DNA Repair, Cell Cycle Checkpoints and Apoptosis as Targets for Anticancer Drugs

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Because most cancers have alterations in the cell cycle checkpoint pathways (p53, pRb) and cell cycle machinery (cyclins, cyclin-dependent kinase inhibitors such as p16), we are exploring inhibitors of cell cycle checkpoints as novel anticancer agents. We are investigating the role of Chk2 in cell cycle checkpoint response in cancer cells. We have expressed Chk2 as a recombinant protein and set up a high throughput screen to discover Chk2 inhibitors (collaboration with Drs. Shoemaker and Scudiero, DTP, NCI). We have 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 selected. Cellular assays are being 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. It is also well established that DNA repair defects predispose to cancers (for instance 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 the base excision repair (BER) pathways 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. 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 such as aminoflavone. Aminoflavone is beginning clinical trials and we found that aminoflavone induces replication double-strand breaks and histone H2AX phosphorylation (gamma-H2AX). Hence, gamma-H2AX can be used as a biomarker to monitor aminoflavone activity in tumor samples. Using the NCI 60 cell line database we found that sulfotransferase expression is highly correlated with aminoflavone activity and can be used to select patients who should benefit from aminoflavone. More recently, we found that aminoflavone mediates crosslinking between cytokeratins and RNA. This study suggests the possibility that cytokeratins (which constitute a major component of the cellular microfilament network) could serve as a scaffold for cytoplasmic RNA and potentially for translation. 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. Our studies on apoptosis are focused on chromatin modifications. We found that one of the early events in apoptosis is the induction of apoptotic Top1-DNA complexes. 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 are focusing on nuclear alterations produced by TRAIL, which is in clinical trials.