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

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Deficiencies in DNA repair (mismatch repair deficiencies in colon cancers, nucleotide excision repair in ovarian and testicular cancers, and in melanomas), deficiencies in cell cycle checkpoints (Rb, p53, BRCA1, BRCA2, and Chk2 deficiencies in solid tumors), and apoptosis (APC mutations in colon cancers, Bcr-Abl recombinations in leukemia, Bcl-2 overexpression in lymphomas) promote cancers. They also contribute to therapeutic responses as normal cells have more robust alternative (redundant) pathways. The Mre11-Rad50-Nbs1 (MRN) complex binds DNA double-strand breaks to repair DNA and activate checkpoints. We recently reported MRN deficiency in 3 of the 7 colon carcinoma cell lines of the NCI Anticancer Drug Screen. To study MRNs involvement in replication-mediated DNA double-strand breaks, we examined checkpoint responses to camptothecin, which induces replication-mediated DNA double-strand breaks after replication forks collide with topoisomerase I cleavage complexes. MRN-deficient cells were deficient for Chk2 activation, whereas Chk1 activation was independent of MRN. Chk2 activation was ATM-dependent, and associated with phosphorylation of Mre11 and Nbs1. Mre11 complementation in MRN-deficient HCT116 cells restored Chk2 activation as well as Rad50 and Nbs1 levels. Conversely, Mre11 downregulation by siRNA in HT29 cells inhibited Chk2 activation and downregulated Nbs1 and Rad50. Proteasome inhibition also restored Rad50 and Nbs1 levels in HCT116 cells suggesting that Mre11 stabilizes Rad50 and Nbs1. Chk2 activation was also defective in 3 out of the 4 MRN-proficient colorectal cell lines because of low Chk2 levels. Thus, 6 out of the 7 colon carcinoma cell lines from the NCI Anticancer Drug Screen are functionally Chk2-deficient in response to replication-mediated DNA double-strand breaks. We propose that Mre11 stabilizes Nbs1 and Rad50 and that MRN activates Chk2 downstream from ATM in response to replication-mediated DNA double-strand breaks. Chk2 deficiency in HCT116 is associated with defective S-phase checkpoint, prolonged G2 arrest and hypersensitivity to camptothecin. The high frequency of MRN and Chk2 deficiencies may contribute to genomic instability and therapeutic response to camptothecins in colorectal cancers. To integrate these alterations within the emerging cellular network of molecular pathways, we have developed a mapping convention that can be visually represented as molecular interaction maps (MIMs). These maps are being presented by different members of the LMP (Dr. Kohn, Dr. Aladjem, and Dr. Pommier) at different meetings. They are published in international journals with high impact factors and we have developed an interactive Website in collaboration with the LMP Bioinformatic group (Dr. Weinstein) (http://discover.nci.nih.gov/mim). 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. We are continuing our studies with ecteinascidin 743 (Et743 - Yondelis) (NSC 648766). Clinical responses to Et743 have been observed in sarcomas, which are notoriously resistant to therapy, as well as in ovarian and breast cancer. Et743 differs from other clinically used anticancer agents because it forms covalent adducts at specific guanines in the DNA minor groove and because it selectively Et743 traps the transcription-coupled NER (TC-NER). Thus, Et743 defines a novel class of anticancer drugs in which enhanced antiproliferative activity parallels enhanced cellular DNA-repair capability. The complementary between the activities of Et-743 and cisplatin with respect to TC-NER suggests the use of Et743 in cisplatin-resistant tumors and vice-versa. A clinical protocol has been proposed for a clinical trial of Et743 in pediatric cancers (Collaboration with Dr. Frank Balis, CCR, NCI). Further molecular studies are planned to determine the transcription- and the strand-specific-dependence of the DNA single-strand breaks induced by Et-743. We are also looking at TC-NER-dependent transcription inhibition by microarray analyses using NER-deficient, XPD, and XPD-complemented cells and Ewing sarcoma cell lines. 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 also set up another mid- high-throughput screen for inhibitors of Tdp1. Tdp1 inhibitors should be synergistic in combination with Top1 inhibitors. Our studies on apoptosis are focused on chromatin modifications. We found that one of the early events in apoptosis is the induction of apoptotic Topoisomerase I-DNA complexes. The apoptotic Topoisomerase I-DNA complexes are induced by a variety of apoptotic stimuli: arsenic trioxide, etoposide, camptothecin, platinum derivatives, taxol, vinblastine. Our working hypothesis that these apoptotic Topoisomerase I-DNA complexes are produced by oxidative lesion of genomic DNA, which trap Topoisomerase I bound to chromatin. Apoptotic Topoisomerase I-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