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 topoisomerase I- and topoisomerase II-mediated DNA damage, respectively. We are identifying TDP1 and TDP2 inhibitors with the goal of finding new drugs with therapeutic potential. High throughput screens have been set up 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). We have also shown that TDP1 inactivation is synergistic with Top1-targeted agents and a range of anticancer drugs including bleomycin, etoposide, alkylating agents, 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 poly(ADPribose)polymerase I (PARP1), which stabilize TDP1 and promotes its binding to the DNA repair base excision factor, XRCC1 and recruitment to DNA damage sites. In a recent study, 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 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 proteosome activity. We also showed that TDP2 functions in coordination with Ku and DNA-dependent protein kinase. PARP inhibitors are in clinical development. We showed that PARP inhibitors differ among each other based on their ability to trap PARP on DNA and thereby induce replicative DNA damage. We provided the first ranking of PARP inhibitors based on their DNA damaging activity: 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). We also showed that PARP is epistatic and physically coupled with TDP1, which implies that PARP inhibitors act, in part, by functionally inactivating TDP1, and that TDP1 inhibitors would be more specific than PARP inhibitors. One of our Chk2 inhibitors (discovered in collaboration with Dr. Shoemaker,NCI, and colleagues at Provid Pharma), PV1062 is in preclinical development under the sponsorship the DDC. Cellular assays have been developed to measure Chk2 inhibition in cells and demonstrate the synergy between Chk2 inhibitors and Top1 inhibitors or microtubule inhibitors. Our Chk2 inhibitors are being evaluated in preclinical models with the perspective of licensing it to drug companies. 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 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 18,000 drugs including the FDA-approved anticancer drugs and anticancer drugs in clinical trials. It lead to the discovery of a novel DNA damage response gene, SLFN11, and we actively studying SLFN11 in our laboratory. We are also developing biomarkers to measure SLFN11 in cancers and to determine its value as predictive marker. We recently completed Whole Exome Sequencing (WES) of the NCI-60, well in advance of the Sanger and Broad Institutes. We also released this year, the array CGH database for the NCI-60. Our databases are publicly available at the DTB Genomics &Bioinformatics Group (GBG) web site: http://discover.nci.nih.gov. 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 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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