Specific inhibition of cyclin-dependent kinase 4/6 by PD ... · Specific inhibition of cyclin-dependent kinase 4/6 by PD 0332991 and associated antitumor activity in human tumor xenografts

Specific inhibition of cyclin-dependent kinase 4/6 byPD 0332991 and associated antitumor activityin human tumor xenografts

David W. Fry,1 Patricia J. Harvey,1 Paul R. Keller,1

William L. Elliott,1 MaryAnne Meade,1

Erin Trachet,1 Mudher Albassam,2

XianXian Zheng,3 Wilbur R. Leopold,1

Nancy K. Pryer,5 and Peter L. Toogood4

1Cancer Pharmacology, 2Worldwide Safety Sciences,3Molecular Technologies, and 4Medicinal Chemistry, PfizerGlobal Research and Development, Ann Arbor, Michiganand 5Onyx Pharmaceuticals, Richmond, California

AbstractPD 0332991 is a highly specific inhibitor of cyclin-dependent kinase 4 (Cdk4) (IC50, 0.011 Mmol/L) andCdk6 (IC50, 0.016 Mmol/L), having no activity against apanel of 36 additional protein kinases. It is a potentantiproliferative agent against retinoblastoma (Rb)-positivetumor cells in vitro, inducing an exclusive G1 arrest, witha concomitant reduction of phospho-Ser780/Ser795 on theRb protein. Oral administration of PD 0332991 to micebearing the Colo-205 human colon carcinoma producesmarked tumor regression. Therapeutic doses of PD0332991 cause elimination of phospho-Rb and theproliferative marker Ki-67 in tumor tissue and down-regulation of genes under the transcriptional control ofE2F. The results indicate that inhibition of Cdk4/6 alone issufficient to cause tumor regression and a net reductionin tumor burden in some tumors. [Mol Cancer Ther2004;3(11):1427–37]

IntroductionThe proliferation of eukaryotic cells typically involves anorderly progression through four distinct phases of the cellcycle: G1, S, G2, and M (1–3). Cyclin-dependent kinases(Cdks) play a key role in regulating cell cycle progressionand to a large degree govern cellular transitions fromgrowth phases (G1 and G2) into phases associated withDNA replication (S) and mitosis (M; refs. 4, 5). Regulation

of Cdk catalytic activity occurs at multiple levels includingphosphorylation and dephosphorylation of the Cdk itself;cyclin synthesis and degradation; and expression, degra-dation, and availability of naturally occurring proteininhibitors and subcellular localization of these variousregulatory components (2, 5, 6).

Progression through the G1-S phase requires phosphor-

ylation of the retinoblastoma (Rb) protein by Cdk4 (7, 8) orthe highly homologous enzyme Cdk6 (9, 10) in complexwith their activating subunits, the D-type cyclins, D1, D2,or D3 (11). Hyperphosphorylation of Rb diminishes itsability to repress gene transcription through the E2F familyof transcription factors and consequently allows synthesisof several genes, the protein products of which are nec-essary for DNA replication (12–15). Thus, the catalytic ac-tivity of Cdk4 or Cdk6 regulates a critical checkpoint for theG1-S transition and the commitment to cell division (16).

More than 90% of human tumors abandon the controlmechanisms for this transition point through a variety ofgenetic and biochemical adaptations (1, 17). Examples ofthese abnormalities include up-regulation of Cdk4 itself;amplification of the D-type cyclins; down-regulation of anaturally occurring inhibitor of Cdk4, called p16INK4A;mutations in Cdk4 that prevent p16INK4A binding to theenzyme; and deletion or mutation of Rb itself (17–21). Allof these aberrations can lead to loss of proliferative controlseither through elimination of the checkpoint altogether orthrough inappropriate or enhanced Cdk4 activity resultingin hyperphosphorylation of Rb. The frequency of thesealterations alone clearly implies that abrogation of the G1

checkpoint or acceleration of the Cdk4/cyclin D pathwayprovides a distinct advantage to cancer cells in terms ofproliferation and perhaps survival.

Based on these observations, cyclin D–dependent kinaseshave been considered for many years a prime target for

cancer chemotherapy (22, 23). Experimental evidencesuggests that inhibition of cyclin D–dependent kinaseactivity may prevent tumor growth and/or at least partiallyrevert the transformed phenotype. For example, reductionof cyclin D expression through antisense technology causesa concomitant decline in cyclin D– dependent kinaseactivity and results in inhibition of tumor growth, abolitionof tumorigenicity, or, in some instances, tumor cell death(24–27). Other reports indicate that exogenous expressionof p16INK4A in tumor cells using adenoviral gene deliverysystems or inducible promoters blocks proliferation andtumorigenic potency both in vitro and in vivo (28–31).These observations lend credence to Cdk4/6 as a targetfor cancer treatment and allow a reasonable expectationthat a specific inhibitor of these enzymes would producea meaningful therapeutic response.

Received 5/5/04; revised 8/25/04; accepted 8/31/04.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.

Requests for reprints: Peter L. Toogood, Medicinal Chemistry, PfizerGlobal Research and Development, 2800 Plymouth Road, Ann Arbor,MI 48105. Phone: 734-622-1335; Fax: 734-622-5165.E-mail: [email protected]

Copyright C 2004 American Association for Cancer Research.

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Several drug discovery programs have produced potentsmall molecule Cdk inhibitors (22, 32–35) from a variety ofchemical classes, which include purine analogues (36–40),pyrimidine analogues (41–43), indenopyrazoles (44, 45),pyridopyrimidines (46–48), pyrazolopyridines (49, 50),indolocarbazoles (51), pyrrolocarbazoles (52, 53), oxindoles(54, 55), and aminothiazoles (56). Several compounds arecurrently in clinical trials including flavopiridol, R-rosco-vitine (CYC202), UCN-01 (7-hydroxystaurosporine), andBMS-387032 (57, 58). Most of these compounds, however,inhibit multiple Cdks, with Cdk2 being a particularlycommon target in drug discovery programs because thisenzyme is easily crystallized with inhibitors of varyingmolecular structure (59, 60). Interestingly, several recentreports have provided evidence that mammalian cells cancontinue to proliferate in the absence of Cdk2/cyclin Eactivity (61–63) possibly due to compensation by Cdk4and/or Cdk6. These reports suggest that Cdk2 may be lessattractive than Cdk4 as an anticancer target. Althoughsome reports in the literature claim Cdk4-specific agents,these compounds are generally weakly selective for Cdk4versus Cdk2 in isolated enzyme assays (10–20-fold), areweakly potent (64, 65), or produce a G2 cell cycle arrest atelevated concentrations, which is inconsistent with Cdk4/6inhibition (42, 43, 47, 51–53, 66). Several groups have re-ported significant Cdk4 selectivity versus Cdk2 withoutsupporting biological data (67). Thus, the biological effectsof selective inhibition of Cdk4 by small, drug-like mole-cules have not been shown previously.

The present report discloses PD 0332991 as a potentinhibitor of Cdk4/6 and provides evidence that the solemechanism of action for this compound is inhibition ofthese enzymes.6 Furthermore, we describe the antitumoractivity of this agent and show significant efficacy in abroad spectrum of human tumor xenografts in vivo ,resulting in complete regression in some tumors with noevidence of acquired resistance or ability to circumventthe growth inhibitory properties of this agent.

Materials andMethodsCdk Assays and Other Protein KinasesAll Cdk-cyclin complexes were expressed in insect cells

through baculovirus infection and purified as describedpreviously (68). The substrate for the Cdk assays was aminoacids 792 to 928 of pRb fused to glutathione S-transferase(10). Cdk assays and IC50 determinations were done asdescribed previously (47). Enzyme assays for other proteinkinases were done as described previously (69–71).

Cell CultureAll cell lines were obtained from American Type Culture

Collection (Manassas, VA) and maintained at 37jC at 5%CO2 in DMEM containing 10% fetal bovine serum (FBS;Life Technologies, Inc., Rockville, MD).

Thymidine Incorporation into DNACells were seeded at 2 � 104 per well in a 96-well

Cytostar T plate (Amersham Biosciences, Piscataway, NJ)and incubated overnight to allow cells to attach. Varyingconcentrations of PD 0332991 were added to the wells andincubated for 24 hours at 37jC. [14C]thymidine (0.1 ACi)was added to each well and incorporation of the radiola-bel was allowed to proceed for 72 hours. Incorporatedradioactivity was determined with a h plate counter(Wallac, Inc., Gaithersburg, MD).

Western Blot AnalysisCell or tissue samples were lysed and Western blot

analysis was done as described previously (47). Rbphosphorylation status was assayed with Ser780 and Ser795

phosphospecific antibodies (Cell Signaling Technology,Beverly, MA) and total Rb expression was monitored usingRb 4H1 monoclonal antibody (Cell Signaling Technology).

ImmunohistochemistryRb 4H1, phospho-Rb (Ser780), and Ki-67 immunohisto-

chemical staining were done using the Ventana Discoveryautostainer (Ventana Medical Systems, Tucson, AZ) withheat at 37jC to 46jC. Slides were deparaffinized usingVentana EZ Prep buffer and antigen retrieval was doneusing the Ventana CC1 mild reagent (containing EDTA) at100jC for 20 minutes. Endogenous peroxidase was inacti-vated using the Ventana Enhanced Inhibitor solution andnonspecific antibody binding was blocked using a 3%blocking solution (Roche Diagnostics, Indianapolis, IN) for30 minutes. Primary antibodies [Rb 4H1 (1:100 dilution,catalogue no. 9309, Cell Signaling Technology), phospho-Rb (Ser780, 1:100 dilution, catalogue no. 9307, Cell SignalingTechnology), and Ki-67 (MIB-1, 1:50 dilution, catalogue no.M7240, DAKO, Carpinteria, CA)] were manually appliedseparately for 30-minute incubation(s) followed by appli-cation of Ventana Endogenous Blocker kit solutions(4 minutes each) to block endogenous biotin and avidin.Secondary antibodies [goat anti-rabbit IgG (for pRb) orhorse anti-mouse IgG (for Rb 4H1 and Ki-67) from VectorLaboratories (Burlingame, CA)] were incubated for30 minutes. Following SA-HRPO conjugate (18 minutes),Ventana enhanced 3,3V-diaminobenzidine tetrahydrochlor-ide (8 minutes), and copper sulfate solution (4 minutes)incubations, sections were counterstained with hematoxy-lin and bluing reagent for 4 minutes each. Slides weremanually rinsed in diluted detergent, dehydrated throughgraded alcohols, cleared in xylene, and mounted withPermount.

Flow CytometryCells were harvested and washed in PBS containing

EDTA (5 mmol/L). They were then washed in PBS con-taining 1% FBS (1% FBS/PBS), fixed in 85% ethanol, andstored at 4jC for at least 16 hours and up to 5 days. Cellswere then washed again in 1% FBS/PBS and incubated at37jC for 30 minutes in 1% FBS/PBS containing propidiumiodide (40 mg/mL, Molecular Probes, Eugene, OR) andRNase A (250 mg/mL, Roche Diagnostics). Data werecollected using a Coulter EPICS Elite ESP (Miami, FL)

6 The discovery and preparation of PD 0332991 is described separately. PL Toogood,PJ Harvey, JT Refine, et al. Discovery of PD 0332991, a potent and selective Cdk 4/6inhibitor, submitted for publication.

Anticancer Activity of the Cdk4/6 Inhibitor PD 03329911428




equipped with a Spectraphysics argon ion laser andanalyzed using ModFit (Verity Software House, Inc.,Topsham, ME). Results represent a minimum of 15,000cells assayed for each sample.

Tumor Xenografts and AnimalsSolid human tumor models Colo-205 colon, SW-620 colon,

MDA-MB-435 breast, SF-295 glioblastoma, ZR-75-1 breast,PC-3 prostate, H125 non–small cell lung, H23 non–smallcell lung, and MDA-MB-468 breast were developed from celllines and maintained in severe combined immunodeficientmice. All tumor models were serially passaged as s.c. im-plants of tumor fragments (f30 mg) from tumors weighingf1,000 mg. Tumor models were passaged V10 generationsin vivo before returning to cryopreserved early passage stockmaterial. The mice used in these studies were obtained fromCharles River Breeding Laboratories (Wilmington, MA) andTaconic Farms (Germantown, NY; National Cancer Institutecolonies). All animals were examined prior to the initiationof studies to ensure that they were healthy and acclimated tothe laboratory environment. Mice were housed in barrierfacilities with food and water provided ad libitum on a 12-hour light/dark cycle. Animal care was provided inaccordance with Association for Assessment and Accredi-tation of Laboratory Animal Care International guidelines.All protocols involving animals were reviewed and ap-proved by the Pfizer Laboratories animal care and usecommittee (Ann Arbor, MI).

In vivo ChemotherapyMice (18–22 g) were randomized and then implanted s.c.

with tumor fragments (f30 mg) into the region of the rightaxilla. Treatment was initiated when tumors reached 100 to150 mg. PD 0332991 was given according to the scheduleand dose indicated in the table and figure legends bygavage as a solution in sodium lactate buffer (50 mmol/L,pH 4.0) based on mean group body weight. In allexperiments, there were 12 mice in the control group and8 mice each in the treated groups. Additional details foreach experiment are given in the table legends.

Isolation ofTotal RNA andTaqman Reverse Transcrip-tion-PCR

RNA was isolated from frozen tumor powders fromthree separate mice for each dose using the Trizol method(Invitrogen, Carlsbad, CA) and further purified withDNase treatment and Qiagen column according to QiagenRNA cleanup protocols (Qiagen, Valencia, CA). Quantita-tive real-time PCR was carried out using the Taqman 5Vnuclease assay system with the signal from a gene-specificprobe that is normalized to the signal for the reference geneof h2-microglobulin (72–74). Primers and probes wereordered from Assay-on-Demand Gene expression products(Applied Biosystems, Foster City, CA). Each assay con-sisted of two unlabeled PCR primers and a FAM dye-labeled Taqman MGB probe. The first-strand cDNA wasgenerated using total RNA (5 Ag) and High-Capacity cDNAArchive kit in a 100 AL reaction (Applied Biosystems). PCRwas done in 384-well plates (20 AL) containing equivalentto cDNA (11 ng), primer/probe sets (1 AL) for the gene ofinterest, and 2� Taqman Universal PCR Master mix (10 AL,

Applied Biosystems). Duplicates were done for each cDNA

sample. Thermal cycling was carried out in ABI PRISM

7900 with default cycling conditions and real-time fluores-

cence detection. Expression fold change was calculated

using the manufacturer’s suggested DDCt method.

StatisticsStatistical analysis was done on the times for individual

tumors (both treated and control) to reach 750 mg. Wheretumors did not reach 750 mg, the regrowth curves wereextrapolated to 750 mg. Time to 750 mg was selected as theexamined variable because it is independent of theevaluation size chosen after completion of therapy (75).All data from each experiment were analyzed by ANOVAand, where significant differences were identified, ana-lyzed using the Bonferroni t test for multiple comparisonsversus control. Levels of statistical significance are indicat-ed for individual curves in each figure. Statistical analyseswere done with SigmaStat 3.0 for Windows.

ResultsPD 0332991 Is a Potent and Selective Inhibitor ofPurified Cdk4/6

Inhibition of Cdks by pyridopyrimidines has beenreported previously (46–48). Optimization of this class ofcompounds for selective inhibition of Cdk4 was achievedby testing compounds against a small panel of fourenzymes, including Cdk4/cyclin D1, Cdk2/cyclin A,fibroblast growth factor receptor, and platelet-derivedgrowth factor receptor. Compounds that possessed highselectivity for Cdk4 versus the other kinases were furtherevaluated in an expanded panel of kinases and assessed forantiproliferative potential against MDA-MB-435 humanbreast carcinoma cells. Those compounds with potentantiproliferative properties were evaluated by flow cytom-etry for their ability to produce a clean G1 arrest in tumorcells across a range of concentrations. This strategy led tothe identification of a subset of pyridopyrimidines thatdisplayed unprecedented levels of selectivity for Cdk4, andfrom these compounds, PD 0332991 (Fig. 1) was selectedfor its superior physical and pharmaceutical properties.

PD 0332991 is a potent and highly selective inhibitor ofCdk4/cyclin D1 kinase activity; it exhibits an IC50 value of0.011 Amol/L against this enzyme under the conditionsdescribed in Materials and Methods (Table 1). Anotherenzyme, Cdk6, which also complexes with cyclin Dsubunits and is highly homologous to Cdk4, can performidentical functions to Cdk4 by phosphorylating Rb at thesame sites (9, 10). Consequently, inhibition of both Cdk4and Cdk6 is necessary to ensure complete suppression ofRb phosphorylation and to produce the maximum thera-peutic response with the greatest spectrum of antitumoractivity. PD 0332991 inhibited Cdk6 with equivalentpotency to Cdk4 (Table 1). Within a panel of enzymes,PD 0332991 exhibited absolute selectivity for Cdk4/6 withlittle or no activity against 36 additional protein kinasesincluding other Cdks and a wide variety of tyrosine andserine, threonine kinases (Table 1). The structure of PD





0332991 and its relationship to PD 0183812 (47) suggest thatPD 0332991 is likely to inhibit Cdk4 by binding to the ATPsite; however, it was not possible to show competitiveinhibition of Cdk4 by PD 0332991 using a kinetic analysisdue to the near equivalence of the K I and the enzymeconcentration required to perform this assay. Similarly, ithas not been possible yet to obtain crystal structures ofCdk4 either with or without bound ligands.

PD 0332991 Inhibits Rb Phosphorylation in TumorCells on Cdk4-Specific Sites

The only known natural substrates for Cdk4/6 are the Rbfamily of gene products, p110(Rb), p107, and p130 (76). Ofthe 16 known phosphorylation sites on Rb, 2 are specificallyphosphorylated by Cdk4/6: Ser780 and Ser795 (77–79). Thephosphorylation status of Rb at these specific sites intreated tumors therefore represents an appropriate bio-marker for inhibition of Cdk4/6 by PD 0332991 in tumorcells and tissue. The IC50 for reduction of Rb phosphory-lation at Ser780 in MDA-MB-435 breast carcinoma cells was0.066 Amol/L (Fig. 2A and B). PD 0332991 was equallyeffective at reducing Rb phosphorylation at Ser795 in thistumor with an IC50 of 0.063 Amol/L, and similar effects onboth Ser780 and Ser795 phosphorylation were obtained in theColo-205 colon carcinoma (data not shown). In vitro timecourse experiments indicated that the reduction of Rbphosphorylation began to occur as soon as 4 hours afterexposure to PD 0332991 and reached a maximum at 16hours. The inhibition was completely reversible becausephosphorylation on Ser780 and Ser795 began to return 2hours after removal of the drug and was complete within16 hours, by which time the cells were actively proliferating(data not shown).

PD 0332991 Is a Potent Antiproliferative Agent ThatArrests Rb-PositiveTumors Exclusively in G1

PD 0332991 is a potent inhibitor of cell growth andsuppresses DNA replication by preventing cells fromentering S phase. The compound inhibited thymidineincorporation into the DNA of Rb-positive human breast,colon, and lung carcinomas as well as human leukemias,with IC50 values ranging from 0.040 to 0.17 Amol/L

(Table 2). PD 0332991 had no activity against Rb-negativecells, indicating that it does not interact with antiprolifer-ative targets other than Cdk4/6. The compound was testedagainst the MDA-MB-468 human breast carcinoma andthe H2009 human non–small cell lung carcinoma, both ofwhich have deleted Rb and exhibited no antiproliferativeactivity in these cells at concentrations as high as 3 Amol/L(Table 2), which is 1 to 2 orders of magnitude higher thanthe concentration necessary to inhibit proliferation in Rb-positive tumor cells.

Figure 1. Molecular structure of PD 0332991.

Table 1. Inhibitory activity of PD 0332991 against a panel ofprotein kinases

Protein kinase IC50 (Amol/L)*

Cdk4/cyclin D1 0.011Cdk4/cyclin D3 0.009Cdk6/cyclin D2 0.015Cdk2/cyclin E2 >10Cdk2/cyclin A >10Cdk1/cyclin B >10Cdk5/p25 >10Epidermal growth factor receptor >10Fibroblast growth factor receptor >10Platelet-derived growth factor receptor >10Insulin receptor >10Lymphocyte kinase >10Vascular endothelial growth factor receptor >10AMP-activated protein kinase >10Checkpoint kinase-1 >10Casein kinase-1 >10Casein kinase-2 >10c-Src kinase >10C-terminal Src kinase >12Dual-specificity tyrosine

phosphorylation-regulated kinase 1A2.0

Glycogen synthase kinase-3h >10c-Jun NH2-terminal kinase >10Mitogen-activated protein kinase 2/Erk2 >10Mitogen-activated

protein kinase–activated protein kinase 1a8.0

Mitogen-activatedprotein kinase–activated protein kinase 2

>10

Mitogen-activated protein kinase kinase >10Mitogen and stress-activated protein kinase 1 >10p70 Ribosomal protein S6 kinase >103-Phosphoinositide-dependent protein kinase 1 >10Phosphorylase kinase >10Cyclic AMP-dependent protein kinase >10Protein kinase B >10Protein kinase C >10p38-Regulated/activated kinase >10Rho-dependent protein kinase >10Stress-activated protein kinase 2a >10Stress-activated protein kinase 3 >10Stress-activated protein kinase 4 >10Serum and glucocorticoid induced kinase >10

*Concentration of PD 0332991 necessary to inhibit activity by 50%. Mean ofat least two separate determinations.





A selective Cdk4/6 inhibitor should cause a specificaccumulation of cells in G1 but have no effect on otherphases of the cell cycle in which cells should continue toprogress and eventually decline in number. MDA-MB-453breast carcinoma cells exposed to varying concentrationsof PD 0332991 for 24 hours showed a significant increasein the percentage of cells in G1 in the presence of as littleas 0.04 Amol/L PD 0332991 with a concomitant declinein other phases of the cell cycle (Fig. 3). Maximum ef-fects were attained at 0.08 Amol/L and an exclusive G1

arrest was maintained even at concentrations as high as10 Amol/L, consistent with the complete absence of anyother effects on the cell cycle. Similar results were obtainedwith Colo-205 cells (data not shown).

PD0332991HasBroad-SpectrumAnticancerActivityand Can Cause Regression of CertainTumors

PD 0332991 exhibits significant antitumor efficacyagainst multiple human tumor xenograft models. In micebearing Colo-205 colon carcinoma xenografts (p16 deleted),daily p.o. dosing for 14 days with PD 0332991 (150 or 75mg/kg) produced rapid tumor regressions (Fig. 4A) and acorresponding tumor growth delay of f50 days with >1log of tumor cell kill at the highest dose tested (Table 3). At37.5 mg/kg, the tumor slowly regressed during treatment.Even at doses as low as 12.5 mg/kg, a 13-day growth delaywas obtained indicating a 90% inhibition of tumor growthrate. Likewise, robust antitumor activity was seen in theMDA-MB-435 breast carcinoma (p16 deleted) where com-plete tumor stasis was apparent at 150 mg/kg (Fig. 4B) andsome cell kill was evident at the highest dose (Table 3).

PD 0332991 was efficacious against a variety of humantumor xenografts (Table 3). Similar to the Colo-205,significant tumor regression was observed in mice bearingthe SF-295 glioblastoma xenografts at doses of 150 mg/kg,and responses nearing stasis (complete suppression oftumor growth) were attained in the ZR-75-1 breast andPC-3 prostate tumor models (Table 3). Modest activity wasachieved in H125 non–small cell lung carcinoma. Thecompound was completely inactive, however, against theRb-negative MDA-MB-468 breast carcinoma, which isconsistent with inhibition of Cdk4/6 as the sole mechanismby which PD 0332991 produces antitumor activity. Alldoses reported in Table 3 were well tolerated by theanimals, with no obvious clinical signs and either minimalweight loss or some weight gain. Treated animals weremaintained for up to 50 days after dosing to facilitate theobservation of any delayed clinical toxicities. At all dosesreported, the animals succumbed to tumor-related death orwere sacrificed once their tumor burden reached f1,000mg. Based on the data shown, 150 mg/kg is defined as amaximum tolerated dose of PD 0332991 given to mice dailyfor 14 or 28 days.

Despite complete regression of certain tumors in responseto PD 0332991, the tumors eventually grew back afterthese short treatment periods. Since in recent years some

Figure 2. Inhibition of Rb phosphorylation at Ser780 by PD 0332991. A,MDA-MB-435 human breast carcinoma cells were treated for 24 hourswith varying concentrations of PD 0332991. Extracts and Western blotswere generated using Ser780 phosphospecific antibodies as described inMaterials and Methods. B, scanning densitometry values of the results inA expressed as a percentage of the control. These data were used togenerate IC50 values.

Table 2. Inhibition of thymidine incorporation into DNA in human tumor cell lines treated with PD 0332991

Cell line Cell type Rb status IC50 (Amol/L)*

MDA-MB-435 Breast carcinoma Positive 0.16ZR-75-1 Breast carcinoma Positive 0.17T-47D Breast carcinoma Positive 0.04MCF-7 Breast carcinoma Positive 0.10H1299 Lung carcinoma Positive 0.12Colo-205 Colon carcinoma Positive 0.13MDA-MB-468 Breast carcinoma Negative >3H2009 Lung carcinoma Negative >3CRRF-CEM Acute lymphoblastic leukemia Positive 0.25K562 Chronic myelogenous leukemia Positive 0.40

*Concentration of PD 0332991 necessary to inhibit cell proliferation by 50%. Mean of at least two separate determinations.





in vitro evidence has suggested that tumors may havethe potential to circumvent a Cdk4 block through activationor elevation of downstream control elements of the cellcycle (80–82), we wanted to determine if the new tumorsthat appeared in treated animals remained sensitive tofurther treatment with PD 0332991 or whether a tumorvariant had evolved exhibiting acquired resistance to thecompound. To address this possibility, Colo-205 colontumors that had emerged after substantial regression frominitial treatment with PD 0332991 in the experimentdescribed in Fig. 4A were harvested and reimplanted intonaı̈ve mice. After the tumors grew to 100 to 150 mg,these tumor-bearing mice were treated with PD 0332991using a schedule identical to the original experiment. Thetumors responded with equal sensitivity to the drugand fully regressed, indicating that no resistance haddeveloped during the initial treatment (Fig. 4C). Similarresults were obtained with MDA-MB-435 tumors (datanot shown).

PD 0332991 Causes a Sustained Suppression ofTumor Rb Phosphorylation In vivo

In parallel with the in vivo efficacy tests, additionaltumors were harvested for pharmacodynamic analysis totest if antitumor activity correlated with modulation of thetarget and to investigate whether the proposed biomarkermight predict for efficacy. Efficacious and nonefficaciousdoses of PD 0332991 were given to mice bearing the MDA-MB-435 breast carcinoma and the phosphorylation statusof Ser780 on Rb in tumor tissue was monitored over time.

Figure 3. PD 0332991 causes an exclusive G1 arrest. MDA-MB-453human breast carcinoma cells were exposed to varying concentrations ofPD 0332991 for 24 hours. Cells were harvested and fixed as described inMaterials and Methods. The DNA histograms were generated by flowcytometry and the percentage of cells in each phase of the cell cycle wasdetermined using ModFit. Additional details are given in Materials andMethods.

Figure 4. In vivo antitumor activity of PD 0332991 given p.o. A,regression of the Colo-205 human colon carcinoma. Tumor fragments(f30 mg) were implanted s.c. in SCID mice and allowed to grow to atleast 100 mg. PD 0332991 was given daily p.o. for 14 days by gavageat the indicated drug doses in lactate buffer (50 mmol/L) at pH 4.0.Untreated control (.), 12.5 mg/kg (o), 37.5 mg/kg (E), 75 mg/kg (4), and150 mg/kg (n). B, growth suppression of the MDA-MB-435 human breastcarcinoma. Tumor implantation and administration of PD 0332991 wasdone as in A, except drug was given daily for 28 days. Untreated control(.), 36 mg/kg (o), 58 mg/kg (E), 93 mg/kg (4), and 150 mg/kg (n). C,tumors retain sensitivity to PD 0332991 after extended treatment. Colo-205 tumors in A that grew back after regression during treatment wereharvested and implanted into naı̈ve mice. Details of drug treatmentare similar to A. Untreated control of tumors treated previously in A with150 mg/kg (.), tumors treated previously with 150 mg/kg and thenretreated with 150 mg/kg (4), untreated control of tumors treatedpreviously in A with 37.5 mg/kg (E), and tumors treated previously with37.5 mg/kg retreated with 150 mg/kg (o). Data in A to C are shownstarting from the first day of dosing. Statistical analysis was done asdescribed in Materials and Methods. Ps are relative to the appropriatecontrol in each case.





The results show that all doses caused a reduction in thebiomarker shortly after drug administration but thatphosphorylation returned at nonefficacious doses (12.5and 37.5 mg/kg) over the 24-hour interval before the nextdose (Fig. 5A). The highly efficacious dose of 150 mg/kgsuppressed Rb Ser780 phosphorylation over the full 24-hourperiod, implying that, with this particular breast tumor,complete suppression of the biomarker needs to bemaintained between drug doses to achieve maximumefficacy. Target modulation in this tumor by PD 0332991given p.o. was also established by immunohistochemistrywhere animals treated with an efficacious dose of PD0332991 were characterized by complete elimination ofphospho-Ser780 from their tumor (Fig. 5B). This effect alsowas associated with a substantial reduction in staining forKi-67, a well-known marker of proliferation (83). Similarexperiments with the highly sensitive Colo-205 coloncarcinoma showed that complete suppression of the bio-marker between doses is not necessary to produce growthinhibition in this tumor. However, total inhibition must bemaintained between doses to achieve tumor regression(Fig. 5C). Comparing the results of efficacy experimentsusing the highly sensitive colon tumor and the moderatelysensitive breast tumor reveals a 7- to 8-fold difference in thedose necessary to produce comparable efficacy. The phar-macodynamic results show a similar difference betweenthe two tumors in the dose required to suppress Rb Ser780

phosphorylation, indicating that modulation of this bio-marker is strongly associated with efficacy.

Down-Regulation of Genes under the Control of E2FinTumors fromMiceTreated with PD 0332991

Hypophosphorylated Rb has been proposed to inhibitcellular proliferation partly by suppressing the transcrip-tion of genes under the control of the E2F transcriptionfactors (12–15). Four E2F-regulated genes were selectedand their expression levels were monitored by reverse

transcription-PCR in Colo-205 tumors from mice treatedp.o. with PD 0332991. The genes include CDC2 that codesfor Cdk1, CCNE2 that codes for cyclin E2, TK1 that codesfor thymidine kinase, and TOP2A that codes for top-oisomerase 2A. All four genes were down-regulated in adose-dependent manner, with maximum reductions rang-ing from 13- to 87-fold (Fig. 5D). The extent of the genechanges paralleled the magnitude of the therapeuticresponse in this tumor, providing additional evidence thatPD 0332991 functions through inhibition of Cdk4/6.

DiscussionIn this report, we describe PD 0332991 as a potent andhighly selective inhibitor of Cdk4 and Cdk6 and show thatsuppression of these enzymes in human tumor xenograftsresults in significant antitumor activity. Given that a majorobstacle to establishing the usefulness of a Cdk4/6 inhibitorhas been the difficulty in obtaining a molecule with com-plete specificity for these enzymes versus other Cdks andprotein kinases, considerable effort was taken to establishthe selectivity of this compound. PD 0332991 was testedagainst 39 individual serine, threonine, and tyrosinekinases, representing most of the primary protein kinasefamilies (84). Other than Cdk4 and Cdk6, the compoundhad little or no activity against any of these enzymes. Basedon the understood role of Cdk4/6 in cell cycle progression,a specific Cdk4/6 inhibitor is predicted to produce anexclusive G1 arrest. Consistent with this expectation, cellstreated with concentrations of PD 0332991 as high as 200-fold above the IC50 for growth inhibition maintained anunequivocal G1 block as the sole perturbation in their DNAhistograms. In contrast, previously reported Cdk4/Dinhibitors have produced a G2-M block at high concen-trations (47, 52). PD 0332991 had no effect on proliferationin tumor cells that were Rb deficient at concentrations >50

Table 3. Spectrum of antitumor activity for PD 0322991 against a panel of advanced stage human tumor xenografts

Tumor* Dose (mg/kg),c MTDb

or highest dose testedDosing schedule % Weight changex T-Ck Net log10 kill{

Colo-205 colon 150b Days 18– 31 �6 48.4 +1.20MDA-MB-435 breast 150b 12– 39 + 32.7 +0.25SF-295 glioblastoma 150b 7 – 20 �11 30.1 +2.24ZR-75-1 breast 150b 7 – 20 �4 12.4 �0.06PC-3 prostate 130 8 – 21 �13 12.8 +0.02H125 lung 150b 19– 32 �12 7.3 �0.31SW-620 colon 150b 10– 23 �3 5.9 �0.55H23 lung 130 9 – 22 + 4.8 �1.07MDA-MB-468 breast (Rb negative) 150b 22– 35 �2 �4.3 —

*All tumors are Rb positive, except MDA-MB-468 in which Rb is deleted.cThe vehicle was 50 mmol/L lactate buffer at pH 4.0. PD 0332991 was given daily p.o. for 14 days, except for MDA-MB-435, which was given for 28 days.Treatments were started when the tumor weights were between 100 and 150 mg.bMTD, maximum tolerated dose.x A negative number is the percentage weight loss seen during treatment. A plus indicates a weight gain through treatment.k T-C is the difference (in days) for the treated and control tumors to reach an evaluation size of 750 mg. A T-C of 0 would indicate no effect.{Net log10 tumor cell kill represents the change in tumor burden during therapy. A negative value indicates a net increase in tumor mass during therapy,whereas a positive value indicates a net reduction in tumor burden. Values near 0 indicate tumor stasis during therapy.





times the levels that cause growth inhibition in Rb-positivecells, consistent with selective inhibition of Cdk4/6.Initiation of the G1 block, emergence of antiproliferativeactivity, and dephosphorylation of Rb at the Cdk4/6-specific site of Ser780 all occurred at similar concentrationsof PD 0332991. Collectively, these data show that thebiochemical activities and biological effects of PD 0332991are in accord with exclusive inhibition of its target enzymesand suggest a high degree of selectivity in the cellularenvironment.

Oral administration of PD 0332991 to mice bearinghuman tumor xenografts clearly showed that inhibition ofCdk4/6 can result in a significant antitumor effect. Thespectrum of responses ranged from partial growth inhibi-tion and tumor stasis to complete regressions, dependingon the tumor, with a therapeutic index as high as 12 in the

Colo-205 model. [The therapeutic index is defined as themaximum tolerated dose divided by the lowest dose to givea tumor growth delay of >50%.] The anticancer activity wasassociated with biochemical changes that are consistentwith the mechanism of action for PD 0332991. Thus,significant antitumor activity was detected only at doseswhere there was a sustained and significant reduction ofphospho-Ser780 on Rb. The decline in Rb Ser780 phosphor-ylation in tumor tissue was confirmed by immunohisto-chemistry techniques, which showed substantial reductionof this marker in tumors from treated animals. Moreover,there was a concomitant elimination of staining for Ki-67, acommonly measured marker of proliferation (83). Further-more, reverse transcription-PCR of selected genes in tumortissue known to be under the control of E2F and down-

regulated in cells expressing a nonphosphorylatable Rb (85)

Figure 5. Therapeutically active doses of PD 0332991 in vivo cause down-regulation of proteins and genes in tumor tissue that are consistent withinhibition of Cdk4/6. A to D, designated tumors were implanted into nude mice as described in Fig. 4 and allowed to grow to f200 mg. Mice were doseddaily for 2 days and tumor tissue was harvested at the designated times after the last dose and processed as described in the individual panels.A, inhibitionof Rb Ser780 phosphorylation in MDA-MB-435 human breast carcinomas in mice treated with PD 0332991. Tumors were excised at the designated timesafter the last dose of drug and snap frozen in liquid nitrogen. Extracts of the tissue and Western blots were done as described in Materials and Methods. B,inhibition of Rb Ser780 phosphorylation in MDA-MB-435 human breast carcinoma and suppression of Ki-67 as visualized by immunohistochemistry. Tumorswere identical toA, except that part of the tumor was fixed in formaldehyde and processed for staining as described in Materials and Methods.C, inhibitionof Rb Ser780 phosphorylation in Colo-205 human colon carcinoma in mice treated with PD 0332991. Tumors were excised at the designated times after thelast dose of drug and snap frozen in liquid nitrogen. Extracts of the tissue and Western blots were done as described in Materials and Methods. D, down-regulation of selected genes under the transcriptional control of E2F in the Colo-205 human colon carcinoma. Tumors were excised 3 hours after the lastdose of drug and snap frozen in liquid nitrogen. RNA was isolated and relative gene expression levels were determined by reverse transcription-PCR asdescribed in Materials and Methods. TK1 , thymidine kinase (.); CDC2 , Cdk1 (E); TOP2A , topoisomerase 2A (n); and CCNE2, cyclin E2 (!).





showed a 13- to 87-fold reduction in expression in tumors

from animals treated with therapeutically active doses of PD

0332991. Finally, PD 0332991 was completely inactive

against Rb-negative tumor xenografts, which is expected

based on its proposed mechanism of action. Taken together,

these in vivo data are consistent with inhibition of Cdk4/6

as the sole basis for the observed antitumor activity.The results of this study address two issues that have

been raised in the past concerning inhibitors of Cdk4/6 astherapeutic agents for oncology, the first being an assump-tion that inhibition of Cdk4/6 may result in a cytostaticphenotype, causing little more than temporary tumorgrowth arrest. Indeed, cytostasis was the phenotypeobserved in tissue culture experiments in vitro, whereincells exposed to PD 0332991 ceased proliferating but didnot die even after prolonged exposure to the compound.In vivo, however, we show that inhibition of Cdk4/6 notonly produces a robust growth suppressive effect in manyhuman tumor xenografts but also is capable of causingcomplete regressions in some tumors. Secondly, based onresults with in vitro culture models, it has been suggestedthat tumors might be able to circumvent the antiprolifer-ative effects of Cdk4/6 inhibition through activation orelevation of downstream control elements of the cell cyclesuch as Cdk2/cyclin E activity or c-myc (80–82). The resultsof this study not only show a robust and sustainedtherapeutic response to Cdk4/6 inhibition but also showthat tumors do not readily develop an ability to overcome acell cycle block incurred through inhibition of Cdk4/6.Colo-205 human tumor xenografts treated with PD 0332991for 14 days and then allowed to regrow over several weeksdo not develop resistance and remain sensitive to inhibitionof Cdk4/6. These results complement recent data suggest-ing that Cdk2 may not be generally required for prolifer-ation in mammalian cells and that Cdk4/6 alone might besufficient to regulate the G1-S phase transition (61). In fact,Cdk2 knockout mice have been reported to be entirelyviable (86). Taken together, the results support Cdk4/6 as arelevant target for cancer chemotherapy.

We can only speculate on how an inhibitor that appearscytostatic in vitro can cause tumor regressions in vivo. Onepossibility is that Cdk4/6 activity is required for tumorsurvival. The fact that Cdk4 disruption renders MEF cellsresistant to transformation by certain oncogenes (87) impliesthat the transformed phenotype requires Cdk4 activity.Furthermore, cyclin D1 is a true oncogene capable oftransforming cells itself, and p16, a natural inhibitor ofCdk4 that is commonly absent or inactivated in tumors,has all the characteristics of a tumor suppressor. Both ofthese observations imply that Cdk4 activity is a criticalcomponent of oncogenesis in certain tumors. Anotherpotential explanation for the observed tumor regressionsmight be that most solid tumors contain a significantpopulation of spontaneously dying cells in addition to aproliferating fraction, with the latter having a slightadvantage in an expanding tumor mass. If proliferationwere slowed to the point that the naturally dying population

was dominant, the net result would be regression of thetumor. Because Rb interacts with well over 100 proteins ofvarious function (88) and because the response to PD0332991 is tumor specific, sensitivity is likely governed bymultiple factors.

In conclusion, the striking therapeutic response toinhibition of Cdk4/6 observed in the current study makesthis approach attractive for clinical application because aninhibitor of Cdk4/6 could have the potential to cause tumorregressions or cures as a single agent. Pragmatically,conventional clinical means of assessing antitumor activityshould be appropriate for PD 0332991 and mechanisticallyrelated compounds. Furthermore, we have shown thatseveral robust biomarkers are measurable by immunohisto-chemistry and could possibly be used to detect drug-relatedtarget modulation in patients or perhaps eventually topredict therapeutic response. PD 0332991 is scheduled forclinical trials in 2004 and should help provide an answerto whether selective inhibitors of Cdk4/6 can provide atherapeutic benefit in cancer patients.

Acknowledgments

We thank Joseph Repine, Hairong Zhou, Dennis McNamara, John Quin,Cathlin Flamme, Michael Waldo, and Vladimir Beylin for contributions andAlexander Bridges, Seth Sadis, and Ellen Dobrusin for support.

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2004;3:1427-1438. Mol Cancer Ther David W. Fry, Patricia J. Harvey, Paul R. Keller, et al. xenografts0332991 and associated antitumor activity in human tumor Specific inhibition of cyclin-dependent kinase 4/6 by PD

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Specific inhibition of cyclin-dependent kinase 4/6 by PD ... · Specific inhibition of cyclin-dependent kinase 4/6 by PD 0332991 and associated antitumor activity in human tumor xenografts