Because most cancers have alterations in DNA repair pathways, cell cycle checkpoint pathways (p53, pRb, Chk2) and cell cycle machinery (BLM, cyclins, cyclin-dependent kinase inhibitors, such as p16), we are dissecting the alterations that are most relevant for DNA targeted anticancer agents, especially topoisomerase inhibitors, and developing inhibitors of DNA repair and cell cycle checkpoints as novel anticancer agents. DNA repair defects not only predispose to cancers (for instance BLM, Mre11, Xeroderma Pigmentosum and ataxia Telangiectasia), but also play an important role in the response of cancer cells to treatments that target DNA and chromatin. We have set up high-throughput screens for inhibitors of tyrosyl-DNA phosphodiesterase 1 (Tdp1), an enzyme that repairs topoisomerase I-mediated DNA damage. We are identifying Tdp1 inhibitors with the goal of finding new drugs with therapeutic potential. High throughput screens have been set up with the NIH National Chemical Genomic Center (NCGC;Dr. Christopher Austin) and the CCR Molecular Therapeutics Drug Discovery Program (MTDP;Dr. Barry O Keefe). We have also shown that Tdp1 inhibitors should be synergistic in combination with Top1-targeted agents and a range of anticancer drugs including bleomycin, etoposide and alkylating agents. We have also shown that Tdp1 is regulated in response to DNA damage. Tdp1 phosphorylation by ATM and DNA-PK stabilizes Tdp1 and promotes the binding of Tdp1 to the DNA repair base excision factor, XRCC1. In a more recent study, we have also shown that Tdp1 enters mitochondria and is critical for the repair of mitochondrial DNA. This is especially important because mitochondria contain their own topoisomerase (Top1mt), which was discovered in our laboratory, and because mitochondria produce oxygen radicals and produce mitochondrial DNA damage, which is repaired by Tdp1. We are investigating the role and cancer-specific alterations of Chk2 in cell cycle checkpoint response and genomic stability. We just finished a study (available online at Oncogene) showing for the first time the differential status of Chk2 (determined by proteomic, phosphoproteomic, gene expression and exome sequencing) in the 60 cell lines of the NCI-DTP screen. Those data demonstrate that cancer cells belong to one of at least 2 groups depending on their Chk2 status. One group consists of cells with Chk2 inactivation. Those cells include all the p53 wild-type cells. The other group consists of cells with endogenous Chk2 activation/phosphorylation by ATM/ATR/DNA-PK. Those cells are all p53 mutants and probably require Chk2 to survive. We have also set up a high throughput screen to discover Chk2 inhibitors (collaboration with Drs. Shoemaker and Scudiero, DTP, NCI, and colleagues at Provid Pharma) and discovered a novel family of Chk2 inhibitors, the bis-guanidylhydrazones. These new drugs act as competitive ATP inhibitors against Chk2. Analogs have been synthesized and co-crystallized with Chk2 in collaboration with David Waugh (CCR). Cellular assays have been developed to measure Chk2 inhibition in cells and to determine whether Chk2 inhibitors can be used to synergize with Top1 inhibitors and other currently available chemotherapeutic agents (microtubule inhibitors). We have proposed that Chk2 inhibitors could be selectively active against tumor cells overexpressing activated Chk2. To approach and study the pathways involved in cancer from a global system biology viewpoint, we are investigating and charactering the 60 cancer cell lines (The NCI-60) from NCI Developmental Therapeutics Program (DTP) in collaboration with our colleagues at DTP and the Meltzer group in CCR. This database is unique in the world because it includes the activity patterns of more than 16,000 drugs including the FDA-approved anticancer drugs. We just finished the full sequencing of all the coding sequences of all genes (exome) for the NCI-60. Our databases will be made available at the LMP Genomics &Bioinformatics Group (GBG) web site: http://discover.nci.nih.gov following publication of our first manuscript. This project takes advantage of the unique databases for the 60 cancer cell lines that constitute the DTP Drug Screen. These databases include several gene expression platforms (Affymetrix and Agilent) for all the genes and all the exons. They also include high resolution SNIPs, array CGH, SKY and chromosome parameters. This year, we implemented two novel databases: a z-score tool that encompasses in one parameter the entire mRNA expression data for any given gene, and a high resolution array CGH (NimbleGen platform). This project is a collaboration between CCR and DCTD. Its uniqueness resides in the high quality genomic databases in the LMP-GBG and the drug responses generated by DCTD. Crossing these various databases (vectors) enables the comparison between gene expression, genomic copy number variants (CNV), mutations (exome sequencing) and drug response. This provide unique ways to correlate drug response with specific genes and genes to genes. We are studying several new drugs in preclinical and early clinical development including agents from the NCI-Developmental Therapeutics Program (DTP). We are focusing on drugs that alter chromatin and cell cycle progression. We are currently looking for the molecular target of lasonolide A, a marine natural product that produces chromatin condensation. We have also characterized the molecular pharmacology of the novel poly(ADPribose) polymerase (PARP) inhibitor, Veliparib (ABT-888) and elucidated the close relationship between PARP and Tdp1 in the repair of topoisomerase-induced DNA damage. Our studies on apoptosis are focused on chromatin modifications. We were the first to demonstrate that one of the early events in apoptosis is the induction of apoptotic topoisomerase I-DNA complexes. We, and others have found that the apoptotic Top1-DNA complexes are induced by a variety of apoptotic stimuli: arsenic trioxide, etoposide, camptothecin, platinum derivatives, taxol, and vinblastine. Our working hypothesis that these apoptotic Top1-DNA complexes are produced by oxidative lesion of genomic DNA, which trap Top1 bound to chromatin. Apoptotic Top1-DNA complexes in turn activate additional apoptotic responses/pathways and might represent an irreversible apoptotic activation loop. To further elucidate the molecular events induced by the apoptotic program, we have focused our recent studies on nuclear alterations produced by TRAIL, which is in clinical trials. We were the first to report the induction of a novel chromatin alteration in early apoptosis: the apoptotic ring. We have demonstrated that the apoptotic ring contains a subset of the DNA damage response (DDR) proteins. This could have two implications. From a basic standpoint, the apoptotic ring may be used to better understand the chromatin changes that take place during programmed cell death. From a translational standpoint, the apoptotic ring could be used to score tumors that respond to TRAIL and other agents that act against cancer cells by inducing apoptosis.
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