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

Division Of Basic Sciences - Nci

Investigators

Linked publications, trials & patents

Abstract

We are pursuing complementary approaches to elucidate the molecular pharmacology of clinically relevant inhibitors of topoisomerases, DNA repair and cell cycle checkpoints. Project #1. Repair of topoisomerase cleavage complexes (TOPccs) by tyrosyl-DNA-phosphodiesterases (TDPs), endonucleases and SUMOylation/ubiquitylation/PARylation Aim 1:Post-translational modifications of TOPccs: TOPccs are excised from DNA by two main mechanisms: 1/ hydrolysis of the covalent linkage between the catalytic tyrosine of topoisomerases and the DNA broken end by tyrosyl-DNA-phosphodiesterases (TDP1 and TDP2); 2/ endonuclease cleavage of the DNA fragment adjacent to the TOPcc by nucleases (Mre11, XPF-ERCC1, XPG, FEN1, APE2...). Because the covalent topoisomerase-tyrosyl-DNA bonds to be cleaved by TDP1 and TDP2 are deep within the TOPccs, TOPccs need to be proteolyzed and/or denatured to provide access to the TDPs (TDP1 and TDP2). We are studying the proteolytic pathways for TOPccs. Our results demonstrate the rapid engagement of the SUMOylation and ubiquitylation pathways, which, in turn drive proteasome-mediated topoisomerase degradation. We have also discovered that, unique to TOP1, TOP1ccs are rapidly PARylated by PARP1 and de-PARylated by PARG (PolyADPribose Glycohydrolase). Thus, we revealed that PARylation regulates the proteolytic digestion of TOP1ccs by preventing their excessive proteasomal degradation while recruiting TDP1. Aim 2: Pharmacology and targeting of TDPs: The rationale for targeting TDPs is rooted in the emerging importance of TDPs for DNA repair and viral replication, and the potential of TDP inhibitors for anticancer drug combinations. We are using biochemical assays with recombinant TDP enzymes and TDP1 and TDP2 knockout cell lines, crystallographic determinations, and molecular modeling to discover novel inhibitors and elucidate their molecular pharmacology. We published the first crystal structures of several TDP1 inhibitors in complex with their target enzyme. This will pave the way for developing clinical candidate drug molecules Project #2. PARP trapping by PARP inhibitors: molecular mechanisms and translational implications PARP inhibitors represent the most advanced cancer therapeutics targeting the DNA damage response. PARP inhibitors (olaparib, rucaparib, niraparib and talazoparib) are FDA-approved. PARP inhibitors are the first drugs to exploit the concept of synthetic lethality for homologous recombination deficiency (HRD) in the clinic. Our studies revealed 'PARP trapping' as a key mechanism explaining the molecular mechanism of action of PARP inhibitors as anticancer agents. This discovery and our work with talazoparib contributed to the approval of talazoparib for breast and ovarian cancer in 2018. Our studies focus on 1/ the most synergistic combinations with temozolomide and with TOP1 inhibitors, including our non-camptothecin indenoisoquinoline TOP1 inhibitors; 2/ the repair mechanisms and determinants of response to PARP inhibitors beyond homologous recombination (HR; BRCAness). We recently showed that the DNA-protein crosslink protease (Spartan: SPTN), TDPs and ubiquitination are involved for the removal of trapped PARP1 and that ubiquitylation of PARP1 by the SUMO-dependent ubiquitin ligase RNF4 precedes the eviction of PARP1 from chromatin. Our ongoing studies demonstrate that endogenous and exogenous carcinogenic DNA-protein crosslinks are rapidly and reversibly PARylated. Project #3. Patient-derived cancer cell lines and organoids to discover and validate novel genomic predictive biomarkers for patient selection and rational drug combinations with TOP1, PARP and DNA damage response (DDR) and cell cycle checkpoint (ATR) inhibitors as part of CellMiner The current lack of predictive biomarkers for widely used anticancer therapies targeting DNA and its replication, and the lack of direct correlation between their primary targets and cellular response warrant the need to identify DNA damage response (DDR) determinants for predicting drug responses and rationalizing drug combinations. Taking advantage of the extensive NCI-60 drug database (> 40,000 drugs including FDA approved and investigational clinical drugs), whole genomic data and our CellMiner facility, we discovered several novel predictive biomarkers for DNA-targeted agents: SLX4 (FANCP) mutations, ATAD5 (ELG1) mutations, and SLFN11 (Schlafen 11) expression. We have extended these analyses to tissue-specific cancer cell line databases (NCI Small Cell Lung Cancers), sarcoma cell lines and adrenal cancer (ACC) cell lines, and larger databases (CCLE: MIT-Broad Institute and CGP: MGH-Sanger), and to CCR clinical trials to test predictive biomarker signatures. Those cancer cell line databases have been made widely and freely available to the research community via CellMiner web-based application (http://discover.nci.nih.gov/). We have generated novel database and web-based pharmacogenomic tools for patient-derived small cell lung cancers (SCLC): SCLC-CellMiner in collaboration with the NCI-DTP (Beverly Teicher Molecular Pharmacology group) and John Minna (UTSW). The manuscript and resource have been published at Cell Press. We have also integrated in the CellMinerCDB database the response of 183 cancer cell lines to > 2,650 drugs tested at the National Center for Advancing Translational Sciences (NCATS. We also published and made available a website for adrenocortical cancers (ACC), a rare disease with poor prognosis, and for which the patients are seen in our clinic by Dr. Jaydira Del River. These publicly available database and website have been published in Cancer Research and highlighted by the CCR Press Office. In September, we will publish an update of our Discover websites in Nucleic Acids Research. Project #4. Schlafen 11 (SLFN11) a predictive biomarkers of response to DNA damaging drugs: We discovered SLFN11 as the most dominant predictor of response to anticancer drugs targeting DNA replication. Indeed, SLFN11 determines response to TOP1, TOP2, PARP inhibitors, DNA synthesis inhibitors (hydroxyurea, gemcitabine. Cytarabine) and platinum derivatives (olaparib, rucaparib, niraparib, talazoparib) but not to tubulin or protein kinase inhibitors or apoptosis-inducing drugs. SLFN11 is inactivated in approximately 50% of cancer cells, making them resistant to DNA damaging agents. Our aims are to elucidate the molecular mechanism of action of SLFN11 and its regulation and relevance for patient responses and rationale drug combinations. We discovered that SLFN11 is recruited to DNA damage sites and to stressed replication forks by binding to RPA and the replicative CMG complex, opening chromatin, inducing the immediate early response (EIR) stress genes, and promoting the degradation of the replication licensing factor CDT1. Last year, we showed that SLFN11 regulating protein homeostasis and proteotoxic stress, and this year we have shown that it blocks homologous recombination repair and ALT (Alternative Lengthening of Telomeres. We have proposed that SLFN11 acts as a "Restriction Factor" for cells with replicative stress and as "potential tumor suppressor". We have also demonstrated that SLFN11 inactivation can be reversed by treatment with epigenetic drugs such as histone deacetylase (HDAC) inhibitors to overcome resistance to DNA-targeted anticancer drugs. This year, we have also shown that, in response to replication stress, such as in cancer cells treated with DNA damaging agents, SLFN11 is recruited to chromatin by polyubiquitylation mediated by the ubiquitin ligase RNF168. We have also shown that multiple myeloma express high levels of SLFN11 and that SLFN11 is recruited to nucleoli in response to proteasome inhibitors where it blocks ribosomal RNA synthesis.