Because most cancers have alterations in cell cycle checkpoint pathways (p53, pRb, Chk2) and cell cycle machinery (BLM, cyclins, cyclin-dependent kinase inhibitors such as p16), we are exploring inhibitors cell cycle checkpoints as novel anticancer agents. We are also investigating the role of both Chk1 and Chk2 in cell cycle checkpoint response, genomic stability and combination therapy in cancer cells. We have set up a high throughput screen to discover Chk2 inhibitors (collaboration with Drs. Shoemaker and Scudiero, DTP, NCI) and discovered a novel family of Chk2 inhibitors, the bis-guanidylhydrazones and shown they 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. We are exploring the possibility that Chk2 inhibitors could be selectively active against tumor cells overexpressing activated Chk2. It is also well established that DNA repair defects predispose to cancers (for instance BLM, Mre11, Xeroderma Pigmentosum and ataxia telangiectasia) and may play an important role in the response of cancers to treatments that target DNA and chromatin. We have set up high-throughput screens for inhibitors of tyrosyl-DNA phosphodiesterase (Tdp1), an enzyme of involved in the repair of topoisomerase-mediated DNA damage. We have identified the first Tdp1 inhibitors, and we are searching for new inhibitors 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 OKeefe). Tdp1 inhibitors should be synergistic in combination with Top1 inhibitors. 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 have continued our studies on the molecular pharmacology of trabectedin, which has recently been approved for the treatment soft tissue sarcomas in Europe. We previously found that trabectedin differs from other clinically used anticancer agents because it forms covalent adducts at specific guanines in the DNA minor groove and because it selectively traps the transcription-coupled NER (TC-NER). We have now found that gamma-H2AX could serve as a pharmacodynamic biomarker for trabectedin. The activation of gamma-H2AX led us to show that the trapping of TC-NER by trabectedin induces the formation of Mre11- and transcription-dependent DNA double-strand breaks. We have also shown that cancer cells degrade RNA polymerase II in response to trabectedin and that this effect is related to the Van Hippel Lindau tumor suppressor gene. We are currently studying two other agents that affect chromatin: lasonolide A and the PARP inhibitor, ABT-888. We are looking at the molecular and cellular pharmacology of those two agents. 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 Top1-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 vinblastin. 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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