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

Yves Pommier

4 Collaborator(s)

Funding source

National Cancer Institute (NIH)
Because most cancers have alterations in DNA repair (homologous recombination repair, Fanconi anemia genes, mismatch repair), cell cycle checkpoints (p53, pRb, Chk2), chromatin (SWI/SNF complexes, histone methylation and acetylation), cell cycle and replication machinery (polymerases, 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 mismatch repair, nucleotide excision repair, 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 phosphodiesterases 1 and 2 (TDP1 and TDP2), enzymes that repair Top1- and Top2-mediated DNA damage, respectively. We are screening for TDP1 and TDP2 inhibitors using biochemical screens with recombinant enzymes, genetically altered cell lines and crystallography. High throughput screens have been set up in our laboratory and with the NCATS (National Center for Advancing Translational Sciences; Dr. Christopher Austin) and the CCR Molecular Therapeutics Drug Discovery Program (MTDP; Dr. Barry O Keefe). The rationale for the discovery of TDP inhibitors is that TDP1 inactivation is synergistic with a broad range of DNA targeted agents already used in cancer chemotherapy including not only Top1 inhibitors but also bleomycin, etoposide, temozolomide (DNA alkylating agent), and chain terminating anticancer and antiviral nucleosides (cytarabine, AZT, acyclovir). We have also shown that TDP1 is regulated in response to DNA damage by phosphorylation by ATM and DNA-PK and by PARP1, which binds very tightly to TDP1, stabilizes TDP1 and promotes its binding to the DNA repair base excision factor, XRCC1 and its recruitment to DNA damage sites. We have 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 have also shown that TDP1 is selectively inactivated in lung cancers, suggesting that TDP1 could be a novel tumor suppressor gene for lung cancers and that TDP1 deficiency in lung cancers could be an indication for treatment with Top1 inhibitors. We recently extended our studies and drug screening to TDP2 (previously known as TTRAP). We solved the crystal structure of TDP2, provided evidence that TDP2 functions as a 2-metal enzyme, and showed that TDP2 plays a critical role for the repair of Top2 cleavage complexes trapped by the anticancer drugs, etoposide, doxorubicin and mitoxantrone, and that proteolysis is required before TDP2, suggesting the coupling of TDP2 with ubiquination and proteasome activity. We also showed that TDP2 functions in coordination with Ku and DNA-dependent protein kinase. Olaparib (LynparzaR)has been approved for BRCA-deficient cancer in December 2014 and at least 4 other poly(ADPribose)polymerase (PARP) inhibitors are in clinical development. We made the landmark discovery that PARP inhibitors act as anticancer agents by trapping PARP on DNA, and showed that PARP inhibitors differ among each other based on their ability to trap PARP and thereby induce replicative DNA damage. We provided the first ranking of PARP inhibitors based on their DNA damaging activity: talazoparib (BMN 673) niraparib olaparib rucaparib veliparib. PARP inhibitors are also synergistic with Top1 inhibitors independently of PARP trapping. Our studies demonstrated selective conditional synergy in cells lacking ERCC1-XPF, which implies that the combination of PARP and Top1 inhibitors should be focused on cancer with preexisting ERCC1-XPF deficiencies (such as lung cancers) or Mre11 deficiencies (such as mismatch repair colon cancers). We demonstrated that PARP is epistatic and physically coupled with TDP1, which implies that PARP inhibitors act, in part, by functionally inactivating TDP1. Yet, TDP1 inhibitors would be more specific than PARP inhibitors, which affect chromatin by many other mechanisms. Cell cycle checkpoints inhibitors are remarkably synergistic with Top1 inhibitors. We have extended this synergy to the novel Chk1 inhibitors in clinical development (AZD 7762) and our non-camptothecin Top1 inhibitors (LMP400). We have also shown that ATR inhibitors, which are also in early clinical trials (VE-821 and VX-970) are highly synergistic with Top1 inhibitors and induce a particularly high histone gamma-H2AX response. We have proposed the value of this response as a clinical biomarker for the upcoming clinical trials combining Top1 inhibitors with ATR inhibitors (VX970 and AZD6738). To approach and study the pathways involved in cancer from a global system biology viewpoint, we are investigating the NCI-60 and cancer cell line databases (CCLE and Sanger cancer cell lines) in collaboration with our colleagues at DTP and the Meltzer group in CCR. The NCI-60 database is unique in the world because it includes the activity patterns of more than 20,000 drugs including the FDA-approved anticancer drugs and anticancer drugs in clinical trials. It led to the discovery of a novel DNA damage response gene, SLFN11, and we are actively studying SLFN11 in our laboratory. We recently found that SLFN11 expression is driven by the ETS transcription factors, explaining why SLFN11 is very high in Ewing's sarcoma (with the translocation EWS-FLI1) and in leukemia (with FLI1 overexpression). We are also finding that approximately 20-30% of cancer cell lines inactivate SLFN11 by CpG hypermethylation, which can be reversed by epigenetic drugs (5-azacytidine and HDAC inhibitors). We are also developing biomarkers to measure SLFN11 in cancers and to determine its value as predictive marker. We have made publicly available the NCI-60 pharmacogenic databases and a set of intuitive tools to mine the data by non-bioinformaticists through the CellMiner website hosted by our DTB Genomics & Bioinformatics Group (GBG) web site: These databases include multiple gene expression platforms (Affymetrix and Agilent) for all the genes and whole exome sequencing (WES). They also include high resolution SNIPs, array CGH, SKY and chromosome parameters. This year, we begun the implementation of two novel databases: whole genome methylation and RNA sequencing for the NCI-60. This project is in collaboration with DCTD and the Genomics Branch in CCR (Dr. Paul Meltzer). Crossing these various databases (vectors) enables the comparison between genomics and drug response. This provide unique ways to correlate drug response with specific genes and genes to genes, and platforms to utilize the TCGA data for precision medicine.

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