Investigating DNA repair vulnerabilities in oncometabolite producing cancers

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PROJECT SUMMARY Cancer-associated mutations in tricarboxylic acid cycle genes induce production of 2-hydroxyglutarate , fumarate, or succinate. These oncometabolites suppress the homologous recombination (HR) DNA repair pathway. Nonhomologous end joining (NHEJ) is the other major pathway for double strand break (DSB) repair, which is further sub-divided into classical (cNHEJ) and highly mutagenic alternative end joining (altNHEJ) pathways. Our group’s preliminary data suggests that oncometabolites induce upregulation of NHEJ repair, however, the mechanistic basis for this observation has yet to be elucidated. Multiple inhibitors have been developed that target proteins within these NHEJ repair pathways, including DNA-PK and pol theta inhibitors, which suggests that NHEJ is a clinically relevant target. I hypothesize specific oncometabolites uniquely and dynamically regulate altNHEJ and cNHEJ, which can be targeted for a therapeutic gain against tumor cells. I will investigate how oncometabolites alter DSB repair and evaluate NHEJ pathways as therapeutic targets through two aims. My first aim will elucidate the dynamic balance between NHEJ pathways in oncometabolite producing cells. I will use U2OS cells that express reporters specific for HR, total NHEJ, and altNHEJ to determine how oncometabolites, added exogenously or intrinsically produced by mutations, alter NHEJ frequency. To determine how cNHEJ and altNHEJ protein recruitment is altered at DNA break sites with diverse chromatin states, I will use both immunofluorescence and chromatin immunoprecipitation sequencing (ChIP-seq) in a cell line in which endogenous double strand breaks can be induced at hundreds of sites in the genome. Changes in the timing of protein recruitment will be studied using ChIP-seq for cNHEJ or altNHEJ proteins identified by immunofluorescence. This will establish the extent to which oncometabolites alter various stages of NHEJ, such as DNA end processing or ligation. My second aim will investigate the effect of cNHEJ and altNHEJ inhibition on oncometabolite producing cancers. I will target cNHEJ and altNHEJ with DNA-PK and pol theta inhibitors, respectively, by performing short-term cell viability assays in oncometabolite producing cell lines. This will determine whether oncometabolite producing cancers are more sensitive to DNA-PK or pol theta inhibitors as single agents compared to parental cell lines. Furthermore, these cell lines show exquisite sensitivity to PARP inhibitors. I will evaluate potential therapeutic combinations by testing the sensitivity of these cell lines to DNA-PK inhibitors and pol theta inhibitors in combination with PARP inhibitors. This will determine whether targeting both cNHEJ or altNHEJ and PARP is more effective than single agents alone. Overall, this proposal will lead to a more complete understanding of how oncometabolites affect DSB repair and identify novel therapeutic strategies for treatment of oncometabolite producing cancers.