Posts Tagged ‘cell cycle’

New studies link cell cycle proteins to immunosurveillance of premalignant cells

Posted in Apoptosis, Biological Networks, Biological Networks, Gene Regulation and Evolution, Cancer - General, CANCER BIOLOGY & Innovations in Cancer Therapy, Cancer-Immune Interactions, Cell Biology, Signaling & Cell Circuits, Circulating Tumor Cells (CTC), Disease Biology, DNA repair, Efficacy of Anti-Tumor T Cells, Exosomes, Exosomes: Natural Carriers for siRNA Delivery, Immune Modulatory, Immuno-Oncology & Genomics, Immunology, KRAS Mutation, Modulating Macrophages in Cancer Immunotherapy, NK Cell-Based Cancer Immunotherapy, TP53 - Germline mutations, tagged CDKI, cell cycle, cell cycle arrest, cell cycle protein, cellular senescence, DNA repair or transcription factor genes, Immunology, immunosuppression, immunosurveillance, NF-kB, p21, p53, premalignant, transcriptional, transcriptional regulation, tumor suppressors on December 16, 2021| Leave a Comment »

New studies link cell cycle proteins to immunosurveillance of premalignant cells

Curator: Stephen J. Williams, Ph.D.

The following is from a Perspectives article in the journal Science by Virinder Reen and Jesus Gil called “Clearing Stressed Cells: Cell cycle arrest produces a p21-dependent secretome that initaites immunosurveillance of premalignant cells”. This is a synopsis of the Sturmlechener et al. research article in the same issue (2).

Complex organisms repair stress-induced damage to limit the replication of faulty cells that could drive cancer. When repair is not possible, tissue homeostasis is maintained by the activation of stress response programs such as apoptosis, which eliminates the cells, or senescence, which arrests them (1). Cellular senescence causes the arrest of damaged cells through the induction of cyclin-dependent kinase inhibitors (CDKIs) such as p16 and p21 (2). Senescent cells also produce a bioactive secretome (the senescence-associated secretory phenotype, SASP) that places cells under immunosurveillance, which is key to avoiding the detrimental inflammatory effects caused by lingering senescent cells on surrounding tissues. On page 577 of this issue, Sturmlechner et al. (3) report that induction of p21 not only contributes to the arrest of senescent cells, but is also an early signal that primes stressed cells for immunosurveillance.Senescence is a complex program that is tightly regulated at the epigenetic and transcriptional levels. For example, exit from the cell cycle is controlled by the induction of p16 and p21, which inhibit phosphorylation of the retinoblastoma protein (RB), a transcriptional regulator and tumor suppressor. Hypophosphorylated RB represses transcription of E2F target genes, which are necessary for cell cycle progression. Conversely, production of the SASP is regulated by a complex program that involves super-enhancer (SE) remodeling and activation of transcriptional regulators such as nuclear factor κB (NF-κB) or CCAAT enhancer binding protein–β (C/EBPβ) (4).

Senescence is a complex program that is tightly regulated at the epigenetic and transcriptional levels. For example, exit from the cell cycle is controlled by the induction of p16 and p21, which inhibit phosphorylation of the retinoblastoma protein (RB), a transcriptional regulator and tumor suppressor. Hypophosphorylated RB represses transcription of E2F target genes, which are necessary for cell cycle progression. Conversely, production of the SASP is regulated by a complex program that involves super-enhancer (SE) remodeling and activation of transcriptional regulators such as nuclear factor κB (NF-κB) or CCAAT enhancer binding protein–β (C/EBPβ) (4).

Sturmlechner et al. found that activation of p21 following stress rapidly halted cell cycle progression and triggered an internal biological timer (of ∼4 days in hepatocytes), allowing time to repair and resolve damage (see the figure). In parallel, C-X-C motif chemokine 14 (CXCL14), a component of the PASP, attracted macrophages to surround and closely surveil these damaged cells. Stressed cells that recovered and normalized p21 expression suspended PASP production and circumvented immunosurveillance. However, if the p21-induced stress was unmanageable, the repair timer expired, and the immune cells transitioned from surveillance to clearance mode. Adjacent macrophages mounted a cytotoxic T lymphocyte response that destroyed damaged cells. Notably, the overexpression of p21 alone was sufficient to orchestrate immune killing of stressed cells, without the need of a senescence phenotype. Overexpression of other CDKIs, such as p16 and p27, did not trigger immunosurveillance, likely because they do not induce CXCL14 expression.In the context of cancer, senescent cell clearance was first observed following reactivation of the tumor suppressor p53 in liver cancer cells. Restoring p53 signaling induced senescence and triggered the elimination of senescent cells by the innate immune system, prompting tumor regression (5). Subsequent work has revealed that the SASP alerts the immune system to target preneoplastic senescent cells. Hepatocytes expressing the oncogenic mutant NRAS^G12V (Gly¹²→Val) become senescent and secrete chemokines and cytokines that trigger CD4⁺ T cell–mediated clearance (6). Despite the relevance for tumor suppression, relatively little is known about how immunosurveillance of oncogene-induced senescent cells is initiated and controlled.

References

2. Sturmlechner I, Zhang C, Sine CC, van Deursen EJ, Jeganathan KB, Hamada N, Grasic J, Friedman D, Stutchman JT, Can I, Hamada M, Lim DY, Lee JH, Ordog T, Laberge RM, Shapiro V, Baker DJ, Li H, van Deursen JM. p21 produces a bioactive secretome that places stressed cells under immunosurveillance. Science. 2021 Oct 29;374(6567):eabb3420. doi: 10.1126/science.abb3420. Epub 2021 Oct 29. PMID: 34709885.

More Articles on Cancer, Senescence and the Immune System in this Open Access Online Scientific Journal Include

Bispecific and Trispecific Engagers: NK-T Cells and Cancer Therapy

Natural Killer Cell Response: Treatment of Cancer

Issues Need to be Resolved With ImmunoModulatory Therapies: NK cells, mAbs, and adoptive T cells

New insights in cancer, cancer immunogenesis and circulating cancer cells

Insight on Cell Senescence

Immune System Stimulants: Articles of Note @pharmaceuticalintelligence.com

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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

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

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.

OPEN IN VIEWER

 

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).

OPEN IN VIEWER

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 (Ki), CDK6, 0.16 nM (Ki)	CDK2, 0.09 nM (Ki)	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

Expand for more

OPEN IN VIEWER

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

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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.

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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)

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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

Expand for more

OPEN IN VIEWER

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).

OPEN IN VIEWER

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

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

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

1. 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.
2. 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.

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Notes

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.

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.

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Footnotes

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

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References

1. Toogood PL, et al. J Med Chem. 2005;48:2388–406. doi: 10.1021/jm049354h. [PubMed] [Cross Ref]
2. Fry DW, et al. Mol Cancer Ther. 2004;3:1427–38. [PubMed]
3. Flaherty KT, et al. Clin Cancer Res. 2012;18:568–76. doi: 10.1158/1078-0432.CCR-11-0509. [PubMed] [Cross Ref]
4. Schwartz GK, et al. Br J Cancer. 2011;104:1862–8. doi: 10.1038/bjc.2011.177. [PMC free article] [PubMed] [Cross Ref]
5. Leonard JP, et al. Blood. 2012;119:4597–607. doi: 10.1182/blood-2011-10-388298. [PubMed] [Cross Ref]
6. Dean JL, et al. Oncogene. 2010;29:4018–32. doi: 10.1038/onc.2010.154. [PubMed] [Cross Ref]
7. Stone S, et al. Cancer Res. 1996;56:3199–202. [PubMed]
8. Roberts PJ, et al. J Natl Cancer Inst. 2012;104:476–87. doi: 10.1093/jnci/djs002. [PMC free article] [PubMed] [Cross Ref]
9. 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, ³ Lisa M. Butler, ³ Wayne D. Tilley, ³ Agnieszka K. Witkiewicz, ^{4 , 2 ,*} and Erik S. Knudsen ^{1 , 2 ,*}

Author information ► Copyright and License information ►

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.

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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

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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

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

Author information ► Copyright and License information ►

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.

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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

 

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

 

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

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)

 

 

Other article in this Open Access Journal on Cell Cycle and Cancer Include:

 

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

 

 

 

 

 

 

 

 

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