cyclin-dependent kinase pathways as targets for cancer treatment. · 2019. 2. 14. · 1 arrest in...

Cyclin-Dependent Kinase Pathways As Targets forCancer TreatmentGeoffrey I. Shapiro

A B S T R A C T

Cyclin-dependent kinases (cdks) are critical regulators of cell cycle progression and RNA transcription. A varietyof genetic and epigenetic events cause universal overactivity of the cell cycle cdks in human cancer, and theirinhibition can lead to both cell cycle arrest and apoptosis. However, built-in redundancy may limit the effectsof highly selective cdk inhibition. Cdk4/6 inhibition has been shown to induce potent G1 arrest in vitro andtumor regression in vivo; cdk2/1 inhibition has the most potent effects during the S and G2 phases and inducesE2F transcription factor–dependent cell death. Modulation of cdk2 and cdk1 activities also affects survivalcheckpoint responses after exposure to DNA-damaging and microtubule-stabilizing agents. The transcriptionalcdks phosphorylate the carboxy-terminal domain of RNA polymerase II, facilitating efficient transcriptionalinitiation and elongation. Inhibition of these cdks primarily affects the accumulation of transcripts with shorthalf-lives, including those encoding antiapoptosis family members, cell cycle regulators, as well as p53 andnuclear factor-kappa B–responsive gene targets. These effects may account for apoptosis induced by cdk9inhibitors, especially in malignant hematopoietic cells, and may also potentiate cytotoxicity mediated bydisruption of a variety of pathways in many transformed cell types. Current work is focusing on overcomingpharmacokinetic barriers that hindered development of flavopiridol, a pan-cdk inhibitor, as well as assessingnovel classes of compounds potently targeting groups of cell cycle cdks (cdk4/6 or cdk2/1) with variable effectson the transcriptional cdks 7 and 9. These efforts will establish whether the strategy of cdk inhibition is ableto produce therapeutic benefit in the majority of human tumors.

J Clin Oncol 24:1770-1783. © 2006 by American Society of Clinical Oncology

INTRODUCTION

The cyclin-dependent kinases (cdks) are het-erodimeric complexes composed of a catalytic ki-nase subunit and a regulatory cyclin subunit, andcomprise a family divided into two groups based ontheir roles in cell cycle progression and transcrip-tional regulation.1,2 Members of the first groupcomprise core components of the cell cycle machin-ery, and include cyclin D–dependent kinases 4 and6, as well as cyclin E–cdk2 complexes, which se-quentially phosphorylate the retinoblastoma pro-tein (Rb), to facilitate the G13S transition.3 CyclinA–dependent kinases 2 and 1 and cyclin B–cdk1complexes are required for orderly S-phase progres-sion and the G23M transition, respectively.4 cdksare regulated by positive phosphorylation, directedby cdk-activating kinase (CAK; cyclin H/cdk7/MAT1),5 as well as negative phosphorylationevents,6 and by their association with cyclins andendogenous Cip/Kip or INK4 (inhibitor of cdk4)inhibitors.7 In malignant cells, altered expression ofcdks and their modulators, including overexpres-sion of cyclins and loss of expression of cdk inhibi-tors, results in deregulated cdk activity, providing aselective growth advantage.8,9 In contrast to cdksgoverning the transitions between cell cycle phases,

transcriptional cdks, including cyclin H–cdk7, andcyclin T–cdk9 (pTEFb), promote initiation andelongation of nascent RNA transcripts by phosphor-ylating the carboxy-terminal domain (CTD) ofRNA polymerase II.10-12 Because of their critical rolein cell cycle progression and cellular transcription, aswell as the association of their activities with apopto-tic pathways, the cdks comprise an attractive set oftargets for novel anticancer drug development.

G13S PROGRESSION AND CANCERCELL CYCLES

The G13S Transition

The retinoblastoma susceptibility protein, Rb,plays a central role in the G13S transition (Fig 1).13

In its hypophosphorylated state, Rb prevents pro-gression from G1 to S through its interaction withE2F transcription family members. This interactionnot only blocks transcriptional activation of E2F,but also actively represses transcription by recruit-ing histone deacetylases to the promoters of genesrequired for S-phase entry.14 During cell cycle pro-gression, Rb is inactivated by sequential phosphory-lation mediated by cyclin D–dependent kinases 4and 6 and cyclin E–cdk2 complexes.15,16 In responseto mitogenic stimulation, cells synthesize D-type

From the Department of MedicalOncology, Dana-Farber Cancer Institute;and the Department of Medicine,Brigham and Women’s Hospital andHarvard Medical School, Boston, MA.

Submitted August 6, 2005; acceptedJanuary 25, 2006.

Supported by Grant No. R01 CA90687and the Dana-Farber/Harvard CancerCenter Specialized Program ofResearch Excellence (SPORE) in LungCancer Grant No. P20 CA90578 fromthe National Institutes of Health, and bya Mantle Cell Lymphoma ResearchGrant from the Lymphoma ResearchFoundation.

Terms in blue are defined in the glossary,found at the end of this article and onlineat www.jco.org.

Author’s disclosures of potentialconflicts of interest and author contribu-tions are found at the end of thisarticle.

Address reprint requests to Geoffrey I.Shapiro, MD, PhD, Dana Farber CancerInstitute, Dana 810A, 44 Binney St,Boston, MA 02115; e-mail: [email protected].

© 2006 by American Society of ClinicalOncology

0732-183X/06/2411-1770/$20.00

DOI: 10.1200/JCO.2005.03.7689

JOURNAL OF CLINICAL ONCOLOGY B I O L O G Y O F N E O P L A S I A

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from 165.124.124.16Information downloaded from jco.ascopubs.org and provided by at Galter Health Sciences Library on September 6, 2014

Copyright © 2006 American Society of Clinical Oncology. All rights reserved.

cyclins that assemble with cdks 4 and 6, a process that requirescontribution of a Cip/Kip family member. Although Cip/Kip fam-ily members promote activity of cyclin D– dependent kinases, theyare also potent inhibitors of cdk2.17 Therefore cyclin D– dependentkinases facilitate G1 progression by first phosphorylating Rb, re-lieving transcriptional repression by the Rb-E2F complex, and bysequestering Cip/Kip proteins, facilitating activation of cyclinE–cdk2. Cyclin E–cdk2-mediated Rb phosphorylation disrupts thebinding of Rb to E2F, allowing E2F activation and the transcription ofgenes necessary for S-phase entry and progression, including cyclin Eitself.18,19 Although Rb is the primary target of cyclin D–dependentkinases, cyclin E–cdk2 phosphorylates other targets as well, includingp27Kip1,20,21 which further facilitates S-phase entry, p220 nuclear pro-tein mapped to the Ataxia Telanglectasia locus (NPAT), which stimu-lates replication-dependent histone gene transcription,22,23 andnucleophosmin, which regulates centrosome duplication.24

G1 progression is also regulated by members of the INK4 family,which act as specific inhibitors of cdks 4 and 6. p16INK4A accumulatesas cells age and induces G1 arrest during senescence by associating with

cdks 4 and 6, promoting release of D-type cyclins. The subsequentdestabilization of D-cyclins and the redistribution of Cip/Kip proteinsto cdk2 contributes to G1 arrest.17,25

Altered Expression of Cell Cycle Proteins in

Human Cancer

The D-cyclin–cdk4/6–INK4–Rb pathway is universally dis-rupted in human cancer. Although Rb loss occurs commonly in sometumor types, the majority of human cancers retain wild-type Rb.Instead, most tumors increase activity of cyclin D–dependent kinasesby multiple mechanisms. Most commonly, p16INK4A is inactivated bygene deletion, point mutation, or transcriptional silencing by methyl-ation.26,27 This permits escape from senescence during the evolutionto malignancy, and leaves the cancer cell with increased cyclin D–dependent kinase activity and a growth advantage. p16INK4A has beenproved definitively to be a tumor suppressor, given that mice withtargeted disruption of p16INK4A, and retention of the tumor suppres-sor p19ARF, encoded by an overlapping reading frame, are predisposedto tumorigenesis.28 Alternatively, both amplification of cdk4 and

Fig 1. The G13S transition. In response to mitogenic signals, cyclin D/cyclin-dependent kinase (cdk) 4/6/Cip/Kip complexes assemble, sequestering Cip/Kip proteinsfrom cyclin E–cdk2. Cyclin D– and E–dependent kinases phosphorylate the retinoblastoma protein (Rb), resulting in release of E2F, which is necessary for transcriptionof genes required for S-phase progression, including cyclin E itself, creating a positive feedback loop at the G1-S boundary. Cyclin E–cdk2 also phosphorylates p220NPAT,nucleophosmin, and p27Kip1, the latter ubiquitinated and degraded. As cells age, p16INK4 is induced, which inhibits cdk4/6, causing release and degradation of D cyclinsand redistribution of Cip/Kip proteins to cyclin E–cdk2. Antiproliferative signals induce Cip/Kip proteins that also inhibit cyclin E–cdk2. Recently identified complexesare outlined in blue. Cells exit quiescence and proliferate in the absence of cdk4/6, compensated by cyclin D–cdk2. In the absence of cdk2, cyclin D–dependent kinasesphosphorylate Rb at cdk2 sites; cyclin E–cdk1 complexes may also functionally substitute. NPAT, nuclear protein mapped to the Ataxia Telanglectasia locus.

Cyclin-Dependent Kinase Pathways

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cdk4R24C mutation resulting in the loss of INK4 binding also occur.The cdk4R24C mutation was first described in malignant melanoma,29

and knock-in mice expressing this mutant develop tumors with al-most complete penetrance30 and melanoma with high frequency.31

Loss of Rb, inactivation of p16INK4A, and amplification or mutation ofcdk4 are usually mutually exclusive events32-35; in mouse models,expression of cdk4R24C and loss of INK4 proteins do not cooperate intumor development, which is consistent with cdk4 inhibition as theprimary function of INK4 family members.36

Overexpression of cyclin D1 is also common. Mammary hyper-plasia and carcinoma occur in mouse mammary tumor virus(MMTV) –cyclin D1 transgenic mice,37,38 and cyclin D1 is a criticalmediator of breast cancer induction by Ras or Neu oncogenes, giventhat MMTV-v-Ha-ras and MMTV-c-neu transgenic mice are pro-tected from breast cancer in a cyclin D1 knockout background.39

Cyclin D1 overexpression can occur as a result of gene rearrangement,including the chromosome 11p15;q13 inversion first described in aparathyroid adenoma40 or the t11;14(q13;q32) translocation inmantle-cell lymphoma in which the coding region of cyclin D1 isjuxtaposed to the immunoglobulin heavy chain gene, resulting in highlevels of cyclin D1 in lymphoid cells, where normally only cyclins D2and D3 are expressed.41 Similar translocations targeting D cyclinshave been described in multiple myeloma.42,43 Gene amplificationis another common mechanism leading to aberrant overexpressionof cyclin D1, although overexpression occurs commonly in theabsence of amplification as well.44 Alternative splicing of cyclin D1creates a transcript encoding cyclin D1b, which lacks the carboxy-terminal sequence containing the Thr286 phosphorylated by glyco-gen synthase kinase-3 �.45,46 Because Thr286 phosphorylation isrequired for nuclear export, cyclin D1b is constitutively nuclearand exhibits enhanced transforming ability in vitro47; its presencein the majority of mantle-cell lymphomas suggests the importanceof persistent nuclear expression for oncogenicity rather than sim-ple overexpression.48 Finally, mutations in cyclin D1 at the Thr286 residueor small adjacent deletions that prevent nuclear export, and also stabilizecyclin D1, have been reported in endometrial carcinomas.49

Cyclin D1 overexpression often accompanies loss of p16INK4A,suggesting that these events may cooperate in promoting transforma-tion. Cyclin D1 may have cdk-independent transcriptional functionsof potential tumorigenic relevance.50,51 Nonetheless, mice are pro-tected by MMTV-neu–driven tumorigenesis not only in the absenceof cyclin D1, but also in the absence of cdk452 or the presence ofp16INK4A,53 indicating that in this instance, the cdk-dependent effectsof cyclin D1 are critical. Loss of p16INK4A or overexpression of cyclinD1 increases the amount of cdk4 available for assembly with cyclin Dand Cip/Kip proteins; the sequestration of Cip/Kip proteins in cyclinD–dependent kinase complexes promotes activation of cyclin E–cdk2and hence augments phosphorylation of and inactivation of Rb.Therefore, increased activity of cyclin E–cdk2 is a consequence ofcyclin D–cdk4/6–INK4 pathway alterations. High levels of cyclin E,54

including hyperactive low molecular weight isoforms55 generated byelastase56 or calpain,57 as well as low levels of p27Kip1 resulting fromincreased proteasomal degradation, also contribute to increased cyclinE–cdk2 activity in transformed cells and tend to define tumors thatcarry a worse prognosis.58-62

Targeting the Cyclin D–cdk4/6–INK4 Pathway

The frequency of cyclin D–cdk4/6–INK4 pathway alterationssuggests that acceleration of G1 progression provides a proliferative

and perhaps survival advantage to cancer cells. Preclinical data suggestthat inhibition of cyclin D–dependent kinase activity may have ther-apeutic benefit. For example, antisense-mediated reduction of cyclinD expression results in inhibition of tumor growth, reversal of onco-genicity, and in some cases, transformed cell death.63-66 Similarly,ectopic expression of p16INK4A using inducible promoters or adeno-viral gene delivery systems has been shown to induce Rb-dependentG1 arrest,67,68 as well as apoptosis in vitro.69 In some but not allinstances, cooperation with p53 appears necessary for the apoptoticresponse.70 In vivo, adenovirus vector-mediated expression ofp16INK4A in non–small-cell lung cancer cell lines lacking p16INK4A

potently inhibits their growth when they are injected as xenografts innude mice, and also slows tumor growth when injected into estab-lished xenografts.71 Similar experiments with established mesotheli-oma xenografts resulted in tumor regression.72

These observations have motivated the development of cdk4/6inhibitors that might achieve selectivity for transformed cells. In cellsthat retain Rb, a cdk4/6 inhibitor should reduce Rb phosphorylationand induce G1 arrest; in tumors lacking Rb, in which p16INK4A ispresent at high levels and already associated with cdk4/6, such an agentwould be ineffective. Until recently, compounds with cdk4/6 inhibi-tory activity also potently inhibited other cdks. However, compoundsspecific for cdk4/6 have now been described. Of particular interest isPD 0332991,73 of the pyridopyrimidine class, members of which wereoptimized by testing against cyclin D1–cdk4, cyclin A–cdk2, fibro-blast growth factor receptor, and platelet-derived growth factor recep-tor. Compounds with high selectivity for cdk4 were then further testedfor their ability to induce G1 arrest (without arrest in other cell cyclephases) in an Rb-positive breast carcinoma line. This strategy identi-fied a subset of pyridopyrimidines with exceptional selectivityfor cdk4; PD 0332991 was selected for its superior pharmacokineticproperties and has entered phase I clinical trial.74,75 As expectedfor a specific cdk4/6 inhibitor, this compound induced exclusiveconcentration-dependent G1 arrest in Rb-positive cell lines in vitro,with dephosphorylation of Rb at known cdk4-specific phosphoryla-tion sites including Ser780 and Ser795. The concentrations that inhib-ited cellular proliferation by 50% (IC50) against a panel of Rb-positivecell lines ranged from 40 to 400 nmol/L, whereas IC50 values againsttwo Rb-negative cell lines was more than 3 �mol/L.

In breast and colon carcinoma cells, the drug is cytostatic; after G1

arrest and cessation of proliferation, cells did not die, even after pro-longed exposure. Strikingly, however, in mice bearing Colo-205 coloncarcinoma xenografts, PD 0332991 produced rapid tumor regres-sion.73 It is possible that the tumor represents a balance of proliferatingand apoptotic cells, and inhibition of the proliferative compartmentallows the naturally dying cells to predominate. p16INK4A replacementhas also been associated with downregulation of vascular endothelialgrowth factor,76 and suppression of angiogenesis could have also con-tributed to the in vivo effects. Suppression of Rb phosphorylation atSer780 was also demonstrated in vivo, with a corresponding decrease inexpression of E2F-1–dependent genes and Ki67 staining. As expected,PD 0332991 was inactive against Rb-negative tumor xenografts.

New Insights Into Cell Cycle Biology

Recent experiments with knockout mouse embryonic fibroblasts(MEFs), as well as antisense and siRNA-mediated depletion of cdks intumor cells, have challenged multiple aspects of the model for G1

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progression depicted in Figure 1.77 For example, quiescent, serum-starved cdk4/cdk6-null fibroblasts enter S phase with kinetics similarto those of wild-type MEFs (albeit with lower efficiency), and early-passage double-mutant MEFs displayed a doubling time similar tothat of wild-type MEFs.78 Therefore, cyclin D–cdk4/6 complexes arenot essential for exit from quiescence after exposure to mitogenicstimuli or for the proliferation of embryonic fibroblasts. Compensa-tion for the loss of cdk4/6 occurred with accumulation of cyclinD–cdk2 complexes capable of phosphorylating Rb and inducing cellproliferation. Furthermore, introduction of a retrovirus encodingsmall interfering of RNA (siRNA) targeting cdk2 with approximately70% knockdown did not affect proliferation of wild-type MEFs butefficiently blocked proliferation of cdk4/cdk6-null MEFs, which pro-vides further evidence indicating that cdk2 can compensate for the lossof cdk4 and cdk6.

These results suggest that a similar bypass may occur in tumorcells after continuous exposure to a specific cdk4/6 inhibitor. Al-though continuous dosing beyond 14 days was not performed inpreclinical in vivo experiments with PD 0332991, rechallenge experi-ments were performed in responding xenografts. Colo-205 colonxenografts that had undergone complete regression during 14 days ofdosing were permitted to grow back; tumors that re-emerged werecollected and reimplanted into naı̈ve mice. After tumors grew to 100 to150 mg, mice were treated with PD 0332991. Importantly, tumorsresponded with equal sensitivity and again underwent substantialregression, demonstrating that no resistance had emerged during theinitial brief treatment period.73

The model in Figure 1 places cdk2 in a central role at the G1-Sboundary. However, several lines of evidence now indicate that cdk2 isnot essential for cell proliferation. This evidence was first demon-strated in a variety of cancer cell lines that were able to proliferate afterspecific and acute depletion of cdk2 by siRNA or antisense oligonu-cleotides.79 In addition, cdk2 knockout mice are viable.80,81 Althoughthere was a defect in germ cell meiotic cell division, the survival of micefor up to 2 years indicated that cdk2 is not required for mitotic celldivision or survival of most cell types. Interestingly, cdk2�/� MEFsdid display delayed entry into S phase, consistent with a role of cdk2 inthe timing of S-phase entry80,81 Nonetheless, the effects of cdk2 loss atthe G1-S boundary are less potent than anticipated, with little effect onoverall proliferation of either normal or malignant mammalian cells.Furthermore, Cip/Kip proteins are able to induce G1 arrest incdk2�/� cells, again suggesting that cdk2 is dispensable and easilybypassed.82 In the absence of cdk2, there is compensation from other cdkfamily members. For example, cdk4 can phosphorylate Rb even at cdk2-preferred sites, including Thr821.80,83 G1 arrest by Cip/Kip inhibitors incdk2�/� cells is likely mediated by inhibition of cyclin E–cdk1 com-plexes, recently demonstrated in both wild-type and knockout cells.84

These data suggest that highly selective inhibition of cdk2 maynot be useful therapeutically. One exception may be malignant mela-noma. In melanocytes, cdk2 undergoes transcriptional regulation bythe melanocyte lineage transcription factor microphthalmia-associated transcription factor ( MITF).85 Microarray data sets reveal atight correlation in expression for MITF and cdk2 in primary humanmelanomas, but not other malignancies, defining melanomas withhigh versus low levels of cdk2. Low levels of MITF and cdk2 expressionin melanoma cell lines accurately predict increased susceptibility to G1

arrest induced by siRNA targeting cdk2 or decreased proliferation in

response to roscovitine, a cdk2 inhibitor.85 Therefore, subsets of mel-anomas may be sensitive to a selective cdk2 inhibitor.

S-PHASE PROGRESSION AND E2F-1–DEPENDENT APOPTOSIS

S-Phase Events

After cdk-mediated phosphorylation of Rb during G1, E2F activ-ity is derepressed and E2F is released. E2F bound to its heterodimericpartner, DP-1, directs transcription of genes required for S phase.However, this transcription is activated only transiently. OrderlyS-phase progression requires the downregulation of E2F-1 activity,accomplished in part by cdk-mediated phosphorylation86-89 (Fig 2A).Inhibition of cdk activity during S phase results in inappropriatelypersistent E2F, which is known to cause S-phase delay and apoptosis.E2F-1–induced apoptosis can occur by both p53-dependent and p53-independent mechanisms,90 the latter involving the activities of E2F-1transcriptional targets, such as p73, Apaf-1, or caspase 3; repression ofMcl-191; or the ability of E2F-1 to interact with death receptor andnuclear factor-kappa B (NF-�B) pathways.92

E2F-1 Phosphorylation

Several cdk holoenzymes phosphorylate E2F-1 during the S andG2 phases and participate in the appropriately timed neutralization ofits activity. Cyclin A–cdk2 stably interacts with the N-terminus ofE2F-1 and directs the phosphorylation of both E2F-1 (likely at Ser307)and DP-1, which inhibits the DNA binding activity of the dimer.86,89

Phosphorylation by cyclin A–cdk1 at Ser375 may promote the forma-tion of Rb–E2F-1 complexes, contributing to the turning off of E2F-1activity late in the cell cycle, so that inhibition of cdk1 would permit thepersistence of E2F-1 free of Rb.93 Finally, the kinase activity associatedwith the general RNA polymerase transcription factor IIH (TFIIH)multisubunit protein complex, cyclin H–cdk7/MAT-1, phosphory-lates E2F-1 at Ser408 and Thr433, which is a prerequisite for ubiquiti-nation and degradation. Mutation of these sites to alanine greatlyenhances E2F-1 stability.94

The targeting of E2F-1 phosphorylation via cdk inhibition maylead to death selectively in transformed cells. The disrupted cyclinD–cdk4/6–INK4–Rb pathway in tumor cells produces high levels ofE2F activity. A small reduction in cdk activity during S phase may leadto persistence of E2F activity that has little effect on normal cells, butmay leave transformed cells with inappropriately persistent E2F activ-ity at a high enough level to surpass the threshold required to induceapoptosis95 (Fig 2B).

Inhibition of cdk2 and cdk1

It is of interest that several approaches aimed at targeting cdk2induce S and G2 arrest, often followed by apoptosis. In fact, themajority of these approaches (ie, in which cdk4/6 is not concomi-tantly inhibited) have produced only weak G1 arrest, so that theabsence of potent G1 arrest in cdk2�/� MEFs80,81 or after siRNAablation in established tumor cell lines79 should not be surprising.The inducible expression of a dominant negative cdk2 mutant(dn-cdk2) in U2OS osteosarcoma cells is particularly instructive.In exponentially growing cells, low-level expression of dn-cdk2resulted in G2 arrest; induction of higher levels caused arrest duringboth the S and G2 phases. Effects on G1 progression were onlyobserved when cells were synchronized and released from anocodazole-induced mitotic block.96 Similarly, the overall weak


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effect on the G1-S boundary can be unmasked when cells aresynchronized by passage directly before transient introduction ofthe dominant negative mutant.97 S and G2 cell cycle effects havealso been described after ectopic expression of p27Kip1 98 or intro-duction of peptides capable of inhibiting cdk2 activity.95

Although cell death was not observed in U2OS cells induciblyexpressing dn-cdk2, experiments only characterized the effects for 24hours and may not have been long enough to detect apoptosis. How-ever, other approaches targeting cdk2 have resulted in profound apo-ptosis, including the introduction of cdk2 inhibitory peptides,95 orafter proteasomal degradation of cyclin A–cdk2.99,100 Ectopic expres-sion of p27Kip1 has also induced apoptosis.98,101 In addition, the inhib-itory peptides, capable of blocking the interaction of cyclin A–cdk2with E2F-1, caused abrupt cell death in tumor cells but not in non-transformed cells. Furthermore, rat fibroblasts engineered to induc-ibly express ectopic E2F-1 underwent apoptosis after peptidetreatment only when the transgene was induced, indicating that ele-vated E2F-1 was sufficient to sensitize nontransformed cells to apo-ptosis after cyclin A-cdk2 inhibition.95

The profound effects on S and G2 cell cycle progression andapoptosis afforded by cdk2 inhibitory peptides, targeted cyclin A deg-radation, or ectopic expression of p27Kip1 still need to be reconciledwith the absence of similar cell cycle effects or cell death after intro-duction of antisense and siRNA targeting cdk2. One possibility is thatthe former approaches are targeting both cdk1 and cdk2. Ectopicp27Kip1 expression would be expected to inhibit both cdks. The re-ported cdk2 inhibitory peptides were capable of cdk1 inhibition athigh concentration, and the proteasomal degradation of cyclin Ashould affect cyclin A–cdk1 activity as well.95,99 The scenario that bothcdk2 and cdk1 inhibition are required also makes sense because bothof these cdks play critical roles in the phosphorylation and modulationof E2F-1 activity. This hypothesis ultimately can be tested by addi-

tional work with cell lines engineered to express reduced levels of bothcdk2 and cdk1 together.84 In the absence of concomitant cdk4/6inhibition, the primary effects of cdk2/cdk1 ablation may be during Sand G2 and associated with apoptosis.

The principles emerging from these approaches have beenborne out with small-molecule cdk inhibitors. In one set of exper-iments, the pan-cdk inhibitor flavopiridol has been used. Becausethis compound inhibits multiple cdks, including cdks 4/6, 2, 1, and7, it induces G1 and G2 arrest in many exponentially growingtumor cell types.102 However, if cells are first recruited to S phase,either by synchronization or by chemotherapy-induced S phasedelay, they are sensitized to flavopiridol, so that cytotoxicity oc-curs.103 Cell death is E2F-1 dependent, given that it is inhibited inE2F-1�/� cells,104,105 and is also selective for transformed cells.103

These results have provided rationale for clinical trials using thesequential combination of gemcitabine and flavopiridol, in whichgemcitabine is administered at a fixed-dose rate to maximize both theincorporation of difluorodeoxycytidine triphosphate into DNA andthe possibility of retardation of S-phase progression. After 16 to 24hours, flavopiridol is administered.106 Interestingly, such chemother-apy/flavopiridol combinations may be less sequence dependent inRb-negative cells, in which G1 arrest induced by flavopiridol is inher-ently less potent. In experiments in which adriamycin and flavopiridolwere applied simultaneously to osteosarcoma cells, the drug combi-nation sensitized Rb-negative SAOS-2 cells, but not a derivative inwhich Rb expression had been restored.107

Several compounds that demonstrate nanomolar or low mi-cromolar potency for inhibition of both cdk2 and cdk1, withsignificantly lower activity against cdk4/6, have been reported, in-cluding seliciclib (Cyclacel Ltd, Dundee, United Kingdom; CYC202,Cyclacel; R-roscovitine, Cyclacel),108,109 BMS-387032 (SNS-032),110,111 SU9516,112 AZ703,113,114 and amino imidazopyridine

Fig 2. S-phase progression and E2F-1–dependent apoptosis. (A) After retinoblas-toma protein (Rb) phosphorylation, E2F,along with a heterodimeric DP family mem-ber partner, directs transcription of S-phasegenes. Transcription is activated transiently.E2F-1 activity is in part limited by phosphor-ylation, mediated by cyclin A– cyclin-dependent kinase (cdk) 2, cyclin A–cdk1,and cyclin H–cdk7. Appropriately timed neu-tralization of E2F activity is required forproper S-phase progression. (B) Inhibition ofcdk activity during S phase results in inappro-priate persistence of E2F activity. In normalcells, this is tolerable, and ultimately cells stillprogress through the cycle. However, trans-formed cells have high baseline levels of E2Factivity, so that even a small reduction in cdkactivity results in accumulation and persis-tence of high enough E2F activity to surpassthe threshold required to induce apoptosis.Targeting of cyclin A or cdk2/1 during Sphase has induced apoptosis selectively intransformed cells.

Geoffrey I. Shapiro




1d.115 As expected, these compounds have been reported to induce Sand G2 arrest followed by apoptosis, with variable effects at the G1-Sboundary that are weak and more prominent after synchronization.Several of these compounds cause induction of E2F-1,116 and apopto-sis is compromised in E2F-1 �/� cells. In the case of AZ703, intro-duction of a dominant negative E2F-1 mutant, capable of bindingDNA and blocking transactivation, inhibited the apoptotic response,suggesting a dependence on E2F-1 transcriptional targets.117

Alteration of cdk activity during the S and G2 phases mayinduce cell death by mechanisms other than the E2F-1– centricmechanism detailed here, including an interface with pathwaysmediating DNA damage and repair, which contribute to the cyto-toxic synergy of chemotherapy agents and cdk modulators. Thesepathways are briefly presented in the Appendix (online only).

CDK1 PARTICIPATES IN THE MITOTIC CHECKPOINT

Cdk1 Inhibition Compromises Survival After

Engagement of the Mitotic Checkpoint

In addition to its known role at the G2-M boundary, cyclinB–cdk1 recently has been implicated in cell survival during mitoticcheckpoint (also known as spindle assembly checkpoint) activation.In response to microtubule stabilization by paclitaxel, spindle assem-bly checkpoint activation and mitotic arrest are associated with anincrease in expression of survivin, an inhibitor of apoptosis proteinand a mitotic regulator. Survivin is expressed in a cell cycle–dependentmanner and is localized to various components of the mitotic appara-tus where it contributes to the regulation of spindle microtubulefunction and cell viability. The increased expression of survivin duringspindle checkpoint activation is related to its stabilization, afforded byphosphorylation on Thr34 by cyclin B–cdk1.118 Expression of a dom-inant negative kinase–dead cdk1 mutant suppressed phosphorylationof survivin at Thr34 and led to massive apoptosis in paclitaxel-treatedcells. Similar data were obtained using cdk1 conditional knockoutcells; overexpression of survivin reversed these effects.

It has also been proposed that after engagement of the mitoticcheckpoint by taxanes, vinca alkaloids, or inhibitors of KSP (hsEg5 orkinesin-5, a member of the kinesin family of mitotic spindle motorproteins), cell death requires an outcome known as adaptation, inwhich cells exit mitotic arrest in the presence of drug, fail cytokinesis,and enter G1.119,120 Escape to G1 despite continued mitotic checkpointsignalingprovokesapoptosis.BecausereductionincyclinB–cdk1activityis required for exit from mitosis, its inhibition after mitotic checkpointengagement facilitates mitotic slippage and exit, and speeds cell death.

These concepts have been tested using small-molecule cdkinhibitors, including purvalanol A, flavopiridol, NU6140, androscovitine. For example, pharmacologic ablation of cdk1 activity inmitotically arrested cells resulted in diminished phosphorylationon Thr34 and survivin depletion followed by apoptosis.118,121,122 Inaddition, flavopiridol has been shown to accelerate mitotic exit ofpaclitaxel-treated cells, promoting a pronounced decline in MPM-2antibody reactivity, indicating depletion of mitotic phosphoproteinmarkers.123 Similarly, application of purvalanol or roscovitine to mi-totically arrested transformed cells promoted mitotic slippage, with arapid reduction in expression of phospho-histone H3 and cyclin B,indicating passage to interphase of cells maintaining a 4N DNA con-tent, followed by induction of substantial apoptosis with no recov-

ery.124,125 In contrast, release from a mitotic block in the absence ofroscovitine induced cell death in only a proportion of cells, withnormal recycling of the remainder of cells over time. The sameevents were not observed in nonmalignant cells, in which roscovi-tine treatment delayed the M3G1 transition after release frommitotic arrest without compromising cell survival. This may reflectthe inherent full competence of the mitotic checkpoint in nontrans-formed cells, more likely to remain engaged and less susceptible toslippage.120 This difference suggests that cell death associated withcdk1 inhibition after activation of the mitotic checkpoint may beselective for transformed cells.

The proposed mechanisms account for previous observationsthat demonstrated sequence-dependent cytotoxic synergy in vitro be-tween paclitaxel and flavopiridol.123,126 When flavopiridol is appliedfirst, arrest at the G2-M boundary prevents mitotic entry and subse-quent paclitaxel-mediated cell death, but when applied after cells arearrested in mitosis, there is dramatic enhancement of apoptosis. Theseresults have been verified in vivo; when treatment of mice bearingbreast or gastric cancer xenografts with paclitaxel or docetaxel is fol-lowed by purvalanol A or flavopiridol,118,127 there is significant en-hancement of chemotherapy-induced anticancer activity, with bothtumor growth delay and tumor regression observed. Paclitaxel anddocetaxel have been studied in phase I and II combinations withflavopiridol,128,129 although the optimal schedules of these agents, andthe most appropriate interval between them, may be critical to theclinical outcome.127,130

TRANSCRIPTIONAL CDKS AND THE POTENTIAL OFCDK7/9 INHIBITION

Phosphorylation of the CTD of RNA Polymerase II

The CTD of RNA polymerase II is regulated by phosphoryla-tion mediated by cdks. The human RNA polymerase II CTD con-tains 52 tandem repeats of the consensus heptapeptide sequenceN-Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7-C. Cyclin T-cdk9 (also calledP-TEFb) preferentially phosphorylates the Ser2 sites of this sequenceto promote transcriptional elongation. It is likely that cyclin T-cdk9can phosphorylate the Ser5 position as well.11 Cyclin H–cdk7/MAT1,in the complex of transcription factor TFIIH, preferentially phosphor-ylates Ser5, which facilitates promoter clearance and transcriptionalinitiation.12 Cyclin H–cdk7 therefore plays a role both as a cell cycleand a transcriptional cdk; it acts as both CAK and a CTD kinase.

Flavopiridol and Seliciclib

Flavopiridol is the most potent known inhibitor of cdk9.131-133

Whereas the IC50 values for other cdks range from 100 to 400 nmol/Lwith Ki values between 40 and 70 nmol/L, the binding of flavopiridolto the adenosine triphosphate (ATP) binding site of cdk9 is signifi-cantly tighter, with 1:1 stoichiometry and a Ki value of 3 nmol/L. Infact, competition with ATP could not be demonstrated. Therefore, theinhibition by flavopiridol of cdk 9, as well as cdk7, has profound effectson cellular transcription.134,135

The transcripts that are most sensitive to flavopiridol-mediatedcyclin T–cdk9 P-TEFb inhibition and cyclin H–cdk7/MAT1 inhibi-tion are those with short half-lives.134,136 The rapid degradation ofthese mRNAs allows their levels to decrease when transcriptionalinitiation and elongation are inhibited. Several gene classes are rela-tively enriched for unstable mRNAs, including immediate early


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transcription factor genes and cytokine genes. Other gene classes en-coding labile mRNAs include transcripts for apoptosis regulators,such as those encoding the antiapoptotic proteins Bcl-2, Mcl-1, andXIAP; cell cycle regulators, which encode cyclin D1, c-myc, and mi-totic regulatory kinases; as well as NF-�B–responsive gene transcriptsand those contributing to the p53 pathway (Fig 3).134

The effect on expression of antiapoptotic proteins may in partaccount for the activity of flavopiridol recently described in chroniclymphocytic leukemia (CLL). Flavopiridol inhibits phosphorylationof the CTD of RNA polymerase II in CLL cells, primarily at ser2, butalso at ser5, and causes decreased transcription,137 associated withreduction of the transcripts of genes with antiapoptotic functions, andwith the consequent decline of cellular levels of their proteins.137-140

Apoptosis correlated best with depletion of Mcl-1 and XIAP; althoughmRNA levels of Bcl-2 declined, protein levels were stable overall,indicating that the outcome of flavopiridol-mediated transcriptionalinhibition is related to half-lives of not only transcripts, but also of theproteins they encode. Given that survival of CLL cells is dependent onthe continuous expression of antiapoptotic proteins such as Mcl-1,their reduction by flavopiridol may in part account for the drug-induced apoptotic response. CLL cells are largely quiescent, and donot require the cell cycle–specific cdks, suggesting that the effect ontranscriptional cdks is largely responsible. Of note, Mcl-1 depletion byflavopiridol in CLL cells treated in vitro has not always been associatedwith reduction in CTD phosphorylation, suggesting that flavopiridolmay also influence an alternative transcription factor or other signal-ing pathways responsible for Mcl-1 expression.141,142

Seliciclib (R-roscovitine) also inhibits cyclin T-cdk9 and cyclinH-cdk7 in addition to cyclin E-cdk2,108,109 and affects RNA polymer-ase II CTD phosphorylation, which is associated with a decrease inMcl-1 as well as other antiapoptotic proteins in CLL cells.143,144 Theproapoptotic activity of both flavopiridol and seliciclib in multiplemyeloma cell lines occurs by a similar mechanism.145-147 Of note,Mcl-1 is also a target of drugs capable of prematurely terminatingtranscription, including 8-chloro-adenosine and fludarabine.148,149

Currently, these results are being translated clinically in trials offlavopiridol and seliciclib in CLL, lymphoma, and multiple myeloma.For flavopiridol, novel drug schedules appear to be overcoming phar-macokinetic barriers.150 Initial trials, both in hematopoietic malig-nancies and solid tumors, used 24- to 72-hour continuous infusions toreflect preclinical observations that prolonged exposure enhancedapoptotic effects in vitro and repeated low-concentration drug treat-ment demonstrated antitumor activity in vivo.102,151,152 Althoughthese schedules produced nanomolar concentrations, consistent withconcentrations capable of producing preclinical effects, prolongedflavopiridol infusions have been largely inactive in multiple set-tings.129,153,154 Subsequent data suggested the superiority of bolusadministration designed to achieve high micromolar concentrations,which led to cures of lymphoma xenografts.155 This prompted thedevelopment of 1-hour infusions, which have achieved micromolarmaximal plasma drug concentrations, although with a short half-life.156 Interestingly, although 24- or 72-hour continuous infusionswere inactive in both CLL and mantle-cell lymphoma, 1-hour bolusinfusions achieved low response rates, suggestive of activity.157-160

Higher than expected concentrations of flavopiridol may be requiredin vivo because of 92% to 95% plasma protein binding.161,162

More recently, a phase I study in relapsed CLL patients used a30-minute bolus dose followed by a 4-hour infusion, pharmacoki-netically modeled to achieve and sustain micromolar concentra-tions for several hours.163 In a preliminary presentation of thisstudy, a 41% response rate was achieved in 22 assessable patients;eight of nine responders had fludarabine-refractory disease, bulkylymphadenopathy, and either 11q or 17p deletion. Dose-limitingtoxicity was life-threatening and fatal tumor lysis syndrome. Activeinvestigation into the development of predictors for this hyper-acute syndrome is ongoing. Considering the improvement inresponse rate between continuous infusion and bolus flavopiridolin mantle-cell lymphoma, trials of bolus/infusion flavopiridol inthis disease are anticipated. Combinations of flavopiridol with

Fig 3. The transcriptional cdks 7 and 9phosphorylate the carboxy-terminal do-main (CTD) of RNA polymerase II (Pol II),facilitating the initiation of transcription andefficient elongation. Inhibition of thesecyclin-dependent kinases (cdks) preferen-tially affects mRNAs with short half-lives,including those encoding antiapoptotic pro-teins, cell cycle regulators, and p53 andnuclear factor-kappa B (NF-�B) pathwaycomponents. VEGF, vascular endothelialgrowth factor.

Geoffrey I. Shapiro




fludarabine and rituximab have demonstrated substantial butmanageable toxicity, with high response rates.164

Although the reduction in antiapoptotic proteins by inhibitorsof cdks 7 and 9 may be adequate to induce significant cell death insome instances, their depletion may sensitize cancer cells to otherapoptotic stimuli emanating from damage of DNA, modulation of mi-crotubular stability, effects of tumor necrosis factor–related apoptosisinducing ligand or from signal transduction inhibition. For example,flavopiridol has been shown to potentiate imatinib-mediated apoptosisin BCR-ABL–positive leukemia cells165; flavopiridol-mediated reduc-tion in levels of Mcl-1 may have contributed to the observed syner-gism, and a phase I combination trial of imatinib and flavopiridol isongoing. Similarly, flavopiridol-mediated downregulation of XIAPunderlies its synergism with tumor necrosis factor–related apoptosisinducing ligand in leukemia cells,166 and the attenuation of expressionof mRNAs and proteins encoding multiple antiapoptosis regulatorsaccounts for synergism with epothilones in breast cancer cells.167

In addition to antiapoptotic genes, cell cycle regulators are alsotranscriptional targets of inhibitors of cdks 7 and 9. For example,cyclin D1 is readily depleted in flavopiridol-168 and seliciclib-treatedcells.169 It also will be important to demonstrate the repression oftranscription of cyclin D1b; nonetheless, depletion of D-cyclins addsrationale for the use of these agents in mantle-cell lymphoma andmultiple myeloma, as well as other tumors that may be particularlydependent on cyclin D1, such as Her-2–positive breast cancer39 oresophageal cancer.170 Similarly, transcriptional repression of c-myc byinhibitors of cdk7 and cdk9 may be particularly pertinent in Burkittlymphoma and small-cell lung cancer.

The inhibition of CTD phosphorylation may also have pro-found effects on p53-dependent and -independent expression ofp21Waf1/Cip1. For example, flavopiridol has been shown to reduceexpression of mdm2, causing p53 stabilization.171,172 In wild-typep53-expressing cells prone to apoptosis, including acute lymphocyticleukemia or germ cell tumor cells,173 induction of p53 may lead to celldeath. In addition, transcriptional repression can also prevent theinduction of p21Waf1/Cip1, so that p53 induction can occur without aconcomitant increase in p21Waf1/Cip1. Although this combination ofevents would be expected to produce apoptosis even in cells prone top53-mediated cell cycle arrest,174 flavopiridol-induced cell death isp53 independent,172,175 indicating that other mechanisms or tran-scriptional targets must be contributing when the drug is used alone.

Interestingly, however, flavopiridol can also prevent induc-tion of p21Waf1/Cip1 in response to DNA damage. p21Waf1/Cip1 me-diates cell cycle arrest and also prevents apoptosis by binding tocaspase 3. Therefore, in p53 wild-type colon carcinoma cell linesthat arrest after irinotecan-mediated stimulation of p53 andp21Waf1/Cip1, flavopiridol-mediated inhibition of the transcrip-tional induction of p21Waf1/Cip1 can result in cell death.176 In thecontext of a phase I trial of irinotecan and flavopiridol, patientswith responsive or stable disease tended to express wild-type p53and have no change or decrease in p21Waf1/Cip1 levels post-treatment.177 Therefore, in this setting, clinical benefit was corre-lated with p53 status and the ability of flavopiridol to inhibit acritical p53 effector than can prevent apoptosis.

p53-independent induction of p21Waf1/Cip1 is characteristic ofhistone deacetylase inhibitors178 and contributes to cytostatic anddifferentiation responses to these agents. In both leukemia and solidtumor models, abrogation of p21Waf1/Cip1 by flavopiridol has resulted

in enhanced cytotoxicity when it was combined with sodium bu-tyrate,179,180 suberoylanilide hydroxamic acid,181 or depsipeptide182;the latter two combinations are also under clinical evaluation.

Additional cdk7/9-related targets of interest in flavopiridol-treated cells include suppression of NF-�B–mediated transcription,perhaps underlying the preclinical synergism observed when fla-vopiridol is combined with either tumor necrosis factor alpha172,183

or bortezomib.184 Finally, flavopiridol has been shown to preventthe hypoxia-mediated induction of vascular endothelial growth fac-tor, which may translate to antiangiogenic effects, and may accountfor responses and stable disease that have been observed in renalcell carcinoma.185-187

SMALL-MOLECULE CDK INHIBITORS

A growing number of cdk inhibitors representing multiple chemicalclasses currently are in clinical trial. The assessment of these drugs,along with pharmacokinetically superior schedules of flavopiridol,will help determine whether preclinical predictions regarding cell cy-cle inhibition will translate therapeutically. These drugs may be classi-fied based on their effects against the cell cycle cdks as either pan-cdkinhibitors or more selective cdk inhibitors, with varying potencyagainst the transcriptional cdks (Fig 4).

In solid tumor studies, flavopiridol, seliciclib, and BMS-387032(SNS-032) have been the most extensively tested. With 1-hour bolusschedules, the dose-limiting toxicity of flavopiridol is neutropenia,156

as opposed to the diarrhea that had occurred on continuous infu-sion schedules,151,152 providing evidence of an antiproliferative effect.Trials using load/infusion schedules, designed to achieve and sustainmicromolar levels, are more mature in hematologic malignancies andare just beginning evaluation in solid tumors. Preliminary data fromphase I trials of seliciclib and BMS-387032 have been reported, sug-gesting that these agents are tolerable, with fatigue, GI adverse effects,elevated aminotransferases, and increased creatinine described.188-192

Antiproliferative toxicity was rare. Although large numbers of subjectshave not been treated at the highest tolerable doses, antitumor activityin these trials was modest, with some outcomes of prolonged stabledisease reported.

In the evaluation of all of these drugs, it will be important toinclude pharmacodynamic measures of whether the cdk targets arehit. This could include assessment of Rb and p27Kip1 phosphoryla-tion193 as markers of cdk4 and/or cdk2 inhibition, as well as CTDphosphorylation, depletion of cyclin D1 or Mcl-1, or induction ofp53 as markers of cdk7/9 inhibition. Such analyses have been donein recent trials including flavopiridol194 and E7070, another cellcycle modulatory agent.195 Results of these studies suggest that cdkinhibition can be achieved in tumor cells. However, correlation ofeffects on cell cycle–related phosphorylation events and effects onstandard antiproliferative markers requires further clarification.194-198

In addition, fluorothymidine positron emission tomography scan-ning may represent a noninvasive technology for monitoring cell cyclearrest in response to these agents.199

Results on the group of compounds currently under study willdetermine whether more promiscuous cdk inhibition is preferable toselective cdk inhibition. For example, both seliciclib and BMS-387032are relatively selective for cdk2. However, these and the majority ofcompounds that inhibit cdk2 also inhibit cdk1; compounds more


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equipotent for cdk2 and cdk1 may be superior. Whether a selectivecdk2/cdk1 inhibitor will be therapeutically superior to a cdk2/cdk1/cdk4/6 inhibitor remains to be determined. In addition, given thatinhibition of cdk2 and cdk1 may potentiate apoptosis induced byDNA-damaging agents that affect S-phase progression or by microtu-bule stabilizing agents, chemotherapy combination trials will continue

to be important. Although this approach requires commitment torandomized studies, cdk inhibitors may ultimately find their place incombination regimens. General principles of cdk inhibition are sum-marized in Table 1.

Several of the compounds under investigation are oral agentsthat may permit continuous daily dosing, rather than the more

Fig 4. Small-molecule cyclin-dependentkinase (cdk) inhibitors may be classifiedbased on effects on the cell cycle cdks.Pan-cdk inhibitors, including flavopiridol andAG-024322,203,204 inhibit cdks 4/6, 2, and 1.Other compounds are highly selective, in-cluding the pyridopyrimidine PD 0332991,73

and triaminopyrimidine, CINK4,205 and inhib-itors of cdk4/6. Several other compoundsinhibit cdk2 and cdk1 more selectively, in-cluding seliciclib109; BMS-387032, an aminothiazole111; PNU-252808, another thiazolederivative

200,201; imidazo[1,2-a]pyridines, in-

cluding AZ703114,117 and aminoimidazo[1,2-a]pyridine-1d115; and the purine-basedNU6102202 and NU6140.122 Seliciclib,BMS-387032, and NU6140 are most selec-tive for cdk2, whereas AZ703 and NU6102inhibit cdk2 and cdk1 with similar poten-cies. Many compounds inhibit cdk9, includ-ing flavopiridol, seliciclib, BMS-387032,and AZ703. CINK4, PNU-252808, AZ703,NU6102, and NU6140 have been studiedpreclinically; the others listed have enteredclinical trials.206

Table 1. Principles of cdk Inhibition

Inhibition of cdk4/6 leads to potent Rb-dependent G1 arrest; however, compensation by cdk2 is possibleInhibition of cdk4/6, cdk2, and cdk1 frequently results in arrest at the G1-S and G2-M boundariesMore selective inhibition of cdk2 and cdk1 leads to less potent G1 arrest, S3G2 effects, and E2F-1–dependent apoptosisRecruitment to S phase by chemotherapy agents may sensitize cells to cdk inhibition; cdk inhibitor–mediated death may occur by E2F-1–dependent apoptosis,

augmentation of DNA damage, or inhibition of DNA repairCdk1 inhibition may augment cell death after mitotic checkpoint activation by taxanes or KSP inhibitorsHighly selective cdk2 inhibition does not have antiproliferative effects in many cancer cell typesInhibition of cdk9 preferentially depletes mRNAs with short half-lives (eg, Mcl-1, cyclin D1, c-myc, p53-induced p21Waf1/Cip1, and hypoxia-induced VEGF)

Abbreviations: cdk, cyclin-dependent kinase; Rb, retinoblastoma protein; KSP, hsEg5 or kinesin-5; VEGF, vascular endothelial growth factor.

Geoffrey I. Shapiro




intermittent schedules used in evaluation of flavopiridol, seliciclib,and BMS-387032 to date. Continuous dosing with maintenance ofconcentrations that afford target inhibition may be critical. Withintermittent dosing, theoretical concerns exist. For example, ATP-competitive cdk inhibition can be associated with negative feed-back that downregulates the inhibitory phosphorylations of cdks atThr14 and Tyr15. After the drug is no longer present in the ATPbinding site, a hyperactive cdk, lacking inhibitory phosphates, maybe poised to drive cell cycle progression, so that even initial potenttarget inhibition cannot translate to growth suppression.207 Thetight binding of a drug such as flavopiridol to the ATP-binding siteof cdk9 must also be considered. In preclinical experiments, lowconcentrations of flavopiridol are easily exhausted, so that thatthere is restoration of transcription with superinduction of theinitial mRNA targets,172 emphasizing the importance of pharma-cokinetic modeling aimed at achieving concentrations that will berapidly cytotoxic.

One of the lessons from the flavopiridol experience is that for newcompounds, it will be important to determine their relative potenciesagainst both the cell cycle and the transcriptional cdks. Effects oncdk7/9 may be particularly relevant to inducing apoptosis in malig-nant hematopoietic cells and may sensitize solid tumor cells to a

variety of apoptotic stimuli, including those induced by inhibitionof cell cycle–related cdks. For example, it has recently been shownthat cell death induced by the imidazo[1,2-a]pyridine cdk1/2 in-hibitor AZ703 is potentiated in cells depleted of cdk9,117 indicatingthat cdks 1, 2, and 9 represent a promising cdk subset, inhibition ofwhich may induce potent cytotoxic effects. Combined inhibitionof cell cycle and transcriptional cdk activities may also be achievedwith an inhibitor of cyclin H– cdk7,208,209 a component of cdk-activating kinase that also phosphorylates E2F-1, and is a CTDkinase as well. This combination of activities may generate cyto-toxic responses by causing perturbations of cell cycle progression,stabilization of E2F-1, and compromised expression of transcriptsrequired by dividing cells, including those encoding anti-apoptoticproteins.207 Alternatively, detailed knowledge of the structure ofcdk7 may pave way to inhibitors that can selectively target CAK orCTD kinase activity, which may help define therapeutic opportu-nities that are either cell cycle or transcription directed, each withvalue in specific disease contexts.211 Finally, the continued discov-ery of critical transcripts, repression of which mediates responsesto transcriptional cdk inhibitors, either alone or in combinationwith other agents, will also identify novel targets for anticancerdrug development.135

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Geoffrey I. Shapiro




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201. Lantham VM, Shapiro GI: Selective inhibitionof cyclin-dependent kinase (cdk)2 by PNU252808induces cell cycle arrest, apoptosis and cytotoxicsynergy with DNA-damaging agents in non-smallcell lung cancer cell lines. Proc Am Assoc CancerRes 44:634, 2003 (abstr 2778)

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■ ■ ■

AcknowledgmentI thank Takeshi Shimamura, PhD, for assistance with the design and preparation of schematic figures.

Appendix

The Appendix is included in the full-text version of this article, available online at www.jco.org. It is not included in the PDF version(via Adobe® Reader®).

Author’s Disclosures of Potential Conflicts of InterestThe author or immediate family members indicated a financial interest. No conflict exists for drugs or devices used in a study if they are not being evaluated as part of

the investigation. For a detailed description of the disclosure categories, or for more information about ASCO’s conflict of interest policy, please refer to the Author DisclosureDeclaration and the Disclosures of Potential Conflicts of Interest section in Information for Contributors.

Authors Employment Leadership Consultant Stock Honoraria Research Funds Testimony Other

Geoffrey Shapiro Sanofi-Aventis (A);Eisai Global

Research (A);AstraZeneca (A)

Sanofi-Aventis (C);AstraZeneca (C);

Eisai GlobalResearch (C)

Dollar Amount Codes (A) � $10,000 (B) $10,000-99,999 (C) � $100,000 (N/R) Not Required

Author Contributions

Conception and design: Geoffrey ShapiroFinancial support: Geoffrey ShapiroAdministrative support: Geoffrey ShapiroData analysis and interpretation: Geoffrey ShapiroManuscript writing: Geoffrey ShapiroFinal approval of manuscript: Geoffrey Shapiro

GLOSSARY

Cdk (cyclin-dependent kinase): Cdk is a protein kinaseinvolved in regulating the cell cycle. It is activated by associatingwith a cyclin, forming a cdk complex.

Cdk (cyclin-dependent kinase) inhibitor: A group ofproteins that interact and inhibit the cdk complex, which affectscell cycle progression.

Cip/Kip inhibitor: A family of cdk inhibitors that includesp21Waf1/Cip1/Sdi1, p27Kip1, and p57Kip2.

E2F transcription family members: Transcription fac-tors that control genes essential for cell cycle progression. Retino-blastoma susceptibility protein prevents progression from the G1phase to S phase through its interaction with E2F transcriptionfamily members.

IAP (inhibitor of apoptosis protein): IAPs are proteinsthat bind to and inhibit the activation of caspases and thereforesuppress apoptosis.

INK4 (inhibitor of the cyclin-dependent kinase 4): INK4genes cause cell cycle arrest through interaction with and inhibition ofcyclin-dependent kinases CDK4 and CDK6.

Mitotic checkpoint: The mitotic (or spindle assembly) checkpointcan delay cell-cycle progression until all chromosomes have successfullymade spindle-microtubule attachments. Defects in the mitotic check-point generate an abnormal balance of chromosomes and might facili-tate tumorigenesis.

Retinoblastoma protein: The first tumor suppressor gene iden-tified in children with hereditary retinoblastomas, its phosphoryla-tion state has important implications for cell cycle progression.Hypophosphorylated RB tightly binds the transcriptional factor E2F(also important for cell cycle regulation), thus preventing E2F-mediated cell cycle entry.

RNA polymerase II: The multiprotein complex which carries outtranscription, reading the DNA template to yield mRNA.


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