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 associ- ated 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 mM in a CDK4 kinase assay. In 50 lioma, and head and neck squamous carcinoma, whereas trans- addition, four compounds structurally related to 3-ATA m 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 ade- novirus 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 3-ATA (K 5 5.5 mM) and a linear mixed mode for benzo- i p16-like inhibitory activity against the CDK4:cyclin D kinase thiadiazine (NSC 645787; K 5 0.73 mM). We have success- 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. Downloaded from clincancerres.aacrjournals.org on October 1, 2021. © 1999 American Association for Cancer Research. 4280 Small Molecule Inhibitors of CDK4 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 mlof125I-protein A (.30 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 ml) 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.