The P16 Status of Tumor Cell Lines Identifies Small Molecule Inhibitors Specific for Cyclin-Dependent Kinase 41

Vol. 5, 4279–4286, December 1999 Clinical Cancer Research 4279

The p16 Status of Tumor Cell Lines Identifies Small Molecule Inhibitors Specific for Cyclin-dependent Kinase 41

Akihito Kubo,2 Kazuhiko Nakagawa,2, 3 CDK4 kinase inhibitors that may selectively induce growth Ravi K. Varma, Nicholas K. Conrad, inhibition of p16-altered tumors. Jin Quan Cheng, Wen-Ching Lee, INTRODUCTION Joseph R. Testa, Bruce E. Johnson, INK4A 4 The p16 gene (also known as CDKN2A) encodes p16 , Frederic J. Kaye, and Michael J. Kelley which inhibits the CDK45:cyclin D and CDK6:cyclin D com- Medicine Branch [A. K., K. N., N. K. C., F. J. K., B. E. J.] and plexes (1). These complexes mediate phosphorylation of the Rb Developmental Therapeutics Program [R. K. V.], National Cancer Institute, Bethesda, Maryland 20889; Department of Medical protein and allow cell cycle progression beyond the G1-S-phase Oncology, Fox Chase Cancer Center, Philadelphia, Pennsylvania checkpoint (2). Alterations of p16 have been described in a wide 19111 [J. Q. C., W-C. L., J. R. T.]; and Department of Medicine, variety of histological types of human cancers including astro- Duke University Medical Center, Durham, North Carolina 27710 cytoma, melanoma, leukemia, breast cancer, head and neck [M. J. K.] squamous cell carcinoma, malignant mesothelioma, and lung cancer. Alterations of p16 can occur through homozygous de- ABSTRACT letion, point mutation, and transcriptional suppression associated with hypermethylation in cancer cell lines and primary Loss of p16 functional activity leading to disruption of tumors (reviewed in Refs. 3–5). the p16/cyclin-dependent kinase (CDK) 4:cyclin D/retino- Whereas the Rb gene is inactivated in a narrow range of blastoma pathway is the most common event in human tumor cells, the pattern of mutational inactivation of Rb is tumorigenesis, suggesting that compounds with CDK4 ki- inversely correlated with p16 alterations (6–8), suggesting that nase inhibitory activity may be useful to regulate cancer cell a single defect in the p16/CDK4:cyclin D/Rb pathway is suffi- growth. To identify such inhibitors, the 60 cancer cell lines cient for tumorigenesis. Genetic alteration or overexpression of of the National Cancer Institute drug screen panel were CDK4 and cyclin D1 has also been observed in various tumor examined for p16 alterations (biallelic deletion, intragenic cells, which supports the model that all tumor cells must cir- mutations, or absent p16 protein), and the growth-inhibitory cumvent this tumor suppressor pathway (1, 9). activity of more than 50,000 compounds against these 60 cell Transfection of the p16 gene into cultured cell lines with lines was compared with their p16 status. One compound, p16 alterations (biallelic deletion or transcriptional suppression) 3-amino thioacridone (3-ATA; NSC 680434), whose growth- causes G arrest and growth suppression in a range of tumor cell inhibitory activity correlated with the p16 status of the cell 1 types including osteosarcoma, esophageal carcinoma, mesothe- lines had an IC of 3.1 ␮M in a CDK4 kinase assay. In 50 lioma, and head and neck squamous carcinoma, whereas trans- addition, four compounds structurally related to 3-ATA ␮ fection of this gene does not induce G1 arrest in Rb-negative inhibited CDK4 kinase with IC50s ranging from 0.2–2.0 M. All five of these compounds were less potent inhibitors of cell cells (10–12). In addition, p16 expression mediated by an adenovirus vector induces G1 arrest and inhibits tumor cell prolif- division cycle 2 and CDK2 kinases, with IC50s 30- to 500-fold higher than that for CDK4. ATP competition experiments eration in NSCLC cell lines with homozygous deletion of the demonstrated a noncompetitive mode of inhibition for p16 gene, but not in NSCLC cell lines expressing functional p16INK4A (13). These data suggest that an agent possessing -␮M) and a linear mixed mode for benzo 5.5 ؍ 3-ATA (K i p16-like inhibitory activity against the CDK4:cyclin D kinase -␮M). We have success 0.73 ؍ thiadiazine (NSC 645787; K i complex might have selective antitumor activity in patients with fully demonstrated a novel approach to identify specific p16-altered tumors. The CDK4 kinase is a member of the evolutionarily con- served family of CDKs, which includes CDC2 and CDK2. Received 10/15/97; revised 7/21/99; accepted 8/30/99. However, alterations of CDC2, CDK2, and their associated The costs of publication of this article were defrayed in part by the cyclins and inhibitors are not common in human cancers (14). payment of page charges. This article must therefore be hereby marked The frequent defects in p16 with deregulated CDK4 activity advertisement in accordance with 18 U.S.C. Section 1734 solely to suggest that pharmacological inhibitors specific for CDK4 may indicate this fact. 1 A portion of this work was supported by Subcontract #6S-1602 from Program Research, Inc.-National Cancer Institute Frederick Cancer Re- search and Development Center. 2 A. K. and K. N. contributed equally to this work. 3 Present address: The Fourth Department of Internal Medicine, Kinki 5 The abbreviations used are: CDK, cyclin-dependent kinase; Rb, reti- University School of Medicine, Osaka-Sayama 589, Japan. noblastoma; NSCLC, non-small cell lung cancer; CDC, cell division 4 To whom requests for reprints should be addressed, at Hematology/ cycle; NCI, National Cancer Institute; SSCP, single-strand conformation Oncology (111G); Duke University/VA Hospital; 508 Fulton Street, polymorphism; GST, glutathione S-transferase; 3-ATA, 3-amino-9-

Durham, NC 27705. Phone: (919) 286-0411, ext. 7331; Fax: (919) thio(10H)-acridone; BTD, benzothiadiazine; GI50, 50% growth inhibi- 286-6896; E-mail: [email protected]. tion.

be more promising as anticancer agents than nonspecific CDK 5% powdered milk, and 1% BSA). The membranes were then inhibitors. To date, several families of chemical inhibitors with incubated with a mixture of 40 ␮lof125I-protein A (Ͼ30 specificity against different CDK activities have been described mCi/mg) in 20 ml of blocking solution and subjected to auto- (15–17), all of which are ATP competitors (15, 16, 18). In radiography. addition, selective peptide inhibitors of CDK2 and CDK4 have COMPARE Analysis. The COMPARE algorithm was been synthesized and evaluated (19, 20). However, no chemical performed as described previously (25, 26). For the identifica- inhibitors specific for CDK4/CDK6 have been reported. tion of agents with differential activity, GI50s of 0 and 1 were We hypothesized that a small molecule with specific in- used for p16-normal and for p16-altered cell lines, respectively. hibitory activity for CDK4:cyclin D kinase would induce greater p16-altered cell lines were those with biallelic deletion, intra- growth suppression among p16-altered cell lines than among genic mutation, or transcriptional suppression of p16, and p16- p16-normal cell lines. The NCI drug screen program has deter- normal cell lines were those without these abnormalities. Pear- mined the growth-inhibitory properties of over 50,000 com- son correlation coefficients were calculated by the SAS pounds of diverse molecular structure against 60 human tumor procedure PROC CORR (SAS Institute, Inc., Cary, NC). cell lines of nine histological groups (21). We determined the Production and Purification of CDKs. Active CDK: p16 status of these 60 cell lines to identify pharmacological cyclin complexes were produced in Sf9 cells coinfected with agents that preferentially inhibited the growth of p16-altered cell baculoviruses encoding human CDK (CDK1, CDK2, or CDK4) lines. Using this method, we identified several novel CDK4 or cyclin (cyclin A, cyclin D1, or cyclin E) gene (the generous inhibitors, some of which exhibit marked selectivity for CDK4 gifts of D. Beach) at a multiplicity of infection of 10, and cell kinase as compared to CDC2 and CDK2 kinases. lysates were prepared as described previously (27, 28). CDK: cyclin complexes were purified by immunoprecipitation using each cyclin antibody (cyclin A, BF-683; cyclin D1, M-20; MATERIALS AND METHODS cyclin E, C-19; Santa Cruz Biotechnology). The purity of im- Cell Lines, Compounds, and in Vitro Sensitivity Test- munoprecipitated complexes was estimated by silver staining ing. Exponentially growing cultures of the 60 cell lines used in and Coomassie Blue staining, followed by Western blotting. the NCI drug screen panel (21) were generously provided by Dr. The concentration of CDK subunit in the holoenzyme immuno- A. Monks. All compounds were obtained from the Drug Syn- precipitated by each cyclin antibody was estimated by Coomas- thesis and Chemistry Branch, NCI. In vitro antitumor activity sie Blue staining of electrophoretically separated proteins in

(GI50) of compounds was determined as described previously in comparison to protein standards of known concentrations (29). the 60 human cancer cell lines of the NCI drug screen panel After quantitation of the CDK subunits, CDK:cyclin complexes (21). Compounds structurally related to 3-ATA (NSC 680434) were titrated for Rb kinase activity using 300 ng of GST-Rb to or BTD (NSC 645787) were selected by a substructure search of determine the optimal amount of the enzyme for each reaction. the entire NCI database (approximately 500,000 compounds) for The estimated amount of CDK used in each assay was 25, 20, structures with two benzene rings fused to a middle ring of any 16, and 60 ng for CDK1:cyclin A, CDK2:cyclin A, CDK2: size or with a sulfone similar to BTD, respectively. This sub- cyclin E, and CDK4:cyclin D1, respectively. structure search identified 77 structurally related compounds; 45 CDK Inhibition Assays. Crude lysate (5 ␮l) containing of these 77 compounds (24 for 3-ATA and 21 for BTD) were CDK:cyclin or the optimized amount of purified CDK:cyclin available for in vitro kinase assay. These 45 compounds have complexes was mixed with test compounds in 30 ␮l of kinase

not been tested for growth-inhibitory activity in the NCI drug buffer [20 mM Tris-HCl (pH 8.0), 10 mM MgCl2,and1mM screen panel. EGTA] and incubated at 30°C for 30 min. The kinase reaction Analysis of the p16 Status of the 60 Cell Lines of the was started by adding 300 ng of GST-Rb protein and 5 ␮Ci of NCI Drug Screen Panel. PCR-SSCP, DNA sequencing, and [␥-32P]ATP to the mixture and incubation at 30°C for 30 min reverse transcription-PCR analysis of p16 in the 60 cell lines of (30). Reactions were stopped by adding 7.5 ␮lof5ϫ SDS the NCI drug screen panel were performed as described previ- sample buffer [312.5 mM Tris-HCl (pH 6.8), 50% glycerol, 10% ously (22, 23). For Southern blot hybridization analysis, reverse SDS, 12.5% 2-mercaptoethanol, and 0.0125% bromphenol transcription-PCR products were separated by agarose gel elec- blue]; samples were separated on 8–16% Tris-glycine denatur- tropheresis, transferred to a nylon membrane, and hybridized ing gels (Novex), and radioactivity incorporated into labeled with a 388-bp p16 exon 1 genomic fragment defined by oligo- substrate was measured by liquid scintillation of the excised nucleotides 2F and 1108R (24). Expression of the glyceralde- bands of GST-Rb. To examine the effect of compounds on the hyde-3-phosphate dehydrogenase gene (GAPDH) was examined initial velocity of the enzyme, reactions were performed for 5 to assure the presence of intact mRNA in each sample. For min without preincubation. To assess the effect of ATP on the immunoblot analysis, 1 ϫ 107 cells were washed with PBS, inhibitory effect of a compound, 20–100 ␮Ci of [␥-32P]ATP resuspended in 0.4 ml of lysis buffer [50 mM Tris-HCl (pH 7.4), were added to the reaction containing 3–200 ␮M ATP. 35 250 mM NaCl, 5 mM EDTA, 0.1% NP40, 50 mM NaF, and 1 mM CDK4 Binding Assays. In vitro-translated, S-labeled phenylmethylsulfonyl fluoride], and centrifuged at 14,000 rpm CDK4 and cyclin D1 were synthesized using plasmids contain- for 20 min at 4°C. Total protein (50 ␮g) was subjected to ing the human CDK4 gene or cyclin D1 gene, a coupled tran- SDS-PAGE, followed by electroblotting to nitrocellulose. The scription-translation system (TNT lysate; Promega), and nitrocellulose membranes were incubated overnight at 4°C with [35S]methionine (Amersham). For p16 binding, 1 ␮g of each a 1:1000 dilution of polyclonal antihuman p16 antiserum GST-p16 or GST protein was mixed with 5 ␮lofin vitro- (PharMingen, San Diego, CA) in blocking solution (1ϫ PBS, translated CDK4 in 100 ␮l of kinase buffer. After incubation at

Table 1 p16 status in the 60 tumor cell lines of the NCI drug screen panel p16-altered cells (47/60) p16 wild-type cells (13/60) Cell line Alterationa Cell line Alterationa Cell line Alterationa Cell line CCRF-CEM HD SK-MEL-5 HD HL-60 PM2 NCI-H522 K-562 HD UACC-62 HD HCT-116 PM3 HCC-2998 MOLT-4 HD OVCAR-5 HD UACC-257 PM4 SF-539 SR HD SK-OV-3 HD DU-145 PM5 SNB-75 A549 HD 786-0 HD RPMI-8226 TD SK-MEL-2 HOP-62 HD A498 HD EKVX TD SK-MEL-28 HOP-92 HD ACHN HD NCI-H23 TD IGROV1 NCI-H226 HD CAKI-1 HD COLO205 TD OVCAR-3 NCI-H322M HD RXF-393 HD HCT-15 TD OVCAR-4 NCI-H460 HD UO-31 HD HT29 TD OVCAR-8 SF-268 HD MCF7 HD KM12 TD SN12C SF-295 HD MDA-MB-231 HD SW-620 TD MCF/ADR-RES SNB-19 HD HS 578T HD TK-10 TD BT-549 U251 HD M14 PM1 PC-3 TD LOX IMVI HD MDA-MB-435 PM1 T-47D TD MALME-3M HD MDA-MB-N PM1 a HD, homozygous deletion; PM, point mutation; PM1, second nucleotide of the first intron (AGgt-AGgc); PM2, premature termination at codon 80 (CGA-TGA); PM3, 1-bp insertion at codon 22; PM4, proline to leucine substitution at codon 81 (CCC-CTC); PM5, aspartate to tyrosine substitution at codon 84 (GAC-TAC); TD, transcriptional defect.

30°C for 30 min, GST fusion proteins were separated by glutathione-Sepharose, resolved on an 8–16% Tris-glycine gel, and stained with Coomassie Blue to observe the recovery of GST- p16 fusions from each binding reaction. Quantitation of the binding reactions was then carried out by phosphorimaging. To test the effect of compounds on CDK4 binding of wild-type p16, up to 300 ␮M of each compound was premixed with CDK4 before adding GST-p16 protein. To examine the compound effect on CDK4:cyclin D1 binding, in vitro-translated cyclin D1, instead of GST-p16, was added and incubated at 30°C for 30 min, and then cyclin D1 was recovered by immunoprecipitation using cyclin D1 antibody (M-20; Santa Cruz Biotechnol- ogy). Fig. 1 Impaired CDK4 binding of p16 mutants. Five ␮l of a CDK4 in vitro translation reaction were mixed with 1 ␮g of GST-p16 wild type RESULTS or mutant [splice site (I1ϩ2T-C) and two missense mutations (P81L and Characterization of the p16 Status of the Cell Lines of D84Y)]. GST fusion proteins were recovered with glutathione-Sepha- the NCI Drug Screen Panel. The 60 cell lines of the NCI rose and separated on an 8–16% denaturing polyacrylamide gel, and 35S drug screen panel were examined for alterations of p16. To signal of CDK4 was quantified by autoradiography using a phospho- detect genetic alterations, PCR-SSCP analysis was performed rimager. Values are the means of triplicate assays. Before autoradiography, equal recovery of GST fusion proteins was confirmed by Coo- for exons 1 and 2 of the p16 gene using genomic DNA (data not massie Blue staining (data not shown). shown). Of the 60 cell lines, 29 were found to lack amplifiable genomic sequences of one or both exons, indicative of a biallelic deletion involving p16 (Table 1). Seven of the 60 cell lines contained abnormally migrating SSCP bands on repeated anal- ther p16 mRNA nor protein (Table 1). In total, 47 of the 60 yses that were not previously reported polymorphisms by DNA (78%) cell lines of the NCI drug screen panel had an alteration sequence analysis. The functional effects of these sequence of p16 (Table 1). variants were assessed by measuring the binding of GST-p16 Identification of CDK4 Inhibitors. To identify com- fusion proteins to CDK4. Binding of mutant GST-p16 fusion pounds that are selectively cytotoxic or cytostatic for p16- proteins (I1ϩ2T-C, P81L, and D84Y) to CDK4 was 13%, 14%, altered cells compared to p16-normal cells, the p16 status of the and 13% of the binding ability of normal p16, respectively (Fig. 60 cell lines was matched to the GI50 of the compounds of the 1). Thus, 36 of 60 (60%) cell lines of the NCI drug screen panel NCI drug screen program and ranked according to Pearson contained a genetic alteration (homozygous deletion or intra- correlation coefficients using the COMPARE algorithm (25, INK4A genic mutation) of p16 that disrupted the function of p16 26). The GI50 of cephalostatin 1, a disteroidal alkaloid extracted (Table 1). To detect epigenetic alterations associated with loss from the marine worm Cephalodiscus gilchristi (31), correlated ϭ of p16 function, p16 mRNA and protein expression were ex- best with p16 status (r 0.599; Table 2). The GI50s of five amined, which revealed 11 additional cell lines expressing nei- related compounds (cephalostatins 7, 9, 8, 4, and 3) were also

Table 2 Candidate kinase inhibitors of CDK4:cyclin D complexes by COMPARE analysis Pearson corr. Classification CDK4 inhibition a b ␮ Rank NSC no. Chemical name coeff. of drugs IC50 ( M) 1 363979 Cephalostatin 1 0.599 Disteroidal alkaloid 20 2 D1 0.571 3 680434 3-Amino-9-thio(10H)-acridone 0.555 Acridone 6.8 4 378736 Cephalostatin 7 0.504 Disteroidal alkaloid NAc 5 D2 0.496 6 378735 Cephalostatin 9 0.493 Disteroidal alkaloid NA 7 378734 Cephalostatin 8 0.491 Disteroidal alkaloid NA 8 650931 2Ј-Bromo-4Ј-epi-daunorubicin 0.488 Anthracyclin NA 9 629487 7-Phenyl-5H-pyrazolo[3,4-e]-1,3,4-Triazin-3-amine 0.485 Pyrazolotriazine Ͼ100 10 674107 2-Acetylimidazo[4,5-b]pyridin-4-Benzyl-3-thiosemicarbazone 0.482 Thiosemicarbazone Ͼ100 11 D3 0.482 12 339555 Bryostatin 1 0.469 Macrocyclic lactone Ͼ100 13 D4 0.462 14 D5 0.462 15 378727 Cephalostatin 4 0.461 Disteroidal alkaloid NA 16 363981 Cephalostatin 3 0.458 Disteroidal alkaloid NA a D1–D5, discrete compounds are provided to the NCI under the terms of a confidentiality agreement. b Corr. coeff., correlation coefficient. c NA, compound not available.

positively correlated with p16 status (r ϭ 0.504, 0.493, 0.491, 100 ␮M using crude Sf9 cell lysate containing CDK4:cyclin D1. 0.461, and 0.458, respectively; Table 2). Bryostatin 1, a protein These candidate compounds for CDK4-specific inhibitors were kinase C activator isolated from the marine bryozoan Bugula tested for CDK inhibitory activity using purified enzymes. neritina (32), had a correlation coefficient of 0.469 (Table 2). Characterization of CDK4 Inhibitors. To remove other Aliquots of 26 of the 40 compounds with the highest factors in crude lysates that may affect the enzymatic activity or Pearson correlation rankings were available for biochemical the effect of inhibitor compounds, we purified baculovirus- analysis. These compounds were assessed for CDK4:cylin D expressed CDK:cyclin complexes by immunoprecipitation. In kinase inhibitory activity using crude Sf9 insect cell lysate addition, we tested flavopiridol (NSC 649890) to compare its containing baculovirus-expressed CDK4:cyclin D1 and a inhibitory kinetics with those of the novel compounds we iden- GST-Rb fusion protein as substrate. Six of the 26 compounds tified. Flavopiridol showed the most potent inhibition in the examined inhibited CDK4:cyclin D1-mediated phosphorylation compounds we tested on CDK4:cyclin D1, CDC2:cyclin A,

of Rb protein with IC50 (50% kinase inhibition) values ranging CDK2:cyclin A, and CDK2:cyclin E, with IC50s of 0.14, 0.1, from 6.8 to more than 100 ␮M (Table 2). No inhibition of 0.08, and 0.32 ␮M, respectively (Fig. 2A). The mechanism of GST-Rb phosphorylation by CDK4:cyclin D1 was observed in inhibition of flavopiridol on CDK4:cyclin D1 is proposed to be the presence of the other 20 compounds at concentrations of up mediated by competition with ATP (Fig. 2B), as described ␮ to 100 M. The most potent inhibitor was 3-ATA, with an IC50 previously on CDC2 kinase (15). 3-ATA, BTD, and compounds of 6.8 ␮M, which shows moderate growth-inhibitory activity structurally related to 3-ATA (NSC 625987, NSC 645153, and ␮ with a mean GI50 of 30 M in the 2-day growth assay of the NCI NSC 521164) inhibited immunopurified CDK4:cyclin D1 with ␮ drug screen. Cephalostatin 1, which has potent antitumor activ- IC50s ranging from 0.2–3.1 M. These five compounds were Ϫ7 Ϫ9 ␮ ity in vitro (ED50,10 to 10 g/ml; Ref. 31), had an IC50 for significantly less potent inhibitors of CDC2:cyclin A, CDK2: ␮ CDK4:cyclin D1 of 20 M, and bryostatin 1 had no inhibitory cyclin A, and CDK2:cyclin E ,with IC50s at least 30-fold higher activity at the highest concentration examined (Table 2). compared to the IC50s for CDK4:cyclin D1 (Table 3, Fig. 2A). To identify compounds in the NCI drug screen that may Kinetic studies using purified CDK4:cyclin D1 and GST-Rb have a mechanism of action similar to that of 3-ATA, we protein as a specific substrate showed that 3-ATA does not

compared the pattern of the GI50 of 3-ATA with the GI50 of all compete with ATP for the inhibition of CDK4 kinase activity compounds tested previously. Six compounds not examined and that BTD has a mixed pattern of inhibition with respect to

previously for CDK4 kinase inhibitory activity had similar ATP (Fig. 2B). The Ki values of 3-ATA, BTD, and flavopiridol patterns of growth-inhibitory activity, with correlation coeffi- on CDK4 kinase against ATP were calculated to be 5.5, 0.73, cients greater than 0.6. Among these six compounds, two BTD and 0.076 ␮M, respectively. To assess the effect of the com- compounds (NSC 645787 and NSC 645788) inhibited CDK4: pounds (3-ATA, BTD, NSC 625987, NSC 645153, NSC ␮ cyclin D1 kinase activity in vitro with IC50s of 5.0 and 17 M, 521164, and flavopiridol) on the binding of CDK4 to p16 or respectively. cyclin D1, binding assays using in vitro-translated CDK4 and Forty-five additional compounds with structural similarity bacterially expressed GST-p16 or in vitro-translated cyclin D1 to 3-ATA and BTD were analyzed to identify additional CDK4- were performed in the presence of these compounds. No inhi- specific inhibitors and obtain preliminary structure-activity re- bition of CDK4 binding to GST-p16 was observed in the pres- lationship information. Nineteen of these compounds inhibited ence of up to 300 ␮M of these compounds (data not shown). For

CDK4 kinase activity with IC50s ranging from 1.1 to more than CDK4-cyclin D1 binding, two compounds (BTD and NSC

Fig. 2 Inhibitory kinetics of 3-ATA, BTD, and flavopiridol on CDKS. A, inhibition of purified CDK4:cyclin D1 (f), CDC2:cyclin A (Ⅺ), CDK2:cyclin A (F), and CDK2:cyclin E (E)by 3-ATA, BTD, and flavopiridol. Values are the means of at least three replicate assays. B, Lin- eweaver-Burk plots for purified CDK4:cyclin D1 in the presence of 300 ng of GST-Rb protein, varied concentrations of ATP, and (a) 3-ATA at 0(Ⅺ),3(f),6(E), and 10 ␮M (F); (b) BTD at 0(Ⅺ), 0.5 (f),1(E), and 2 (F); and (c) flavopiridol at 0 (Ⅺ) and 0.1 ␮M (f). Values are the means of duplicate assays. See the text for ex- perimental conditions.

625987) inhibited CDK4:cyclin D1 binding only at concentra- tors of CDK4 kinase activity. From among the large number of tions of at least 300 ␮M (data not shown), which is nearly 3 logs compounds of diverse molecular structure in this database, we higher than their IC50s on CDK4 kinase inhibitory assay. selected for further study compounds with greater growth inhibitory activity against p16-altered cells than against p16- DISCUSSION normal cells. Genetic alterations of the p16 gene in primary tumors, Using this approach and further biochemical analyses, we including homozygous deletions and intragenic mutations, are identified five compounds (3-ATA, NSC 625987, NSC 645153, observed in 35% of NSCLC and 60% of glioma, head and neck NSC 521164, and BTD) that inhibit CDK4 kinase activity in squamous carcinoma, and pancreatic cancer (reviewed in Ref. vitro, with an IC50 of 30-fold to more than 500-fold lower than

4). In addition, hypermethylation of the 5Ј CpG island of the p16 the IC50 required to inhibit CDC2 and CDK2 kinases. The gene, correlating with complete transcriptional suppression, is inhibitory activity of the parent compound, 3-ATA, was not observed in 25% of NSCLC, 31% of breast cancer, and 40% of attenuated with increasing concentrations of ATP, unlike other colon cancer (reviewed in Ref. 5). Thus, inactivation of the p16 chemical inhibitors of CDKs described to date (15–18). How- gene is a frequent event in various histological types of primary ever, BTD, which has a growth-inhibitory pattern similar to that tumors and tumor cell lines. of 3-ATA, inhibited CDK4 in a linear mixed fashion with The observation of growth suppression after expression of respect to ATP. Because the ATP-binding pocket of CDKs is p16INK4A in tumor cells with deletion of the p16 gene (13, 33) likely to accommodate various structures (34), BTD might par- has suggested that inhibition of CDK4 kinase activity may be a tially compete with ATP by binding to the ATP-binding pocket useful therapeutic strategy for patients whose tumors have p16 or by interfering with ATP binding. defects. We used the existing database of the NCI drug screen These five compounds, as well as flavopiridol, did not program (21, 25) to identify potential pharmacological inhibi- affect p16 binding to CDK4 in vitro, suggesting that the mech-

anism of CDK4 inhibition by these compounds is not mimicking Through COMPARE analysis and biochemical screening, the tumor suppressor p16. In addition, only two compounds the cephalostatins were also found to have greater growth- (BTD and NSC 625987) inhibited in vitro cyclin D1 binding to inhibitory activity against p16-altered cells than against p16-

CDK4 at nearly 1000-fold higher concentration than their IC50 normal cells. However, CDK4 kinase inhibitory activity by on CDK4 kinase. This observation suggests that direct inhibition cephalostatin 1 occurs at concentrations at least 1000-fold

of cyclin D1 binding to CDK4 is not the central mechanism of higher than the GI50 of cephalostatin 1, suggesting that the inhibition of CDK4 kinase activity by these compounds. INK4 growth-inhibitory activity of the cephalostatins is not predomi- inhibitors bind next to the ATP binding site of the catalytic cleft nantly due to CDK4 kinase inhibition. Our analysis also iden- of CDK6 and interfere with ATP binding by causing conforma- tified bryostatin 1 as being more active against p16-altered cells tional changes (35, 36). Our data suggest that there may be than p16-normal cells. However, there was no direct inhibition additional mechanisms mediating specific CDK4 inhibition. of CDK4 kinase activity in vitro. The addition of bryostatin 1 to

cells has been shown to result in decreased CDK2 activity, cycle inhibition by the tumour suppressor p16. Nature (Lond.), 375: which is due, at least in part, to dephosphorylation of CDK2 503–506, 1995. (37). Bryostatin 1 may similarly decrease CDK4 activity 10. Guan, K. L., Jenkins, C. W., Li, Y., Nichols, M. A., Wu, X., through dephosphorylation of CDK4, thus explaining its greater O’Keefe, C. L., Matera, A. G., and Xiong, Y. Growth suppression by p18, a p16INK4/MTS1- and p14INK4B/MTS2-related CDK6 inhibitor, activity against p16-altered cells. correlates with wild-type pRb function. Genes Dev., 8: 2939–2952, The therapeutic index of agents in vivo is thought to be 1994. related to the specificity of their actions on molecular targets. In 11. Okamoto, A., Demetrick, D. J., Spillare, E. A., Hagiwara, K., our initial attempt to improve upon the approximately 10-fold Hussain, S. P., Bennett, W. P., Forrester, K., Gerwin, B., Serrano, M., specificity of 3-ATA for CDK4 compared to CDC2 and CDK2, Beach, D. H., and Harris, C. C. Mutations and altered expression of we were able to identify compounds structurally related to p16INK4 in human cancer. Proc. Natl. Acad. Sci. USA, 91: 11045– 3-ATA with at least 100-fold higher specificity for CDK4 than 1109, 1994. for CDC2 and CDK2. These compounds may provide a step to 12. Liggett, W. H., Jr., Sewell, D. A., Rocco, J., Ahrendt, S. A., Koch, W., and Sidransky, D. p16 and p16 ␤ are potent growth suppressors of develop structure-based chemical libraries to identify or synthe- head and neck squamous carcinoma cells in vitro. Cancer Res., 56: size more potent inhibitors of CDK4 kinase activity. Also, 4119–4123, 1996. determination of the crystal structure of CDK4 or CDK6 bound 13. Jin, X., Nguyen, D., Zhang, W. W., Kyritsis, A. P., and Roth, J. A. to these compounds will allow us to better understand the Cell cycle arrest and inhibition of tumor cell proliferation by the mechanism of inhibition specific for CDK4 or CDK6. p16INK4 gene mediated by an adenovirus vector. Cancer Res., 55: In conclusion, we have identified specific small molecule 3250–3253, 1995. inhibitors of CDK4 by comparing the growth-inhibitory activity 14. Sherr, C. J. Cancer cell cycles. Science (Washington DC), 274: of more than 50,000 compounds with the p16 status of the cell 1672–1677, 1996. lines in the NCI drug screen panel. This approach may ulti- 15. Losiewicz, M. D., Carlson, B. A., Kaur, G., Sausville, E. A., and Worland, P. J. Potent inhibition of CDC2 kinase activity by the fla- mately lead to the development of a useful therapeutic strategy vonoid L86–8275. Biochem. Biophys. Res. Commun., 201: 589–595, for patients with p16-altered tumors. 1994. 16. Kitagawa, M., Okabe, T., Ogino, H., Matsumoto, H., Suzuki-Taka- ACKNOWLEDGMENTS hashi, I., Kokubo, T., Higashi, H., Saitoh, S., Taya, Y., Yasuda, H., Ohba, Y., Nishimura, S., Tanaka, N., and Okuyama, A. Butyrolactone I, We thank George Johnson, Jill Johnson, Edward Sausville, and a selective inhibitor of cdk2 and cdc2 kinase. Oncogene, 8: 2425–2432, Kenneth Paull of the Developmental Therapeutics Program, NCI for 1993. advice and assistance in performing COMPARE analyses. We thank 17. Gray, N. S., Wodicka, L., Thunnissen, A. M., Norman, T. C., Anne Monks and her laboratory for providing cell lines and John Kwon, S., Espinoza, F. H., Morgan, D. O., Barnes, G., LeClerc, S., Weinstein for helpful discussion. We are grateful to David Beach for Meijer, L., Kim, S. H., Lockhart, D. J., and Schultz, P. G. 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Akihito Kubo, Kazuhiko Nakagawa, Ravi K. Varma, et al.

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