Posts Tagged ‘Biology’

Heat Shock Proteins (HSP) and Molecular Chaperones

Posted in Cancer - General, Proteins, Proteomics, tagged Aging, Biology, Cancer - General, cataract, heat shock proteins, immunity, lens proteins, medicine, Molecular chaperones on April 13, 2016| Leave a Comment »

Heat Shock Proteins (HSP) and Molecular Chaperones

Curator: Larry H. Bernstein, MD, FCAP

 

 

Report on the VIIth International Symposium on Heat Shock Proteins in Biology & Medicine

Stuart K. Calderwood and Lawrence E. Hightower
Cell Stress Chaperones. 2015 Mar; 20(2): 213–216.doi:  10.1007/s12192-014-0562-z

The major themes of this meeting were: new properties of heat shock proteins (HSPs) and heat shock factor (HSF) and role in the etiology of cancer, molecular chaperones in aging, extracellular HSPs in inflammation and immunity, role of heat shock and the heat shock response in immunity and cancer, protein aggregation disorders and HSP expression, and Hsp70 in blood cell differentiation.

This symposium was the seventh symposium in a series presided over by Dr Stuart Calderwood aimed at exploring the association of molecular chaperones, heat shock proteins, and the heat shock response in physiological/pathological processes. The biochemistry and ultrastructure of molecular chaperones was not emphasized, as these topics are well represented at other meetings. The major themes were: new properties of heat shock proteins (HSPs) and heat shock factor (HSF) and role in the etiology of cancer, molecular chaperones in aging, extracellular HSPs in inflammation and immunity, role of heat shock and the heat shock response in immunity and cancer, protein aggregation disorders and HSP expression, and Hsp70 in blood cell differentiation. This report gives a thematic overview and does not include all the topics presented.

NEW PROPERTIES OF HSPS AND HSF, AND ROLE IN THE ETIOLOGY OF CANCER 

One of the exciting aspects of the meeting involved advances made in understanding the biology of Hsp90. In recent years, we have understood the molecular chaperone activities of Hsp90 mostly in terms of its biochemistry, cooperative interactions with cochaperones. However, Dr Len Neckers (NCI/NIH), the conference keynote speaker, has opened up new areas in our understanding of this chaperone by characterizing the role of posttranslational modification (PTM) in terms of phosphorylation, acetylation, and sumoylation in Hsp90 biology. One particularly intriguing possibility is that altered signaling mechanisms characteristic of cancer may target such PTMs, and this could contribute to the “addiction to chaperones” observed in malignant cells. (Also discussed later by Dr Mehdi Mollapour, SUNY Upstate Medical University).

In addition, interesting differences in properties of the two Hsp90 isoforms have been detected. Dr Wei Li (University of Southern California) has shown that Hsp90a can be released into the extracellular environment and there take part in cell regulation, mediating for instance wound healing effects. In addition, proteomic studies carried out by Thomas Prince (NCI/NIH) in the Neckers lab indicate that Hsp90β may be more dedicated to “housekeeping” molecular chaperone functions while Hsp90α may play more glamorous roles in cell regulation. These distinctions might not be anticipated based on the rather minimal sequence differences between the Hsp90s but offer keen insights into the biology of this chaperone. Finally, Dr Tim Haystead (Duke University) discussed the approach of targeting ectopically expressed Hsp90 for imaging and treatment.

Another PTM with implications in the stress response is the modification of intracellular proteins by monosaccharides of O-linked β-N- acetylglucosamine (O-GlcNAc). Dr Natasha Zachara (Johns Hopkins University School of Medicine) discussed targets for this modification and roles in cytoprotection.

The poster session was also rich in Hsp90 studies, mostly from the Neckers lab—presentations by Kristin Beebe et al. (NCI/NIH) Posttranslational modification state of Hsp90 differentially affects binding of small molecule inhibitors; Toshiki Kijima et al. (NCI/NIH), Defined interactions between HSF1 and Hsp90; T. Prince et al. (NCI/NIH) Hsp90 and tyrosine kinase inhibitors: A synergistic approach towards combating cancer; Andrew W. Truman (University of Chicago)Quantitive ptoteomics of the yeast Hsp70/Hsp90 interactomes during DNA damage reveals chaperone–dependent regulation of ribonucleotide reductase. Inhibition of Hsp90 via C–domain induces temporally distinct phosphorylation patterns; and Diana M. Dunn (SUNY Upstate Medical University)Phosphorylation of human Hsp90 threonine 115 modulates chaperone function and drug sensitivity.

Hsp70 is also emerging as a factor in cell regulation, exhibiting properties beyond a narrow role in chaperoning. Dr Michael Sherman (Boston University) showed a key role for Hsp72 in mammary cancer, and this property did not seem to depend on alterations in protein folding. Instead, Hsp72 appeared to function through its co-chaperone Bag3, a major regulatory molecule in cell signaling. In addition, a presentation by Stuart Calderwood (Harvard Medical School) that included work by Jianlin Gong showed that Hsp72 is required for tumor initiation and metastasis in murine spontaneous breast cancer. These effects appeared to be partially mediated through regulation of expression of the protoooncogene c–Met, a key player in invasion and metastasis in cancer. We anticipate advances in understanding of the roles of individual members of the Hsp70 family, as is currently emerging for Hsp90. The prospect of targeting Hsp70 with small molecule inhibitors was elegantly discussed by Maureen Murphy (The Wistar Institute), who introduced a novel class of drugs that could selectively kill cancer cells by inhibiting Hsp70 function. In a related topic, Dr Mathias P. Mayer (University of Heidelberg) showed a detailed analysis of the activities of inhibitors targeting various domains in Hsp70.

Dr Takanori Eguchi (Harvard Medical School) then described his studies showing an unconventional role for the extracellular protease MMP3 as a nuclear protein that could trigger molecular chaperone synthesis (HspA7) in mammary cancer. Interestingly, a role in cancer for the Hsp70 co-chaperone Hsp40 was also shown by Dr Jane Trepel (NCI/NIH).

One presentation that stood apart was that of Dr Carmen Garrido (INSERM U866) who has shown very impressive studies indicating a key role for Hsp70 in hematopoiesis, acting through the factor GATA1. This role appeared to depend on nuclear localization of Hsp70, and Dr Garrido is attempting to study the role of PTM, particularly phosphorylation in this function/localization of Hsp70. This continued the theme of HSP PTM and regulation in the cell.

MOLECULAR CHAPERONES IN AGING

A symposium on molecular chaperones in aging was organized by Dr Shelley Buffenstein (University of Texas Health Science Center San Antonio). This symposium featured some fascinating studies on the naked mole rat (NMR), a rodent with a remarkable lifespan based on size (32 years compared to 3 years in the comparably sized mouse). This has permitted comparative biology studies that have uncovered important aspects of the aging process in mammals. Dr Buffenstein showed that one aspect of the proteotoxic response was enhanced in NMR—proteasome activity that was resistant to oxidative stress as well as conventional proteasome inhibitors. Such proteasome resistance appeared to be conferred by Hsp70 and Hsp40. Karl Rodriguez (also from the UTHSC San Antonio) stressed the importance of Hsp25 in the longevity of NMR. This small HSP is expressed to very high levels in this organism. Kenneth B. Storey (Carleton University) finally gave an encyclopedic presentation entitled “HeatShock Proteins and Hypometabolism in Nature”, discussing the multiple roles of chaperones in hibernation and other processes involving a step down in metabolism.

PROTEIN AGGREGATION DISORDERS AND HSP EXPRESSION

Michael Sherman (Boston University) chaired a lively and highly diverse session on protein aggregation disorders and HSPs. Gary Jones (Maynooth University) discussed his studies on the roles of Hsp104, Hsp70, and Hsp40 in prion propagation in yeast, concentrating on Hsp70. The Hsp complex was able to dissolve prions in yeast. Daniel Kaganovich (Hebrew University) then continued in a yeast theme, discussing a further strategy for resolving proteotoxic stress involving asymmetric cell division in which damaged proteins and mitochondria remain with the mother cell after mitosis. Nava Zaarur (Boston University) then discussed the role of aggresome particles in resolving aggregated proteins, in this case in eucaryotes. Alberto Macario (University of Maryland School of Medicine) discussed the role of chaperonins in proteotoxic disorders dealing with the effects of a pathogenic mutation of human CCT5 on its intrinsic properties. Dr Elaine C. Lee (University of Connecticut) discussed another type of stress. She showed significant roles for chaperones in osmotic stress responses of Caenorhabditis elegans models of polyglutamine diseases.

EXTRACELLULAR HSPS, INFLAMMATION, AND IMMUNITY

Although it is now generally accepted that HSPs can escape the confines of the cell, many questions still remain regarding their extracellular properties, particularly with regard to their immune effects. These questions include: whether HSPs are mostly immunostimulatory or immunosuppressive, whether they can induce sterile inflammation, and what structures on the immune cells recognize the HSPs. Dr Cristina Bonorino (Pontifícia Universidade Católica do Rio Grande do Sul) chaired a symposium “HSP as modulators of immunity: prokaryotic meets eukaryotic” featuring presentations by Robert Binder (University of Pittsburgh), Eckhart R. Podack (University of Miami), Renata Pasqualini (University of New Mexico Medical School), and Cristina Bonorino. In short, the talks indicated that while the prokaryotic chaperone DNA-PK can be immunosppressive and prolong the lifetime of transplanted tissues and reduce the morbidity of arthritis (Drs Bonorino and Kamal Moudgil (University of Maryland School of Medicine)), HSPs can also be immunostimulatory and act as cancer vaccines when associated with cancer antigens (Drs Binder and Podack). In the discussion, it was stressed that these effects may be related to HSP dose, with low doses of HSP antigen complex favoring immunity while higher doses may lead to immunoregulatory effects (Dr Binder). Most parties agreed that much future study is required to resolve all these issues. It was also suggested, inspired by the presentation of Dr Neckers, that HSP PTMs might also be playing roles in shading the immune effects of HSPs (Dr Bonorino). In the next session, Drs Shawn Wang (Virginia Commonwealth University School of Medicine) and John Subjeck (Roswell Park Cancer Institute) discussed the molecular foundations of their highly effective large HSP vaccines that are now in clinical trial for tumor immunotherapy. They indicated that the high avidity for antigen of the larger HSPs might be key for effectiveness. Although the nature of HSP receptors is still not fully resolved, Ayesha Murshid (Harvard Medical School) made a strong case for the scavenger receptor SREC-I as a key molecule in the effects of HSPs on immune cells. As many of the HSPs are in large families, it has not been clear whether all members of Hsp90 or Hsp70 can function outside the cell. Dr Wei Li (University of Southern California) showed that HSP90 family member Hsp90α is the major secreted factor while Dr John Williams (University of Chester) showed potent extracellular effects for human HSP70 isoform HSPA1A. Extracellular roles are not restricted to Hsp90, and Edward O’Brien (Libin Cardiovascular Institute of Alberta/University of Calgary) discussed the extracellular role of heat shock protein 27 (HSPB1) in inflammatory vascular disease. Another lively issue is whether HSPs are released as free proteins, packaged in exosomes, or whether both forms co-exist. This issue was discussed by Monika Fleshner (University of Colorado) and Antonio De Maio (University of California San Diego). Dr De Maio brought up the interesting scenario of Hsp70 binding directly to lipid membranes and perhaps forming membrane channels (Ryan White, University of Maryland).

HSPs are evidently not the only types of stress proteins that can function in the extracellular milieu, as indicated by Dr Michael A. Lynes (University of Connecticut). In a presentation entitled Therapeutic manipulation of the stress response during inflammatory disease, Dr Lynes showed a significant role for extracellular metallothionen in inflammatory bowel disease. Along those lines, Dr George Perdrizet (University of California San Diego) discussed the use of hyperbaric oxygen for enhanced wound healing in diabetic neuropathy, showing impressive clinical findings.

see more at — doi:  10.1007/s12192-014-0562-z

Lens Intermediate Filaments

Paul FitzGerald

Exp Eye Res. 2009 Feb; 88(2): 165–172.      doi:  10.1016/j.exer.2008.11.007

 The ocular lens assembles two separate Intermediate Filament systems sequentially with differentiation. Canonical 8–11 nm IFs composed of Vimentin are assembled in lens epithelial cells and younger fiber cells, while the fiber cell-specific Beaded Filaments are switched on as fiber cell elongation initiates. Some of the key features of both filament systems are reviewed. Actin filaments and microtubules are essential to the most elemental functions of eukaryotic cells. These filamentous structures are assembled from proteins derived from small, highly conserved gene families. Though tissue specificity exists in the expression of some actin and tubulin family members, they are generally expressed in a ubiquitous manner, and are required for eukaryotic cell survival and replication. In contrast, the family of proteins that comprise the cytoplasmic Intermediate Filaments (IFs) is one of the largest in the human genome with greater than 60 members. IFs are generally not required for cell survival, and are absent from single cell eukaryotes, suggesting a more recent appearance on the evolutionary stage, and a less-essential role in the biology of the cell.

The IF family also differs sharply from actins and tubulins in that there is great variation in both size and sequence among the IF proteins, with sequence identity falling below 30% between more distant members of the human IF family. However, despite the large number of IF proteins available for the construction of IFs, any given cell typically expresses only 1–3 IF proteins, with expression tightly restricted to cell type and state of differentiation. This suggests a considerable degree of cell-specific specialization.

While IF proteins show considerable sequence and size variation, they are unified into a family on the basis of three major features:

1. Predicted domain structure (see figure 1): Algorithms that predict coiled-coil structure show a common predicted domain structure consisting of a) head and tail domains which are quite variable in both size and sequence, and b) a central rod domain where the size (~310 amino acids) and predicted secondary structure is strongly conserved. The rod domain consists of large regions of alpha helical structure (coil domains) interrupted by short non-helical regions (“linkers”) that connect the coil domains. The size, number, and placement of linkers and coils are well-conserved. Moreover, the coil domains exhibit a heptad repeat pattern where the 1, 4 positions in the heptad are dominated by hydrophobic amino acids. Because the 1,4 positions are aligned on one  side of the helix, they form a largely hydrophobic “stripe” that runs along one side of the alpha helix. This stripe mediates the dimerization of two matched coil domains. The hydrophobic stripe gently twists around the axis of the helix, giving rise to a supercoiling of two alpha helices, hence the “coiled-coil”.

The requisite hydrophobicity at the 1, 4 positions of the heptad can be conferred by any of several amino acids, thus the central rod domain of IF proteins, while showing conserved secondary structure, also exhibits a generally high degree of sequence variation. The exceptions to this are two short motifs found at either end of the central rod domain, commonly called the Helix Initiation Motif (HIM) and the Helix termination Motif (HTM). At these two sites the primary sequence among IF proteins is well conserved. Not surprisingly, the HIM and HTM motifs are intolerant of mutations, with the majority of known IF diseases arising from point mutations in these sites (http://www.interfil.org).

2. Conserved gene structure: Sequence analysis of IF proteins has allowed clustering of IF proteins into several major classes. Sequence conservation in the rod domain is high within a class (typically greater than 70%) but low between classes. Analysis of the IF genes shows that there is conservation of gene structure as well within the IF family, with the number and placement of introns and exons well conserved, especially in the central rod domain. The degree of gene structure conservation correlates well with the degree of primary sequence conservation, and reinforces the grouping of IF proteins into classes on the basis of primary sequence.

The Type I and II IF classes are called cytokeratins, and these comprise the IFs of epithelia. These begin assembly as an obligate heterodimer of one Type I and one Type II cytokeratin. The Type III IF proteins include vimentin, desmin, GFAP, and peripherin, and these are commonly found in tissues of mesenchymal origin. While Type III IF proteins can heterodimerize, they are more commonly found as homomeric filaments. The Type IV IF proteins are the neurofilament proteins Heavy (NFH), Medium (NFM) and Light (NFL), which assemble collectively into the IFs of neurons.

3. IF proteins form 8–11 nm diameter IFs. Ultimately, despite the differences in head/ tail size and sequence, and variation in the rod domain sequence, all cytoplasmic IF proteins typically assemble into 8–11 nm IFs.

The mechanism by which vimentin is removed as the cell transitions to the organelle-free state is unknown. In cells undergoing mitosis, vimentin IFs are routinely dismantled by phosphorlylation (Inagaki, Nishi et al. 1987), a modification that causes the relatively stable IF polymer break up into smaller subunits, thought to be tetramers. These are subsequently reassembled after cell division is complete. However, vimentin in lens fiber cells appears to removed, and not simply dismantled. IFs are known to be among the first targets of calcium activated proteases (calpains) in cells that are damaged, and many investigators have demonstrated the calcium-activated degradation of both vimentin and BFs in lens(Yoshida, Murachi et al. 1984; Truscott, Marcantonio et al. 1990; Marcantonio and Duncan 1991; Bettelheim, Qin et al. 1995; Andersson, Sjostrand et al. 1996; Sanderson, Marcantonio et al. 1996; Sanderson, Marcantonio et al. 2000). The dismantling of organelles implies a potential release of calcium from organelles in which it is otherwise routinely sequestered. Whether this release occurs, and whether it alters cytoplasmic calcium levels to a degree that would activate those calpains present in the fiber cell is not known.

Caspases activated in the apoptotic cascade can target conserved sites in IF proteins(Caulin, Salvesen et al. 1997). The elimination of organelles from the fiber cell represents an incomplete apoptotic event, and thus those enzymes responsible for organelle elimination may also represent a viable mechanism for explanation of vimentin’s suggested disappearance(Oshima 2002; Omary, Coulombe et al. 2004).

The loss or reduction of vimentin levels does not leave the mature lens fiber cell devoid of an IF system, however. In a manner that emulates IF switching seen in stratified epithelia, a second generation IF system is switched on in the lens as vimentin is being switched off. It is here where the story of the lens IF system takes the most unusual turn yet described in the IF field.

The initial recognition that the mature lens fiber cells departed from the IF dogma was made when Maisel and co-workers noted the presence of “Beaded Chain Filaments” or Beaded Filaments (BFs) in an electron microscopic analysis of chick lens homogenates (Maisel and Perry 1972; Maisel 1977; Bradley and Maisel 1978; Bradley, Ireland et al. 1979). These studies noted a clearly filamentous structure that was distinct from thin filaments, microtubules, and IFs, which at that time were emerging as the universal cytoskeletal structures common to essentially all vertebrate cells. Speculation emerged that these structures were thin filaments with bound alpha crystallin particles, or nucleosomes on DNA, but these explanations were ruled out experimentally (Bloemendal, Berbers et al. 1984; Ireland and Maisel 1984).

Consistent with the emerging role of IFAPs in modulating and adapting IF function is the observation that fiber cell vimentin IFs interact with the N Cadherin-gamma catenin complex (Leonard, Chan et al. 2008), lengsin(Wyatt, Gao et al. 2008), MIP(Lindsey Rose, Gourdie et al. 2006), periplakin(Yoon and FitzGerald 2008), tropomodulin(Fischer, Quinlan et al. 2003) and possibly other complexes which are present in lens(Bagchi, Katar et al. 2002; Straub, Boda et al. 2003; Bagchi, Katar et al. 2004). The number of candidate linker proteins demonstrated in lens leads to the expectation that the BF and IF are likely to accomplish multiple functions, and that these may be modulated as differentiation progresses, and as the fiber cell proteome changes, either by expression or proteolysis. Similarly, the small heat shock proteins, whose chaperone function appears essential to IF/BF assembly and maintenance, must be considered as critical parts of the biology of IFs in lens. Mutations in the small heat shock proteins have been shown to precipitate a failure in the IF systems in many tissues, and in lens specifically, and to subsequently emulate IF diseases (FitzGerald and Graham 1991; Nicholl and Quinlan 1994; Carter, Hutcheson et al. 1995; Vicart, Caron et al. 1998; Muchowski, Valdez et al. 1999; Perng, Cairns et al. 1999; Perng, Muchowski et al. 1999; Evgrafov, Mersiyanova et al. 2004; Treweek, Rekas et al. 2005; Song, Hanson et al. 2008). The growing multiplicity of IF interactions underscores the need to expect that failure in “IF function” in the lens can result from failure in a wide spectrum of proteins that affect assembly, phosphorylation, proteolytic modification, stability, removal, or linkage to other cellular structures, and that “IF failure” is likely to show considerable variability in phenotype.

Morphological characterization of the AlphaA- and AlphaB-crystallin double knockout mouse lens    Edited by Harry Maisel

Daniel L Boyle, Larry Takemoto, James P Brady and Eric F Wawrousek
BMC Ophthalmology 2003; 3:3   http://dx.doi.org:/10.1186/1471-2415-3-3

Background: One approach to resolving some of the in vivo functions of alpha-crystallin is to generate animal models where one or both of the alpha-crystallin gene products have been eliminated. In the single alpha-crystallin knockout mice, the remaining alpha-crystallin may fully or partially compensate for some of the functions of the missing protein, especially in the lens, where both alphaA and alphaB are normally expressed at high levels. The purpose of this study was to characterize gross lenticular morphology in normal mice and mice with the targeted disruption of alphaA- and alphaB-crystallin genes (alphaA/BKO). Methods: Lenses from 129SvEvTac mice and alphaA/BKO mice were examined by standard scanning electron microscopy and confocal microscopy methodologies. Results: Equatorial and axial (sagittal) dimensions of lenses for alphaA/BKO mice were significantly smaller than age-matched wild type lenses. No posterior sutures or fiber cells extending to the posterior capsule of the lens were found in alphaA/BKO lenses. Ectopical nucleic acid staining was observed in the posterior subcapsular region of 5 wk and anterior subcapsular cortex of 54 wk alphaA/BKO lenses. Gross morphological differences were also observed in the equatorial/bow, posterior and anterior regions of lenses from alphaA/BKO mice as compared to wild mice. Conclusion: These results indicated that both alphaA- and alphaB-crystallin are necessary for proper fiber cell formation, and that the absence of alpha-crystallin can lead to cataract formation.

Dogfish a-Crystallin Sequences COMPARISON WITH SMALL HEAT SHOCK PROTEINS AND SCHISTOSOMA EGG ANTIGEN*

Wilfried W. de JongS, Jack A. M. Leunissen, Pieter J. M. Leenen, Anneke Zweers, and Marlies Versteeg
J BIOL CHEM  1988; 263(11):5141-5149

The amino acid sequences of the a-crystallin A and B chains of the dogfish, Squalus acanthias, have been determined. Comparison with a-crystallins from other species reveals that charged amino acid replacements have been strongly avoided in the evolution of this lens protein. The homology of a-crystallins with the small heat shock proteins is pronounced throughout the major part of the proteins, starting from the position of the first intron in the a-crystallin genes, but is also detectable in the amino-terminal sequences of human, Xenopus, and Drosophila small heat shock proteins. In addition, a remarkable short sequence similarity is present only in the amino termini of dogfish aB and Drosophila HSP22. The Schistosoma egg antigen p40 turns out to have a tandomly repeated region of homology with the common sequence domain of a-crystallins and small heat shock proteins. Comparison of hydropathy profiles indicates the conservation of conformation of the common domains in these three families of proteins. Construction of phylogenetic trees suggests that the aA and aB genes apparently originated from a single ancestral small heat shock protein gene and indicates that introns have been lost during the evolution of the heat shock protein.

Acknowledgment – Maisel, H. (ed) (1985) The Ocular Lens, Marcel Dekker, New. York

 

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Novel Mechanisms of Resistance to Novel Agents

Posted in Cancer - General, Cancer and Current Therapeutics, CANCER BIOLOGY & Innovations in Cancer Therapy, Cell Biology, Cell Biology, Signaling & Cell Circuits, Curation, Gene Regulation, Personalized and Precision Medicine & Genomic Research, Pharmacogenomics, tagged Aviva Lev-Ari, Biology, BRAF, Cancer - General, chemoresistance, Chemotherapy, CML, Conditions and Diseases, DNA, Imatinib, Larry H. Bernstein, Personalized medicine, research on January 12, 2016| Leave a Comment »

Novel Mechanisms of Resistance to Novel Agents

 

Curators: Larry H. Berstein, M.D. FACP & Stephen J. Williams, Ph.D.

For most of the history of chemotherapy drug development, predicting the possible mechanisms of drug resistance that ensued could be surmised from the drug’s pharmacologic mechanism of action. In other words, a tumor would develop resistance merely by altering the pathways/systems which the drug relied on for mechanism of action. For example, as elucidated in later chapters in this book, most cytotoxic chemotherapies like cisplatin and cyclophosphamide were developed to bind DNA and disrupt the cycling cell, thereby resulting in cell cycle arrest and eventually cell death or resulting in such a degree of genotoxicity which would result in great amount of DNA fragmentation. These DNA-damaging agents efficacy was shown to be reliant on their ability to form DNA adducts and lesions. Therefore increasing DNA repair could result in a tumor cell becoming resistant to these drugs. In addition, if drug concentration was merely decreased in these cells, by an enhanced drug efflux as seen with the ABC transporters, then there would be less drug available for these DNA adducts to be generated. A plethora of literature has been generated on this particular topic.

However in the era of chemotherapies developed against targets only expressed in tumor cells (such as Gleevec against the Bcr-Abl fusion protein in chronic myeloid leukemia), this paradigm had changed as clinical cases of resistance had rapidly developed soon after the advent of these compounds and new paradigms of resistance mechanisms were discovered.

Imatinib resistance can be seen quickly after initiation of therapy

Speed of imatinib resistance a result of rapid gene amplification of BCR/ABL target, thereby decreasing imatinib efficacy

 

 

 

 

 

 

 

 

 

 

Although there are many other new mechanisms of resistance to personalized medicine agents (which are discussed later in the chapter) this post is a curation of cellular changes which are not commonly discussed in reviews of chemoresistance and separated in three main categories:

Cellular Diversity and Adaptation

Identifying Cancers and Resistance

Cancer Drug-Resistance Mechanism

p53 tumor drug resistance gene target

Variability of Gene Expression and Drug Resistance

 

Expression of microRNAs and alterations in RNA resulting in chemo-resistance

Drug-resistance Mechanism in Tumor Cells

Overexpression of miR-200c induces chemoresistance in esophageal cancers mediated through activation of the Akt signaling pathway

 

The miRNA–drug resistance connection: a new era of personalized medicine using noncoding RNA begins

 

Gene Duplication of Therapeutic Target

 

The advent of Gleevec (imatinib) had issued in a new era of chemotherapy, a personalized medicine approach by determining the and a lifesaver to chronic myeloid leukemia (CML) patients whose tumors displayed expression of the Bcr-Abl fusion gene. However it was not long before clinical resistance was seen to this therapy and, it was shown amplification of the drug target can lead to tumor outgrowth despite adequate drug exposure. le Coutre, Weisberg and Mahon^{23, 24, 25} all independently generated imatinib-resistant clones through serial passage of the cells in imatinib-containing media and demonstrated elevated Abl kinase activity due to a genetic amplification of the Bcr–Abl sequence. However, all of these samples were derived in vitro and may not represent a true mode of clinical resistance. Nevertheless, Gorre et al.²⁶ obtained specimens, directly patients demonstrating imatinib resistance, and using fluorescence in situ hybridization analysis, genetic duplication of the Bcr–Abl gene was identified as one possible source of the resistance. Additional sporadic examples of amplification of the Bcr–Abl sequence have been clinically described, but the majority of patients presenting with either primary or secondary imatinib resistance fail to clinically demonstrate Abl amplification as a primary mode of treatment failure.

This is seen in the following papers:

Clinical resistance to STI-571 cancer therapy caused by BCR-ABL gene mutation or amplification.Gorre ME, Mohammed M, Ellwood K, Hsu N, Paquette R, Rao PN, Sawyers CL. Science. 2001 Aug 3;293(5531):876-80. Epub 2001 Jun 21.

and in another original paper by le Coutre et. al.

Induction of resistance to the Abelson inhibitor STI571 in human leukemic cells through gene amplification. le Coutre P¹, Tassi E, Varella-Garcia M, Barni R, Mologni L, Cabrita G, Marchesi E, Supino R, Gambacorti-Passerini C. Blood. 2000 Mar 1;95(5):1758-66

The 2-phenylaminopyrimidine derivative STI571 has been shown to selectively inhibit the tyrosine kinase domain of the oncogenic bcr/abl fusion protein. The activity of this inhibitor has been demonstrated so far both in vitro with bcr/abl expressing cells derived from leukemic patients, and in vivo on nude mice inoculated with bcr/abl positive cells. Yet, no information is available on whether leukemic cells can develop resistance to bcr/abl inhibition. The human bcr/abl expressing cell line LAMA84 was cultured with increasing concentrations of STI571. After approximately 6 months of culture, a new cell line was obtained and named LAMA84R. This newly selected cell line showed an IC50 for the STI571 (1.0 microM) 10-fold higher than the IC50 (0.1 microM) of the parental sensitive cell line. Treatment with STI571 was shown to increase both the early and late apoptotic fraction in LAMA84 but not in LAMA84R. The induction of apoptosis in LAMA84 was associated with the activation of caspase 3-like activity, which did not develop in the resistant LAMA84R cell line. LAMA84R cells showed increased levels of bcr/abl protein and mRNA when compared to LAMA84 cells. FISH analysis with BCR- and ABL-specific probes in LAMA84R cells revealed the presence of a marker chromosome containing approximately 13 to 14 copies of the BCR/ABL gene. Thus, overexpression of the Bcr/Abl protein mediated through gene amplification is associated with and probably determines resistance of human leukemic cells to STI571 in vitro. (Blood. 2000;95:1758-1766)

This is actually the opposite case with other personalized therapies like the EGFR inhibitor gefinitib where actually the AMPLIFICATION of the therapeutic target EGFR is correlated with better response to drug in

Molecular mechanisms of epidermal growth factor receptor (EGFR) activation and response to gefitinib and other EGFR-targeting drugs.Ono M, Kuwano M. Clin Cancer Res. 2006 Dec 15;12(24):7242-51. Review.

Abstract

The epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases, including EGFR, HER2/erbB2, and HER3/erbB3, is an attractive target for antitumor strategies. Aberrant EGFR signaling is correlated with progression of various malignancies, and somatic tyrosine kinase domain mutations in the EGFR gene have been discovered in patients with non-small cell lung cancer responding to EGFR-targeting small molecular agents, such as gefitinib and erlotinib. EGFR overexpression is thought to be the principal mechanism of activation in various malignant tumors. Moreover, an increased EGFR copy number is associated with improved survival in non-small cell lung cancer patients, suggesting that increased expression of mutant and/or wild-type EGFR molecules could be molecular determinants of responses to gefitinib. However, as EGFR mutations and/or gene gains are not observed in all patients who respond partially to treatment, alternative mechanisms might confer sensitivity to EGFR-targeting agents. Preclinical studies showed that sensitivity to EGFR tyrosine kinase inhibitors depends on how closely cell survival and growth signalings are coupled with EGFR, and also with HER2 and HER3, in each cancer. This review also describes a possible association between EGFR phosphorylation and drug sensitivity in cancer cells, as well as discussing the antiangiogenic effect of gefitinib in association with EGFR activation and phosphatidylinositol 3-kinase/Akt activation in vascular endothelial cells.

 

Mutant Variants of Therapeutic Target

 

resistant subclones in tissue samples and Tyrosine Kinase tumor activity

 

Mitochondrial Isocitrate Dehydrogenase and Variants

Mutational Landscape of Rare Childhood Brain Cancer: Analysis of 60 Intercranial Germ Cell Tumor Cases using NGS, SNP and Expression Array Analysis – Signaling Pathways KIT/RAS are affected by mutations in IGCTs

 

AND seen with the ALK inhibitors as well (as seen in the following papers

Acquisition of cancer stem cell-like properties in non-small cell lung cancer with acquired resistance to afatinib.

Hashida S, Yamamoto H, Shien K, Miyoshi Y, Ohtsuka T, Suzawa K, Watanabe M, Maki Y, Soh J, Asano H, Tsukuda K, Miyoshi S, Toyooka S. Cancer Sci. 2015 Oct;106(10):1377-84. doi: 10.1111/cas.12749. Epub 2015 Sep 30.

In vivo imaging models of bone and brain metastases and pleural carcinomatosis with a novel human EML4-ALK lung cancer cell line.

Nanjo S, Nakagawa T, Takeuchi S, Kita K, Fukuda K, Nakada M, Uehara H, Nishihara H, Hara E, Uramoto H, Tanaka F, Yano S. Cancer Sci. 2015 Mar;106(3):244-52. doi: 10.1111/cas.12600. Epub 2015 Feb 17.

Identification of a novel HIP1-ALK fusion variant in Non-Small-Cell Lung Cancer (NSCLC) and discovery of ALK I1171 (I1171N/S) mutations in two ALK-rearranged NSCLC patients with resistance to Alectinib. Ou SH, Klempner SJ, Greenbowe JR, Azada M, Schrock AB, Ali SM, Ross JS, Stephens PJ, Miller VA.J Thorac Oncol. 2014 Dec;9(12):1821-5

Reports of chemoresistance due to variants have also been seen with the BRAF inhibitors like vemurafenib and dabrafenib:

The RAC1 P29S hotspot mutation in melanoma confers resistance to pharmacological inhibition of RAF.

Watson IR, Li L, Cabeceiras PK, Mahdavi M, Gutschner T, Genovese G, Wang G, Fang Z, Tepper JM, Stemke-Hale K, Tsai KY, Davies MA, Mills GB, Chin L.Cancer Res. 2014 Sep 1;74(17):4845-52. doi: 10.1158/0008-5472.CAN-14-1232-T. Epub 2014 Jul 23

 

 

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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.

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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).

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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

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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

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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.

Go to:

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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Science Teaching in Math and Technology

Posted in Math, Uncategorized, tagged Biochemistry and Molecular Biology, Biology, Cell Biology, Science education, STEM on September 25, 2015| Leave a Comment »

Science Teaching in Math and Technology

Larry H. Bernstein, MD, FCAP, Curator

LPBI

2015 Best High Schools for STEM Rankings Methodology

U.S. News looked at 500 public high schools to identify the best in math and science education.

By Robert Morse May 11, 2015

U.S. News & World Report’s Best High Schools for STEM rankings methodology is based on the key principle that students at the Best High Schools for STEM must participate in and pass a robust curriculum of college-level math and science courses. STEM stands for science, technology, engineering and math.

To be included in the U.S. News Best High Schools for STEM rankings, a public high school first had to be listed as a gold medal winner in the 2015 U.S. News Best High Schools rankings. That meant that the top 500 nationally ranked high schools were eligible for the STEM rankings.

Those eligible schools were next judged nationally on their level of math and science participation and success, using Advanced Placement STEM test data for 2013 graduates as the benchmark to conduct the analysis. The U.S. News Best High Schools for STEM rankings methodology does not rely on any data from the U.S. Department of Education.

AP is a College Board program that offers college-level courses at high schools across the country. College Board defines STEM Math as AP courses in Calculus AB, Calculus BC, Computer Science A and Statistics; and STEM Science as AP courses in Biology, Chemistry, Environmental Science, Physics B, Physics C: Electricity and Magnetism and Physics C: Mechanics.

Math and science success at the high school level was assessed by computing a STEM Achievement Index for each school that ranked in the top 500 of the 2015 Best High Schools. The index was based on the percentage of all the AP test-takers in a school’s 2013 graduating class who took and passed college-level AP STEM Math and AP STEM Science tests. The higher a high school scored on the STEM Achievement Index, the better it placed in the Best High Schools for STEM rankings.

The maximum STEM Achievement Index value is 100. No public high school evaluated achieved that top score. The highest index was 98.3.

The first step in the rankings process was to compute the STEM Math Achievement Index. It was derived from two variables. The first was the percentage of AP test-takers in the 2013 graduating class who took at least one AP STEM Math course during high school, which was weighted 25 percent. The second was the percentage of those AP STEM Math test-takers who passed at least one AP STEM Math test during high school, receiving an AP score of 3 or higher. This variable was weighted 75 percent.

The next step was to calculate a STEM Science Achievement Index. Much like the math index, it was derived from the percentage of AP test-takers in the 2013 graduating class who took at least one AP STEM Science course during high school – weighted 25 percent – and the percentage of those AP STEM Science test-takers who passed at least one AP STEM Science test during high school, receiving an AP score of 3 or higher – weighted 75 percent.

This means that the methodology weights students taking AP math and science STEM courses at the high school level at 25 percent and passing those same AP STEM courses at 75 percent. In other words, passing both AP math and science tests was three times as important in the rankings as simply taking AP math and science courses.

The final step in the rankings process was to calculate the overall STEM Achievement Index, a combination of the STEM Math Achievement Index and the STEM Science Achievement Index. Each index was weighted at 50 percent, and then added together to create a composite value that is the STEM Achievement Index score.

The STEM rankings were based on sorting the unrounded – to many decimal places – STEM Achievement Index in descending order, with the top-ranked schools having the highest index values. The STEM Achievement Index was then rounded to the nearest 10th for online publication.

The top 250 high schools that achieved a value of greater than or equal to 66.8 in their STEM Achievement Index scored high enough to be numerically ranked. That high index cutoff point was used since it meant that all the high schools in the STEM rankings had, on average, more than two-thirds of the AP test-takers in their 2013 graduating class take and pass at least one AP STEM Math and one AP STEM Science test.

AP® and Advanced Placement® are registered trademarks of the College Board. Used with permission.

Top 50 Science Teacher Blogs

Bringing the subject of science to life for students is the challenge shared by the teachers who author these 50 amazing and insightful science education blogs. Sharing narratives set within and beyond the classroom walls, these next generation educators embrace technology but are never so dazzled by it that they lose sight of their common goal.

Action-Reaction
Physics Teacher Frank Noschese discusses science education topics like whether Khan Academy is effective at teaching physics, applying Angry Birds in physics lessons, and the idea of pseudoteaching.

Teaching High School Psychology
Teaching High School Psychology is a joint collaboration that explores the deeper lesson of the Stanford Prison Experiment, Gamification and its implications as a behavioral motivator, and opportunities for teaching Operant Conditioning with TV’s Big Bang Theory.

Little Miss Hypothesis
Inspiring Kindergarten scientists and giving a too-often neglected subject its due is the aim of Little Miss Hypothesis, where Mrs. Coe chronicles activities with growing brains, harvesting Spirit Garden salads and the development of science centers in a classroom that is home to Bluebonnet the Betta fish and the crab shack’s resident hermits.

Science for Kids
Sue Cahalane shares her passion for teaching science to elementary students in grades PK-4 on Science for Kids with ideas for classroom experiments, tutorials for science lessons, updates on science education news, and photos of students engaged in science activities.

Science Education on the Edge
Chris Ludwig, a high school science teacher from Colorado, writes about improving assessment and instruction in science and education technology.

Teach Science for All
Kirk Robbins shares helpful resources and tools for science teachers including reports, useful websites, and online tools.

Teaching | chemistry
Ellena Bethea, a high school chemistry teacher, writes about grading practices, online tools, and lab activities.

Adventures with the Lower Level
Tracie Schroeder shares her experiences teaching science, teaching methods, and thoughts on learning.

Physics in Flux
Dan Fullerton provides a resource for teachers with details of his successes and failures, technology guides, and physics book reviews.

Think Thank Thunk
On his blog, Think Thank Thunk, Shawn Cornally celebrates the Merlin within every teacher, the need for repackaging education, the debate surrounding Standards Based Grading and the dread of being dull as he chronicles his plight as an educator.

nashworld
Marine biology teacher Sean Nash gets inspired by WordFoto and invites educators to appreciate and aim for “Whoa” moments on his blog, nashworld.

Science Teacher
A science teacher and former pediatrican finds an exemplary model in Dr. Seuss, challenges technophiles to understand deeply, and explains why he has made a tradition of culminating each school year with a field trip to watch horseshoe crabs in the throes of romance.

Teach Science
At Teach Science, Ed Hitchcock muses on the DNA shared by Socrates and explains why science’s greatest appeal is the unexpected.

Quantum Progress
At Quantum Progress, 9th grade Atlanta physics teacher John Burk relives a childhood tradition at Physics Teacher Camp, promotes blogging as a tool for professional development, and ponders why physics buildings never win campus beauty contests.

Pedagogue Padawan
At Pedagogue Padawan, Geoff Schmit shares innovative ideas for using Sudoku to teach Circuit Analysis, Angry Birds as a lesson in holography, and wikispaces as a tool for science projects.

Re:thinking
Re:thinking blends personal reflection with a challenge to rethink school culture and policy as 9th grade teacher Ben Wildeboer finds teachable moments in events like the Japanese quake and explains the importance of “hard fun” for students.

Journey in Technology
At Journey in Technology a Dallas Physics teacher discusses implementing Khan Academy, discovering community and deep connections at Educon, and transforming the pseudoteaching of “cookbook” lab projects into real learning in the classroom.

Always Formative
Jason Buell is a middle school science teacher from California who writes about standards-based grading, education conferences, education books and more.

Stretching Forward
At Stretching Forward, Earth science teacher Janelle Wilson shares experiences from the Space Academy for Educators, discusses class blogging, and shares thoughts on engaging students and parents in science.

Tearing Down Walls
Derrick Willard teaches AP Environmental Science and discusses using social media and online tools to extend lessons outside the walls of the physical classroom.

Teaching Computer Science
Alfred Thompson is a former high school computer science teacher who currently works at Microsoft and writes about computer science education and resources.

A+ Computer Science Blog
High school computer science teacher Stacey Armstrong discusses why computer science is cool, game programming, career options in computer science, and computer science resources.

Teaching CS in Dallas
Kathleen Weaver writes about teaching robotics, Android development, and computer science education topics on her blog.

In Need of a Base Case
This blog discusses the need for change in computer science education, computer science project ideas, and the value of learning computer science.

Hélène Martin
Hélène Martin muses on the power of childhood playthings to fuel tech career ambitions and describes how lost airport luggage is a reminder to look for ways to leverage computing to solve real-life problems in this blog from the perspective of a computer science teacher.

Garth’s CS Education Blog
A computer science and programming teacher at a private school writes about teaching fun and important concepts and preparing students for computer science careers.

The Blog of Phyz
The Blog of Phyz is California teacher Dean Baird’s platform for debunking “Magnet Boys” and magic wristbands, and touting a 75 cent investment guaranteed to wow even the most cynical student.

Mr. Gonzalez’s Classroom
An Olympic Odyssey customarily culminates the academic year for middle school teacher Alfonso Gonzalez, who explores the challenge of giving terms like “on-task” and “structured learning” 21st century relevance on his blog, Mr. Gonzalez’s Classroom.

Free/Libre Open Source Science Education
Pseudoteaching and trends like the “reverse lecture” are hot topics on Free/Libre Open Source Science Education, where Steve Dickie shares his own innovative methods, including cartooning with GoAnimate and creating his own textbooks.

The Science Classroom
Oklahoma physics teacher Jody Bowie reports on the thrill of seeing students connect classroom lessons in everyday life, explains why everyone needs a whetstone to hone their thinking and divulges his identification with the Wizard in Wicked on The Science Classroom blog.

Jacobs Physics
On his blog Greg Jacobs calls course evaluations brutal but vital and bucks a few trends by advocating daily work and disparaging summer assignments in favor of starting each year “from the ground up”.

New Physics Modeler
Bryan Battaglia explains the appeal of professional conferences, the career changing power of blogging, and reflects that teachers gain as many lessons by year’s end as their students.

Just Call Me Ms Frizzle
Becky offers a distinctive first-year teacher perspective on Just Call Me Ms Frizzle, contrasting the low of leaving the room in frustration with the high of a Friday classroom on its best behavior, along with the challenge of teaching a non-traditional class.

And Yet it Moves
On his blog, And Yet it Moves, Ben Chun explains why problem-solving skills trump smarts, tackles the debate over doing away with honors classes, and challenges the AP curriculum.

Reflections of a Science Teacher
Sandra McCarron dismisses the notion of a rubric for thinking, believes that a successful classroom starts out with a vision and ponders the merits of science fairs that have been sacrificed to make way for education reforms on her blog, Reflections of a Science Teacher.

Hurricane Maine
A veteran teacher discusses ideas in education and technology, interesting articles, and how to make school more like play rather than work.

The Physics of Learning
Doug Smith authors this physics education blog that discusses topics like whether to use iPads in the classroom, the myths of merit pay, and scientific literacy.

Room 611
Mr. Young teaches Earth science and other subjects in Canada and provides insights into class by outlining what is covered in class almost every school day.

Using Blogs in Science Education
Stacey Baker is a high school biology teacher and writes about how to use classroom blogs to help students learn science.

Physics! Blog!
Physics! Blog! shares results of The No Homework Experiment and discusses standards based grading, the goal of testing, and teaching students how to learn from mistakes.

Ideas for Teaching Computer Technology to Kids
A blog sharing ideas and resources for teaching computer technology including robots for computer science education, programming resources, and computer science teaching tools.

MrReid.org
A physics teacher shares interesting science articles like Nobel prize winning sentences, things from movies that cannot exist, and cool science videos.

Teach. Brian. Teach.
Brian discusses what makes for a good science conversation, reflects on teaching, shares observations of students, and explains why it is important to point out when students are having fun doing science.

The Skeptical Teacher
A high school physics teacher discusses science education and promotes critical thinking on his blog.

Physics & Physical Science Demos, Labs, & Projects for High School Teachers
A physics teacher provides a resource for science teachers to share ideas for labs and demonstrations and commentary on what works.

The Art of Teaching Science
Jack Hassard is a professor of science education and explores issues in teaching science, shares resources for science teachers, and discusses why teaching science is important.

SuperFly Physics
At SuperFly Physics, Andy Rundquist shares ideas for teaching physics, fun science experiments, and interesting physics problems.

Newton’s Minions
A physics blog sharing student work, anecdotes from the classroom, thoughts on student assessment, and ideas for teaching complex physics lessons.

Mr. Barlow’s Blog
Mr. Barlow is a high school science teacher and podcaster from Melbourne, Australia who shares interesting science studies, cool science news, and optical illusions at his blog.

http://www.teachercertificationdegrees.com/top-blogs/science-teacher/

What is JASON?

We are a non-profit organization that connects students, in the classroom and out, to real science and exploration to inspire and motivate them to study and pursue careers in Science, Technology, Engineering and Math (STEM).

We embed exciting STEM professionals and cutting-edge research into award-winning, standards-aligned in and out-of-school curricula. Live webcasts connect students with inspirational STEM role models. Student materials include reading selections with read-to-me functionality, inquiry-based labs, videos, and online games. For teachers and informal educators, we provide lesson plans, assessments, and comprehensive professional development programs.

http://www.jason.org/sites/default/files/images/rotators/website%20klein%20feature.jpg

Ten Websites for Science Teachers

Originally Published: February 7, 2012 | Updated: October 10, 2014

www.nsta.org/about/awards.aspx

http://www.edutopia.org/sites/default/files/styles/feature_image_breakpoints_theme_edutopia_desktop_1x/public/slates/Science_Teachers_0.jpg?itok=i6yFGrRK

We all know that the web is full of excellent web resources for science teachers and students. However, unless you live on the web, finding the best websites can become quite a challenge. This isn’t a “Top Ten” list — instead, it is a list of websites that I either use on a regular basis or just find interesting. From teaching resources for the nature of science and authentic field journals to wacky videos about numbers, I am sure that you will find something in the following list the works for you!

1) Understanding Science

UC Berkeley’s Understanding Science website is a “must use” for all science teachers. It is a great resource for learning more about the process of science. The resource goes much deeper than the standard “PHEOC” model of the scientific method by emphasizing peer review, the testing of ideas, a science flowchart and “what is science?” checklist. Understanding Science also provides a variety of teaching resources including case studies of scientific discoveries and lesson plans for every grade level.

2) Field Research Journals

The Field Book Project from the National Museum of Natural History and the Smithsonian Institution Archives intends to create a “one stop” archive for field research journals and other documentation. You can find plenty of examples from actual field research journals for your classes.

3) Evolution

Berkeley’s Understanding Evolution website is the precursor to their Understanding Science efforts. The Understanding Evolution website provides a plethora of resources, news items and lessons for teaching about evolution. Lessons provide appropriate “building blocks” to help students at any grade level work towards a deeper understanding of evolution. The Evo 101 tutorial provides a great overview of the science behind evolution and the multiple lines of evidence that support the theory.

4) PhET Simulations

PhET from the University of Colorado provides dozens of fantastic simulations for physics, chemistry and biology. The website also includes a collection of teacher contributed activities, lab experiences, homework assignments and conceptual questions that can be used with the simulations.

5) Earth Exploration

The Earth Exploration Toolbook provides a series of activities, tools and case studies for using data sets with your students.

6) EdHead Interactives

Edheads is an organization that provides engaging web simulations and activities for kids. Current activities focus on simulated surgical procedures, cell phone design (with market research), simple and compound machines, and weather prediction.

7) Plant Mentors

Do you teach about plants? Check out Planting Science to connect your middle or high school students to science mentors and a collaborative inquiry project. From the project:

Planting Science is a learning and research resource, bringing together students, plant scientists, and teachers from across the nation. Students engage in hands-on plant investigations, working with peers and scientist mentors to build collaborations and to improve their understanding of science.

8) Periodic Table of Videos

Check out The Periodic Table of Videos for a wide array of videos about the elements and other chemistry topics.

9) More Videos!

Students can read and watch video about 21 Smithsonian scientistsincluding a volcano watcher, fossil hunter, art scientist, germinator and zoo vet.

10) Even More Videos!

How many videos were watched on YouTube in 2010? If you said 22 billion, you are sort of correct… Those 22 billion views only represent the number of times education videos were watched! In addition to this list of science and math YouTube channels, here are two of my favorites:

• SciShow is all about teaching scientific concepts in an accessible and easy-to-understand manner. This channel includes a variety of short (3 minute) and long (10 minute) videos. New videos are released weekly.
• Former BBC journalist Brady Haran is crazy about math and science. If you love numbers, you will love his Numberphile channel, dedicated to exploring the stories behind numbers.
• And let’s close with a particularly good SciShow on Climate Change:

https://youtu.be/M2Jxs7lR8ZI

Best High Schools

http://www.usnews.com/education/best-high-schools/national-rankings

	School for the Talented and Gifted 1201 EAST EIGHTH ST DALLAS, TX 75203 Dallas Independent School District GOLD Medal		15:1 Near National Avg 253 Students 17 Teachers	100.0 Above National Avg 100% Tested (AP®) 100% Passed (AP®)
#2	BASIS Scottsdale 11440 NORTH 136TH ST SCOTTSDALE, AZ 85259 BASIS Schools Inc. GOLD Medal		N/A N/A 698 Students N/A Teachers	100.0 Above National Avg 100% Tested (AP®) 100% Passed (AP®)
#3	Thomas Jefferson High School for Science and Technology 6560 BRADDOCK RD ALEXANDRIA, VA 22312 Fairfax County Public Schools GOLD Medal		17:1 Near National Avg 1,846 Students 111 Teachers	100.0 Above National Avg 100% Tested (AP®) 100% Passed (AP®)
#4	Gwinnett School of Mathematics, Science and Technology 970 MCELVANEY LN LAWRENCEVILLE, GA 30044 Gwinnett County Public Schools GOLD Medal		18:1 Near National Avg 851 Students 48 Teachers	100.0 Above National Avg 100% Tested (AP®) 100% Passed (AP®)
	School of Science and Engineering Magnet 1201 EAST EIGHTH ST DALLAS, TX 75203 Dallas Independent School District GOLD Medal		16:1 Near National Avg 386 Students 24 Teachers	100.0 Above National Avg 100% Tested (AP®) 100% Passed (AP®)
#6		Carnegie Vanguard High School 1501 TAFT HOUSTON, TX 77019 Houston Independent School District GOLD Medal	17:1 Near National Avg 590 Students 34 Teachers	100.0 Above National Avg 100% Tested (AP®) 100% Passed (AP®)
#7		Academic Magnet High School 5109A WEST ENTERPRISE ST NORTH CHARLESTON, SC 29405 Charleston County School District GOLD Medal	14:1 Near National Avg 610 Students 44 Teachers	100.0 Above National Avg 100% Tested (AP®) 100% Passed (AP®)
#8		University High School 9419 WEST VAN BUREN ST TOLLESON, AZ 85353 Tolleson Union High School District GOLD Medal	34:1 Larger than National Avg 460 Students 14 Teachers	100.0 Above National Avg 100% Tested (AP®) 100% Passed (AP®)
#9		Lamar Academy 1009 NORTH 10TH ST MCALLEN, TX 78501 Mcallen Independent School District GOLD Medal	6:1 Smaller than National Avg 106 Students 19 Teachers	100.0 Above National Avg 100% Tested (IB) 100% Passed (IB)
#10		Gilbert Classical Academy High School 55 NORTH GREENFIELD RD GILBERT, AZ 85234 Gilbert Unified District GOLD Medal	11:1 Smaller than National Avg 220 Students 20 Teachers	100.0 Above National Avg 100% Tested (AP®) 100% Passed (AP®)
#11		The High School of American Studies at Lehman College 2925 GOULDEN AVE BRONX, NY 10468 New York City Public Schools GOLD Medal	16:1 Near National Avg 393 Students 25 Teachers	100.0 Above National Avg 100% Tested (AP®) 100% Passed (AP®)
#12		American Indian Public High School 3637 MAGEE AVE OAKLAND, CA 94619 Oakland Unf GOLD Medal	19:1 Larger than National Avg 243 Students 13 Teachers	100.0 Above National Avg 100% Tested (AP®) 100% Passed (AP®)
#13		International Studies Charter High School 2480 SW 8TH ST MIAMI, FL 33135 Miami-Dade County Public Schools GOLD Medal	13:1 Near National Avg 359 Students 27 Teachers	100.0 Above National Avg 100% Tested (AP®) 100% Passed (AP®)
#14		High School for Dual Language and Asian Studies 350 GRAND ST NEW YORK, NY 10002 New York City Public Schools GOLD Medal	16:1 Near National Avg 392 Students 25 Teachers	100.0 Above National Avg 100% Tested (AP®) 100% Passed (AP®)
#15		Northside College Preparatory High School 5501 NORTH KEDZIE AVE CHICAGO, IL 60625 Chicago Public Schools GOLD Medal	14:1 Near National Avg 1,069 Students 74 Teachers	99.7 Above National Avg 100% Tested (AP®) 100% Passed (AP®)
#16		Oxford Academy 5172 ORANGE AVE CYPRESS, CA 90630 Anaheim Union High GOLD Medal	30:1 Larger than National Avg 1,152 Students 38 Teachers	99.5 Above National Avg 100% Tested (AP®) 99% Passed (AP®)
#17		University High School 421 NORTH ARCADIA BLVD TUCSON, AZ 85711 Tucson Unified School District GOLD Medal	21:1 Larger than National Avg 934 Students 44 Teachers	99.3 Above National Avg 100% Tested (AP®) 99% Passed (AP®)
#18		Pacific Collegiate School 255 SWIFT ST SANTA CRUZ, CA 95060 Santa Cruz County Office Of Education GOLD Medal	19:1 Larger than National Avg 515 Students 28 Teachers	99.1 Above National Avg 100% Tested (AP®) 99% Passed (AP®)
#19		Biotechnology High School 5000 KOZLOSKI RD FREEHOLD, NJ 07728 Monmouth County Vocational School District GOLD Medal	14:1 Near National Avg 311 Students 23 Teachers	99.1 Above National Avg 100% Tested (IB) 99% Passed (IB)
#20		High Technology High School 765 NEWMAN SPRINGS RD LINCROFT, NJ 07738 Monmouth County Vocational School District GOLD Medal	13:1 Near National Avg 280 Students 22 Teachers	98.5 Above National Avg 99% Tested (AP®) 99% Passed (AP®)

The United States has developed as a global leader, in large part, through the genius and hard work of its scientists, engineers, and innovators. In a world that’s becoming increasingly complex, where success is driven not only by what you know, but by what youcan do with what you know, it’s more important than ever for our youth to be equipped with the knowledge and skills to solve tough problems, gather and evaluate evidence, and make sense of information. These are the types of skills that students learn by studying science, technology, engineering, and math—subjects collectively known as STEM.

Yet today, few American students pursue expertise in STEM fields—and we have an inadequate pipeline of teachers skilled in those subjects. That’s why President Obama has set a priority of increasing the number of students and teachers who are proficient in these vital fields.

http://www.ed.gov/sites/default/files/stem-infographic.jpg

The need

All young people should be prepared to think deeply and to think well so that they have the chance to become the innovators, educators, researchers, and leaders who can solve the most pressing challenges facing our nation and our world, both today and tomorrow. But, right now, not enough of our youth have access to quality STEM learning opportunities and too few students see these disciplines as springboards for their careers.expand/collapse

The goals

President Obama has articulated a clear priority for STEM education: within a decade, American students must “move from the middle to the top of the pack in science and math.” The Obama Administration also is working toward the goal of fairness between places, where an equitable distribution of quality STEM learning opportunities and talented teachers can ensure that all students have the chance to study and be inspired by science, technology, engineering, and math—and have the chance to reach their full potential.expand/collapse

The plan

The Committee on STEM Education (CoSTEM), comprised of 13 agencies—including all of the mission-science agencies and the Department of Education—are facilitating a cohesive national strategy, with new and repurposed funds, to increase the impact of federal investments in five areas: 1.) improving STEM instruction in preschool through 12th grade; 2.) increasing and sustaining public and youth engagement with STEM; 3.) improving the STEM experience for undergraduate students; 4.) better serving groups historically underrepresented in STEM fields; and 5.) designing graduate education for tomorrow’s STEM workforce.expand/collapse

Supporting Teachers and Students in STEM

At the Department of Education, we share the President’s commitment to supporting and improving STEM education. Ensuring that all students have access to high-quality learning opportunities in STEM subjects is a priority, demonstrated by the fact that dozens of federal programs have made teaching and learning in science, technology, engineering, and math a critical component of competitiveness for grant funding. Just this year, for the very first time, the Department announced that its Ready-to-Learn Television grant competition would include a priority to promote the development of television and digital media focused on science.

The Department’s Race to the Top-District program supports educators in providing students with more personalized learning—in which the pace of and approach to instruction are uniquely tailored to meet students’ individual needs and interests—often supported by innovative technologies. STEM teachers across the country also are receiving resources, support, training, and development through programs like Investing in Innovation (i3), the Teacher Incentive Fund, the Math and Science Partnershipsprogram, Teachers for a Competitive Tomorrow, and the Teacher Quality Partnerships program.

Because we know that learning happens everywhere—both inside and outside of formal school settings—the Department’s 21st Century Community Learning Centers program is collaborating with NASA, the National Park Service, and the Institute of Museum and Library Services to bring high-quality STEM content and experiences to students from low-income, high-need schools. This initiative has made a commitment to Native-American students, providing about 350 young people at 11 sites across six states with out-of-school STEM courses focused on science and the environment.

And in higher education, the Hispanic-Serving Institutions-STEM program is helping to increase the number of Hispanic students attaining degrees in STEM subjects.

This sampling of programs represents some of the ways in which federal resources are helping to assist educators in implementing effective approaches for improving STEM teaching and learning; facilitating the dissemination and adoption of effective STEM instructional practices nationwide; and promoting STEM education experiences that prioritize hands-on learning to increase student engagement and achievement.

Learn more

• Five-Year Strategic Plan for STEM Education [PDF]
• STEM Programs at ED
• Green Strides Program
• Women in STEM
• 2015 White House Science Fair
  • President Obama’s Remarks
• Educate to Innovate
• Civil Rights Data Collection
  • College- and Career-Ready/ STEM Access Snapshot [PDF]

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seri_scores

http://i.livescience.com/images/i/000/017/835/i02/seri_scores.png?1310070712

A new ranking of how well the United States’ schools are preparing students for science and engineering careers shows that although there’s a small number of high performers, most states are doing a poor job of educating students in these subjects.

According to the ranking of schools teaching kindergarten through 12th grade, Massachusetts leads the pack with a score of 4.82 on a scale of 1 to 5, while Mississippi trails behind as “worst in the United States” with a 1.11 score. Twenty-one states in total, including California, earned what the ranking classified as “below average” or “far below average” scores, and only 10 states earned scores above the national average of 2.82.

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