT03809988, NCT02764541, NCT03007979, NCT03633331
ER	Selective estrogen receptor degraders (SERDs): G1T48, ZN-c5, SAR439859, AZD9833, GDC-9545		HR⁺ breast cancer	NCT03455270, NCT04546009, NCT04436744, NCT04478266, NCT03560531, NCT03616587, NCT03284957, NCT03332797
ER	Selective estrogen receptor modulator (SERM): bazedoxifene		HR⁺ breast cancer	NCT03820830, NCT02448771
Aromatase + PD-1	Letrozole, anastrozole	Pembrolizumab, nivolumab	Stage IV ER⁺ breast cancer	NCT02778685, NCT04075604
PD-1		Nivolumab, pembrolizumab, MGA012	Liposarcoma	NCT04438824
PD-L1		Avelumab	AR⁺ breast cancer, TNBC, ER⁺/HER2^– metastatic breast cancer	NCT04360941, NCT03147287
EGFR + PD-L1	Cetuximab	Avelumab	Squamous cell carcinoma of the head and neck	NCT03498378
HER2	Tucatinib, trastuzumab, pertuzumab, T-DM1, ZW25		HER2⁺ breast cancer	NCT03530696, NCT03054363, NCT02448420, NCT03709082, NCT03304080, NCT02947685
EGFR/HER2	Neratinib		Advanced solid tumors with EGFR mutation/amplification, HER2 mutation/amplification, HER3/4 mutation, or KRAS mutation	NCT03065387
EGFR	Cetuximab		Metastatic colorectal cancer, squamous cell carcinoma of the head and neck	NCT03446157, NCT02499120
FGFR	Erdafitinib		ER⁺/HER2^–/FGFR-amplified metastatic breast cancer	NCT03238196
FGFR1-3	Rogaratinib		FGFR1-3⁺/HR⁺ breast cancer	NCT04483505
IGF-1R	Ganitumab		Ewing sarcoma	NCT04129151
VEGF1-3 receptors + PD-L1	Axitinib	Avelumab	NSCLC	NCT03386929
RAF	Sorafenib		Leukemia	NCT03132454
MEK	PD-0325901, binimetinib		KRAS mutant NSCLC, TNBC, KRAS and NRAS mutant metastatic or unresectable colorectal cancer	NCT02022982, NCT03170206, NCT04494958, NCT03981614
ERK	Ulixertinib		Advanced pancreatic cancer and other solid tumors	NCT03454035
PI3K	Copanlisib		HR⁺ breast cancer	NCT03128619
PI3K	Taselisib, pictilisib, GDC-0077		PIK3CA mutant advanced solid tumors, PIK3CA mutant and HR⁺ breast cancer	NCT02389842, NCT04191499, NCT03006172
PI3K/mTOR	Gedatolisib		Metastatic breast cancer, advanced squamous cell lung, pancreatic, head and neck cancer and other solid tumors	NCT02684032, NCT03065062, NCT02626507
mTOR	Everolimus, vistusertib		HR⁺ breast cancer	NCT02871791
AKT	Ipatasertib		HR⁺ breast cancer, metastatic breast cancer, metastatic gastrointestinal tumors, NSCLC	NCT03959891, NCT04060862, NCT04591431
BTK	Ibrutinib		Mantle cell lymphoma	NCT03478514
BCL-2	Venetoclax		ER⁺/BCL-2⁺ advanced or metastatic breast cancer	NCT03900884
AR	AR antagonists: bicalutamide		AR⁺ metastatic breast cancer	NCT02605486
Lysosome + aromatase	Hydroxychloroquine + letrozole		ER⁺ breast cancer	NCT03774472
Proliferating cells	Standard chemotherapy		Stage IV ER⁺ breast cancer	NCT03355157
Proliferating cells	Radiation		Stage IV ER⁺ breast cancer	NCT03870919, NCT03691493, NCT04605562
BCR-ABL	Bosutinib		HR⁺ breast cancer	NCT03854903
*Ribociclib*
Aromatase	Letrozole, anastrozole, exemestane		HR⁺ breast cancer, metastatic breast cancer, ovarian cancer	NCT04256941, NCT03425838, NCT03822468, NCT02712723, NCT03673124, NCT02941926, NCT03248427, NCT03671330, NCT02333370, NCT01958021, NCT03425838
LHRH	LHRH agonists: goserelin, leuprolide		HR⁺ breast cancer	NCT03944434
ER	ER antagonists: fulvestrant		HR⁺ breast cancer, advanced breast cancer	NCT03227328, NCT02632045, NCT02632045, NCT03555877
PD-1		Spartalizumab	Breast cancer and ovarian cancer, recurrent and/or metastatic head and neck squamous cell carcinoma, melanoma	NCT03294694, NCT04213404, NCT03484923
HER2	Trastuzumab, pertuzumab, T-DM1		HER2⁺ breast cancer	NCT03913234, NCT02657343
EGFR	Nazartinib (EGF816)		EGFR mutant NSCLC	NCT03333343
RAF	Encorafenib, LXH254		NSCLC, BRAF mutant melanoma	NCT02974725, NCT03333343, NCT04417621, NCT02159066
MEK	Binimetinib		BRAF V600-dependent advanced solid tumors, melanoma	NCT01543698, NCT02159066
PI3K	Alpelisib		Breast cancer with PIK3CA mutation	NCT03439046
mTOR	Everolimus		Advanced dedifferentiated liposarcoma, leiomyosarcoma, glioma, astrocytoma, glioblastoma, endometrial carcinoma, pancreatic cancer, neuroendocrine tumors	NCT03114527, NCT03355794, NCT03834740, NCT03008408, NCT02985125, NCT03070301
mTOR + inflammation	Everolimus + dexamethasone		ALL	NCT03740334
SHP2	TNO155		Advanced solid tumors	NCT04000529
AR	AR antagonists: bicalutamide, enzalutamide		TNBC, metastatic prostate carcinoma	NCT03090165, NCT02555189
HDAC	Belinostat		TNBC, ovarian cancer	NCT04315233
proliferating cells	Standard chemotherapy		Ovarian cancer, metastatic solid tumors, soft tissue sarcoma, hepatocellular carcinoma	NCT03056833, NCT03237390, NCT03009201, NCT02524119
*Abemaciclib*
Aromatase	Letrozole, anastrozole, exemestane		HR⁺ breast cancer, metastatic breast cancer, endometrial cancer	NCT04256941, NCT03425838, NCT04227327, NCT04393285, NCT04305236, NCT03643510, NCT03675893, NCT04352777, NCT04293393, NCT02057133
ER	ER antagonists: fulvestrant		Advanced breast cancer, low-grade serous ovarian cancer	NCT03227328, NCT03531645, NCT04158362, NCT01394016
PD-1		Nivolumab, pembrolizumab	Head and neck cancer, g astroesophageal cancer, NSCLC, HR⁺ breast cancer	NCT04169074, NCT03655444, NCT03997448, NCT02779751
ER + PD-L1	ER antagonists: fulvestrant	Atezolizumab	HR⁺ breast cancer, metastatic breast cancer	NCT03280563
AKT + ER + PD-L1	Ipatasertib + ER antagonists: fulvestrant	Atezolizumab	HR⁺ breast cancer	NCT03280563
PD-L1		LY3300054	Advanced solid tumors	NCT02791334
HER2	Trastuzumab		HER2⁺ metastatic breast cancer	NCT04351230
Receptor tyrosine kinases	Sunitinib		Metastatic renal cell carcinoma	NCT03905889
IGF-1/IGF-2	Xentuzumab		HR⁺ breast cancer	NCT03099174
VEGF-A	Bevacizumab		Glioblastoma	NCT04074785
PI3K	Copanlisib		HR⁺ breast cancer, metastatic breast cancer	NCT03939897
PI3K/mTOR	LY3023414		Metastatic cancer	NCT01655225
ERK1/2	LY3214996		tumors with ERK1/2 mutations, glioblastoma, metastatic cancer	NCT04534283, NCT04391595, NCT02857270
*Trilaciclib*
Proliferating cells	Chemotherapy		SCLC: This trial evaluates the potential clinical benefit of trilaciclib in preventing chemotherapy-induced myelosuppression in patients receiving chemotherapy	NCT04504513
Proliferating cells + PD-L1	Carboplatin + etoposide	Atezolizumab	SCLC: This trial investigates the potential clinical benefit of trilaciclib in preserving the bone marrow and the immune system, and enhancing antitumor efficacy when administered with chemotherapy	NCT03041311
Proliferating cells	Topotecan		SCLC: This trial investigates the potential clinical benefit of trilaciclib in preserving the bone marrow and the immune system, and enhancing the antitumor efficacy of chemotherapy when administered prior to chemotherapy	NCT02514447
Proliferating cells	Carboplatin + gemcitabine		Metastatic TNBC: This study investigates the potential clinical benefit of trilaciclib in preserving the bone marrow and the immune system, and enhancing the antitumor efficacy of chemotherapy when administered prior to chemotherapy	NCT02978716
*Lerociclib*
ER	ER antagonist: fulvestrant		HR⁺/HER2^– metastatic breast cancer	NCT02983071
EGFR	Osimertinib		EGFR mutant NSCLC	NCT03455829
*SHR6390*
ER	ER antagonist: fulvestrant		HR⁺/HER2^– recurrent/ metastatic breast cancer	NCT03481998
Aromatase	Letrozole, anastrozole		HR⁺/HER2^– recurrent/ metastatic breast cancer	NCT03966898, NCT03772353
EGFR/HER2	Pyrotinib		HER2⁺ gastric cancer, HER2⁺ metastatic breast cancer	NCT04095390, NCT03993964
AR	AR antagonists: SHR3680		metastatic TNBC	NCT03805399
*PF-06873600*
Endocrine therapy	Single agent and then in combination with endocrine therapy		HR⁺/HER2^– metastatic breast cancer, ovarian and fallopian tube cancer, TNBC and other tumors	NCT03519178
*FCN-473c*
Aromatase	Letrozole		ER⁺/HER2^– advanced breast cancer	NCT04488107

Table 4. Ongoing clinical trials testing new combinations with CDK4/6 inhibitors.

HR⁺, hormone receptor–positive; LHRH, luteinizing hormone–releasing hormone; ER⁺, estrogen receptor–positive; PD-1, programmed cell death protein 1; PD-L1, programmed cell death 1 ligand 1; AR⁺, androgen receptor–positive; TNBC, triple-negative breast cancer; EGFR, epidermal growth factor receptor; HER2⁺, human epidermal growth factor receptor 2–positive; FGFR, fibroblast growth factor receptor; IGFR, insulin-like growth factor receptor; VEGF, vascular endothelial growth factor receptor; PI3K, phosphoinositide 3-kinase; NSCLC, non–small-cell lung cancer; ALL, acute lymphoblastic leukemia; SCLC, small-cell lung cancer.

http://newdrugapprovals.org/2015/02/05/fda-approves-ibrance-for-postmenopausal-women-with-advanced-breast-cancer/

Resistance to CDK4/6 inhibitors

Although CDK4/6 inhibitors represent very effective agents in cancer treatment, nearly all patients eventually develop resistance and succumb to the disease. Moreover, a substantial fraction of tumors show intrinsic resistance to treatment with CDK4/6 inhibitors (Fig. 3).

Fig. 3. Mechanisms of cancer cell resistance to CDK4/6 inhibition.

Known mechanisms include loss of RB1, activation of pathways impinging on CycD-CDK4/6, amplification of the *CDK4/6* genes and overexpression of CDK6 protein, activation of CycE-CDK2, and lysosomal sequestration of CDK4/6 inhibitors. Blank pieces of the puzzle denote additional mechanisms that remain to be discovered.

The best-documented mechanism of preexisting and acquired resistance is the loss of RB1 (71, 81, 140). Acquired RB1 loss has been detected in PDXs (141), in circulating tumor DNA (ctDNA) (142, 143), and in tumors from patients treated with CDK4/6 inhibitors (144, 145). However, RB1 mutations are likely subclonal and are seen in only 5 to 10% of patients (143, 145).

Increased expression of CDK6 was shown to underlie acquired resistance to CDK4/6 inhibitors. Amplification of the CDK6 gene and the resulting overexpression of CDK6 protein were found in abemaciclib-resistant ER⁺ breast cancer cells (146) and in ctDNA of patients with ER⁺ breast cancers that progressed during treatment with palbociclib plus endocrine therapy (147). Also, CDK4 gene amplification conferred insensitivity to CDK4/6 inhibition in GBM and sarcomas (148–150), whereas overexpression of CDK4 protein was associated with resistance to endocrine therapy in HR⁺ breast cancers (79).

Resistant breast cancer cells can also up-regulate the expression of CDK6 through suppression of the TGF-β/SMAD4 pathway by the microRNA miR-432-5p. In this mechanism, exosomal expression of miR-432-5p mediates the transfer of the resistance phenotype between neighboring cell populations (151). Another mechanism of CDK6 up-regulation in ER⁺ breast cancers is the loss of FAT1, which represses CDK6 expression via the Hippo pathway. Loss of FAT1 triggers up-regulation of CDK6 expression by the Hippo pathway effectors TAZ and YAP. Moreover, genomic alterations in other components of the Hippo pathway, although rare, are also associated with reduced sensitivity to CDK4/6 inhibitors (81).

Genetic lesions that activate pathways converging on D-type cyclins can cause resistance to CDK4/6 inhibitors. These include (i) FGFR1/2 gene amplification or mutational activation, detected in ctDNA from patients with ER⁺ breast cancers that progressed upon treatment with palbociclib plus endocrine therapy (147); (ii) hyperactivation of the MAPK pathway in resistant prostate adenocarcinoma cells, possibly due to increased production of EGF by cancer cells (152); and (iii) increased secretion of FGF in palbociclib-resistant KRAS-mutant NSCLC cells, which stimulates FGFR1 signaling in an autocrine or paracrine fashion, resulting in activation of ERK1/2 and mTOR as well as up-regulation of D-cyclin, CDK6, and cyclin E expression (153). Analyses of longitudinal tumor biopsies from a melanoma patient revealed an activating mutation in the PIK3CA gene that conferred resistance to ribociclib plus MEK inhibitor treatment (154). It is possible that these lesions elevate the cellular levels of active cyclin D–CDK4/6 complexes, thereby increasing the threshold for CDK4/6 inhibition.

Formation of a noncanonical cyclin D1–CDK2 complex was shown to represent another mechanism of acquired CDK4/6 inhibitor resistance. Such a complex was observed in palbociclib-treated ER⁺ breast cancer cells and was implicated in overcoming palbociclib-induced cell cycle arrest (141). Also, depletion of AMBRA1 promoted the interaction of D-cyclins with CDK2, resulting in resistance to CDK4/6 inhibitors (20, 22); it remains to be seen whether this represents an intrinsic or acquired resistance mechanism in human tumors.

Genetic analyses revealed that activation of cyclin E can bypass the requirement for cyclin D–CDK4/6 in development and tumorigenesis (155, 156). Hence, it comes as no surprise that increased activity of cyclin E–CDK2 is responsible for a large proportion of intrinsic and acquired resistance to CDK4/6 inhibitors. Several different mechanisms can activate cyclin E–CDK2 kinase in resistant tumor cells: (i) Down-regulation of KIP/CIP inhibitors results in increased activity of cyclin E–CDK (54, 157). (ii) Loss of PTEN expression, which activates AKT signaling, leads to nuclear exclusion of p27^KIP1. This in turn prevents access of p27^KIP1 to CDK2, resulting in increased CDK2 kinase activity (144). (iii) Activation of the PI3K/AKT pathway causes decreased levels of p21^CIP1. Co-treatment of melanoma PDXs with MDM2 inhibitors (which up-regulate p21^CIP1 via p53) sensitized intrinsically resistant tumor cells to CDK4/6 inhibitors (158). (iv) Up-regulation of cyclin D1 levels triggers sequestration of KIP/CIP inhibitors from cyclin E–CDK2 to cyclin D–CDK4/6, thereby activating the former (158). (v) Amplification of the CCNE1 gene and increased levels of cyclin E1 protein result in elevated activity of E-CDK2 kinase (141). (vi) mTOR signaling has been shown to up-regulate cyclin E1 (and D1) in KRAS-mutated pancreatic cancer cells; CDK2 activity was essential for CDK4/6 inhibitor resistance in this setting (159). (vii) Up-regulation of PDK1 results in activation of the AKT pathway, which increases the expression of cyclins E and A and activates CDK2 (160). (viii) In CDK4/6 inhibitor–resistant melanoma cells, high levels of RNA-binding protein FXR1 increase translation of the amino acid transporter SLC36A1. Up-regulation of SLC36A1 expression activates mTORC1, which in turn increases CDK2 expression (161). All these lesions are expected to allow cell proliferation, despite CDK4/6 inhibition, as a consequence of the activation of the downstream cell cycle kinase CDK2.

The role for cyclin E–CDK2 in CDK4/6 inhibitor resistance has been confirmed in clinical trials. In patients with advanced ER⁺ breast cancer treated with palbociclib and letrozole or fulvestrant, the presence of proteolytically cleaved cytoplasmic cyclin E in tumor tissue conferred strongly shortened progression-free survival (71). Moreover, analyses of PALOMA-3 trial for patients with ER⁺ breast cancers revealed lower efficacy of palbociclib plus fulvestrant in patients displaying high cyclin E mRNA levels in metastatic biopsies (80). Amplification of the CCNE1 gene was detected in ctDNA of patients with ER⁺ breast cancers that progressed on palbociclib plus endocrine therapy (147). Also, amplification of the CCNE2 gene (encoding cyclin E2) was seen in a fraction of CDK4/6 inhibitor–resistant HR⁺ mammary carcinomas (145, 162).

Collectively, these analyses indicate that resistant cells may become dependent on CDK2 for cell cycle progression. Indeed, depletion of CDK2 or inhibition of CDK2 kinase activity in combination with CDK4/6 inhibitors blocked proliferation of CDK4/6 inhibitor–resistant cancer cells (111, 141, 158–161). Recently, two CDK2-specific inhibitors, PF-07104091 (163) and BLU0298 (164), have been reported. PF-07104091 is now being tested in a phase 2 clinical trial in combination with palbociclib plus antiestrogens. Another recent study identified a novel compound, PF-3600, that inhibits CDK4/6 and CDK2 (165). PF3600 had potent antitumor effects against xenograft models of intrinsic and acquired resistance to CDK4/6 inhibition (165). A phase 2 clinical trial is currently evaluating this compound as a single agent and in combination with endocrine therapy in patients with HR⁺/HER2^– breast cancer and other cancer types.

Whole-exome sequencing of 59 HR⁺/HER2^– metastatic breast tumors from patients treated with CDK4/6 inhibitors and anti-estrogens revealed eight alterations that likely conferred resistance: RB1 loss; amplification of CCNE2 or AURKA; activating mutations or amplification of AKT1, FGFR2, or ERBB2; activating mutations in RAS genes; and loss of ER expression. The frequent activation of AURKA (in 27% of resistant tumors) raises the possibility of combining CDK4/6 inhibitors with inhibitors of Aurora A kinase to overcome resistance (145).

In contrast to ER⁺ mammary carcinomas, TNBCs are overall resistant to CDK4/6 inhibition (45). A subset of TNBCs display high numbers of lysosomes, which causes sequestration of CDK4/6 inhibitors into the expanded lysosomal compartment, thereby preventing their action on nuclear CDK4/6. Preclinical studies revealed that lysosomotropic agents that reverse the lysosomal sequestration (such as chloroquine, azithromycin, or siramesine) render TNBC cells fully sensitive to CDK4/6 inhibition (71, 111). These observations now need to be tested in clinical trials for TNBC patients.

Outlook

Although D-cyclins and CDK4/6 were discovered 30 years ago, several aspects of cyclin D–CDK4/6 biology, such as their role in antitumor immunity, are only now starting to be appreciated. The full range of cyclin D–CDK4/6 functions in tumor cells remains unknown. It is likely that these kinases play a much broader role in cancer cells than is currently appreciated. Hence, the impact of CDK4/6 inhibition on various aspects of tumorigenesis requires further study. Also, treatment of patients with CDK4/6 inhibitors likely affects several aspects of host physiology, which may be relevant to cancer progression.

In the next years, we will undoubtedly witness the development and testing of new CDK4/6 inhibitors. Because activation of CDK2 represents a frequent CDK4/6 inhibitor resistance mechanism, compounds that inhibit CDK4/6 and CDK2 may prevent or delay the development of resistance. Conversely, selective compounds that inhibit CDK4 but not CDK6 may allow more aggressive dosing, as they are expected not to result in bone marrow toxicity caused by CDK6 inhibition. New, less basic CDK4/6 inhibitor compounds (111) may escape lysosomal sequestration and may be efficacious against resistant cancer types such as TNBC. Degrader compounds, which induce proteolysis of cyclin D rather than inhibit cyclin D–CDK4/6 kinase, may have superior properties, as they would extinguish both CDK4/6-dependent and -independent functions of D-cyclins in tumorigenesis. Moreover, dissolution of cyclin D–CDK4/6 complexes is expected to liberate KIP/CIP inhibitors, which would then inhibit CDK2. D-cyclins likely play CDK-independent functions in tumorigenesis—for example, by regulating gene expression (166). However, their role in tumor biology and the utility of targeting these functions for cancer treatment remain largely unexplored.

An important challenge will be to test and identify combinatorial treatments involving CDK4/6 inhibitors for the treatment of different tumor types. CDK4/6 inhibitors trigger cell cycle arrest of tumor cells and, in some cases, senescence. It will be essential to identify combination treatments that convert CDK4/6 inhibitors from cytostatic compounds to cytotoxic ones, which would unleash the killing of tumor cells. Genome-wide high-throughput screens along with analyses of mouse cancer models and PDXs will help to address this point. Another largely unexplored area of cyclin D–CDK4/6 biology is the possible involvement of these proteins in other pathologies, such as metabolic disorders. Research in this area may extend the use of CDK4/6 inhibitors to treatment of other diseases. All these unresolved questions ensure that CDK4/6 biology will remain an active area of basic, translational, and clinical research for several years to come.

CDK inhibitors and Breast Cancer

The U.S. Food and Drug Administration today granted accelerated approval to Ibrance (palbociclib) to treat advanced (metastatic) breast cancer inr postmenopausal women with estrogen receptor (ER)-positive, human epidermal growth factor receptor 2 (HER2)-negative metastatic breast cancer who have not yet received an endocrine-based therapy. It is to be used in combination with letrozole, another FDA-approved product used to treat certain kinds of breast cancer in postmenopausal women.

See Dr. Melvin Crasto’s blog posts on the announcement of approval of Ibrance (palbociclib) at

and about the structure and mechanism of action of palbociclib

http://newdrugapprovals.org/2014/01/05/palbociclib/

From the CancerNetwork at http://www.cancernetwork.com/aacr-2014/cdk-inhibitors-show-impressive-activity-advanced-breast-cancer

CDK Inhibitors Show Impressive Activity in Advanced Breast Cancer

News | April 08, 2014 | AACR 2014, Breast Cancer

By Anna Azvolinsky, PhD

Chemical structure of palbociclib

Palbociclib and LY2835219 are both cyclin-dependent kinase (CDK) 4/6 inhibitors. CDK4 and CDK6 are kinases that, together with cyclin D1, facilitate the transition of dividing cells from the G1 to the S (synthesis) phase of the cell cycle. Preclinical studies have shown that breast cancer cells rely on CDK4 and CDK6 for division and growth, and that selective CDK4/6 inhibitors can arrest the cells at this G1/S phase checkpoint.

The results of the phase II trial of palbociclib and phase I trial of LY2835219 both indicated that hormone receptor (HR)-positive disease appears to be the best marker to predict patient response.

LY2835219 Phase I Trial Demonstrates Early Activity

The CDK4/6 inhibitor LY2835219 has demonstrated early activity in heavily pretreated women with metastatic breast cancer. Nineteen percent of these women (9 out of 47) had a partial response and 51% (24 out of 47) had stable disease following monotherapy with the oral CDK4/6 inhibitor. Patients had received a median of seven prior therapies, and 75% had metastatic disease in the lung, liver, or brain. The median age of patients was 55 years.

All of the partial responses were in patients with HR-positive disease. The overall response rate for this patient subset was 25% (9 of 36 patients). Twenty of the patients with stable disease had HR-positive disease, with 13 patients having stable disease lasting 24 weeks or more.

Despite treatment, disease progression occurred in 23% of the patients.

These results were presented at a press briefing by Amita Patnaik, MD, associate director of clinical research at South Texas Accelerated Research Therapeutics in San Antonio, Texas, at the 2014 American Association for Cancer Research (AACR) Annual Meeting, held April 5–9, in San Diego.

The phase I trial of LY2835219 enrolled 132 patients with five different tumor types, including metastatic breast cancer. Patients received 150-mg to 200-mg doses of the oral drug every 12 hours.

The overall disease control rate was 70% for all patients and 81% among the 36 HR-positive patients.

The median progression-free survival (PFS) was 5.8 months for all patients and 9.1 months for HR-positive patients. Patnaik noted that the median PFS is still a moving target, as 18 patients, all with HR-positive disease, remain on therapy.

“The data are rather encouraging for a very heavily pretreated patient population,” said Patnaik during the press briefing.

Even though the trial was not designed to compare efficacy based on breast cancer subpopulations, the results in HR-positive tumors are particularly encouraging, according to Patnaik.

Common adverse events thought to be treatment-related were diarrhea, nausea, fatigue, vomiting, and neutropenia. These adverse events occurred in 5% or less of patients at grade 3 or 4 toxicity, except neutropenia, which occurred as a grade 3 or 4 toxicity in 11% of patients. Patnaik noted during the press briefing that the neutropenia was uncomplicated and did not result in discontinuation of therapy by any of the patients.

Palbociclib Phase II Data “Impressive”

The addition of the oral CDK4/6 inhibitor palbociclib resulted in an almost doubling of PFS in first-line treatment of postmenopausal metastatic breast cancer patients with HR-positive disease compared with a control population. The patients in this trial were not previously treated for their metastatic breast cancer, unlike the patient population in the phase I LY2835219 trial.

Patients receiving the combination of palbociclib at 125 mg once daily plus letrozole at 2.5 mg once daily had a median PFS of 20.2 months compared with a median of 10.2 months for patients treated with letrozole alone (hazard ratio = 0.488; P = .0004).

Richard S. Finn, MD, assistant professor of medicine at the University of California, Los Angeles, presented the data from the phase II PALOMA-1 trial at a press briefing at the AACR Annual Meeting.

A total of 165 patients were randomized 1:1 to either the experimental arm or control arm.

Forty-three percent of patients in the combination arm had an objective response compared with 33% of patients in the control arm.

Overall survival (OS), a secondary endpoint in this trial, was encouraging but the results are still preliminary, said Finn during the press briefing. The median OS was 37.5 months in the palbociclib arm compared with 33.3 months in the letrozole alone arm (P = .21). Finn noted that long-term follow-up is necessary to establish the median OS. “This first look of the survival data is encouraging. This is a front-line study, and it is encouraging that there is early [separation] of the curves,” he said.

No new toxicities were reported since the interim trial results. Common adverse events included leukopenia, neutropenia, and fatigue. The neutropenia could be quickly resolved and was uncomplicated and not accompanied by fever, said Finn.

Palbociclib is currently being tested in two phase III clinical trials: The PALOMA-3 trial is testing the combination of palbociclib with letrozole and fulvestrant in late-stage metastatic breast cancer patients who have failed endocrine therapy. The PENELOPE-B trial is testing palbociclib in combination with standard endocrine therapy in HR-positive breast cancer patients with residual disease after neoadjuvant chemotherapy and surgery.

References

Patnaik A, Rosen LS, Tolaney SM, et al. Clinical activity of LY2835219, a novel cell cycle inhibitor selective for CDK4 and CDK6, in patients with metastatic breast cancer. American Association for Cancer Research Annual Meeting 2014; April 5–9, 2014; San Diego. Abstr CT232.
Finn RS, Crown JP, Lang I, et al. Final results of a randomized phase II study of PD 0332991, a cyclin-dependent kinase (CDK)-4/6 inhibitor, in combination with letrozole vs letrozole alone for first-line treatment of ER+/HER2-advanced breast cancer (PALOMA-1; TRIO-18). American Association for Cancer Research Annual Meeting 2014; April 5–9, 2014; San Diego. Abstr CT101.

– See more at: http://www.cancernetwork.com/aacr-2014/cdk-inhibitors-show-impressive-activity-advanced-breast-cancer#sthash.f29smjxi.dpuf

The Cell Cycle and Anti-Cancer Targets

From Cell Cycle in Cancer: Cyclacel Pharmaceuticals™ (note dotted arrows show inhibition of steps e.g. p21, p53)

For a nice video slideshow explaining a bit more on cyclins and the cell cycle please see video below:

Cell Cycle. 2012 Nov 1; 11(21): 3913.

doi: 10.4161/cc.22390

PMCID: PMC3507481

Cyclin-dependent kinase 4/6 inhibition in cancer therapy

Neil Johnson and Geoffrey I. Shapiro^*

See the article “Therapeutic response to CDK4/6 inhibition in breast cancer defined by ex vivo analyses of human tumors” in volume 11 on page 2756.

See the article “CDK4/6 inhibition antagonizes the cytotoxic response to anthracycline therapy” in volume 11 on page 2747.

This article has been cited by other articles in PMC.

Cyclin-dependent kinases (CDKs) drive cell cycle progression and control transcriptional processes. The dysregulation of multiple CDK family members occurs commonly in human cancer; in particular, the cyclin D-CDK4/6-retinoblastoma protein (RB)-INK4 axis is universally disrupted, facilitating cancer cell proliferation and prompting long-standing interest in targeting CDK4/6 as an anticancer strategy. Most agents that have been tested inhibit multiple cell cycle and transcriptional CDKs and have carried toxicity. However, several selective and potent inhibitors of CDK4/6 have recently entered clinical trial. PD0332991, the first to be developed, resulted from the introduction of a 2-aminopyridyl substituent at the C2-position of a pyrido(2,3-d)pyrimidin-7-one backbone, affording exquisite selectivity toward CDK4/6.¹ PD0332991 arrests cells in G₁ phase by blocking RB phosphorylation at CDK4/6-specfic sites and does not inhibit the growth of RB-deficient cells.² Phase I studies conducted in patients with advanced RB-expressing cancers demonstrated mild side effects and dose-limiting toxicities of neutropenia and thrombocytopenia, with prolonged stable disease in 25% of patients.³^,4 In cyclin D1-translocated mantle cell lymphoma, PD0332991 extinguished CDK4/6 activity in patients’ tumors, resulting in markedly reduced proliferation, and translating to more than 1 year of stability or response in 5 of 17 cases.⁵

Two recent papers from the Knudsen laboratory make several important observations that will help guide the continued clinical development of CDK4/6 inhibitors. In the study by Dean et al., surgically resected patient breast tumors were grown on a tissue culture matrix in the presence or absence of PD0332991. Crucially, these cultures retained associated stromal components known to play important roles in cancer pathogenesis and therapeutic sensitivities, as well as key histological and molecular features of the primary tumor, including expression of ER, HER2 and K_i-67. Similar to results in breast cancer cell lines,⁶ the authors demonstrate that only RB-positive tumors have growth inhibition in response to PD0332991, irrespective of ER or HER2 status, while tumors lacking RB were completely resistant. This result underscores RB as the predominant target of CDK4/6 in breast cancer cells and the primary marker of drug response in primary patient-derived tumors. As expected, RB-negative tumors routinely demonstrated robust expression of p16^INK4A; however, p16^INK4A expression was not always a surrogate marker for RB loss, supporting the importance of direct screening of tumors for RB expression to select patients appropriate for CDK4/6 inhibitor clinical trials.

In the second study, McClendon et al. investigated the efficacy of PD0332991 in combination with doxorubicin in triple-negative breast cancer cell lines. Again, RB functionality was paramount in determining response to either PD0332991 monotherapy or combination treatment. In RB-deficient cancer cells, CDK4/6 inhibition had no effect in either instance. However, in RB-expressing cancer cells, CDK4/6 inhibition and doxorubicin provided a cooperative cytostatic effect, although doxorubicin-induced cytotoxicity was substantially reduced, assessed by markers for mitotic catastrophe and apoptosis. Additionally, despite cytostatic cooperativity, CDK4/6 inhibition maintained the viability of RB-proficient cells in the presence of doxorubicin, which repopulated the culture after removal of drug. These results reflect previous data demonstrating that ectopic expression of p16^INK4A can protect cells from the lethal effects of DNA damaging and anti-mitotic chemotherapies.⁷ Similar results have been reported in MMTV-c-neu mice bearing RB-proficient HER2-driven tumors, where PD0332991 compromised carboplatin-induced regressions,⁸ suggesting that DNA-damaging treatments should not be combined concomitantly with CDK4/6 inhibition in RB-proficient tumors.

To combine CDK4/6 inhibition with cytotoxics, sequential treatment may be considered, in which CDK4/6 inhibition is followed by DNA damaging chemotherapy; cells relieved of G₁ arrest may synchronously enter S phase, where they may be most susceptible to agents disrupting DNA synthesis. Release of myeloma cells from a prolonged PD0332991-mediated G₁ block leads to S phase synchronization; interestingly, all scheduled gene expression is not completely restored (including factors critical to myeloma survival such as IRF4), further favoring apoptotic responses to cytotoxic agents.⁹ Furthermore, in RB-deficient tumors, CDK4/6 inhibitors may be used to maximize the therapeutic window between transformed and non-transformed cells treated with chemotherapy. In contrast to RB-deficient cancer cells, RB-proficient non-transformed cells arrested in G₁ in response to PD0332991 are afforded protection from DNA damaging agents, thereby reducing associated toxicities, including bone marrow suppression.⁸

In summary, the current work provides evidence for RB expression as a determinant of response to CDK4/6 inhibition in primary tumors and highlights the complexity of combining agents targeting the cell cycle machinery with DNA damaging treatments.

Dean JL, McClendon AK, Hickey TE, Butler LM, Tilley WD, Witkiewicz AK, Knudsen ES. Therapeutic response to CDK4/6 inhibition in breast cancer defined by ex vivo analyses of human tumors Cell Cycle 2012 11 2756 61 doi: 10.4161/cc.21195.

Notes

McClendon AK, Dean JL, Rivadeneira DB, Yu JE, Reed CA, Gao E, Farber JL, Force T, Koch WJ, Knudsen ES. CDK4/6 inhibition antagonizes the cytotoxic response to anthracycline therapy Cell Cycle 2012 11 2747 55 doi: 10.4161/cc.21127.

Footnotes

Previously published online: www.landesbioscience.com/journals/cc/article/22390

Author information ► Copyright and License information ►

References

Toogood PL, et al. J Med Chem. 2005;48:2388–406. doi: 10.1021/jm049354h. [PubMed] [Cross Ref]
Fry DW, et al. Mol Cancer Ther. 2004;3:1427–38. [PubMed]
Flaherty KT, et al. Clin Cancer Res. 2012;18:568–76. doi: 10.1158/1078-0432.CCR-11-0509. [PubMed] [Cross Ref]
Schwartz GK, et al. Br J Cancer. 2011;104:1862–8. doi: 10.1038/bjc.2011.177. [PMC free article] [PubMed] [Cross Ref]
Leonard JP, et al. Blood. 2012;119:4597–607. doi: 10.1182/blood-2011-10-388298. [PubMed] [Cross Ref]
Dean JL, et al. Oncogene. 2010;29:4018–32. doi: 10.1038/onc.2010.154. [PubMed] [Cross Ref]
Stone S, et al. Cancer Res. 1996;56:3199–202. [PubMed]
Roberts PJ, et al. J Natl Cancer Inst. 2012;104:476–87. doi: 10.1093/jnci/djs002. [PMC free article] [PubMed] [Cross Ref]
Huang X, et al. Blood. 2012;120:1095–106. doi: 10.1182/blood-2012-03-415984. [PMC free article] [PubMed] [Cross Ref]

Cell Cycle. 2012 Jul 15; 11(14): 2756–2761.

doi: 10.4161/cc.21195

PMCID: PMC3409015

Therapeutic response to CDK4/6 inhibition in breast cancer defined by ex vivo analyses of human tumors

Jeffry L. Dean,^{1 , 2}A. Kathleen McClendon,^{1 , 2}Theresa E. Hickey,³Lisa M. Butler,³Wayne D. Tilley,³Agnieszka K. Witkiewicz,^{4 , 2 ,*} and Erik S. Knudsen^{1 , 2 ,*}

See commentary “Cyclin-dependent kinase 4/6 inhibition in cancer therapy” in volume 11 on page 3913.

This article has been cited by other articles in PMC.

Abstract

To model the heterogeneity of breast cancer as observed in the clinic, we employed an ex vivo model of breast tumor tissue. This methodology maintained the histological integrity of the tumor tissue in unselected breast cancers, and importantly, the explants retained key molecular markers that are currently used to guide breast cancer treatment (e.g., ER and Her2 status). The primary tumors displayed the expected wide range of positivity for the proliferation marker Ki67, and a strong positive correlation between the Ki67 indices of the primary and corresponding explanted tumor tissues was observed. Collectively, these findings indicate that multiple facets of tumor pathophysiology are recapitulated in this ex vivo model. To interrogate the potential of this preclinical model to inform determinants of therapeutic response, we investigated the cytostatic response to the CDK4/6 inhibitor, PD-0332991. This inhibitor was highly effective at suppressing proliferation in approximately 85% of cases, irrespective of ER or HER2 status. However, 15% of cases were completely resistant to PD-0332991. Marker analyses in both the primary tumor tissue and the corresponding explant revealed that cases resistant to CDK4/6 inhibition lacked the RB-tumor suppressor. These studies provide important insights into the spectrum of breast tumors that could be treated with CDK4/6 inhibitors, and defines functional determinants of response analogous to those identified through neoadjuvant studies.

Keywords: ER, PD0332991, breast cancer, cell cycle, ex vivo

Author information ► Copyright and License information ►

Introduction

Breast cancer is a highly heterogeneous disease.¹^–⁴ Such heterogeneity is known to influence patient response to both standard of care and experimental therapeutics. In regards to biomarker-driven treatment of breast cancers, it was initially recognized that the presence of the estrogen receptor α (ER) in a fraction of breast cancer cells was associated with the response to tamoxifen and similar anti-estrogenic therapies.⁵^,⁶ Since this discovery, subsequent marker analyses and gene expression profiling studies have further divided breast cancer into a series of distinct subtypes that harbor differing and often divergent therapeutic sensitivities.¹^–³ While clearly important in considering the use of several current standard of care therapies, these markers, or molecular sub-types, do not necessarily predict the response to new therapeutic approaches that are currently undergoing clinical development. Thus, there is the continued need for functional analyses of drug response and the definition of new markers that can be used to direct treatment strategies.

Currently, all preclinical cancer models are associated with specific limitations. It is well known that cell culture models lack the tumor microenvironment known to have a significant impact on tumor biology and therapeutic response.⁷^–⁹ Xenograft models are dependent on the host response for the engraftment of tumor cells in non-native tissues, which do not necessarily recapitulate the nuances of complex tumor milieu.¹⁰ In addition, genetically engineered mouse models, while enabling the tumor to develop in the context of the host, can develop tumors that do not mirror aspects of human disease.¹⁰ Furthermore, it remains unclear whether any preclinical model truly represents the panoply of breast cancer subtypes that are observed in the clinic. Herein, we utilized a primary human tumor explant culture approach to interrogate drug response, as well as specific determinants of therapeutic response, in an unselected series of breast cancer cases.

Cell Cycle. 2012 Jul 15; 11(14): 2747–2755.

doi: 10.4161/cc.21127

PMCID: PMC3409014

CDK4/6 inhibition antagonizes the cytotoxic response to anthracycline therapy

Kathleen McClendon,^{1 , †}Jeffry L. Dean,^{1 , †}Dayana B. Rivadeneira,¹Justine E. Yu,¹Christopher A. Reed,¹Erhe Gao,²John L. Farber,³Thomas Force,²Walter J. Koch,²and Erik S. Knudsen^{1 ,*}

See commentary “Cyclin-dependent kinase 4/6 inhibition in cancer therapy” in volume 11 on page 3913.

This article has been cited by other articles in PMC.

http://www.onclive.com/publications/contemporary-oncology/2014/november-2014/targeting-cell-cycle-progression-cdk46-inhibition-in-breast-cancer/1

Abstract

Triple-negative breast cancer (TNBC) is an aggressive disease that lacks established markers to direct therapeutic intervention. Thus, these tumors are routinely treated with cytotoxic chemotherapies (e.g., anthracyclines), which can cause severe side effects that impact quality of life. Recent studies indicate that the retinoblastoma tumor suppressor (RB) pathway is an important determinant in TNBC disease progression and therapeutic outcome. Furthermore, new therapeutic agents have been developed that specifically target the RB pathway, potentially positioning RB as a novel molecular marker for directing treatment. The current study evaluates the efficacy of pharmacological CDK4/6 inhibition in combination with the widely used genotoxic agent doxorubicin in the treatment of TNBC. Results demonstrate that in RB-proficient TNBC models, pharmacological CDK4/6 inhibition yields a cooperative cytostatic effect with doxorubicin but ultimately protects RB-proficient cells from doxorubicin-mediated cytotoxicity. In contrast, CDK4/6 inhibition does not alter the therapeutic response of RB-deficient TNBC cells to doxorubicin-mediated cytotoxicity, indicating that the effects of doxorubicin are indeed dependent on RB-mediated cell cycle control. Finally, the ability of CDK4/6 inhibition to protect TNBC cells from doxorubicin-mediated cytotoxicity resulted in recurrent populations of cells specifically in RB-proficient cell models, indicating that CDK4/6 inhibition can preserve cell viability in the presence of genotoxic agents. Combined, these studies suggest that while targeting the RB pathway represents a novel means of treatment in aggressive diseases such as TNBC, there should be a certain degree of caution when considering combination regimens of CDK4/6 inhibitors with genotoxic compounds that rely heavily on cell proliferation for their cytotoxic effects.

Click on Video Link for Dr. Tolaney slidepresentation of recent data with CDK4/6 inhibitor trial results https://youtu.be/NzJ_fvSxwGk

Audio and slides for this presentation are available on YouTube: http://youtu.be/NzJ_fvSxwGk

Sara Tolaney, MD, MPH, a breast oncologist with the Susan F. Smith Center for Women’s Cancers at Dana-Farber Cancer Institute, gives an overview of phase I clinical trials and some of the new drugs being tested to treat breast cancer. This talk was originally given at the Metastatic Breast Cancer Forum at Dana-Farber on Oct. 5, 2013.

A great article on current clinical trials and explanation of cdk inhibitors by Sneha Phadke, DO; Alexandra Thomas, MD at the site OncoLive

cdk4/6 inhibitor Ibrance Has Favorable Toxicity and Adverse Event Profile

As discussed in earlier posts and the Introduction to this chapter on Cytotoxic Chemotherapeutics, most anti-cancer drugs developed either to target DNA, DNA replication, or the cell cycle usually have similar toxicity profile which can limit their therapeutic use. These toxicities and adverse events usually involve cell types which normally exhibit turnover in the body, such as myeloid and lymphoid and granulocytic series of blood cells, epithelial cells lining the mucosa of the GI tract, as well as follicular cells found at hair follicles. This understandably manifests itself as common toxicities seen with these types of agents such as the various cytopenias in the blood, nausea vomiting diarrhea (although there are effects on the chemoreceptor trigger zone), and alopecia.

It was felt that the cdk4/6 inhibitors would show serious side effects similar to other cytotoxic agents and this definitely may be the case as outlined below:

(Side effects of palbociclib) From navigatingcancer.com

Palbociclib may cause side effects. Tell your doctor if any of these symptoms are severe or do not go away:

nausea
diarrhea
vomiting
decreased appetite
tiredness
numbness or tingling in your arms, hands, legs, and feet
sore mouth or throat
unusual hair thinning or hair loss

Some side effects can be serious. If you experience any of these symptoms, call your doctor immediately or get emergency medical treatment:

fever, chills, or signs of infection
shortness of breath
sudden, sharp chest pain that may become worse with deep breathing
fast, irregular, or pounding heartbeat
rapid breathing
weakness
unusual bleeding or bruising
nosebleeds

The following is from FDA Drug Trials Snapshot of Ibrance™:

See PDF on original submission and CDER review

original FDA Ibrance submission

original FDA Ibrance submission

CDER Review Ibrance

4.3 Preclinical Pharmacology/Toxicology

For full details, please see Pharmacology/Toxicology review by Dr. Wei Chen The nonclinical studies adequately support the safety of oral administration of palbociclib for the proposed indication and the recommendation from the team is for approval. Non-clinical studies of palbociclib included safety pharmacology studies, genotoxicity

studies, reproductive toxicity studies, pharmacokinetic studies, toxicokinetic studies and repeat-dose general toxicity studies which were conducted in rats and dogs. The pivotal toxicology studies were conducted in compliance with Good Laboratory Practice regulation.

Pharmacology:

As described above, palbociclib is an inhibitor of CDK4 and CDK6. Palbociclib modulates downstream targets of CDK4 and CDK6 in vitro and induces G1 phase cell cycle arrest and therefore acts to inhibit DNA synthesis and cell proliferation. Combination of palbociclib with anti-estrogen agents demonstrated synergistic inhibition

of cell proliferation in ER+ breast cancer cells. Palbociclib showed anti-tumor efficacy in animal tumor model studies. Safety pharmacology studies with palbociclib demonstrated adverse effects on both the respiratory and cardiovascular function of dogs at a dose of 125mg/day (four times and 50-times the human clinical exposure

respectively) based on mean unbound Cmax.

General toxicology:

Palbociclib was studied in single dose toxicity studies and repeated dose studies in rats and dogs. Adverse effects in the bone marrow, lymphoid tissues, and male reproductive organs were observed at clinically relevant exposures. Partial to complete reversibility of toxicities to the hematolymphopoietic and male reproductive systems was demonstrated following a recovery period (4-12 weeks), with the exception of the male reproductive organ findings in dogs. Gastrointestinal, liver, kidney, endocrine/metabolic (altered glucose metabolism), respiratory, ocular, and adrenal effects were also seen.

Genetic toxicology:

Palbociclib was evaluated for potential genetic toxicity in in vitro and in vivo studies. The Ames bacterial mutagenicity assay in the presence or absence of metabolic activation demonstrated non-mutagenicity. In addition, palbociclib did not induce chromosomal aberrations in cultured human peripheral blood lymphocytes in the presence or absence of metabolic activation. Palbociclib was identified as aneugenic based on kinetochore analysis of micronuclei formation in an In vitro assay in CHO-WBL cells. In addition, palbociclib was shown to induce micronucleus formation in male rats at doses 100

mg/kg/day (10x human exposure at the therapeutic dose) in an in vivo rat micronucleus assay.

Reproductive toxicology: No effects on estrous cycle and no reproductive toxicities were noticed in standard assays.

Pharmacovigilance (note please see PDF for more information)

Deaths Associated With Trials: Although a few deaths occurred during some trials no deaths were attributed to the drug.

Non-Serious Adverse Events:

(note a reviewers comment below concerning incidence of pulmonary embolism is a combination trial with letrazole)

Tumor Suppressor Pathway, Hippo pathway, is responsible for Sensing Abnormal Chromosome Numbers in Cells and Triggering Cell Cycle Arrest, thus preventing Progression into Cancer

Nonhematologic Cancer Stem Cells [11.2.3]

New methods for Study of Cellular Replication, Growth, and Regulation

Multiple Lung Cancer Genomic Projects Suggest New Targets, Research Directions for Non-Small Cell Lung Cancer

Proteomics, Metabolomics, Signaling Pathways, and Cell Regulation: a Compilation of Articles in the Journal http://pharmaceuticalintelligence.com

In Focus: Targeting of Cancer Stem Cells

Viruses; dsDNA viruses, no RNA stage; Papillomaviridae; unclassified Papillomaviridae; Human papillomavirus types

Observations on Human Papilloma Virus and Cancer

Posted in Biological Networks, Biomarkers & Medical Diagnostics, Cancer and Current Therapeutics, Pharmacogenomics, tagged biomarkers, Cancer research, Cell Biology, Clinical trial, genetics on October 13, 2015| Leave a Comment »

Observations on Human Papilloma Virus and Cancer

Curator: Demet Sag, PhD, CRA, GCP

What is Human Papilloma Virus?

Human papillomavirus

Taxonomy ID: 10566
Inherited blast name: viruses
Rank: species
Genetic code: Translation table 1 (Standard)
Host: vertebrates| human
Other names:

synonym:	human papillomavirus HPV
synonym:	Human Papilloma Virus

Lineage( full )

Entrez records
Database name	Subtree links	Direct links
Nucleotide	7,782	7,775
Protein	2,611	2,604
Structure	3	3
Genome	1	1
Popset	34	34
PubMed Central	4,742	4,742
Gene	21	21
SRA Experiments	43	43
Probe	12	12
Assembly	1	1
Bio Project	6	6
Bio Sample	53	53
PubChem BioAssay	5	5
Taxonomy	8	1

Human papillomavirus
Specialty	Infectious disease, gynecology HPV_ WHO_RHR_08.14_eng-Cervical cancer, human papillomavirus (HPV), and HPV vaccines WHO= papilloma virus info
ICD–10	B97.7
ICD–9-CM	078.1 079.4
DiseasesDB	6032
eMedicine	med/1037
MeSH	D030361

ICTV homepage

WHO= papilloma virus info

WHO_RHR_08.14_eng-Cervical cancer, human papillomavirus (HPV), and HPV vaccines

Why is it related to Human Cancer?

Since its first presumed diagnosis in women by an Italian Physician back in 1800s many developments took place to identify the real causative agents (PMID:19135222). Especially in 1970s the full discovery and relation between HPV and cancer established. Human papilloma virus (HPV) is the second common cancer death in women, although HPV vaccines helped to decrease the morbidity rate there are complications due to vaccines. Still there is an increase with cervical cancer estimated to be 490,000.

CDC also provided simple information for public on HPV since there is a misunderstanding that some people think it is like herpes or HIV viruses. Yet, pathology is much different and changes based on age since younger women or girls can fight off but after age 30 predisposition of HPV as a cancer increases. (http://www.cdc.gov/cancer/hpv/pdf/HPV_Testing_2012_English.pdf)

Cervical cancer is responsible for 10–15% of cancer-related deaths in women worldwide^1,2. The etiological role of infection with high-risk human papilloma viruses (HPV) in cervical carcinomas is well established.

Relationship of mutational spectrum and rates with clinicopathological characteristics in cervical carcinoma presented at http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4161954/bin/nihms610939f1.jpg

All panels are aligned with vertical tracks representing 115 individuals. The data is sorted in order by histology (middle panel) and total mutational rate (top panel). The relative frequencies of nucleotide mutations occurring at cytosines preceeded by thymines (Tp*C) or at cytosines followed by guanines (*CpG) sites are depicted in red and orange respectively, on the second panel. The bottom heatmap shows the distribution of mutations in significantly mutated genes (q<0.1) in squamous cell carcinomas and adenocarcinomas in the order listed in the following Table, TP53, ERBB2 and KRAS were significant recurrence (q<0.1) among cancer driver genes reported in COSMIC.

Nature. Author manuscript; available in PMC 2014 Sep 12. Published in final edited form as: Nature. 2014 Feb 20; 506(7488): 371–375.

Genes with Significantly Recurrent Somatic Mutations in Cervical Carcinomas

Gene	Description	Nonsilent mutations	Relative frequency	Patients	Unique sites	Silent mutations	Indel + null	q
SQUAMOUS CELL CARCINOMA (N=79)
FBXW7^**	F-box and WD repeat domain containing 7	12	15%	12	8	0	2	4.03E-12
PIK3CA	phosphoinositide-3-kinase, catalytic, alpha polypeptide	11	14%	10	5	0	1	<9.08e-12
MAPK1^**	mitogen-activated protein kinase 1	6	8%	6	3	0	0	0.000671
HLA-B⁺	major histocompatibility complex, class I, B	7	9%	6	7	1	3	0.00169
STK11	serine/threonine kinase 11	3	4%	2	2	0	1	0.012
EP300⁺	E1A binding protein p300	13	16%	12	13	1	4	0.0354
NFE2L2⁺	nuclear factor (erythroid-derived 2)-like 2	3	4%	3	2	0	0	0.0597
PTEN	phosphatase and tensin homolog (mutated in multiple advanced cancers 1)	5	6%	5	5	0	3	0.0693
ADENOCARCINOMA (N=24)
ELF3^*	E74-like factor 3 (ets domain transcription factor, epithelial-specific)	3	13%	3	3	0	3	0.03
CBFB^*	core-binding factor, beta subunit	2	8%	2	2	0	1	0.0342

Indel: insertions or deletions;

Null: nonsense, frameshft or splice-site mutations;

q: q value, false discovery rate (Benjamini-Hochberg procedure).

^**Genes with mutations observed in only squamous cell carcinomas

^*Genes with mutations observed in only adenocarcinomas

⁺Genes with a majority of mutations occurring in squamous cell carcinomas.

Following figure (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4161954/bin/nihms610939f2.jpg)

Novel recurrent somatic mutations in cervical carcinoma

The locations of somatic mutations in novel significantly mutated genes in 115 cervical carcinoma, FBXW7, MAPK1, HLA-B, EP300, NFE2L2 and ELF3 are shown in the context of protein domain models derived from UniProt and Pfam annotations. Numbers refer to amino acid residues. Each filled circle represents an individual mutated tumor sample: missense and silent mutations are represented by filled black and grey circles, respectively while nonsense, frameshift, and splice site mutations are represented by filled red circles and red text. Domains are depicted with various colors with an appropriate key located on the right hand of each domain model.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4161954/bin/nihms610939f3.jpg

Relationships between HPV integration, copy number amplifications and gene expression in cervical carcinoma

Panel (a) shows comparative histograms of true and simulated genomic distances between HPV integration sites and the nearest copy number amplification (log segmean difference >0.5). Panel (b) shows boxplots of gene expression levels across 79 cervical tumors for 41 genes with chimeric human-HPV read pairs. The expression levels for tumors with HPV integration in the respective genes are highlighted in red circles. Panel (c) shows scatter plots comparing copy number alterations and gene expression levels across 79 tumors in selected integration site genes. The red circles represent data for the tumors with HPV integration events involving the respective genes.

Table. Diseases Associated With Specific HPV Types (e-Medicine)

Nongenital Cutaneous Disease	HPV Type
Common warts (verrucae vulgaris)	1, 2, 4, 26, 27, 29, 41, 57, 65, 75-78
Plantar warts (myrmecias)	1, 2, 4, 60, 63
Flat warts (verrucae planae)	3, 10, 27, 28, 38, 41, 49
Butcher’s warts (common warts of people who handle meat, poultry, and fish)	1-4, 7, 10, 28
Mosaic warts	2, 27, 57
Ungual squamous cell carcinoma	16
Epidermodysplasia verruciformis (benign)	2, 3, 10, 12, 15, 19, 36, 46, 47, 50
Epidermodysplasia verruciformis (malignant or benign)	5, 8-10, 14, 17, 20-25, 37, 38
Nonwarty skin lesions	37, 38
Nongenital Mucosal Disease	HPV Type
Respiratory papillomatosis	6, 11
Squamous cell carcinoma of the lung	6, 11, 16, 18
Laryngeal papilloma (recurrent respiratory papillomatosis)^[17]	2, 6, 11, 16, 30, 40, 57
Laryngeal carcinoma	6, 11
Maxillary sinus papilloma	57
Squamous cell carcinoma of the sinuses	16, 18
Conjunctival papillomas	6, 11
Conjunctival carcinoma	16
Oral focal epithelial hyperplasia (Heck disease)	13, 32
Oral carcinoma	16, 18
Oral leukoplakia	16, 18
Squamous cell carcinoma of the esophagus	16, 18
Anogenital Disease	HPV Type
Condylomata acuminata	1-6, 10, 11, 16, 18, 30, 31, 33, 35, 39-45, 51-59, 70, 83
Bowenoid papulosis	16, 18, 34, 39, 40, 42, 45
Bowen disease	16, 18, 31, 34
Giant condylomata (Buschke-Löwenstein tumors)	6, 11, 57, 72, 73
Unspecified intraepithelial neoplasia	30, 34, 39, 40, 53, 57, 59, 61, 62, 64, 66-69
Low-grade squamous intraepithelial lesions (LGSIL)	6, 11, 16, 18, 26, 27, 30, 31, 33-35, 40, 42-45, 51-58, 61, 62, 67-69, 71-74, 79, 81-84
High-grade squamous intraepithelial lesions (HGSIL)	6, 11, 16, 18, 31, 33, 35, 39, 42, 44, 45, 51, 52, 56, 58, 59, 61, 64, 66, 68, 82
Carcinoma of vulva	6, 11, 16, 18
Carcinoma of vagina	16
Carcinoma of cervix^{[18, 19]}	16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, 70, 73, 82
Carcinoma of anus	16, 31, 32, 33
Carcinoma in situ of penis (erythroplasia of Queyrat)	16
Carcinoma of penis	16, 18

Epidemiology

“Human papillomavirus (HPV) has become synonymous with cervical cancer, but its actual footprint is much bigger” said James Mitchell Crow. (PMID: 229324377 James Mitchell Crow. “HPV: The global burden”. Nature 488 S2–S3 (30 August 2012) doi:10.1038/488S2a Published online 29 August 2012).

Every year, over 27,000 women and men are affected by a cancer caused by HPV— that’s a new case every 20 minutes.

Persistent HPV infection can cause cervical and other cancers including:

Cervical cancer: The most common HPV cancer. Almost all cervical cancer is caused by HPV.
Vulvar cancer: About 69% are linked to HPV.
Vaginal cancer: About 75% are linked to HPV.
Penile cancer: About 63% are linked to HPV.
Anal cancer: About 91% are linked to HPV.
Cancer of the back of the throat: About 72% are linked to HPV. [Note: Many of these cancers may be related to tobacco and alcohol use]

Pathology:

Virus Diseases [C02]
DNA Virus Infections [C02.256]

Papillomavirus Infections [C02.256.650]

Warts [C02.256.650.810] +

Virus Diseases [C02]

Tumor Virus Infections [C02.928]

Papillomavirus Infections [C02.928.725]

(PMID: 229324377)

Diagnostics:

In the lab few places propagating HPV. There are measures that need to be taken by the laboratory personnel. CDC as well as WHO published various articles to inform public.

Sensitivity and testing for Pap smear and HPV DNA testing in the detection of CIN2+

Test	Sensitivity	Specificity
Pap smear	53-55.4%	96.3-96.8%
High-risk HPV DNA testing	94.6-96.1%	90.7-94.1%
Pap smear + high-risk HPV testing	100%	92.5%

Cuzick J, Clavel C, Petry KU, Meijer CJ, Hoyer H, Ratnam S, Szarewski A, Birembaut P, Kulasingam S, Sasieni P, Iftner T. Overview of the European and North American studies on HPV testing in primary cervical cancer screening. Int J Cancer. 2006; 119(5):1095.

Mayrand MH, Duarte-Franco E, Rodrigues I, Walter SD, Hanley J, Ferenczy A, Ratnam S, Coutlée F, Franco EL, Canadian Cervical Cancer Screening Trial Study Group.

Human papillomavirus DNA versus Papanicolaou screening tests for cervical cancer N Engl J Med. 2007;357(16):1579.

Best Pract Res Clin Obstet Gynaecol. Author manuscript; available in PMC 2013 Apr 22. (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3632360/)

HPV Genotyping tests¹

HPV genotyping test	HPV types detected
Cervista® HPV 16/18 (Hologic, Inc; Marlborough, MA)^a	HR HPV types 16 and 18
Digene HPV Genotyping PS Test (Qiagen; Hilden, Germany)	HR HPV types 16, 18, and 45
Roche LINEAR ARRAY HPV Genotyping Test (Roche; Basel, Switzerland)	37 LR and HR HPV types
Innogenetics INNO-LiPA HPV Genotyping Extra (Innogenetics; Gent, Belgium)	28 LR and HR HPV types
SPF10 Line Probe Assay HPV-typing System (Roche; Basel, Switzerland)	Recognizes most genital tract HPV types
Papillocheck1 (Greiner Bio-One; Frickenhausen Germany)	18 HR and 6 LR HPV types
RealTime High Risk HPV Assay (Abbott Laboratories;Abbott Park, IL)	HPV types 16 and 18
HPV Genotyping LQ Test (Qiagen Inc; Valencia, CA)	18 HR HPV types
Seeplex HPV4A ACE (Seegene; Rockville, MD)	HPV types 16 and 18
CLART HPV 2 (Genomica; Madrid, Spain)	35 LR and HR HPV types
GenoFlow HPV Array (DiagCor; North Point, Hong Kong)	33 LR and HR HPV types
fHPV Typing (molGENTIX; Barcelona, Spain)	15 LR and HR HPV types

HPV, human papillomavirus; HR, high-risk; LR, low-risk.

^aFDA-approved test.

¹Schutzbank TE, Ginocchio CC. Assessment of clinical and analytical performance characteristics of an HPV genotyping test. Diagn Cytopathol. 2011 Apr 6. doi:10.1002/dc.21661.

Most papillomas are sufficiently distinct to be clinically recognizable. Bowenoid papulosis may be mistaken for lichen planus, psoriasis, seborrheic keratoses, or condylomata acuminata.

In additions to the conditions listed in the differential diagnosis, other problems to be considered include the following:

Acanthosis nigricans
Acrochordon
Actinic keratoses
Anogenital malignancy
Anogenital warts in children
Bowenoid papulosis
Carbon dioxide laser surgery for intraepithelial cervical neoplasms
Cervical polyp
Condyloma latum
Corns and calluses
Dermatitis papillaris
Endoscopic gynecologic surgery
Epidermodysplasia verruciformis
Fordyce spots
Hymenal remnants
Hypopigmentation
Keloid and hypertrophic scar
Keratoacanthoma
Laryngeal papillomatosis of neonates and infants
Malignant tumors of the mobile tongue
Micropapillomatosis labialis
Nevi
Pap test
Pityriasis versicolor
Psoriasis (plaque)
Recurrent respiratory papillomatosis
Seborrheic keratosis
Sinonasal papillomas, treatment
Skin tags (fibroepithelial polyps)
Verrucous carcinoma
Vestibular papillomatosis

Differential Diagnoses

Basal Cell Carcinoma
Benign Cervical Lesions
Benign Vulvar Lesions
Bowen Disease
Carbon Dioxide Laser Surgery of the Lower Genital Tract
Cervical Cancer
Chancroid
Conization of Cervix
Erythroplasia of Queyrat (Bowen Disease of the Glans Penis)
Giant Condylomata Acuminata of Buschke and Lowenstein
Gynecologic Cryosurgery
Gynecologic Laparoscopy
Head and Neck Cutaneous Squamous Cell Carcinoma
Hemorrhoids
Herpes Simplex
Lichen Planus
Malignant Dermatologic Diseases of the Male Genitalia
Malignant Vulvar Lesions
Molluscum Contagiosum
Nonmalignant Dermatologic Diseases of the Male Genitalia
Pearly Penile Papules
Pediatric Syphilis
Penile Cancer
Rectal Cancer
Recurrent Respiratory Papillomatosis
Rehabilitation for Paget Disease
Sebaceous Hyperplasia
Surgical Treatment of Vulvar Cancer
Urethral Warts
Vaginal Cancer

Treatment:

1. Immunomodulators

Class Summary

Immune response modifiers have immunomodulatory effects and are used for treatment of external anogenital warts (EGWs) or condylomata acuminata. Interferon alfa, beta, and gamma may be administered topically, systemically, and intralesionally. They stimulate production of cytokines and demonstrate strong antiviral activity.

Imiquimod (Aldara, Zyclara)

Imiquimod is an imidazoquinolinamine derivative that has no in vitro antiviral activity but does induce macrophages to secrete cytokines such as interleukin (IL)-2 and interferon alfa and gamma. Its mechanisms of action are unknown. Imiquimod has been studied extensively and is a new therapy relative to other EGW treatments. It may be more effective in women than in men.

Imiquimod is dispensed as an individual dose. Patients are advised to wash the affected area with mild soap and water upon awakening and to remove residual drug.

Interferon alfa-n3 (Alferon N)

Interferon alfa is a protein product either manufactured from a single-species recombinant DNA process or obtained from pooled units of donated human leukocytes that have been induced by incomplete infection with a murine virus.

The mechanisms by which interferon alfa exerts antiviral activity are not understood clearly. However, modulation of the host immune response may play an important role. This agent is indicated for intralesional treatment of refractory or recurring external condyloma acuminatum and is particularly useful for patients who have not responded satisfactorily to other treatment modalities (eg, podophyllin, surgical excision, laser therapy, or cryotherapy).

Interferon alfa-2b (Intron A)

This is a protein product manufactured by recombinant DNA technology. Its mechanism of antitumor activity is not clearly understood; however, direct antiproliferative effects against malignant cells and modulation of host immune response may play important roles. Its immunomodulatory effects include suppression of tumor cell proliferation, enhancement of macrophage phagocytic activity, and augmentation of lymphocyte cytotoxicity.

This agent is indicated for intralesional treatment of refractory or recurring external condyloma acuminatum and is particularly useful for patients who have not responded satisfactorily to other treatment modalities (eg, podophyllin, surgical excision, laser therapy, or cryotherapy).

2. Keratolytic Agents

Class Summary

Antimitotic drugs arrest dividing cells in mitosis, resulting in the death of proliferating cells. They cause cornified epithelium to swell, soften, macerate, and then desquamate. Many of them are chemotherapeutic agents. The drugs listed below are used specifically for treatment of EGWs or condylomata acuminata.

Keratolytic agents are used to aid in removal of keratin in hyperkeratotic skin disorders, including corns, ichthyoses, common warts, flat warts, and other benign verrucae.

Podofilox (Condylox)

Podofilox is a topical antimitotic that can be synthesized chemically or purified from the plant families Coniferae and Berberidaceae (eg, species of Juniperus and Podophyllum). It is the active agent of podophyllin resin and is available as a 0.5% solution. Treatment results in necrosis of visible wart tissue; the exact mechanism of action is unknown. Treatment should be limited to no more than 10 cm2 of wart tissue, and no more than 0.5 mL/day of solution should be given. This is a patient-applied therapy.

Podophyllum resin (Podocon-25)

Podophyllin is derived from May apple (Podophyllum peltatum Linné) and contains the active agent podophyllotoxin, a cytotoxic substance that arrests mitosis in metaphase. American podophyllum contains one fourth the amount of podophyllotoxin that Indian podophyllum does. The potency of podophyllin varies considerably between batches. The exact mechanism of action is unknown.

Podophyllin is used as a topical treatment for benign growths, including external genital and perianal warts, papillomas, and fibroids. It results in necrosis when applied to anogenital warts. Only a trained medical professional can apply it, and it cannot be dispensed to a patient.

Trichloroacetic acid 85% (Tri-Chlor)

Trichloroacetic acid (TCA) is a highly corrosive desiccating agent that cauterizes skin, keratin, and other tissues and is used to burn lesions. Although it is caustic, it causes less local irritation and systemic toxicity than other agents in the same class. However, response often is incomplete, and recurrence is common.

Most clinicians use 25-50% TCA, although some use concentrations as high as 85% and then neutralize with either water or bicarbonate. Tissue sloughs and subsequently heals in 7-10 days. TCA therapy is less destructive than laser surgery, electrocautery, or cryotherapy.

Salicylic acid (Compound W, Dr. Scholl’s Clear Away Warts, Freezone)

By dissolving the intercellular cement substance, salicylic acid produces desquamation of the horny layer of skin without affecting the structure of viable epidermis. It is used for removal of nongenital cutaneous warts, particularly common or plantar warts. Before application, wash the affected area. The wart may be soaked in warm water for 5 minutes. Dry the area thoroughly.

3. Antineoplastics, Antimetabolite

Class Summary

Antimetabolites interfere with nucleic acid synthesis and inhibit cell growth and proliferation. These are topical preparations that contain the fluorinated pyrimidine 5-fluorouracil (5-FU). Although these chemotherapeutic agents are not formally approved for use against warts, some studies have demonstrated a benefit against EGWs or condylomata acuminata.

Fluorouracil topical (Efudex, Carac, Fluoroplex)

Topical 5-FU interferes with DNA synthesis by blocking the methylation of deoxyuridylic acid and inhibits thymidylate synthetase, which subsequently reduces cell proliferation. Its primary indication is for topical treatment of actinic keratoses. Although it is not approved by the US Food and Drug Administration (FDA) for the treatment of warts, it has been used in adults, particularly for warts resistant to other forms of treatment. It is used for management of superficial basal cell carcinomas.

The solution contains either 2% or 5% 5-FU in propylene glycol, tris (hydroxymethyl) aminomethane, hydroxypropyl cellulose, paraben, and disodium edetate. The cream contains 5% 5-FU in white petrolatum, stearyl alcohol, propylene glycol, polysorbate 60, and paraben. When topical 5-FU is applied to the lesion, the area undergoes a sequence of erythema, vesiculation, desquamation, erosion, and reepithelialization.

4. Topical Skin Products

Class Summary

Sinecatechins is another topical product that has gained FDA approval for genital warts.

Sinecatechins (Veregen)

Sinecatechins ointment is a botanical drug product for topical use that consists of extract from green tea leaves. It contains 15% sinecatechins and is available in 15- and 30-g tubes. Its mode of action is unknown, but it does elicit antioxidant activity in vitro. Sinecatechins ointment is indicated for topical treatment of external genital and perianal warts (condylomata acuminata) in immunocompetent patients.

5. Vaccines, Inactivated, Viral

Class Summary

Three vaccines are available for the prevention of HPV-associated dysplasias and neoplasia, including cervical, vulvar, vaginal, and anal cancer; genital warts (condylomata acuminata); and precancerous genital lesions.

Human papillomavirus vaccine, nonavalent (Gardasil 9)

Recombinant vaccine that targets 9 HPV types (6, 11, 16, 18, 31, 33, 45, 52, and 58). It is indicated for females aged 9-26 years to prevent cervical, vulvar, vaginal, and anal cancer. It is also indicated to prevent genital warts and dysplastic lesions (eg, cervical, vulvar, vaginal, anal).

It is also indicated for boys aged 9-15 years for prevention of anal cancer, genital warts, and anal intraepithelial neoplasia. In addition to the approved indications, the CDC recommends vaccinating males aged 16 through 21 years not previously vaccinated. CDC recommendations also include men through age 26 years not previously vaccinated. Vaccination is also recommended by the CDC among men who have sex with men and among immunocompromised persons (including those with HIV infection) if not vaccinated previously through age 26 years.

Human papillomavirus vaccine, quadrivalent (Gardasil)

The quadrivalent HPV recombinant vaccine was the first vaccine indicated to prevent cervical cancer, genital warts (condylomata acuminata), and precancerous genital lesions (eg, cervical adenocarcinoma in situ; cervical intraepithelial neoplasia grades I-III; vulvar intraepithelial neoplasia grades II and III; and vaginal intraepithelial neoplasia grades II and III) due to HPV types 6, 11, 16, and 18. Its efficacy is mediated by humoral immune responses following immunization series.

The quadrivalent vaccine is FDA-approved for females aged 9-26 years and is under FDA priority review to evaluate efficacy in women aged 27-45 years. It is indicated for boys and men aged 11-26 years for prevention of condylomata acuminata caused by HPV types 6 and 11. It is also indicated in people aged 9-26 years for prevention of anal cancer and associated precancerous lesions.

Zaravinos A¹. An updated overview of HPV-associated head and neck carcinomas.

Human papillomavirus vaccine, bivalent (Cervarix)

The bivalent HPV vaccine is a recombinant vaccine prepared from the L1 protein of HPV types 16 and 18. It is indicated in girls and women aged 10-25 years for the prevention of diseases caused by oncogenic HPV types 16 and 18 (eg, cervical cancer, cervical intraepithelial neoplasia grade II or higher, adenocarcinoma in situ, and cervical intraepithelial neoplasia grade I).

HPV Vaccines: Indications Approved and HPV Types by Specific Vaccines

Indicated to Prevent	HPV 9-valent*	HPV 4-valent	HPV 2-valent
Girls and Women
Approved ages	9-26 y	9-26 y	9-25 y
Cervical cancer	HPV types 16, 18, 31, 33, 45, 52, and 58	HPV types 16 and 18	HPV types 16 and 18
Vulvar cancer	HPV types 16, 18, 31, 33, 45, 52, and 58	HPV types 16 and 18	Not approved
Vaginal cancer	HPV types 16, 18, 31, 33, 45, 52, and 58	HPV types 16 and 18	Not approved
Anal cancer	HPV types 16, 18, 31, 33, 45, 52, and 58	HPV types 16 and 18	Not approved
Genital warts (condyloma acuminata)	HPV types 6 and 11	HPV types 6 and 11	Not approved
Cervical intraepithelial neoplasia (CIN) grade 2/3 and cervical adenocarcinoma in situ (AIS)	HPV types 6, 11, 16, 18, 31, 33, 45, 52, and 58	HPV types 6, 11, 16, and 18	HPV types 16 and 18
Cervical intraepithelial neoplasia (CIN) grade 1	HPV types 6, 11, 16, 18, 31, 33, 45, 52, and 58	HPV types 6, 11, 16, and 18	HPV types 16 and 18
Vulvar intraepithelial neoplasia (VIN) grades 2 and 3	HPV types 6, 11, 16, 18, 31, 33, 45, 52, and 58	HPV types 6, 11, 16, and 18	Not approved
Vaginal intraepithelial neoplasia (VaIN) grades 2 and 3	HPV types 6, 11, 16, 18, 31, 33, 45, 52, and 58	HPV types 6, 11, 16, and 18	Not approved
Anal intraepithelial neoplasia (AIN) grades 1, 2, and 3	HPV types 6, 11, 16, 18, 31, 33, 45, 52, and 58	HPV types 6, 11, 16, and 18	Not approved
Boys and Men
Approved ages	9-15 y*	9-26 y	Not approved
Anal cancer	HPV types 16, 18, 31, 33, 45, 52, and 58	HPV types 16 and 18	Not approved
Genital warts (condyloma acuminata)	HPV types 6 and 11	HPV types 6 and 11	Not approved
Anal intraepithelial neoplasia (AIN) grades 1, 2, and 3	HPV types 6, 11, 16, 18, 31, 33, 45, 52, and 58	HPV types 6, 11, 16, and 18	Not approved
*The CDC recommends vaccinating males 16-21 y not previously vaccinated, and through age 26 y among men who have sex with men and among immunocompromised persons (including those with HIV infection) if not vaccinated previously

Clinical Trials:

Two trials of clinically approved human papillomavirus (HPV) vaccines, Females United to Unilaterally Reduce Endo/Ectocervical Disease (FUTURE I/II) and the Papilloma Trial Against Cancer in Young Adults (PATRICIA), reported a 22% difference in vaccine efficacy (VE) against cervical intraepithelial neoplasia grade 2 or worse in HPV-naïve subcohorts; however, serological testing methods and the HPV DNA criteria used to define HPV-unexposed women differed between the studies.

The risk of newly detected human papillomavirus (HPV) infection and cervical abnormalities in relation to HPV type 16/18 antibody levels at enrollment in PATRICIA (Papilloma Trial Against Cancer in Young Adults; NCT00122681).

The control arm of PATRICIA (PApilloma TRIal against Cancer In young Adults,NCT00122681) was used to investigate the risk of progression from cervical HPV infection to cervical intraepithelial neoplasia (CIN) or clearance of infection, and associated determinants.

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Herrero R, Quint W, Hildesheim A, Gonzalez P, Struijk L, Katki HA, Porras C, Schiffman M, Rodriguez AC, Solomon D, Jimenez S, Schiller JT, Lowy DR, van Doorn LJ, Wacholder S, Kreimer AR. CVT Vaccine Group. Reduced Prevalence of Oral Human Papillomavirus (HPV) 4 Years after Bivalent HPV Vaccination in a Randomized Clinical Trial in Costa Rica. PLoS One. 2013 Jul 17;8(7):e68329. ClinicalTrials.gov, Registry number NCT00128661.

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Hildesheim A, Herrero R, Wacholder S, Rodriguez AC, Solomon D, Bratti MC, Schiller JT, Gonzalez P, Dubin G, Porras C, Jimenez SE, Lowy DR. Costa Rican HPV Vaccine Trial Group. Effect of human papillomavirus 16/18 L1 viruslike particle vaccine among young women with preexisting infection: a randomized trial. JAMA. 2007;298(7):743–753. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00128661.

PMCID: PMC282145

McCance D, Kopan R, Fuchs E, et al. Human papillomavirus type 16 alters human epithelial cell differentiation in vitro. Proc. Natl. Acad. Sci. 1988;85:7169–7173.

PMCID: PMC3137403

Castellsague X, Munoz N, Pitisuttithum P, Ferris D, Monsonego J, Ault K, et al. End-of-study safety, immunogenicity, and efficacy of quadrivalent HPV (types 6, 11, 16, 18) recombinant vaccine in adult women 24–45 years of age. Br J Cancer. 2011;105:28–37.

PMID:20508593

CDC. FDA Licensure of Bivalent Human Papillomavirus Vaccine (HPV2, Cervarix) for Use in Females and Updated HPV Vaccination Recommendations from the Advisory Committee on Immunization Practices (ACIP) MMWR. 2010;59(20):626–629.

PMID:22189893

CDC. Recommendations on the Use of Quadrivalent Human Papillomavirus Vaccine in Males-Advisory Committee on Immunization Practices (ACIP) MMWR. 2011;60(50):1705–1708.

CDC. [accessed October 28, 2013];Biosafety in Microbiological and Biomedical Laboratories (BMBL) (5th Edition). http://www.cdc.gov/biosafety/publications/bmbl5/

PMID:19342877

Chow LT, Duffy AA, Wang HK, Broker TR. A highly efficient system to produce infectious human papillomavirus. Cell Cycle. 2009;8:1319–1323.

Clinical Trials Publications:

Kreimer AR, Rodriguez AC, Hildesheim A, Herrero R, Porras C, Schiffman M, González P, Solomon D, Jiménez S, Schiller JT, Lowy DR, Quint W, Sherman ME, Schussler J, Wacholder S; CVT Vaccine Group. Proof-of-principle evaluation of the efficacy of fewer than three doses of a bivalent HPV16/18 vaccine. J Natl Cancer Inst. 2011 Oct 5;103(19):1444-51. doi: 10.1093/jnci/djr319. Epub 2011 Sep 9.

Kemp TJ, Hildesheim A, Safaeian M, Dauner JG, Pan Y, Porras C, Schiller JT, Lowy DR, Herrero R, Pinto LA. HPV16/18 L1 VLP vaccine induces cross-neutralizing antibodies that may mediate cross-protection. Vaccine. 2011 Mar 3;29(11):2011-4. doi: 10.1016/j.vaccine.2011.01.001. Epub 2011 Jan 15.
Additional publications automatically indexed to this study by ClinicalTrials.gov Identifier (NCT Number):

Kreimer AR, Struyf F, Del Rosario-Raymundo MR, Hildesheim A, Skinner SR, Wacholder S, Garland SM, Herrero R, David MP, Wheeler CM; Costa Rica Vaccine Trial and PATRICIA study groups. Efficacy of fewer than three doses of an HPV-16/18 AS04-adjuvanted vaccine: combined analysis of data from the Costa Rica Vaccine and PATRICIA trials. Lancet Oncol. 2015 Jul;16(7):775-86. doi: 10.1016/S1470-2045(15)00047-9. Epub 2015 Jun 9.

Gonzalez P, Hildesheim A, Herrero R, Katki H, Wacholder S, Porras C, Safaeian M, Jimenez S, Darragh TM, Cortes B, Befano B, Schiffman M, Carvajal L, Palefsky J, Schiller J, Ocampo R, Schussler J, Lowy D, Guillen D, Stoler MH, Quint W, Morales J, Avila C, Rodriguez AC, Kreimer AR; Costa Rica HPV Vaccine Trial (CVT) Group. Rationale and design of a long term follow-up study of women who did and did not receive HPV 16/18 vaccination in Guanacaste, Costa Rica. Vaccine. 2015 Apr 27;33(18):2141-51. doi: 10.1016/j.vaccine.2015.03.015. Epub 2015 Mar 18.

Lang Kuhs KA, Porras C, Schiller JT, Rodriguez AC, Schiffman M, Gonzalez P, Wacholder S, Ghosh A, Li Y, Lowy DR, Kreimer AR, Poncelet S, Schussler J, Quint W, van Doorn LJ, Sherman ME, Sidawy M, Herrero R, Hildesheim A, Safaeian M; Costa Rica Vaccine Trial Group. Effect of different human papillomavirus serological and DNA criteria on vaccine efficacy estimates. Am J Epidemiol. 2014 Sep 15;180(6):599-607. doi: 10.1093/aje/kwu168. Epub 2014 Aug 19.

Hildesheim A, Wacholder S, Catteau G, Struyf F, Dubin G, Herrero R; CVT Group. Efficacy of the HPV-16/18 vaccine: final according to protocol results from the blinded phase of the randomized Costa Rica HPV-16/18 vaccine trial. Vaccine. 2014 Sep 3;32(39):5087-97. doi: 10.1016/j.vaccine.2014.06.038. Epub 2014 Jul 10.

Lang Kuhs KA, Gonzalez P, Rodriguez AC, van Doorn LJ, Schiffman M, Struijk L, Chen S, Quint W, Lowy DR, Porras C, DelVecchio C, Jimenez S, Safaeian M, Schiller JT, Wacholder S, Herrero R, Hildesheim A, Kreimer AR; Costa Rica Vaccine Trial Group. Reduced prevalence of vulvar HPV16/18 infection among women who received the HPV16/18 bivalent vaccine: a nested analysis within the Costa Rica Vaccine Trial. J Infect Dis. 2014 Dec 15;210(12):1890-9. doi: 10.1093/infdis/jiu357. Epub 2014 Jun 23.

Lang Kuhs KA, Gonzalez P, Struijk L, Castro F, Hildesheim A, van Doorn LJ, Rodriguez AC, Schiffman M, Quint W, Lowy DR, Porras C, Delvecchio C, Katki HA, Jimenez S, Safaeian M, Schiller J, Solomon D, Wacholder S, Herrero R, Kreimer AR; Costa Rica Vaccine Trial Group. Prevalence of and risk factors for oral human papillomavirus among young women in Costa Rica. J Infect Dis. 2013 Nov 15;208(10):1643-52. doi: 10.1093/infdis/jit369. Epub 2013 Sep 6.

Herrero R, Quint W, Hildesheim A, Gonzalez P, Struijk L, Katki HA, Porras C, Schiffman M, Rodriguez AC, Solomon D, Jimenez S, Schiller JT, Lowy DR, van Doorn LJ, Wacholder S, Kreimer AR; CVT Vaccine Group. Reduced prevalence of oral human papillomavirus (HPV) 4 years after bivalent HPV vaccination in a randomized clinical trial in Costa Rica. PLoS One. 2013 Jul 17;8(7):e68329. doi: 10.1371/journal.pone.0068329. Print 2013.

Clarke M, Schiffman M, Wacholder S, Rodriguez AC, Hildesheim A, Quint W; Costa Rican Vaccine Trial Group. A prospective study of absolute risk and determinants of human papillomavirus incidence among young women in Costa Rica. BMC Infect Dis. 2013 Jul 8;13:308. doi: 10.1186/1471-2334-13-308.

Castro FA, Quint W, Gonzalez P, Katki HA, Herrero R, van Doorn LJ, Schiffman M, Struijk L, Rodriguez AC, DelVecchio C, Lowy DR, Porras C, Jimenez S, Schiller J, Solomon D, Wacholder S, Hildesheim A, Kreimer AR; Costa Rica Vaccine Trial Group. Prevalence of and risk factors for anal human papillomavirus infection among young healthy women in Costa Rica. J Infect Dis. 2012 Oct 1;206(7):1103-10. Epub 2012 Jul 30.

Kreimer AR, González P, Katki HA, Porras C, Schiffman M, Rodriguez AC, Solomon D, Jiménez S, Schiller JT, Lowy DR, van Doorn LJ, Struijk L, Quint W, Chen S, Wacholder S, Hildesheim A, Herrero R; CVT Vaccine Group. Efficacy of a bivalent HPV 16/18 vaccine against anal HPV 16/18 infection among young women: a nested analysis within the Costa Rica Vaccine Trial. Lancet Oncol. 2011 Sep;12(9):862-70. doi: 10.1016/S1470-2045(11)70213-3. Epub 2011 Aug 22. Erratum in: Lancet Oncol. 2011 Nov;12(12):1096.

Wacholder S, Chen BE, Wilcox A, Macones G, Gonzalez P, Befano B, Hildesheim A, Rodríguez AC, Solomon D, Herrero R, Schiffman M; CVT group. Risk of miscarriage with bivalent vaccine against human papillomavirus (HPV) types 16 and 18: pooled analysis of two randomised controlled trials. BMJ. 2010 Mar 2;340:c712. doi: 10.1136/bmj.c712.

Dessy FJ, Giannini SL, Bougelet CA, Kemp TJ, David MP, Poncelet SM, Pinto LA, Wettendorff MA. Correlation between direct ELISA, single epitope-based inhibition ELISA and pseudovirion-based neutralization assay for measuring anti-HPV-16 and anti-HPV-18 antibody response after vaccination with the AS04-adjuvanted HPV-16/18 cervical cancer vaccine. Hum Vaccin. 2008 Nov-Dec;4(6):425-34. Epub 2008 Nov 11.

Hildesheim A, Herrero R, Wacholder S, Rodriguez AC, Solomon D, Bratti MC, Schiller JT, Gonzalez P, Dubin G, Porras C, Jimenez SE, Lowy DR; Costa Rican HPV Vaccine Trial Group. Effect of human papillomavirus 16/18 L1 viruslike particle vaccine among young women with preexisting infection: a randomized trial. JAMA. 2007 Aug 15;298(7):743-53.

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Reporter: Aviva Lev-Ari, PhD, RN Head and Neck Cancer Studies Suggest Alternative Markers More Prognostically Useful than HPV DNA Testing September 18, 2012 By a GenomeWeb staff reporter NEW YORK (GenomeWeb News) – The presence or absence of human papillomavirus DNA on its own in an individual’s head or neck cancer does not provide enough information […]

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http://www.medpagetoday.com/HematologyOncology/CervicalCancer/51016

Papilloma viruses for cervical cancer

Larry H. Bernstein, MD, FCAP, Curator

LPBI

Practice Bulletin No. 131: Screening for Cervical Cancer

Obstetrics & Gynecology:

November 2012

doi: http://10.1097/AOG.0b013e318277c92a

The incidence of cervical cancer in the United States has decreased more than 50% in the past 30 years because of widespread screening with cervical cytology. In 1975, the rate was 14.8 per 100,000 women. By 2008, it had been reduced to 6.6 per 100,000 women. Mortality from the disease has undergone a similar decrease from 5.55 per 100,000 women in 1975 to 2.38 per 100,000 women in 2008 (1). The American Cancer Society (ACS) estimates that there will be 12,170 new cases of cervical cancer in the United States in 2012, with 4,220 deaths from the disease (2). Cervical cancer is much more common worldwide, particularly in countries without screening programs, with an estimated 530,000 new cases of the disease and 275,000 resultant deaths each year (3, 4). When cervical cancer screening programs have been introduced into communities, marked reductions in cervical cancer incidence have followed (5, 6).

New technologies for cervical cancer screening continue to evolve as do recommendations for managing the results. In addition, there are different risk-benefit considerations for women at different ages, as reflected in age-specific screening recommendations. The ACS, the American Society for Colposcopy and Cervical Pathology (ASCCP), and the American Society for Clinical Pathology (ASCP) have recently updated their joint guidelines for cervical cancer screening (7), and an update to the U.S. Preventive Services Task Force recommendations also has been issued (8). The purpose of this document is to provide a review of the best available evidence regarding screening for cervical cancer.

Study Backs Co-Testing for Cervical Cancer

A positive co-test result was more sensitive than either a positive HPV-only test or a positive Pap-only test.

Charles Bankhead

Cervical cancer screening with a test for human papillomavirus (HPV) resulted in a 50% higher rate of false-negative results versus Pap testing and three times greater versus co-testing, a large retrospective study showed.

Data encompassing more than 250,000 women showed a false-negative rate of 18.6% compared with 12.2% for Pap testing. With a false-negative rate of 5.5%, screening women with the HPV test and Pap test missed the fewest cancers.

The results support clinical guidelines that recommend co-testing, according to authors of a report in Cancer Cytopathology. The results differ dramatically, however, from those of previous studies that have consistently shown greater diagnostic accuracy for the HPV test compared with the Pap test.

“The reason that women are screened is that they want to be protected from cervical cancer,” study author R. Marshall Austin, MD, PhD, of Magee-Women’s Hospital and the University of Pittsburgh, told MedPage Today. “The previous trials have generally focused on cervical intraepithelial neoplasia 2 or 3, so-called precancer. The difference is that most of what we call precancer will actually never develop into cancer.

“The unique thing about this study, and what makes it so important, is that we looked at over 500 invasive cervical cancers, which are what women want to be protected against, and looked at the effectiveness of the methods of testing.”

A year ago, the FDA approved Roche’s cobas assay for HPV DNA as a first-line test for cervical cancer screening, following a unanimous vote for approval by an FDA advisory committee.

The approval was based primarily on a pivotal trial involving 47,200 women at high risk for cervical cancer. The primary endpoint was the proportion of patients who developed grade ≥3 cervical intraepithelial neoplasia (≥CIN3). The results showed a greater than 50% reduction in the incidence of ≥CIN3 with the DNA test versus Pap testing.

Austin and colleagues retrospectively analyzed clinical records for 256,648 average-risk women, ages 30 to 65, all of whom underwent co-testing as a screen for cervical cancer and subsequently had a cervical biopsy within a year of co-testing. The primary objective was to determine the sensitivity of the three screening methods for detection of biopsy-proven ≥CIN3 and invasive cancer.

The results showed that 74.7% of the women had a positive HPV test, 73.8% had an abnormal Pap test (atypical squamous cells of undetermined significance or worse), 89.2% had a positive co-test, and 1.6% had ≥CIN3.

Biopsy results showed that co-testing had the highest sensitivity for ≥CIN3 (98.8% versus 94% for HPV test only and 91.3% for Pap testing alone, P<0.0001). The Pap test had greater specificity versus HPV testing alone or co-testing (26.3% versus 25.6% versus 10.9%, P<0.0001).

Investigators identified 526 patients who developed biopsy-proven invasive cervical cancer. Of those patients, 98 tested negative by HPV assay only, 64 by Pap test only, and 29 by co-testing.

Given the average risk of the patient population included in the study, the results are broadly applicable to women in the age range studied, regardless of baseline risk for cervical cancer, Austin said.

The data also show that co-testing is better than either test alone, which supports current ACS recommendations for cervical cancer screening.

“The current approach, according to the American Cancer Society and 25 other organizations that worked with us on our last guideline, co-testing is the preferred strategy,” Saslow told MedPage Today. “This paper completely backs that up. Even though a Pap alone is acceptable, clearly, co-testing is the best way to go.”

Noting that only half of women in the U.S. do not under go co-testing despite clinical guidelines recommending it for more than a decade, Saslow asked, “What’s taking so long?”

Earlier this year, several organizations released joint “interim guidance” regarding cervical cancer screening. Described as an aid to clinical decision-making until existing guidelines are updated, the interim guidance characterized the HPV-DNA test as an acceptable alternative to Pap testing as a primary screening test.

In their article, Austin and colleagues argued that the HPV assay should be evaluated in comparison with the Pap test but as an alternative to co-testing.

“HPV-only primary screening for cervical cancer presents many challenges for clinicians,” the authors said. “Questions arise regarding its effectiveness, its long-term risk, and when it is the best option for a particular patient.

“Clinicians had similar questions when co-testing was first recommended for women 30 and older in 2006,” they added. “Since then the adoption of co-testing has steadily increased, with approximately 50% of physicians co-testing women 30 and older, but it is still not done at the recommended level.”

The study had some limitations. The authors could not confirm that the cervical biopsy results were from women who did not have an intervening screening test or treatment with a different provider during the study period.

Also, the authors were unable to draw conclusions based on the overall population of women who were screened for cervical cancer because the dataset consisted of screening results of women who underwent biopsies.

A spatial model predicts that dispersal and cell turnover limit intratumour heterogeneity.

Can IntraTumoral Heterogeneity Be Thought of as a Mechanism of Resistance?

Posted in Cancer and Current Therapeutics, CANCER BIOLOGY & Innovations in Cancer Therapy, Cell Biology, Cell Biology, Signaling & Cell Circuits, Clinical & Translational, Clinical Genomics, Conference Coverage with Social Media, Curation, Genetics & Pharmaceutical, Genome Biology, Personalized and Precision Medicine & Genomic Research, Pharmacogenomics, tagged antitumor therapy, breast cancer, Cancer - General, Cancer Genomics, Cell Biology, chemoresistance, Chemotherapy, Clinical trial, clonal heterogeneity, Conditions and Diseases, DNA, health, intratumoral heterogeneity, mechanisms of disease, medicine, Personalized medicine, research, science on October 12, 2015| Leave a Comment »

Can IntraTumoral Heterogeneity Be Thought of as a Mechanism of Resistance?

Curator/Reporter: Stephen J. Williams, Ph.D.

Therapeutic resistance remains one of the most challenging problems for the oncologist, despite the increase of new therapeutics in the oncologist’s toolkit. As new targeted therapies are developed, and new novel targets are investigated as potential therapies, especially cytostatic therapies which it has become evident our understanding of chemoresistance is expanding beyond mechanisms to circumvent a drug’s pharmacologic mechanism of action (i.e. increased DNA repair and cisplatin) or pharmacokinetic changes (i.e. increased efflux by acquisition of a MDR phenotype).

In a talk at the 2015 AACR National Meeting, Dr. Charles Swanton discusses the development of tumor heterogeneity in the light of developing, or acquired, drug resistance. Chemoresistance is either categorized as acquired resistance (where resistance develops upon continued exposure to drug) or inherent resistance (related to a tumor being refractory or unresponsive to drug). Dr Swanton discusses findings where development of this heterogeneity (discussed here in a posting on Issues in Personalized Medicine in Cancer: Intratumor Heterogeneity and Branched Evolution Revealed by Multiregion Sequencing) and here (Notes On Tumor Heterogeneity: Targets and Mechanisms, from the 2015 AACR Meeting in Philadelphia PA) on recent findings on Branched Chain Heterogeneity) is resulting in clones resistant to the initial drug treatment.

To recount a bit of background I list the overall points of the one of previous posts on tumor heterogeneity (and an interview with Dr. Charles Swanton) are as follows:

Multiple biopsies of primary tumor and metastases are required to determine the full mutational landscape of a patient’s tumor

The intratumor heterogeneity will have an impact on the personalized therapy strategy for the clinician

Metastases arising from primary tumor clones will have a greater genomic instability and mutational spectrum than the tumor from which it originates

Tumors and their metastases do NOT evolve in a linear path but have a branched evolution and would complicate biomarker development and the prognostic and resistance outlook for the patient

The following is a curation of various talks and abstracts from the 2015 AACR National Meeting in Philadelphia on effects of clonal evolution and intratumoral heterogeneity of a tumor with respect to development of chemoresistance. As this theory of heterogeneity and clonal evolution is particularly new I attempted to present all works (although apologize for the length upfront) to forgo bias and so the reader may extract any information pertinent to their clinical efforts and research. However I will give a brief highlight summary below:

From the 2015 AACR National Meeting in Philadelphia

PresentationNumber:NGO2

Presentation Title:

Polyclonal and heterogeneous resistance to targeted therapy in leukemia

Presentation Time:

Monday, Apr 20, 2015, 10:40 AM -10:55 AM

Location:

Room 201, Pennsylvania Convention Center

Author Block:

Catherine C. Smith, Amy Paguirigan, Chen-Shan Chin, Michael Brown, Wendy Parker, Mark J. Levis, Alexander E. Perl, Kevin Travers, Corynn Kasap, Jerald P. Radich, Susan Branford, Neil P. Shah. University of California, San Francisco, CA, Fred Hutchinson Cancer Research Center, Seattle, WA, Pacific Biosciences, Menlo Park, CA, Royal Adelaide Hospital, Adelaide, Australia, Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD, Abramson Cancer Center of the University of Pennsylvania, Philadelphia, PA, University of California, San Francisco, CA

Abstract Body:

Genomic studies in solid tumors have revealed significant branching intratumoral clonal genetic heterogeneity. Such complexity is not surprising in solid tumors, where sequencing studies have revealed thousands of mutations per tumor genome. However, in leukemia, the genetic landscape is considerably less complex. Chronic myeloid leukemia (CML) is the human malignancy most definitively linked to a single genetic lesion, the BCR-ABL gene fusion. Genome wide sequencing of acute myeloid leukemia (AML) has revealed that AML is the most genetically straightforward of all extensively sequenced adult cancers to date, with an average of 13 coding mutations and 3 or less clones identified per tumor.
In CML, tyrosine kinase inhibitors (TKIs) of BCR-ABL have resulted in high rates of remission. However, despite excellent initial response rates with TKI monotherapy, patients still relapse, including virtually all patients with Philadelphia-positive acute lymphoblastic leukemia and blast crisis CML. Studies of clinical resistance highlight BCR-ABL as the sole genetic driver in CML as secondary kinase domain (KD) mutations that prevent drug binding are the predominant mechanism of relapse on BCR-ABL TKIs.
In AML, a more diverse panel of disease-defining genetic mutations has been uncovered. However, in individual patients, a single oncogene can still drive disease. This is the case in FLT3 mutant AML, in which the investigational FLT3 TKI quizartinib achieved an initial response rate of ~50% in relapsed/refractory AML patients with activating FLT3 internal tandem duplication (ITD) mutations, though most patients eventually relapsed. Confirming the importance of FLT3 in disease maintenance, we showed that 8 of 8 patients who relapsed on quizartinib did so due to acquired drug-resistant FLT3 KD mutations.
Studies in CML have revealed that sequential TKI therapy is associated with additional complexity where multiple mutations can coexist separately in an individual patient (“polyclonality”) or in tandem on a single allele (“compound mutations”). In AML, we observed polyclonal FLT3-ITD KD mutations in 2 of 8 patients examined in our initial study of quizartinib resistance.
In light of the polyclonal KD mutations observed in CML and AML at the time of TKI relapse, we undertook next generation sequencing studies to determine the true genetic complexity in CML and AML patients at the time of relapse on targeted therapy. We used Pacific Biosciences RS Single Molecule Real Time (SMRT) third generation sequencing technology to sequence the entire ABL KD or the entire FLT3 juxtamembrane and KD on a single strand of DNA. Using this method, we assessed a total of 103 samples from 79 CML patients on ABL TKI therapy and 36 paired pre-treatment and relapse samples from 18 FLT3-ITD+ AML patients who responded to investigational FLT3 TKI therapy.
In CML, using SMRT sequencing, we detected all mutations previously detected by direct sequencing. Of samples in which multiple mutations were detectable by direct sequencing, 85% had compound mutant alleles detectable in a variety of combinations. Compound mutant alleles were comprised of both dominant and minor mutations, some which were not detectable by direct sequencing. In the most complex case, 12 individual mutant alleles comprised of 7 different mutations were identified in a single sample.
For 12 CML patients, we interrogated longitudinal samples (2-4 time points per patient) and observed complex clonal relationships with highly dynamic shifts in mutant allele populations over time. We detected compound mutations arising from ancestral single mutant clones as well as parallel evolution of de novo polyclonal and compound mutations largely in keeping with what would be expected to cause resistance to the second generation TKI therapy received by that patient.
We used a phospho-flow cytometric technique to assesses the phosphorylation status of the BCR-ABL substrate CRKL in as a method to test the ex vivo biochemical responsiveness of individual mutant cell populations to TKI therapy and assess functional cellular heterogeneity in a given patient at a given timepoint. Using this technique, we observed co-existing cell populations with differential ex vivo response to TKI in 2 cases with detectable polyclonal mutations. In a third case, we identified co-existence of an MLL-AF9 containing cell population that retained the ability to modulate p-CRKL in response to BCR-ABL TKIs along with a BCR-ABL containing only population that showed biochemical resistance to all TKIs, suggesting the co-existence of BCR-ABL independent and dependent resistance in a single patient.
In AML, using SMRT sequencing, we identified acquired quizartinib resistant KD mutations on the FLT3-ITD (ITD+) allele of 9 of 9 patients who relapsed after response to quizartinib and 4 of 9 patients who relapsed after response to the investigational FLT3 inhibitor, PLX3397. In 4 cases of quizartinib resistance and 3 cases of PLX3397 resistance, polyclonal mutations were observed, including 7 different KD mutations in one patient with PLX3397 resistance. In 7 quizartinib-resistant cases and 3 PLX3397-resistant cases, mutations occurred at the activation loop residue D835. When we examined non-ITD containing (ITD-) alleles, we surprisingly uncovered concurrent drug-resistant FLT3 KD mutations on ITD- alleles in 7 patients who developed quizartinib resistance and 4 patients with PLX3397 resistance. One additional PLX3397-resistant patient developed a D835Y mutation only in ITD- alleles at the time of resistance, suggesting selection for a non-ITD containing clone. All of the individual substitutions found on ITD- alleles were the same substitutions identified on ITD+ alleles for each individual patient.
Given that the same individual mutations found on ITD- alleles were also found on ITD+ alleles, we sought to determine whether these mutations were found in the same cell or were indicative of polyclonal blast populations in each patient. To answer this question, we performed single cell sorting of viably frozen blasts from 3 quizartinib-resistant patients with D835 mutations identified at the time of relapse and genotyped single cells for the presence or absence of ITD and D835 mutations. This analysis revealed striking genetic heterogeneity. In 2/3 cases, polyclonal D835 mutations were found in both ITD+ and ITD- cells. In all cases, FLT3-ITD and D835 mutations were found in both heterozygous and homozygous combinations. Most surprisingly, in all 3 patients, approximately 30-40% of FLT3-ITD+ cells had no identified quizartinib resistance-causing FLT3 KD mutation to account for resistance, suggesting the presence of non-FLT3 dependent resistance in all patients.
To determine that ITD+ cells lacking FLT3 KD mutations observed in patients relapsed on quizartinib are indeed consistent with leukemic blasts functionally resistant to quizartinib and do not instead represent a population of differentiated or non-proliferating cells, we utilized relapse blasts from another patient who initially achieved clearance of bone marrow blasts on quizartinib and developed a D835Y mutation at relapse. We performed a colony assay in the presence of 20nM quizartinib. As expected, this dose of quizartinib was unable to suppress the colony-forming ability of blasts from this relapsed patient when compared to DMSO treatment. Genotyping of individual colonies grown from this relapse sample in the presence of 20nM quizartinib again showed remarkable genetic heterogeneity, including ITD+ and ITD- colonies with D835Y mutations in homozygous and heterozygous combinations as well as ITD+ colonies without D835Y mutations, again suggesting the presence of blasts with non-FLT3 dependent resistance. Additionally, 4 colonies with no FLT3 mutations at all were identified in this sample, suggesting the presence of a quizartinib-resistant non-FLT3 mutant blast population. To see if we could identify specific mechanisms of off-target resistance, we performed targeted exome sequencing 33-AML relevant genes from relapse and pre-treatment DNA from all four patients and detected no new mutations in any genes other than FLT3 acquired at the time of disease relapse. Clonal genetic heterogeneity is not surprising in solid tumors, where multiple driver mutations frequently occur, but in CML and FLT3-ITD+ AML, where disease has been shown to be exquisitely dependent on oncogenic driver mutations, our studies suggest a surprising amount of clonal diversity. Our findings show that clinical TKI resistance in these diseases is amazingly intricate on the single allele level and frequently consists of both polyclonal and compound mutations that give rise to an complicated pool of TKI-resistant alleles that can change dynamically over time. In addition, we demonstrate that cell populations with off-target resistance can co-exist with other TKI-resistant populations, underscoring the emerging complexity of clinical TKI resistance. Such complexity argues strongly that monotherapy strategies in advanced CML and AML may be ultimately doomed to fail due to heterogeneous cell intrinsic resistance mechanisms. Ultimately, combination strategies that can address both on and off target resistance will be required to effect durable therapeutic responses.

Session Title:

Tumor Heterogeneity and Evolution

Session Type:

Educational Session

Session Start/End Time:

Saturday, Apr 18, 2015, 1:00 PM – 3:00 PM

Location:

Terrace Ballroom II-III (400 Level), Pennsylvania Convention Center

CME:

CME-Designated

CME/CE Hours:

Session Description:

One of the major challenges for both the measurement and management of cancer is its heterogeneity. Recent studies have revealed both extensive inter- and intra-tumor heterogeneity at the genotypic and phenotypic levels. Leaders in the field will discuss this challenge, its origins, dynamics and clinical importance. They will also review how we can best measure and deal with tumor heterogeneity, particularly intra-tumor heterogeneity.

Presentations:
	Chairperson Saturday, Apr 18, 2015, 1:00 PM – 3:00 PM Carlo C. Maley. UCSF Helen Diller Family Comp. Cancer Center, San Francisco, CA
	Universal biomarkers: How to handle tumor heterogeneity Saturday, Apr 18, 2015, 1:00 PM – 1:25 PM Carlo C. Maley. UCSF Helen Diller Family Comp. Cancer Center, San Francisco, CA
	Discussion Saturday, Apr 18, 2015, 1:25 PM – 1:30 PM
	Heterogeneity of resistance to cancer therapy Saturday, Apr 18, 2015, 1:30 PM – 1:55 PM Ivana Bozic. HARVARD UNIV., Cambridge, MA
	Discussion Saturday, Apr 18, 2015, 1:55 PM – 2:00 PM
	Determinants of phenotypic intra-tumor heterogeneity: integrative approach Saturday, Apr 18, 2015, 2:00 PM – 2:25 PM Andriy Marusyk, Michalina Janiszewska, Doris Tabassum. Dana-Farber Cancer Institute, Boston, MA, Dana-Farber Cancer Institute, Boston, MA
	Discussion Saturday, Apr 18, 2015, 2:25 PM – 2:30 PM
	Cancer clonal complexity and evolution at the macro- and microheterogeneity scale Saturday, Apr 18, 2015, 2:30 PM – 2:55 PM Marco Gerlinger. Institute of Cancer Research, London, United Kingdom
	Discussion Saturday, Apr 18, 2015, 2:55 PM – 3:00 PM

From Ivana Bozic:

Waclaw B, Bozic I, Pittman ME, Hruban RH, Vogelstein B, Nowak MA.

Nature. 2015 Sep 10;525(7568):261-4. doi: 10.1038/nature14971. Epub 2015 Aug 26.

PMID:

26308893

Timing and heterogeneity of mutations associated with drug resistance in metastatic cancers.

Select item 253494242.

Bozic I, Nowak MA.

Proc Natl Acad Sci U S A. 2014 Nov 11;111(45):15964-8. doi: 10.1073/pnas.1412075111. Epub 2014 Oct 27.

PMID:

25349424

Free PMC Article

Evolutionary dynamics of cancer in response to targeted combination therapy.

Select item 238053823.

Bozic I, Reiter JG, Allen B, Antal T, Chatterjee K, Shah P, Moon YS, Yaqubie A, Kelly N, Le DT, Lipson EJ, Chapman PB, Diaz LA Jr, Vogelstein B, Nowak MA.

Elife. 2013 Jun 25;2:e00747. doi: 10.7554/eLife.00747.

PMID:

23805382

Free PMC Article

The molecular evolution of acquired resistance to targeted EGFR blockade in colorectal cancers.

Select item 227228434.

Diaz LA Jr, Williams RT, Wu J, Kinde I, Hecht JR, Berlin J, Allen B, Bozic I, Reiter JG, Nowak MA, Kinzler KW, Oliner KS, Vogelstein B.

Nature. 2012 Jun 28;486(7404):537-40. doi: 10.1038/nature11219.

PMID:

22722843

Free PMC Article

Chairperson
Tuesday, Apr 21, 2015, 10:30 AM -12:30 PM
David A. Cheresh. UCSD Moores Cancer Center, La Jolla, CA

Session Title:

Mechanisms of Cancer Therapy Resistance

Session Type:

Educational Session

Session Start/End Time:

Saturday, Apr 18, 2015, 1:00 PM – 3:00 PM

Location:

Room 204, Pennsylvania Convention Center

CME:

CME-Designated

CME/CE Hours:

Session Description:

Despite dramatic advances in the treatment of cancer, therapy resistance remains the most significant hurdle in improving the outcome of cancer patients. In this session, we will discuss many different aspects of therapy resistance, including a summary of our current understanding of therapy resistant tumor cell populations as well as analyses of the challenges associated with intratumoral heterogeneity and adaptive responses to targeted therapies.

Presentations:
	Chairperson Saturday, Apr 18, 2015, 1:00 PM – 3:00 PM Charles Swanton. Cancer Research UK London Research Institute, London, United Kingdom
	Tumor heterogeneity and drug resistance Saturday, Apr 18, 2015, 1:00 PM – 1:30 PM Charles Swanton. Cancer Research UK London Research Institute, London, United Kingdom
	Discussion Saturday, Apr 18, 2015, 1:30 PM – 1:40 PM Discussion Discussion, Discussion
	Principles of resistance to targeted therapy Saturday, Apr 18, 2015, 1:40 PM – 2:10 PM Levi A. Garraway. Dana-Farber Cancer Institute, Boston, MA
	Discussion Saturday, Apr 18, 2015, 2:10 PM – 2:20 PM Discussion Discussion, Discussion
	Adaptive re-wiring of signaling pathways driving drug resistance to targeted therapies Saturday, Apr 18, 2015, 2:20 PM – 2:50 PM Taru E. Muranen. Harvard Medical School, Boston, MA
	Discussion Saturday, Apr 18, 2015, 2:50 PM – 3:00 PM Discussion Discussion, Discussion

Presentation Abstract

Abstract Number:	737

Presentation Title:	Clonal evolution of the HER2 L755S mutation as a mechanism of acquired HER-targeted therapy resistance

Presentation Time:	Sunday, Apr 19, 2015, 1:00 PM – 5:00 PM

Location:	Section 30

Poster Board Number:	29

Author Block:	Xiaowei Xu¹, Agostina Nardone¹, Huizhong Hu¹, Lanfang Qin¹, Sarmistha Nanda¹, Laura Heiser², Nicholas Wang², Kyle Covington¹, Edward Chen¹, Alexander Renwick¹, Tamika Mitchell¹, Marty Shea¹, Tao Wang¹, Carmine De Angelis¹, Alejandro Contreras¹, Carolina Gutierrez¹, Suzanne Fuqua¹, Gary Chamness¹, Chad Shaw¹, Marilyn Li¹, David Wheeler¹, Susan Hilsenbeck¹, Mothaffar Fahed Rimawi¹, Joe Gray², C.Kent Osborne¹, Rachel Schiff¹. ¹Baylor College of Medicine, Houston, TX; ²Oregon Health & Science University, Portland, OR

Abstract Body:	Background: Targeting HER2 with lapatinib (L), trastuzumab (T), or the LT combination, is effective in HER2+ breast cancer (BC), but acquired resistance commonly occurs. In our 12-week neoadjuvant trial (TBCRC006) of LT without chemotherapy in HER2+ BC, the overall pathologic complete response (pCR) rate was 27%. To investigate resistance mechanisms, we developed 10 HER2+ BC cell line models resistant (R) to one or both drugs (LR/TR/LTR). To discover potential predictive markers/therapeutic targets to circumvent resistance, we completed genomic profiling of the cell lines and a subset of pre-treatment specimens from TBCRC006. Methods: Parental (P) and LR/TR/LTR lines of 10 cell line models were profiled with whole exome/RNA sequencing. Mutations detected in R lines but not in P lines of the same model were identified. Mutation-specific Q-PCR was designed for sensitive quantification. Resistant cell and xenograft tumor growth were measured in response to drugs. Whole exome sequencing (>100X) and Ampliseq of 17 baseline tumor/normal pairs from TBCRC006 were performed. Results: We found and validated the HER2 L755S mutation in the BT474/ATCC-LTR line and BT474/AZ-LR line (in ~30% of DNA/RNA), in which the HER pathway was reactivated for resistance. Overexpression of this mutation was previously shown to induce LR in HER2-negative BC cell lines, and resistant growth of BT474/AZ-LR line is significantly inhibited by HER2-L755S-specific siRNA knock-down, suggesting its role as an acquired L/LT resistance driver in HER2+ BC. Sequencing of BT474/AZ-LR single cell clones found the mutation in ~30% of HER2 copies in every cell. Using mutation-specific Q-PCR, we found statistically higher HER2 L755S levels in two BT474 parentals compared to P lines of SKBR3, AU565, and UACC812. These data suggest that HER2 L755S resistant subclones preexist in the BT474 parentals and were selected by L treatment to become the major clone in the two R lines. The HER1/2 irreversible tyrosine kinase inhibitor (TKI) afatinib (Afa) robustly inhibited growth of BT474/AZ-LR and BT474/ATCC-LTR cells (IC₅₀: Afa 0.02µM vs. L 3 µM) and BT474/AZ-LR xenografts. Whole exome sequencing/Ampliseq of TBCRC006 found the HER2 L755S mutation in 1/17 primaries. This patient did not achieve pCR. The variant was present in 2% of DNA on both platforms, indicating a subclonal event of the resistance mutation. Conclusion: Acquired L/LT resistance in the two BT474 R lines is due to selection of HER2 L755S subclones present in parental cells. The higher HER2 L755S levels in BT474 parentals compared with other parentals, and detection of its subclonal presence in a pre-treatment HER2+ BC patient, suggest that sensitive mutation detection methods will be needed to identify patients with potentially actionable HER family mutations in primary tumor. Treating this patient group with an irreversible TKI like Afa may prevent resistance and improve clinical outcome of this subset of HER2+ BC.
Presentation Number:	SY07-04

Presentation Title:	The evolutionary landscape of CLL: Therapeutic implications

Presentation Time:	Sunday, Apr 19, 2015, 2:25 PM – 2:45 PM

Location:	Grand Ballroom (300 Level), Pennsylvania Convention Center

Author Block:	Catherine J. Wu. Dana-Farber Cancer Institute, Boston, MA

Abstract Body:	Clonal evolution is a key feature of cancer progression and relapse. Recent studies across cancers have demonstrated the extensive degree of intratumoral heterogeneity present within individual cancers. We hypothesized that evolutionary dynamics contribute to the variations in disease tempo and response to therapy that are highly characteristic of chronic lymphocytic leukemia (CLL). We have recently investigated this phenomenon by developing a pipeline that estimates the fraction of cancer cells harboring each somatic mutation within a tumor through integration of whole-exome sequence (WES) and local copy number data (Landau et al., Cell 2013). By applying this analysis approach to 149 CLL cases, we discovered earlier and later cancer drivers, uncovered patterns of clonal evolution in CLL and linked the presence of subclones harboring driver mutations with adverse clinical outcome. Thus, our study, generated from a heterogeneous sample cohort, strongly supports the concept that CLL clonal evolution arises from mass extinction and therapeutic bottlenecks which lead to the emergence of highly fit (and treatment resistant) subclones. We further hypothesized that epigenetic heterogeneity also shapes CLL clonal evolution through interrelation with genetic heterogeneity. Indeed, in recent work, we have uncovered stochastic methylation disorder as the primary cause of methylation changes in CLL and cancer in general, and that this phenomena impacts gene transcription, genetic evolution and clinical outcome. Thus, integrated studies of genetic and epigenetic heterogeneity in CLL have revealed the complex and diverse evolutionary trajectories of these cancer cells. Immunotherapy is exquisitely suited for specifically and simultaneously targeting multiple lesions. We have developed an approach that leverages whole-exome sequencing to systematically identify personal tumor mutations with immunogenic potential, which can be incorporated as antigen targets in multi-epitope personalized therapeutic vaccines. We are pioneering this approach in an ongoing trial in melanoma and will now expand this concept to address diverse malignancies. Our expectation is that the choice of tumor neoantigens for a vaccine bypasses thymic tolerance and thus generates highly specific and potent high-affinity T cell responses to eliminate tumors in any cancer, including both ‘trunk’ and ‘branch’ lesions.

Abstract Number:

LB-056

Presentation Title:

TP53 and RB1 alterations promote reprogramming and antiandrogen resistance in advanced prostate cancer

Presentation Time:

Sunday, Apr 19, 2015, 4:50 PM – 5:05 PM

Location:

Room 122, Pennsylvania Convention Center

Author Block:

Ping Mu, Zhen Cao, Elizabeth Hoover, John Wongvipat, Chun-Hao Huang, Wouter Karthaus, Wassim Abida, Elisa De Stanchina, Charles Sawyers. Memorial Sloan Kettering Cancer Center, New York, NY

Abstract Body:

Castration-resistant prostate cancer (CRPC) is one of the most difficult cancers to treat with conventional methods and is responsible for nearly all prostate cancer deaths in the US. The Sawyers laboratory first showed that the primary mechanism of resistance to antiandrogen therapy is elevated androgen receptor (AR) expression. Research based on this finding has led to the development of next-generation antiandrogen: enzalutamide. Despite the exciting clinical success of enzalutamide, about 60% of patients exhibit various degrees of resistance to this agent. Highly variable responses to enzalutamide limit the clinical benefit of this novel antiandrogen, underscoring the importance of understanding the mechanisms of enzalutamide resistance. Most recently, an unbiased SU2C-Prostate Cancer Dream Team metastatic CRPC sequencing project led by Dr. Sawyers and Dr. Chinnaiyan revealed that mutations in the TP53 locus are the most significantly enriched alteration in CRPC tumors when compared to primary prostate cancers. Moreover, deletions and decreased expressions of the TP53 and RB1 loci (co-occurrence and individual occurrence) are more commonly associated with CRPC than with primary tumors. These results established that alteration of the TP53 and RB1 pathways are associated with the development of antiandrogen resistance.
By knockdowning TP53 or/and RB1 in the castration resistant LNCaP/AR model, we demonstrate that the disruption of either TP53 or RB1 alone confers significant resistance to enzalutamide both in vitro and in vivo. Strikingly, the co-inactivation of these pathways confers the most dramatic resistance. Since up-regulation of either AR or AR target genes is not observed in the resistant tumors, loss of TP53 and RB1 function confers enzalutamide resistance likely through an AR independent mechanism. In the clinic, resistance to enzalutamide is increasingly being associated with a transition to a poorly differentiated or neuroendocrine-like histology. Interestingly, we observed significant up-regulations of the basal cell marker Ck5 and the neuroendocrine-like cell marker Synaptophysin in the TP53 and RB1 inactivated cells, as well as down-regulation of the luminal cell marker Ck8. The differences between these markers became even greater after enzalutamide treatment. By using the p53-stabilizing drug Nutlin, level of p53 is rescued and consequently the the decrease of AR protein caused by RB1 and TP53 knockdown is reversed. These results strongly suggest that interference of TP53 and RB1 pathways confers antiandrogen resistance by “priming” prostate cancer cells to reprogramming or transdifferentiation, likely neuroendocrine-like differentiation, in response to treatment. Futher experiments will be performed to assess the molecular mechanism of TP53/RB1 alterations in mediating cell programming and conferring antiandrogen resistance.

Abstract Number:	LB-146

Presentation Title:	TGF-β-induced tumor heterogeneity and drug resistance of cancer stem cells

Presentation Time:	Monday, Apr 20, 2015, 1:00 PM – 5:00 PM

Location:	Section 41

Author Block:	Naoki Oshimori¹, Daniel Oristian¹, Elaine Fuchs². ¹Rockefeller University, New York, NY; ²HHMI/Rockefeller University, New York, NY

Abstract Body:	Among the most common and life-threatening cancers world-wide, squamous cell carcinoma (SCC) exhibit high rates of tumor recurrence following anti-cancer therapy. Subsets of cancer stem cells (CSCs) often escape anti-cancer therapeutics and promote recurrence. However, its sources and mechanisms that generate tumor heterogeneity and therapy-resistant cell population are largely unknown. Tumor microenvironment may drive intratumor heterogeneity by transmitting signaling factors, oxygen and metabolites to tumor cells depending on their proximity to the local sources. While the hypothesis is attractive, experimental evidence is lacking, and non-genetic mechanisms that drive functional heterogeneity remain largely unknown. As a potential non-genetic factor, we focused on TGF-β because of its multiple roles in tumor progression. Here we devise a functional reporter system to monitor, track and modify TGF-β signaling in mouse skin SCC in vivo. Using this approach, we found that perivascular TGF-β in the tumor microenvironment generates heterogeneity in TGF-β signaling in neighboring CSCs. This heterogeneity is functionally important: small subsets of TGF-β-responding CSCs proliferate more slowly than their non-responding counterparts. They also exhibit invasive morphology and a malignant differentiation program compared to their non-responding neighbors. By lineage tracing, we show that although TGF-β-responding CSCs clonally expand more slowly they gain a growth advantage in a remarkable ability to escape cisplatin-induced apoptosis. We show that indeed it is their progenies that make a substantial contribution in tumor recurrence. Surprisingly, the slower proliferating state of this subset of CSCs within the cancer correlated with but did not confer the survival advantage to anti-cancer drugs. Using transcriptomic, biochemical and genetic analyses, we unravel a novel mechanism by which heterogeneity in the tumor microenvironment allows a subset of CSCs to respond to TGF-β, and evade anti-cancer drugs. Our findings also show that TGF-β established ability to suppress proliferation and promote invasion and metastasis do not happen sequentially, but rather simultaneously. This new work build upon the roles of this factor in tumor progression, and sets an important paradigm for a non-genetic factor that produces tumor heterogeneity.

Abstract Number:

LB-129

Presentation Title:

Identifying tumor subpopulations and the functional consequences of intratumor heterogeneity using single-cell profiling of breast cancer patient-derived xenografts

Presentation Time:

Monday, Apr 20, 2015, 1:00 PM – 5:00 PM

Location:

Section 41

Author Block:

Paul Savage1, Sadiq M. Saleh1, Ernesto Iacucci1, Timothe Revil1, Yu-Chang Wang1, Nicholas Bertos1, Anie Monast1, Hong Zhao1, Margarita Souleimanova1, Keith Szulwach2, Chandana Batchu2, Atilla Omeroglu1, Morag Park1, Ioannis Ragoussis1. 1McGill University, Montreal, QC, Canada; 2Fluidigm Corporation, South San Francisco, CA

Abstract Body:

Human breast tumors have been shown to exhibit extensive inter- and intra-tumor heterogeneity. While recent advances in genomic technologies have allowed us to deconvolute this heterogeneity, few studies have addressed the functional consequences of diversity within tumor populations. Here, we identified an index case for which we have derived a patient-derived xenograft (PDX) as a renewable tissue source to identify subpopulations and perform functional assays. On pathology, the tumor was an invasive ductal carcinoma which was hormone receptor-negative, HER2-positive (IHC 2+, FISH average HER2/CEP17 2.4), though the FISH signal was noted to be heterogeneous. On gene expression profiling of bulk samples, the primary tumor and PDX were classified as basal-like. We performed single cell RNA and exome sequencing of the PDX to identify population structure. Using a single sample predictor of breast cancer subtype, we have identified single basal-like, HER2-enriched and normal-like cells co-existing within the PDX tumor. Genes differentially expressed between these subpopulations are involved in proliferation and differentiation. Functional studies distinguishing these subpopulations are ongoing. Microfluidic whole genome amplification followed by whole exome capture of 81 single cells showed high and homogeneous target enrichment with >75% of reads mapping uniquely on target. Variant calling using GATK and Samtools revealed founder mutations in key genes as BRCA1 and TP53, as well as subclonal mutations that are being investigated further. Loss of heterozygocity was observed in 16 TCGA cancer driver genes and novel mutations in 7 cancer driver genes. These findings may be important in understanding the functional consequences of intra-tumor heterogeneity with respect to clinically important phenotypes such as invasion, metastasis and drug-resistance.

Abstract Number:

2847

Presentation Title:

High complexity barcoding to study clonal dynamics in response to cancer therapy

Presentation Time:

Monday, Apr 20, 2015, 4:35 PM – 4:50 PM

Location:

Room 118, Pennsylvania Convention Center

Author Block:

Hyo-eun C. Bhang1, David A. Ruddy1, Viveksagar Krishnamurthy Radhakrishna1, Rui Zhao2, Iris Kao1, Daniel Rakiec1, Pamela Shaw1, Marissa Balak1, Justina X. Caushi1, Elizabeth Ackley1, Nicholas Keen1, Michael R. Schlabach1, Michael Palmer1, William R. Sellers1, Franziska Michor2, Vesselina G. Cooke1, Joshua M. Korn1, Frank Stegmeier1. 1Novartis Institutes for BioMedical Research, Cambridge, MA; 2Dana-Farber Cancer Institute, Boston, MA

Abstract Body:

Targeted therapies, such as erlotinib and imatinib, lead to dramatic clinical responses, but the emergence of resistance presents a significant challenge. Recent studies have revealed intratumoral heterogeneity as a potential source for the emergence of therapeutic resistance. However, it is still unclear if relapse/resistance is driven predominantly by pre-existing or de novo acquired alterations. To address this question, we developed a high-complexity barcode library, ClonTracer, which contains over 27 million unique DNA barcodes and thus enables the high resolution tracking of cancer cells under drug treatment. Using this library in two clinically relevant resistance models, we demonstrate that the majority of resistant clones pre-exist as rare subpopulations that become selected in response to therapeutic challenge. Furthermore, our data provide direct evidence that both genetic and non-genetic resistance mechanisms pre-exist in cancer cell populations. The ClonTracer barcoding strategy, together with mathematical modeling, enabled us to quantitatively dissect the frequency of drug-resistant subpopulations and evaluate the impact of combination treatments on the clonal complexity of these cancer models. Hence, monitoring of clonal diversity in drug-resistant cell populations by the ClonTracer barcoding strategy described here may provide a valuable tool to optimize therapeutic regimens towards the goal of curative cancer therapies.

Abstract Number:

3590

Presentation Title:

Resistance mechanisms to ALK inhibitors

Presentation Time:

Tuesday, Apr 21, 2015, 8:00 AM -12:00 PM

Location:

Section 31

Poster Board Number:

Author Block:

Ryohei Katayama1, Noriko Yanagitani1, Sumie Koike1, Takuya Sakashita1, Satoru Kitazono1, Makoto Nishio1, Yasushi Okuno2, Jeffrey A. Engelman3, Alice T. Shaw3, Naoya Fujita1. 1Japanese Foundation for Cancer Research, Tokyo, Japan; 2Graduate School of Medicine, Kyoto University, Kyoto, Japan; 3Massachusetts General Hospital Cancer Center, Boston, MA

Abstract Body:

Purpose: ALK-rearranged non-small cell lung cancer (NSCLC) was first reported in 2007. Approximately 3-5% of NSCLCs harbor an ALK gene rearrangement. The first-generation ALK tyrosine kinase inhibitor (TKI) crizotinib is a standard therapy for patients with advanced ALK-rearranged NSCLC. Several next-generation ALK-TKIs have entered the clinic and have shown promising antitumor activity in crizotinib-resistant patients. As patients still relapse even on these next-generation ALK-TKIs, we examined mechanisms of resistance to one next-generation ALK-TKI – alectinib – and potential strategies to overcome this resistance.
Experimental Procedure: We established a cell line model of alectinib resistance, and analyzed resistant tumor specimens from patients who had relapsed on alectinib. Cell lines were also established under an IRB-approved protocol when there was sufficient fresh tumor tissue. We established Ba/F3 cells expressing EML4-ALK and performed ENU mutagenesis to compare potential crizotinib or alectinib-resistance mutations. In addition, we developed Ba/F3 models harboring ALK resistance mutations and evaluated the potency of multiple next-generation ALK-TKIs including 3rd generation ALK inhibitor in these models and in vivo. To elucidate structure-activity-relationships of ALK resistance mutations, we performed computational thermodynamic simulation with MP-CAFEE.
Results: We identified multiple resistance mutations, including ALK I1171N, I1171S, and V1180L, from the ENU mutagenesis screen and the cell line model. In addition we found secondary mutations at the I1171 residue from the Japanese patients who developed resistance to alectinib or crizotinib. Both ALK mutations (V1180L and I1171 mutations) conferred resistance to alectinib as well as to crizotinib, but were sensitive to ceritinib and other next-generation ALK-TKIs. Based on thermodynamics simulation, each resistance mutation is predicted to lead to distinct structural alterations that decrease the binding affinity of ALK-TKIs for ALK.
Conclusions: We have identified multiple alectinib-resistance mutations from the cell line model, patient derived cell lines, and tumor tissues, and ENU mutagenesis. ALK secondary mutations arising after alectinib exposure are sensitive to other next generation ALK-TKIs. These findings suggest a potential role for sequential therapy with multiple next-generation ALK-TKIs in patients with advanced, ALK-rearranged cancers.

Session Title:

Mechanisms of Resistance: From Signaling Pathways to Stem Cells

Session Type:

Major Symposium

Session Start/End Time:

Tuesday, Apr 21, 2015, 10:30 AM -12:30 PM

Location:

Terrace Ballroom II-III (400 Level), Pennsylvania Convention Center

CME:

CME-Designated

CME/CE Hours:

Session Description:

Even the most effective cancer therapies are limited due to the development of one or more resistance mechanisms. Acquired resistance to targeted therapies can, in some cases, be attributed to the selective propagation of a small population of intrinsically resistant cells. However, there is also evidence that cancer drugs themselves can drive resistance by triggering the biochemical- or genetic-reprogramming of cells within the tumor or its microenvironment. Therefore, understanding drug resistance at the molecular and biological levels may enable the selection of specific drug combinations to counteract these adaptive responses. This symposium will explore some of the recent advances addressing the molecular basis of cancer cell drug resistance. We will address how tumor cell signaling pathways become rewired to facilitate tumor cell survival in the face of some of our most promising cancer drugs. Another topic to be discussed involves how drugs select for or induce the reprogramming of tumor cells toward a stem-like, drug resistant fate. By targeting the molecular driver(s) of rewired signaling pathways and/or cancer stemness it may be possible to select drug combinations that prevent the reprogramming of tumors and thereby delay or eliminate the onset of drug resistance.

Presentations:

Introduction
Tuesday, Apr 21, 2015, 10:30 AM -10:40 AM

Resistance to tyrosine kinase inhibitors: Heterogeneity and therapeutic strategies.
Tuesday, Apr 21, 2015, 10:40 AM -10:55 AM
Jeffrey A. Engelman. Massachusetts General Hospital, Boston, MA

Discussion
Tuesday, Apr 21, 2015, 10:55 AM -11:00 AM

NG04: Clinical acquired resistance to RAF inhibitor combinations in BRAF mutant colorectal cancer through MAPK pathway alterations
Tuesday, Apr 21, 2015, 11:00 AM -11:15 AM
Ryan B. Corcoran, Leanne G. Ahronian, Eliezer Van Allen, Erin M. Coffee, Nikhil Wagle, Eunice L. Kwak, Jason E. Faris, A. John Iafrate, Levi A. Garraway, Jeffrey A. Engelman. Massachusetts General Hospital Cancer Center, Boston, MA, Dana-Farber Cancer Institute, Boston, MA

Discussion
Tuesday, Apr 21, 2015, 11:15 AM -11:20 AM

SY27-02: Tumour heterogeneity and therapy resistance in melanoma
Tuesday, Apr 21, 2015, 11:20 AM -11:35 AM
Claudia Wellbrock. Univ. of Manchester, Manchester, United Kingdom

Presentation Number:	SY27-02

Presentation Title:	Tumour heterogeneity and therapy resistance in melanoma

Presentation Time:	Tuesday, Apr 21, 2015, 11:20 AM -11:35 AM

Location:	Terrace Ballroom II-III (400 Level), Pennsylvania Convention Center

Author Block:	Claudia Wellbrock. Univ. of Manchester, Manchester, United Kingdom

Abstract Body:	Solid tumors are structurally very complex; they consist of heterogeneous cancer cell populations, other non-cancerous cell types and a distinct extracellular matrix. Interactions of cancer cells with non-cancerous cells is well investigated, and our recent work in melanoma has demonstrated that the cellular environment that surrounds cancer cells has a major impact on the way a patient responds to MAP-kinase pathway targeting therapy. We have shown that intra-tumor signaling within a heterogeneous tumor can have a major impact on the efficacy of BRAF and MEK inhibitors. With the increasing evidence of genetic and phenotypic heterogeneity within tumors, intra-tumor signaling between individual cancer-cell subpopulations is therefore a crucial factor that needs to be considered in future therapy approaches. Our work has identified the ‘melanocyte-lineage survival oncogene’ MITF as an important player in phenotypic heterogeneity (MITFhigh and MITFlow cells) in melanoma, and MITF expression levels are crucial for the response to MAP-kinase pathway targeted therapy. We found that ‘MITF heterogeneity’ can be caused by cell-autonomous mechanisms or by the microenvironment, including the immune-microenvironment. We have identified various mechanisms underlying MITF action in resistance to BRAF and MEK inhibitors in melanoma. In MITFhigh expressing cells, MITF confers cell-autonomous resistance to MAP-kinase pathway targeted therapy. Moreover, it appears that in melanomas heterogeneous for MITF expression (MITFhigh and MITFlow cells), individual subpopulations of resistant and sensitive cells communicate and MITF can contribute to overall tumor-resistance through intra-tumor signaling. Finally, we have identified a novel approach of interfering with MITF action, which profoundly sensitizes melanoma to MAP-kinase pathway targeted therapy.

Discussion
Tuesday, Apr 21, 2015, 11:35 AM -11:40 AM

SY27-03: Breast cancer stem cell state transitions mediate therapeutic resistance
Tuesday, Apr 21, 2015, 11:40 AM -11:55 AM
Max S. Wicha. University of Michigan, Comprehensive Cancer Center, Ann Arbor, MI

Discussion
Tuesday, Apr 21, 2015, 11:55 AM -12:00 PM

SY27-04: Induction of cancer stemness and drug resistance by EGFR blockade
Tuesday, Apr 21, 2015, 12:00 PM -12:15 PM
David A. Cheresh. UCSD Moores Cancer Center, La Jolla, CA

Cellular Reprogramming in Carcinogenesis: Implications for Tumor Heterogeneity, Prognosis, and Therapy

Session Type:

Major Symposium

Session Start/End Time:

Tuesday, Apr 21, 2015, 10:30 AM -12:30 PM

Location:

Room 103, Pennsylvania Convention Center

CME:

CME-Designated

CME/CE Hours:

Session Description:

Cancers, both solid and liquid, consist of phenotypically heterogeneous cell types that make up the full cellular complement of disease. Deep sequencing of bulk cancers also frequently reveals a genetic intratumoral heterogeneity that reflects clonal evolution in space and in time and under the influence of treatment. How the distinct phenotypic and genotypic cells contribute to individual cancer growth and progression is incompletely understood. In this symposium, we will discuss issues of cancer heterogeneity and effects on growth and treatment resistance, with emphasis on cancer cell functional properties and influences of the microenvironment, interclonal genomic heterogeneity, and lineage relationships between cancer cells with stem cell and differentiated properties. Understanding these complex cellular relationships within cancers will have critical implications for devising more effective treatments.

Presentations:
	Chairperson Tuesday, Apr 21, 2015, 10:30 AM -12:30 PM Peter B. Dirks. Univ. of Toronto Hospital for Sick Children, Toronto, ON, Canada
	Introduction Tuesday, Apr 21, 2015, 10:30 AM -10:40 AM
	Origins, evolution and selection in childhood leukaemia Tuesday, Apr 21, 2015, 10:40 AM -11:00 AM Tariq Enver. Cancer Research UK, London, United Kingdom
	Discussion Tuesday, Apr 21, 2015, 11:00 AM -11:05 AM
	Cytokine-controlled stem cell plasticity inintestinal tumorigenesis Tuesday, Apr 21, 2015, 11:05 AM -11:25 AM Florian Greten. Georg-Speyer-Haus, Frankfurt, Germany
	Discussion Tuesday, Apr 21, 2015, 11:25 AM -11:30 AM
	SY23-03: Intratumoural heterogeneity in human serous ovarian carcinoma Tuesday, Apr 21, 2015, 11:30 AM -11:50 AM John P. Stingl. Cancer Research UK Cambridge Research Inst., Cambridge, United Kingdom
	Discussion Tuesday, Apr 21, 2015, 11:50 AM -11:55 AM
	Functional and genomic heterogeneity in brain tumors Tuesday, Apr 21, 2015, 11:55 AM -12:15 PM Proc Natl Acad Sci U S A. 2015 Jan 20;112(3):851-6. doi: 10.1073/pnas.1320611111. Epub 2015 Jan 5. Single cell-derived clonal analysis of human glioblastoma links functional and genomic heterogeneity. Meyer M¹, Reimand J², Lan X³, Head R¹, Zhu X¹, Kushida M¹, Bayani J⁴, Pressey JC⁵, Lionel AC⁶, Clarke ID⁷, Cusimano M⁸, Squire JA⁹, Scherer SW⁶, Bernstein M¹⁰, Woodin MA⁵, Bader GD¹¹, Dirks PB¹². Author information Abstract Glioblastoma (GBM) is a cancer comprised of morphologically, genetically, and phenotypically diverse cells. However, an understanding of the functional significance of intratumoral heterogeneity is lacking. We devised a method to isolate and functionally profile tumorigenic clones from patient glioblastoma samples. Individual clones demonstrated unique proliferation and differentiation abilities. Importantly, naïve patient tumors included clones that were temozolomide resistant, indicating that resistance to conventional GBM therapy can preexist in untreated tumors at a clonal level. Further, candidate therapies for resistant clones were detected with clone-specific drug screening. Genomic analyses revealed genes and pathways that associate with specific functional behavior of single clones. Our results suggest that functional clonal profiling used to identify tumorigenic and drug-resistant tumor clones will lead to the discovery of new GBM clone-specific treatment strategies.

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739: Tumor cell plasticity with transition to a mesenchymal phenotype is a mechanism of chemoresistance that is reversed by Notch pathway inhibition in lung adenocarcinoma
Sunday, Apr 19, 2015, 1:00 PM – 5:00 PM
Khaled A. Hassan. University Of Michigan, Ann Arbor, MI

745: Oncostatin M receptor activation leads to molecular targeted therapy resistance in non-small cell lung cancer
Sunday, Apr 19, 2015, 1:00 PM – 5:00 PM
Kazuhiko Shien¹, Vassiliki A. Papadimitrakopoulou¹, Dennis Ruder¹, Nana E. Hanson¹, Neda Kalhor¹, J. Jack Lee¹, Waun Ki Hong¹, Ximing Tang¹, Roy S. Herbst², Luc Girard³, John D. Minna³, Jonathan M. Kurie¹, Ignacio I. Wistuba¹, Julie G. Izzo¹. ¹University of Texas MD Anderson Cancer Center, Houston, TX; ²Yale Cancer Center, Yale School of Medicine, New Haven, CT; ³Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, Dallas, TX

746: Activation of EGFR bypass signaling through TGFα overexpression induces acquired resistance to alectinib in ALK-translocated lung cancer cells
Sunday, Apr 19, 2015, 1:00 PM – 5:00 PM
Tetsuo Tani, Hiroyuki Yasuda, Junko Hamamoto, Aoi Kuroda, Daisuke Arai, Kota Ishioka, Keiko Ohgino, Ichiro Kawada, Katsuhiko Naoki, Hayashi Yuichiro, Tomoko Betsuyaku, Kenzo Soejima. Keio University, Tokyo, Japan

752: Elucidating the mechanisms of acquired resistance in lung adenocarcinomas
Sunday, Apr 19, 2015, 1:00 PM – 5:00 PM
Sandra Ortiz-Cuarán¹, Lynnette Fernandez-Cuesta¹, Christine M. Lovly², Marc Bos¹, Matthias Scheffler³, Sebastian Michels³, Kerstin Albus⁴, Lydia Meyer⁴, Katharina König⁴, Ilona Dahmen¹, Christian Mueller¹, Luca Ozretić⁴, Lars Tharun⁴, Philipp Schaub¹, Alexandra Florin⁴, Berit Pinther¹, Nike Bahlmann¹, Sascha Ansén³, Martin Peifer¹, Lukas C. Heukamp⁴, Reinhard Buettner⁴, Martin L. Sos¹, Jürgen Wolf³, William Pao², Roman K. Thomas¹. ¹University of Cologne, Cologne, Germany; ²Department of Medicine, Vanderbilt University, Nashville, TN; ³Department of Internal Medicine, Center for Integrated Oncology Köln-Bonn, University Hospital Cologne, Cologne, Germany; ⁴Institute of Pathology, University Hospital Cologne, Cologne, Germany

	760: On the evolution of erlotinib-resistant NSCLC subpopulations Sunday, Apr 19, 2015, 1:00 PM – 5:00 PM Michael E. Ramirez¹, Robert J. Steininger, III¹, Lani F. Wu², Steven J. Altschuler². ¹UT Southwestern, Dallas, TX; ²UCSF, San Francisco, CA
	763: Implications of resistance patterns with NSCLC targeted agents Sunday, Apr 19, 2015, 1:00 PM – 5:00 PM David J. Stewart, Paul Wheatley-Price, Rob MacRae, Jason Pantarotto. University of Ottawa, Ottawa, ON, Canada

768: A kinome-wide siRNA screen identifies modifiers of sensitivity to the EGFR T790M-targeted tyrosine kinase inhibitor (TKI), AZD9291, in EGFR mutant lung adenocarcinoma
Sunday, Apr 19, 2015, 1:00 PM – 5:00 PM
Eiki Ichihara¹, Joshua A. Bauer², Pengcheng Lu³, Fei Ye³, Darren Cross⁴, William Pao¹, Christine M. Lovly¹. ¹Vanderbilt University School of Medicine, Nashville, TN; ²Vanderbilt Institute of Chemical Biology High-Throughput Screening Facility, Nashville, TN; ³Vanderbilt University Medical Center, Nashville, TN; ⁴AstraZeneca Oncology Innovative Medicines, United Kingdom

LB-055: Clinical acquired resistance to RAF inhibitor combinations in BRAF-mutant colorectal cancer through MAPK pathway alterations
Sunday, Apr 19, 2015, 4:35 PM – 4:50 PM
Leanne G. Ahronian¹, Erin M. Sennott¹, Eliezer M. Van Allen², Nikhil Wagle², Eunice L. Kwak¹, Jason E. Faris¹, Jason T. Godfrey¹, Koki Nishimura¹, Kerry D. Lynch³, Craig H. Mermel¹, Elizabeth L. Lockerman¹, Anuj Kalsy¹, Joseph M. Gurski, Jr.¹, Samira Bahl⁴, Kristin Anderka⁴, Lisa M. Green⁴, Niall J. Lennon⁴, Tiffany G. Huynh³, Mari Mino-Kenudson³, Gad Getz¹, Dora Dias-Santagata³, A. John Iafrate³, Jeffrey A. Engelman¹, Levi A. Garraway², Ryan B. Corcoran¹. ¹Massachusetts General Hospital Cancer Center, Boston, MA; ²Dana Farber Cancer Institute, Boston, MA; ³Massachusetts General Hospital Department of Pathology, Boston, MA; ⁴Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA

Notes On Tumor Heterogeneity: Targets and Mechanisms, from the 2015 AACR Meeting in Philadelphia PA

Issues in Personalized Medicine: Discussions of Intratumor Heterogeneity from the Oncology Pharma forum on LinkedIn

Issues in Personalized Medicine in Cancer: Intratumor Heterogeneity and Branched Evolution Revealed by Multiregion Sequencing

CANCER COMPLEXITY: Heterogeneity in Tumor Progression and Drug Response – 2015 Annual Symposium @Koch Institute for Integrative Cancer Research at MIT – W34, 6/12/2015 9:00 AM EDT – 4:30 PM EDT

In vitro Models of Tumor Microenvironment for New Cancer Target and Drug Discovery, 11/17 – 11/19/2014, Hyatt Boston Harbor

What can we expect of tumor therapeutic response?

From the NIH: New NIH breast cancer research to focus on prevention

Posted in CANCER BIOLOGY & Innovations in Cancer Therapy, Cancer Informatics, Cancer Prevention: Research & Programs, Cancer Screening, Evidence-based decision-making, Translational Science, tagged Biology, breast cancer, Cancer - General, cancer risk, Clinical trial, Conditions and Diseases, early detection of cancer, health, National Institutes of Health, prevention, research on October 8, 2015| Leave a Comment »

New NIH breast cancer research to focus on prevention

Reporter: Stephen J. Williams, PhD

A new phase of the Breast Cancer and the Environment Research Program (BCERP), focused on prevention, is being launched at the National Institutes of Health. Grant-funded researchers will now work across scientific disciplines, involve new racially and ethnically diverse communities, and expand the study of risk factors that precede breast cancer, such as breast density.

These new directions reflect recommendations made by the Interagency Breast Cancer and Environmental Research Coordinating Committee (IBCERCC) in 2013. IBCERCC was congressionally mandated to review the state of the science around breast cancer and environmental influences by the Breast Cancer and Environmental Research Act. Recommendations included prioritizing prevention, involving transdisciplinary research teams, engaging public stakeholders, collaborating across federal agencies, and communicating the science to the public.

This broadened research focus will add to the growing knowledge of environmental and genetic factors that may influence breast cancer risk across the lifespan. The six new BCERP projects, plus a new coordinating center promoting cross-project collaboration, are jointly funded by the National Institute of Environmental Health Sciences (NIEHS) and the National Cancer Institute. All projects involve strong partnerships between researchers and organizations focused on breast cancer prevention or environmental health.

The new research will be conducted at the following institutions

Brigham and Women’s Hospital, Boston
City of Hope/Beckman Research Institute, Duarte, California
Columbia University, New York City
Georgetown Lombardi Comprehensive Cancer Center, Washington, D.C.
Michigan State University, Lansing
University of Massachusetts, Amherst
University of Wisconsin – Madison (Coordinating Center)

“The beauty of this research is that scientific discoveries and community observations inform each other, in order to dive deeper into the complex causes of breast cancer,” said Gwen Collman, Ph.D., director of NIEHS Division of Extramural Research and Training.

The focus on minority and socio-economically disadvantaged women is an important step in addressing disparities in breast cancer outcomes. Although African-American women are diagnosed with breast cancer less often than white women, more aggressive cancers and breast cancer deaths are more common among African-American women.

Another new direction for BCERP is research on the role of breast density as a possible intermediate risk factor for breast cancer. Dense breast tissue is one of the most common risk factors for breast cancer. Identifying links between environmental exposures and high breast density may provide new insights into prevention.

“These priorities reflect our continued commitment to breast cancer prevention,” noted Caroline Dilworth, Ph.D., BCERP program lead at NIEHS. “Our goal is to build on the high quality science we’ve been funding for more than a decade, while also being responsive to the expert recommendations of the IBCERCC report.”

Grant Numbers: U01ES026130, U01ES026137, U01ES026122, U01ES026132, U01ES026119, U01ES026140, U01ES026127

NIEHS supports research to understand the effects of the environment on human health and is part of NIH. For more information on environmental health topics, visit www.niehs.nih.gov. Subscribe to one or more of the NIEHS news lists to stay current on NIEHS news, press releases, grant opportunities, training, events, and publications.

The National Cancer Institute leads the National Cancer Program and the NIH’s efforts to dramatically reduce the prevalence of cancer and improve the lives of cancer patients and their families, through research into prevention and cancer biology, the development of new interventions, and the training and mentoring of new researchers. For more information about cancer, please visit the NCI website at http://www.cancer.gov or call NCI’s Cancer Information Service at 1-800-4-CANCER.

About the National Institutes of Health (NIH): NIH, the nation’s medical research agency, includes 27 Institutes and Centers and is a component of the U.S. Department of Health and Human Services. NIH is the primary federal agency conducting and supporting basic, clinical, and translational medical research, and is investigating the causes, treatments, and cures for both common and rare diseases. For more information about NIH and its programs, visit www.nih.gov.

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Curation of Recently Halted Oncology Trials Due to Serious Adverse Events – 2015

Posted in Cancer and Current Therapeutics, Clinical & Translational, Drug Toxicity, FDA, FDA Regulatory Affairs, Health Law & Patient Safety, Pharmaceutical Drug Discovery, Pharmaceutical Industry Competitive Intelligence, Pharmaceutical R&D Investment, Pharmacologic toxicities, tagged Adverse event, breast cancer, Cancer - General, chemotherapeutic toxicity, Chemotherapy, Clinical trial, Conditions and Diseases, FDA, FDA approval, FDA Warning Letter, Food and Drug Administration, health, medicine, National Institutes of Health, pharmacotoxicity, serious adverse event on October 7, 2015| Leave a Comment »

Curation of Recently Halted Oncology Trials Due to Serious Adverse Events – 2015

Curator: Stephen J. Williams, Ph.D.

The following is reports of oncology clinical trials in 2015 which have been halted for Serious Adverse Events (SAE), in most instances of an idiopathic nature. For comparison I have listed (as of this writing) the oncology drug approvals (8) for 2015. (from CenterWatch.com)

Oncology Drugs Approved in 2015

Farydak (panobinostat); Novartis; For the treatment of multiple myeloma, Approved February 2015

Ibrance (palbociclib); Pfizer; For the treatment of ER-positive, HER2-negative breast cancer, Approved February 2015

Lenvima (lenvatinib); Eisai; For the treatment of thyroid cancer, Approved February 2015

Lonsurf (trifluridine and tipiracil); Taiho Oncology; For the treatment of metastatic colorectal cancer , Approved September 2015

Odomzo (sonidegib); Novartis; For the treatment of locally advanced basal cell carcinoma, July 2015

Opdivo (nivolumab); Bristol-Myers Squibb; For the treatment of metastatic squamous non-small cell lung cancer, Approved March 2015

Unituxin (dinutuximab); United Therapeutics; For the treatment of pediatrics with high-risk neuroblastoma, Approved March 2015

Varubi (rolapitant); Tesaro; For the prevention of delayed nausea and vomiting associated with chemotherapy, Approved September 2015

Death Forces FDA to Place Clinical Hold on Advaxis (ADXS) Cancer Drug

from Biospace News

October 7, 2015
By Alex Keown, BioSpace.com Breaking News Staff

PRINCETON, N.J. – Following the death of a patient, the U.S. Food and Drug Administration (FDA) placed a hold on Advaxis (ADXS)’s experimental cancer treatment axalimogene filolisbac, which is currently in mid-stage trials.

In a statement issued this morning, Advaxis maintains the patient’s death was a result of the severity of her cancer and not due to the company’s experimental cancer treatment. It is seeking proof from the FDA that the drug was not a factor in the death. Still, the hold on the experimental cancer drug will cause the company to halt four clinical trials, Advaxis said. Other clinical trials, including those with the experimental ADXS-PSA and ADXS-HER2, are not affected by this hold. The company said it will continue to actively enroll and dose patients.

The FDA placed a hold on the drug on Oct. 2 after the company submitted a safety report to the regulatory agency that week. The drug is being developed to treat patients with persistent or recurrent metastatic (squamous or non-squamous cell) carcinoma of the cervix (PRmCC) who have progressed on at least one prior line of systemic therapy. Phase I trials released at the end of September showed treatment with axalimogene filolisbac resulted in a 38.5 percent 12-month overall survival rate in 26 patients. Patients typically fighting PRmCC who have failed at least one line of therapy have a typical survival rate of four to seven months.

Read full story here

FDA Halts Trial of Halozyme’s PEGPH20 for Pancreatic Cancer

Apr 9, 2014 Alex Philippidis

Halozyme Therapeutics acknowledged today that the FDA placed a formal clinical hold on its troubled Study 202 assessing its experimental drug PEGPH20 in patients with pancreatic cancer—less than a week after the company temporarily halted enrolling and dosing patients in the ongoing Phase II trial.

The agency told Halozyme it placed the clinical hold following the company’s pause in study activity. The trial’s independent data monitoring committee is evaluating data from the trial to learn why patients treated with PEGPH20 as well as nab-paclitaxel and gemcitabine saw a higher rate of blood clots and other thromboembolic events compared with patients treated with nab-paclitaxel and gemcitabine alone.

“We will be providing this information to the data monitoring committee and the FDA in parallel so they can complete their respective assessments,” Helen Torley, M.B. Ch.B., M.R.C.P., Halozyme’s president and CEO, said in a statement.

“Pancreatic cancer has one of the lowest survival rates of any cancer. We remain committed to evaluating PEGPH20 as a possible therapy to address this devastating disease,” Dr. Torley added.

As with Halozyme’s statement last week, the company’s latest remarks did not indicate when Halozyme expects to resume enrolling and dosing patients in Study 202, or how many patients had been enrolled and dosed when the temporary halt occurred.

The trial was envisioned as having 124 subjects, divided evenly between a treatment arm of PEGPH20 and nab-paclitaxel, and a gemcitabine arm, preceded by eight subject “run-in” phase assessing safety and tolerability, according to Study 202’s page on ClinicalTrials.gov (NCT01839487), last updated on January 27.

The study is one of two Phase II trials for PEGPH20; the other, SWOG, also aims to assess the drug for pancreatic cancer.

PEGPH20 is an investigational PEGylated form of Halozyme’s FDA-approved recombinant human hyaluronidase rHuPH20 (marketed as Hylenex®), designed to dramatically increases the half-life of the compound in the blood and allow for intravenous administration.

The temporary halt for Study 202 came two months after Halozyme publicly cited “potential acceleration of the PEGPH20 program” among several R&D programs for which it raised funds through a public offering of common stock that closed in February and generated approximately $107.8 million in net proceeds.

FDA orders CytRx to halt patient enrollment after death of a cancer patient

November 18, 2014 | By FierceBiotech John Carroll

CytRx ($CYTR) has run into an unexpected roadblock with its cancer drug conjugate aldoxorubicin, slamming the brakes on new patient recruitment in all their clinical trials after the FDA dropped a partial clinical hold on the program. According to the biotech the hold was forced by the death of a patient who was given the drug through a compassionate use program.

LA-based CytRx execs say that patients already enrolled in the studies will continue to receive the therapy as investigators added new safety measures, retooling trial protocols to include an “appropriate inclusion/exclusion criteria, an additional patient screening assessment and an evaluation of serum electrolytes prior to aldoxorubicin administration.” The patient who died, they added, had not qualified for any of its studies.

As it stands now, the biotech doesn’t know exactly how long the partial hold will last, but their announcement sought to calm jumpy investors, saying they expected to resolve the FDA’s demands “expeditiously” and can stick to their current timelines. CytRx says it expects to report preliminary results from their mid-stage study of Kaposi’s sarcoma in the second quarter of 2015 and preliminary results from the ongoing Phase II clinical trial of aldoxorubicin in glioblastoma multiforme in the first half of 2015. The company added that it is committed to completing enrollment in their Phase III trial by the end of next year.

hat reassurance appears to have helped with investors, who seemed to count this as more of a temporary setback than a catastrophe. Shares for CytRx were down about 9% in mid-morning trading.

Aldoxorubicin uses a linker molecule to attach to albumin in the blood and concentrate in tumors, where the acidic environment releases the chemotherapy doxorubicin in doses up to four times higher than what’s used now. Late last year their stock soared after their drug scored promising results for progression-free survival in a Phase IIb trial.

This case illustrates one reason why biotechs often quietly squirm under the pressure of compassionate use programs. They can be expensive to operate, time-consuming and raise fresh concerns when a patient dies or experiences a setback. On the other hand, if regulators take action like this following the death of an advanced stage cancer patient, there may have been something about the case that triggered broader concerns for the entire patient population

Clot risk in Lilly lung-cancer drug raises FDA concerns

July 7, 2015

Bloomberg News

KEYWORDS Cancer / Diseases / Health Care / Eli Lilly and Co. / Health Care Businesses / Health Care & Life Sciences / Health Care & Insurance

Eli Lilly and Co.’s experimental lung cancer drug has raised concerns with U.S. regulators that it may increase patients’ risk of suffering potentially deadly blood clots.

The drug, known as necitumumab, improved patients’ overall chances of survival, yet people taking the medicine also experienced more risk, Food and Drug Administration staff said in a report Tuesday. Indianapolis-based Lilly is seeking to sell the medicine to treat a subset of the most common type of lung cancer.

FDA advisers will meet Thursday to discuss the risks and benefits of necitumumab for patients with advanced squamous non-small cell lung cancer, in combination with chemotherapy. The FDA is expected to decide if Lilly can sell the drug by the end of the year.

While the safety of necitumumab reflects that of similar drugs, the increased danger of clotting “in this already high risk population is of concern,” FDA staff wrote.

One study showed that out of 538 patients taking necitumumab and chemotherapy, 9 percent experienced a serious clot, compared with 5 percent of 541 patients given only chemotherapy, according to the staff report.

Squamous lung cancer accounts for 25 percent to 30 percent of all lung cancer, according to the American Cancer Society.

Patients in a clinical trial who took necitumumab lived a median of 11.5 months, 1.6 months longer than those who got only chemotherapy, the FDA staff report said.

Opdivo Side Effects Center (as seen on Rxlist.com) (NOTE:TRIAL NOT HALTED)

Last reviewed on RxList 10/05/2015

Opdivo (nivolumab) is a human monoclonal antibody used to treat patients with unresectable or metastatic melanoma and disease progression following ipilimumab and, if BRAF V600 mutation positive, a BRAF inhibitor; and to treat metastatic squamous non-small cell lung cancer with progression on or after platinum-based chemotherapy. Common side effects of Opdivo include fatigue, rash, itching, cough, upper respiratory tract infection, swelling of the extremities, shortness of breath, muscle pain, decreased appetite, nausea, vomiting, constipation, diarrhea, weakness, swelling, fever, abdominal pain, chest pain, joint pain, and weight loss.

Opdivo FDA Prescribing Information: Side Effects
(Adverse Reactions)

Clinical Trials Experience

Because clinical trials are conducted under widely varying conditions, adverse reaction rates observed in the clinical trials of a drug cannot be directly compared to rates in the clinical trials of another drug and may not reflect the rates observed in clinical practice.

The data described in the WARNINGS AND PRECAUTIONS section and below reflect exposure to OPDIVO in Trial 1, a randomized trial in patients with unresectable or metastatic melanoma and in Trial 3, a single-arm trial in patients with metastatic squamous non-small cell lung cancer (NSCLC).

Clinically significant adverse reactions were evaluated in a total of 691 patients enrolled in Trials 1, 3, or an additional dose finding study (n=306) administering OPDIVO at doses of 0.1 to 10 mg/kg every 2 weeks [see WARNINGS AND PRECAUTIONS].

Unresectable or Metastatic Melanoma

The safety of OPDIVO was evaluated in Trial 1, a randomized, open-label trial in which 370 patients with unresectable or metastatic melanoma received OPDIVO 3 mg/kg every 2 weeks (n=268) or investigator’s choice of chemotherapy (n=102), either dacarbazine 1000 mg/m² every 3 weeks or the combination of carboplatin AUC 6 every 3 weeks plus paclitaxel 175 mg/m² every 3 weeks [see Clinical Studies]. The median duration of exposure was 5.3 months (range: 1 day to 13.8+ months) with a median of eight doses (range: 1 to 31) in OPDIVO-treated patients and was 2 months (range: 1 day to 9.6+ months) in chemotherapy treated patients. In this ongoing trial, 24% of patients received OPDIVO for greater than 6 months and 3% of patients received OPDIVO for greater than 1 year.

In Trial 1, patients had documented disease progression following treatment with ipilimumab and, if BRAF V600 mutation positive, a BRAF inhibitor. The trial excluded patients with autoimmune disease, prior ipilimumab-related Grade 4 adverse reactions (except for endocrinopathies) or Grade 3 ipilimumab-related adverse reactions that had not resolved or were inadequately controlled within 12 weeks of the initiating event, patients with a condition requiring chronic systemic treatment with corticosteroids ( > 10 mg daily prednisone equivalent) or other immunosuppressive medications, a positive test for hepatitis B or C, and a history of HIV.

The study population characteristics in the OPDIVO group and the chemotherapy group were similar: 66% male, median age 59.5 years, 98% white, baseline ECOG performance status 0 (59%) or 1 (41%), 74% with M1c stage disease, 73% with cutaneous melanoma, 11% with mucosal melanoma, 73% received two or more prior therapies for advanced or metastatic disease, and 18% had brain metastasis. There were more patients in the OPDIVO group with elevated LDH at baseline (51% vs. 38%).

OPDIVO was discontinued for adverse reactions in 9% of patients. Twenty-six percent of patients receiving OPDIVO had a drug delay for an adverse reaction. Serious adverse reactions occurred in 41% of patients receiving OPDIVO. Grade 3 and 4 adverse reactions occurred in 42% of patients receiving OPDIVO. The most frequent Grade 3 and 4 adverse reactions reported in 2% to less than 5% of patients receiving OPDIVO were abdominal pain, hyponatremia, increased aspartate aminotransferase, and increased lipase.

FDA Approves Eisai’s LENVIMA™ (lenvatinib) for the Treatment of Patients with Locally Recurrent or Metastatic, Progressive, Radioactive Iodine-Refractory Differentiated Thyroid Cancer

– Press release from Eisai (NOTE: TRIAL NOT HALTED)

Feb 13, 2015

WOODCLIFF LAKE, N.J., Feb. 13, 2015 /PRNewswire/ — Eisai Inc. announced today that the U.S. Food and Drug Administration (FDA) approved the company’s receptor tyrosine kinase inhibitor LENVIMA™ (lenvatinib) for the treatment of locally recurrent or metastatic, progressive, radioactive iodine-refractory differentiated thyroid cancer (RAI-R DTC). LENVIMA was approved following a priority review by the FDA, which is designated for drugs the FDA believes have the potential to provide a significant improvement in the treatment of a serious condition. LENVIMA demonstrated a statistically significant progression-free survival (PFS) prolongation and response rate in patients with progressive, differentiated thyroid cancer who had become refractory to radioactive iodine (RAI) therapy.

In the clinical trial, adverse events led to dose reductions in 68% of patients who received LENVIMA and 5% of patients who received placebo. Some patients will need to discontinue treatment for serious adverse reactions. In the trial, 18% of patients treated with LENVIMA and 5% who received placebo discontinued treatment. The most common adverse reactions (at least 10%) that resulted in dose reductions of LENVIMA were hypertension (13%), proteinuria (11%), decreased appetite (10%), and diarrhea (10%).

AstraZeneca halts a pair of lung cancer trials over a safety scare

From October 9, 2015 | By Damian Garde of FierceBiotech

“AstraZeneca ($AZN) is pressing pause on trials combining two of its most important pipeline cancer treatments after tracking reports of lung disease, halting enrollment as it gathers more information.

The company is testing a combination of AZD9291 and durvalumab, formerly MEDI4736, in two studies involving patients with non-small cell lung cancer. Late last month, AstraZeneca hit the brakes on enrollment in both trials due to an increase in reports of interstitial lung disease, which can lead to dangerous scarring and impaired pulmonary function. The pauses are temporary, the company stressed in an emailed statement, and patients already enrolled in the study will be given new consent forms to ensure they understand the risks before choosing whether keep getting treatment.”

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Horizon Discovery Group plc In-licenses Oncology Programme from Servier and Enters Option Agreement

Posted in Cancer and Current Therapeutics, Clinical & Translational, Clinical Diagnostics, Clinical Genomics, HealthCare IT, Personalized and Precision Medicine & Genomic Research, tagged Aviva Lev-Ari, Cancer - General, Clinical trial, Conditions and Diseases, genomics, health, Personalized medicine on October 7, 2015| Leave a Comment »

Horizon Discovery Group plc In-licenses Oncology Programme from Servier and Enters Option Agreement

Reporter: Aviva-Lev Ari, Ph.D., R.N.; Stephen J. Williams, Ph.D.

Press release

7 October 2015

Horizon Discovery Group plc In-licenses Oncology Programme from Servier and Enters Option Agreement

License programme with milestone payments of up to £50 million plus royalties on product sales
Horizon to in-license novel kinase inhibitor programme from Servier
Horizon will exploit its translational genomics and combination sciences platform to define optimum approaches to treatment and identify cancer patient populations most likely to respond

Cambridge, UK, 7 October 2015: Horizon Discovery Group plc (LSE: HZD) (“Horizon” or “the Company”), the international life science company supplying research tools and services that power genomics research and the development of personalised medicines, announces today that its leveraged business unit has signed a programme in-licensing and option agreement with Servier, the independent French research-based pharmaceutical company. The agreement is potentially worth over £50 million to Horizon in preclinical and clinical milestones, payments linked to net sales, and tiered royalties on future product sales.

Horizon has in-licensed novel kinase inhibitors from Servier that exhibit great promise based on pre-clinical data for treatment of a range of cancer types but do not currently have a biomarker to define a sensitive patient population. Horizon will use its world-leading platform, comprising isogenic cell lines and in vivo models, CRISPR-Cas9 mediated gene editing technology and ultra-high-throughput combination screening, to identify the population of cancer patients most likely to respond to the in-licensed compounds, whether as single agents or in combinations with other drugs. Horizon also has the option to explore the use of the inhibitors in other therapeutic indications.

Under the terms of the agreement, Servier has a first option to license back the assets. Should Servier take up this option, Horizon would receive up to £50 million in milestone payments plus royalties on product sales. If Servier does not take up its option, Horizon will be free to seek another pharma partner and Horizon and Servier would then share in the success of the progression of the programme as it advances into the clinic and registration.

Horizon will evaluate the mechanism of action of the candidate compounds, and will verify the patient stratification hypothesis by both in vitro and in vivo preclinical experiments. Horizon will also define a path towards the development of biomarkers for both patient stratification and drug efficacy.

Dr. Darrin M. Disley, Chief Executive Officer of Horizon Discovery Group plc, said: “The in-license of assets with a strong pre-clinical pedigree but do not yet have a clear clinical development strategy, represents a great opportunity for companies like Horizon. Demonstrating our scientific leadership through our translational genomics, drug combination and biomarker discovery platforms; we seek to identify genetic markers that predict drug sensitivity enabling programmes like this one to be progressed rapidly into the clinic for defined patient populations. This innovative deal, as part of our strategy to drive accelerated growth, offers significant upside potential for our investors built upon the leverage of our intellectual property, technology platforms and know-how.”

Mr Jean Pierre Abastado, Director of Oncology Innovation, Servier, commented: “The long standing collaboration between Servier and Vernalis has led to the discovery of novel kinase inhibitors. Horizon’s technology portfolio and expertise makes them ideally positioned to progress these drug candidates into the clinic and to investigate their potential for therapeutic efficacy both alone and in combination therapies. Servier is committed to driving therapeutic progress for the benefit of patients, with partnerships such as this playing a key role.”

ENDS

For further information from Horizon Discovery Group plc, please contact:

Zyme Communications (Trade and Regional Media)

Katie Odgaard

Tel: +44 (0)7787 502 947

Email: katie.odgaard@zymecommunications.com

Consilium Strategic Communications (Financial Media and Investor Relations)

Amber Fennell / Jessica Hodgson / Matthew Neal / Laura Thornton

Tel: +44 (0) 20 3709 5701

Email: horizon@consilium-comms.com

Panmure Gordon & Co. (NOMAD)

Corporate Finance: Freddy Crossley / Duncan Monteith / Fabien Holler

Broking: Tom Salvesen

Tel: +44 20 7886 2500

Notes for Editors

About Horizon Discovery Group plc www.horizondiscovery.com/

Horizon is a revenue-generating life science group supplying research tools to organisations engaged in genomics research and the development of personalised medicines. Horizon has a diverse and international customer base of over 1,200 organisations across more than 50 countries, including major pharmaceutical, biotechnology and diagnostic companies as well as leading academic research centers. The Group supplies its products and services into multiple markets, estimated to total in excess of £29 billion by 2015.

Horizon’s core capabilities are built around its proprietary translational genomics platform, a high-precision and flexible suite of gene editing tools able to alter almost any endogenous gene sequence of human or mammalian cell-lines. Horizon offers over 20,000 catalogue products, almost all of which are based on the application of gene editing to generate cell lines that accurately model the disease-causing mutations found in genetically based diseases. These ‘patients-in-a-test-tube’ are being used by customers to identify the effect of individual or compound genetic mutations on drug activity, patient responsiveness, and resistance, which may lead to the successful prediction of which patient sub-groups will respond to currently available and future drug treatments.

In addition, Horizon provides custom cell line and in vivo model generation services for research and bioproduction applications, quantitative molecular reference standards, in vivo disease models, and contract research and custom screening services.

Horizon is headquartered in Cambridge, UK, and is listed on the London Stock Exchange’s AIM market under the ticker “HZD”, for further information please visit: www.horizondiscovery.com.

About Servier

Servier is an independent French research-based pharmaceutical company. Its development is driven by the pursuit of innovation in the therapeutic areas of cardiovascular, metabolic, central nervous system, psychiatric, bone, muscle and joint diseases, as well as cancer.

In 2014, the company recorded a turnover of 4 billion euros.
92% of Servier medicines are prescribed outside of France.
28% of turnover from Servier drugs was reinvested in Research and Development in 2014.

With a strong international presence in 146 countries, Servier employs more than 21,400 people worldwide.

Oncology is one of the key priorities of Servier in terms of research and development with currently 8 new molecular entities in clinical development in breast cancer, lung cancer, other solid tumours and various types of lymphomas and leukaemias. This portfolio of innovative cancer treatments is being developed with various partners worldwide, and covers different hallmarks of cancer including cytotoxics, pro-apoptotic, targeted, immune and cellular therapies. Hence, Servier aims at delivering a significant and positive impact on cancer patients’ lives.

Maturation Stage of T-cell Acute Lymphoblastic Leukemia Determines BCL-2 versus BCL-XL Dependence and Sensitivity to ABT-199

Why Does Cytotoxic Chemotherapy Still Remain a Mainstay in Many Chemotherapeutic Regimens?

Posted in Cancer and Current Therapeutics, CANCER BIOLOGY & Innovations in Cancer Therapy, Scientific & Biotech Conferences: Press Coverage, tagged breast cancer, Cancer - General, Chemotherapy, Clinical trial, Conditions and Diseases, DNA, DNA Sequencing, genetics, genome, genomics, Harvard Medical School, health, medicine, Personalized medicine, research, science on September 15, 2015| Leave a Comment »

Why Does Cytotoxic Chemotherapy Still Remain a Mainstay in Many Chemotherapeutic Regimens? [6.1.1]

Reporter: Stephen J. Williams, Ph.D.

At the 2015 AACR National Meeting, Drs. Anthony Letai, Dr. Michael Hermann, Dr. Rene Bernards, and Dr. Guido Kroemer gave The 2015 Stanley J. Korsmeyer Memorial Symposium: Cell Death and Cancer Therapy: Why Has Conventional Chemotherapy Been So Successful?

Cytotoxic chemotherapy, for which the mechanism of action is centered on the ability of the drug to kill a cell by either necrosis, genotoxic, apoptosis, or autophagy mechanisms rather than just halting cell growth, is still, in this era of personalized and cytostatic therapies, is still a mainstay in many treatment regimens for a majority of cancers. Treatment regimens such as MOPP (mechlorethamine, Oncovin, procarbazine, prednisone), CMF (cyclophosphamide, methotrexate, 5-fluorouracil) , carboplatin with taxol, and even with personalized therapies, which usually are given in combination with a cytotoxic agent. However treatment regimens containing these cytotoxic chemotherapeutics show some of the best survival rates. The abstract for the Symposium is given below:

In this current era of precisely targeted therapies and –omics technologies, it is often forgotten that no medical therapy has cured, and continues to cure, more people of cancer than conventional chemotherapy. Notwithstanding its superior performance across many cancer types, the mechanism of the therapeutic index of conventional agents, largely targeting ubiquitous elements like DNA and microtubules, is poorly understood. The textbook explanation of conventional chemotherapy’s working by killing supposedly rapidly dividing cancer cells lacks clinical evidence and flies in the face of many obvious clinical counter-examples. In the session,m the speakers will describe how conventional cytotoxic chemotherapy preferentially kills cancer cells. Moreover, they will describe how clinical response to chemotherapy might be better predicted.

This post is presented as the speakers titles and a brief curation of their papers related to the subject matter.

Anthony G. Letai, Dana-Farber Cancer Institute, Boston, MA. Conventional chemotherapy cures people by exploiting apoptotic priming.

Conventional chemotherapy has an amazing track record that is often under-appreciated in today’s world of genomics and targeted pathway inhibitors. Conventional chemotherapy is responsible for curing millions of cancer patients over the past 5 decades. That is, millions of patients have presented to their doctors with an otherwise fatal malignancy, were given a finite course of chemotherapy (largely DNA and microtubule perturbing agents) and had their cancer eradicated, never to return. Perhaps as remarkable as the magnitude of the achievement of conventional chemotherapy is the magnitude of our ignorance of why it should ever work, and why it works far better in some tumors than in others. Textbook explanations rely on concepts of differential proliferation rates in cancers that are incompletely supported in the clinical literature. Successful chemotherapy treatments usually kill via the mitochondrial pathway of apoptosis. We have found that simple functional measurements of the pre-treatment state of the tumor cell can be rapidly made with BH3 profiling. These measurements demonstrate that a major, if not the major, reason for a therapeutic index for cancer chemotherapy is that chemo-sensitive cancer cells are simply more primed for apoptosis than normal cells. Moreover, apoptotic priming can be measured to make clinical predictions regarding quality of response on an individualized basis. Enhancing pretreatment priming of cancer cells with selectively acting targeted agents is a promising strategy to extend the demonstrated curative power of conventional chemotherapy.

Triona Ni Chonghaile, Justine E. Roderick, Cian Glenfield, Jeremy Ryan, Stephen E. Sallan, Lewis B. Silverman, Mignon L. Loh, Stephen P. Hunger, Brent Wood, Daniel J. DeAngelo, Richard Stone, Marian Harris, Alejandro Gutierrez, Michelle A. Kelliher, Anthony Letai

Cancer Discov. Author manuscript; available in PMC 2015 March 1.

Published in final edited form as: Cancer Discov. 2014 September; 4(9): 1074–1087. Published online 2014 July 3. doi: 10.1158/2159-8290.CD-14-0353

High Mitochondrial Priming Sensitizes hESCs to DNA-Damage-Induced Apoptosis

Julia C. Liu, Xiao Guan, Jeremy A. Ryan, Ana G. Rivera, Caroline Mock, Vishesh Agrawal, Anthony Letai, Paul H. Lerou, Galit Lahav

Cell Stem Cell. Author manuscript; available in PMC 2014 October 3.

Published in final edited form as: Cell Stem Cell. 2013 October 3; 13(4): 483–491. Published online 2013 August 15. doi: 10.1016/j.stem.2013.07.018

Correction in: volume 13 on page 634

Prolonged mitotic arrest triggers partial activation of apoptosis, resulting in DNA damage and p53 induction

James D. Orth, Alexander Loewer, Galit Lahav, Timothy J. Mitchison

Mol Biol Cell. 2012 February 15; 23(4): 567–576. doi: 10.1091/mbc.E11-09-0781

Stem cells: Balancing resistance and sensitivity to DNA damage

Julia C. Liu, Paul H. Lerou, Galit Lahav

Trends Cell Biol. Author manuscript; available in PMC 2015 May 1.

Published in final edited form as: Trends Cell Biol. 2014 May; 24(5): 268–274. Published online 2014 April 7. doi: 10.1016/j.tcb.2014.03.002

Michael T. Hermann, MIT Koch Institute for Integrated Cancer Research, Cambridge MA. Using convential chemotherapy as targeted agents.

Exploiting the Synergy between Carboplatin and ABT-737 in the Treatment of Ovarian Carcinomas

Harsh Vardhan Jain, Alan Richardson, Michael Meyer-Hermann, Helen M. Byrne

PLoS One. 2014; 9(1): e81582. Published online 2014 January 6.

Rene Bernards, Netherlands Cancer Institute, Amsterdam, The Netherlands. Identifying responders to chemotherapies through functional genomics

MED12 Controls the Response to Multiple Cancer Drugs through Regulation of TGF-β Receptor Signaling

Sidong Huang, Michael Hölzel, Theo Knijnenburg, Andreas Schlicker, Paul Roepman, Ultan McDermott, Mathew Garnett, Wipawadee Grernrum, Chong Sun, Anirudh Prahallad, Floris H. Groenendijk, Lorenza Mittempergher, Wouter Nijkamp, Jacques Neefjes, Ramon Salazar, Peter ten Dijke, Hidetaka Uramoto, Fumihiro Tanaka, Roderick L. Beijersbergen, Lodewyk F.A. Wessels, René Bernards

Cell. Author manuscript; available in PMC 2013 June 5.

Published in final edited form as: Cell. 2012 November 21; 151(5): 937–950.

Sorafenib synergizes with metformin in NSCLC through AMPK pathway activation

Floris H Groenendijk, Wouter W Mellema, Eline van der Burg, Eva Schut, Michael Hauptmann, Hugo M Horlings, Stefan M Willems, Michel M van den Heuvel, Jos Jonkers, Egbert F Smit, René Bernards

Int J Cancer. 2015 March 15; 136(6): 1434–1444. Published online 2014 August 1.

The Corepressor CTBP2 Is a Coactivator of Retinoic Acid Receptor/Retinoid X Receptor in Retinoic Acid Signaling

Prashanth Kumar Bajpe, Guus J. J. E. Heynen, Lorenza Mittempergher, Wipawadee Grernrum, Iris A. de Rink, Wouter Nijkamp, Roderick L. Beijersbergen, Rene Bernards, Sidong Huang

Mol Cell Biol. 2013 August; 33(16): 3343–3353. doi: 10.1128/MCB.01213-12

Using Functional Genetics to Understand Breast Cancer Biology

Alan Ashworth, Rene Bernards

Cold Spring Harb Perspect Biol. 2010 July; 2(7): a003327. doi: 10.1101/cshperspect.a003327

SMARCE1 suppresses EGFR expression and controls responses to MET and ALK inhibitors in lung cancer

Andreas I Papadakis, Chong Sun, Theo A Knijnenburg, Yibo Xue, Wipawadee Grernrum, Michael Hölzel, Wouter Nijkamp, Lodewyk FA Wessels, Roderick L Beijersbergen, Rene Bernards, Sidong Huang

Cell Res. 2015 April; 25(4): 445–458. Published online 2015 February 6.

The Good, the Bad, and the Ugly: in search of gold standards for assessing functional genetic screen quality

Bastiaan Evers, Rene Bernards, Roderick L Beijersbergen

Mol Syst Biol. 2014 July; 10(7): 738. Published online 2014 July 1.

An Integrative Genomic and Proteomic Analysis of PIK3CA, PTEN, and AKT Mutations in Breast Cancer

Katherine Stemke-Hale, Ana Maria Gonzalez-Angulo, Ana Lluch, Richard M. Neve, Wen-Lin Kuo, Michael Davies, Mark Carey, Zhi Hu, Yinghui Guan, Aysegul Sahin, W. Fraser Symmans, Lajos Pusztai, Laura K. Nolden, Hugo Horlings, Katrien Berns, Mien-Chie Hung, Marc J. van de Vijver, Vicente Valero, Joe W. Gray, René Bernards, Gordon B. Mills, Bryan T. Hennessy

Cancer Res. Author manuscript; available in PMC 2009 August 1.

Published in final edited form as: Cancer Res. 2008 August 1; 68(15): 6084–6091.

CTF Meeting 2012: Translation of the Basic Understanding of the Biology and Genetics of NF1, NF2, and Schwannomatosis Toward the Development of Effective Therapies

Brigitte C. Widemann, Maria T. Acosta, Sylvia Ammoun, Allan J. Belzberg, Andre Bernards, Jaishri Blakeley, Antony Bretscher, Karen Cichowski, D. Wade Clapp, Eva Dombi, Gareth D. Evans, Rosalie Ferner, Cristina Fernandez-Valle, Michael J. Fisher, Marco Giovannini, David H. Gutmann, C. Oliver Hanemann, Robert Hennigan, Susan Huson, David Ingram, Joe Kissil, Bruce R. Korf, Eric Legius, Roger J. Packer, Andrea I McClatchey, Frank McCormick, Kathryn North, Minja Pehrsson, Scott R. Plotkin, Vijaya Ramesh, Nancy Ratner, Susann Schirmer, Larry Sherman, Elizabeth Schorry, David Stevenson, Douglas R. Stewart, Nicole Ullrich, Annette C. Bakker, Helen Morrison

Am J Med Genet A. Author manuscript; available in PMC 2014 September 1.

Published in final edited form as: Am J Med Genet A. 2014 March; 0(3): 563–578. Published

Analysis of the MammaPrint Breast Cancer Assay in a Predominantly Postmenopausal Cohort

Ben S. Wittner, Dennis C. Sgroi, Paula D. Ryan, Tako J. Bruinsma, Annuska M. Glas, Anitha Male, Sonika Dahiya, Karleen Habin, Rene Bernards, Daniel A. Haber, Laura J. Van’t Veer, Sridhar Ramaswamy Clin Cancer Res. Author manuscript; available in PMC 2011 May 7.

Guido Kroemer, INSERM U848- Institute Gustave-Roussy, Villejuif, France. A hallmark of successful cancer therapies: Reinstatement of immunosurvelliance.

Immune infiltrate in cancer Gautier Stoll, Laurence Zitvogel, Guido Kroemer

Aging (Albany NY) 2015 June; 7(6): 358–359. Published online 2015 June 25.

Corrigendum: “Combinatorial Strategies for the Induction of Immunogenic Cell Death”

Lucillia Bezu, Ligia C. Gomes-da-Silva, Heleen Dewitte, Karine Breckpot, Jitka Fucikova, Radek Spisek, Lorenzo Galluzzi, Oliver Kepp, Guido Kroemer

Front Immunol. 2015; 6: 275. Published online 2015 June 1. doi: 10.3389/fimmu.2015.00275

Corrects: Front Immunol. 2015; 6: 187.

Meta-analysis of organ-specific differences in the structure of the immune infiltrate in major malignancies

Gautier Stoll, Gabriela Bindea, Bernhard Mlecnik, Jérôme Galon, Laurence Zitvogel, Guido Kroemer

Oncotarget. 2015 May 20; 6(14): 11894–11909. Published online 2015 May 19

Novel Approaches to Cancer Therapy [11.1]

Misfolded Proteins – from Little Villains to Little Helpers… Against Cancer

Multiple Lung Cancer Genomic Projects Suggest New Targets, Research Directions for Non-Small Cell Lung Cancer

A Synthesis of the Beauty and Complexity of How We View Cancer

Good and Bad News Reported for Ovarian Cancer Therapy