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Evolution of Anticancer Drug Discovery and the Role of Cell-Based Screening

Frank M. Balis
trated by the development of imatinib, which was discovered by screening compound libraries for inhibitors of the protein kinase activity in vitro (10). Many of the proteins involved in cell cycle regulation, signal transduction, and the regulation of apoptosis are enzymes or receptors and are, therefore, potentially amenable to inhibition by small molecules (1114). Other examples of target-based treatments undergoing clinical evaluation include farnesyltranferase inhibitors, which block post-translational prenylation of ras, cyclin-dependent kinase inhibitors, protein kinase C inhibitors, and epidermal growth factor receptor kinase inhibitors (15). However, target-based screening for discovery of new molecularly targeted cancer treatments has shortcomings. Unlike the Bcr-Abl fusion protein in CML and ras oncogenes that represent mutations leading to a gain in function, most mutations in cancer cells result in a loss or inactivation of a protein (e.g., p53 mutations), and screening a group of drugs by use of the protein product of mutated tumor suppressor genes is unlikely to discover a small molecule that restores protein function. In addition, compounds that are active in a target-based screening assay may not be specific for the protein used in the screen, and, as a result, the pharmacologic effect of the compound at a cellular or organism level may be more closely related to its effect on other unrelated and potentially higher affinity targets. Finally, most molecular targets for new cancer treatments interact with other proteins within pathways or networks in the cell, and the pharmacologic effect resulting from the inhibition of a specific target may be influenced by the expression or relative levels of these interacting proteins. Therefore, target-based screening assays may not be predictive of drug effect within the context of the whole cell (16). As demonstrated by Dunstan et al. (17) in this issue of the Journal, cell-based screening assays will continue to play an important role in drug discovery as we move into the moleculartargeting era. Their three-stage cell-based procedure using yeast cells that can be genetically manipulated is adaptable to highthroughput screening to identify agents that have a selective effect against cells with specific mutations (gene deletions). Unlike target-based assays, this cell-based assay is not a mechanistic screen, but determining the mechanism of action of selectively toxic agents from this screen may identify new molecular targets (e.g., downstream effectors in a pathway that is derepressed by the deletion of a tumor suppressor gene) for subsequent target-based screening. From a pharmacologic perspective, this cell-based screen detects collateral sensitivity. The
Affiliation of author: Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD. Correspondence to: Frank M. Balis, M.D., National Institutes of Health, Bldg. 10, Rm. 13C103, 10 Center Dr., Bethesda, MD 208921920 (e-mail: balisf@

The approach to the discovery of new anticancer drugs has recently evolved from a reliance on empiric cell-based screening for antiproliferative effects to a more mechanistically based approach that targets the specific molecular lesions thought to be responsible for the development and maintenance of the malignant phenotype in various forms of cancer. The ultimate goal of the development of molecularly targeted drugs is to improve the efficacy and selectivity of cancer treatment by exploiting the differences between cancer cells and normal cells. The success of recently developed molecularly targeted agents, such as tretinoin (all-trans-retinoic acid) for acute promyelocytic leukemia (1,2) and imatinib (STI-571) for chronic myelogenous leukemia (CML) (3,4) and gastrointestinal stromal tumors (5), provides early clinical validation for the molecularly targeted approach to drug discovery. Most of the commonly used cytotoxic anticancer drugs were discovered through random high-throughput screening of synthetic compounds and natural products in cell-based cytotoxicity assays. Despite the number and chemical diversity of these agents, the mechanisms of action are limited (Table 1), and most compounds are DNA-damaging agents with a low therapeutic index. With this screening approach, mechanism of action is not a primary determinant in selecting agents for further development, and, as a result, none of the current drugs directly targets the molecular lesions responsible for malignant transformation. The initial National Cancer Institute (NCI) high-throughput screen used the highly chemosensitive P388 leukemia cell line, but this screen failed to identify drugs that were active against the common adult solid tumors. In the mid-1980s, the NCI implemented a new in vitro disease-oriented screen consisting of 60 human tumor cell lines representing nine common forms of cancer (6). It remains to be determined whether selective activity in vitro against cell lines representing a particular histologic form of cancer will be predictive for antitumor activity in vivo (7). As the molecular bases of specific forms of cancer are elucidated, a variety of new potential therapeutic targets are being identified. In the most common forms of cancers, the accumulation of mutations in multiple genes is required for tumorigenesis (8,9). These mutations, such as the ras-activating mutations and mutations that inactivate p53, allow cancer cells to circumvent intrinsic and extrinsic controls that tightly regulate the cell cycle and cell division and apoptosis in normal cells. After identification of the genetic alterations in cancer cells, the critical transforming mutations must be discerned from genetic alterations that are the result of the inherent genetic instability in cancer cells but that do not play a direct role in tumorigenesis (9). This target validation is performed in preclinical cancer models. Identification of valid molecular targets has led to rational target-based drug discovery at the protein level, as illus78 EDITORIAL

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Journal of the National Cancer Institute, Vol. 94, No. 2, January 16, 2002

Table 1. Mechanism of action of the commonly used natural product anticancer drugs* Drug class Anthracyclines Epipodophyllotoxins Dactinomycin Camptothecins Taxanes Vinca alkaloids Mechanism of action Topoisomerase II inhibitors Topoisomerase II inhibitors Topoisomerase II inhibitors Topoisomerase I inhibitors Tubulin-binding agents Tubulin-binding agents

(1) Sun GL, Ouyang RR, Chen SJ, Gu YZ, Huang LA, Lu JX, et al. Treatment of acute promyelocytic leukemia with all-trans retinoic acid. A five-year experience. Chin Med J (Engl) 1993;106:7438. (2) Tallman MS, Andersen JW, Schiffer CA, Appelbaum FR, Feusner JH, Ogden A, et al. All-trans-retinoic acid in acute promyelocytic leukemia. N Engl J Med 1997;337:10218. (3) Druker BJ, Sawyers CL, Kantarjian H, Resta DJ, Reese SF, Ford JM, et al. Activity of a specific inhibitor of the BCR-ABL tyrosine kinase in the blast crisis of chronic myeloid leukemia and acute lymphoblastic leukemia with the Philadelphia chromosome. N Engl J Med 2001;344:103842. (4) Druker BJ, Talpaz M, Resta DJ, Peng B, Buchdunger E, Ford JM, et al. Efficacy and safety of a specific inhibitor of the BCR-ABL tyrosine kinase in chronic myeloid leukemia. N Engl J Med 2001;344:10317. (5) van Oosterom AT, Judson I, Verweij J, Stroobants S, Donato di Paola E, Dimitrijevic S, et al. Safety and efficacy of imatinib (STI571) in metastatic gastrointestinal stromal tumours: a phase I study. Lancet 2001;358:14213. (6) Monks A, Scudiero D, Skehan P, Shoemaker R, Paull K, Vistica D, et al. Feasibility of a high-flux anticancer drug screen using a diverse panel of cultured human tumor cell lines. J Natl Cancer Inst 1991;83:75766. (7) Weinstein JN, Myers TG, OConnor PM, Friend SH, Fornace AJ Jr, Kohn KW, et al. An information-intensive approach to the molecular pharmacology of cancer. Science 1997;275:3439. (8) Renan MJ. How many mutations are required for tumorigenesis? Implications from human cancer data. Mol Carcinog 1993;7:13946. (9) Kaelin WG Jr. Choosing anticancer drug targets in the postgenomic era. J Clin Invest 1999;104:15036. (10) Drucker BJ, Lydon NB. Lessons learned from the development of an abl tyrosine kinase inhibitor for chronic myelogenous leukemia. J Clin Invest 2000;105:37. (11) Drews J. Drug discovery: a historical perspective. Science 2000;287: 19604. (12) Shapiro GI, Harper JW. Anticancer drug targets: cell cycle and checkpoint control. J Clin Invest 1999;104:164553. (13) Gibbs JB. Anticancer drug targets: growth factors and growth factor signaling. J Clin Invest 2000;105:913. (14) Sellers WR, Fisher DE. Apoptosis and cancer drug targeting. J Clin Invest 1999;104:165561. (15) Gibbs JB. Mechanism-based target identification and drug discovery in cancer research. Science 2000;287:196973. (16) Sausville EA, Johnson JI. Molecules for the millennium: how will they look? New drug discovery year 2000. Br J Cancer 2000;83:14014. (17) Dunstan HM, Ludlow C, Goehle S, Cronk M, Szankasi P, Evans DR, et al. Cell-based assays for identification of novel double-strand break-inducing agents. J Natl Cancer Inst 2002;94:8894. (18) Hartwell LH, Szankasi P, Roberts CJ, Murray AW, Friend SH. Integrating genetic approaches into the discovery of anticancer drugs. Science 1997; 278:10648. (19) Weinstein JN, Buolamwini JK. Molecular targets in cancer drug discovery: cell-based profiling. Curr Pharm Des 2000;6:47383.

*The source and chemical structures of these agents are diverse, but the molecular targets of these agents are limited to the topoisomerases and tubulin.

genetic mutation enhances the cells sensitivity to drugs that affect related pathways within the cell. The assay is also a direct application of the concept of synthetic lethality, which has been described previously in yeast (9,18). Two genes are synthetically lethal if mutations in either one is survivable, but mutations in both genes are lethal. For this cell-based assay, disruption of the function of the genes product by a drug is only lethal in cells that have a mutation in a second related gene. Cell-based assays are also used to confirm the activity of agents discovered in target-based screening assays and to assess the drugs pharmacologic effects at the cellular level. Unexpected effects in cellular systems may suggest other targets for the agent or interactions of the primary molecular target with other vital proteins within the cell. The NCIs panel of 60 human tumor cell lines has also been adapted to provide information about the mechanism of action or molecular target of new agents that are tested based on the drugs profile of activity in the screen (19). As new targets are identified, their expression in each of the 60 cell lines in the panel can be characterized and correlated with the activity profile of the 70 000 compounds screened previously without having to retest each agent. The identification of the topoisomerases as targets for drugs that were selectively active in yeast cells deficient in DNA double-strand break-repair proteins was apparently based on their activity profile in the human tumor cell line panel. Target-based and cell-based screening for new anticancer drugs in the molecular targeting era are complementary methods of identifying more selective anticancer drugs. They represent a dramatic shift in the drug discovery process that is likely to have an impact not only on the pharmacologic properties of new anticancer agents reaching the clinic but also on our approach to clinical drug development and the treatment of cancer.

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Journal of the National Cancer Institute, Vol. 94, No. 2, January 16, 2002