DNA double strand breaks (DSBs) are the most lethal type of DNA damage. Defects in DSB repair by homologous recombination-directed (HDR) DNA repair sensitizes cancer cells to inhibitors of poly (ADP ribose) polymerase (PARP), an enzyme facilitating single strand base repair (SSB). In metastatic triple negative breast cancer (TNBC) patients carrying HDR-inactivating germline mutations in the HDR genes BRCA1 and BRCA2 (gBRCAm), the EMBRACA trial of the PARP inhibitor (PARPi) olapaprib has shown life-prolonging effects. Thus, the olapaprib is now a FDA-approved monotherapy for gBRCAm TNBC patients. These findings are highly significant, as they endorse the concept that genetic defects in HDR pave the way to cancer cell killing by PARPi that prevent single strand DNA repair. Only 15% of all TNBC patients are gBRCAm carriers, with the remaining 85% of gBRCAm-negative TNBC showing inconsistent responses to PARPi despite BRCA-like phenotypes (BRCAness). This signifies the Research Plan presented herein as it proposes to induce HDR-deficiency through Rad51 inhibition, which then in turn amplifies PARPi efficacy. We have identified a novel class of HDR-inhibitors that are fatty acids (NFA) nitroalkenes, which are well tolerated and readily deployable in humans. Our research has identified a unique regulatory domain on the essential HDR gene Rad51 that is controlled by reducing- oxidation (redox) post-translational modifications (PTM) and can be readily targeted as a novel chemotherapeutic strategy for TNBC. The reversible and site-specific alkylation-mediated PTM of Rad51 by a lipid electrophile nitro fatty acid (NFA) severely compromises nuclear Rad51 foci and TNBC cell survival, especially when combined with PARPis in vitro and in vivo. Thus, specific focus is placed on tow different perspectives. First, detailed mechanistic understanding will come from characterizing the specificity of Rad51 alkylation by the NFAs and the impact on HDR. In addition, an efficacious NFA regioisomers designed from X- ray structure-based modeling studies, that already showed increased TNBC cell killing, will be further evaluated for extents of Rad51 targeting, inhibition of HDR repair in combination with PARPi and net effects on TNBC killing in a TNBC cell line panel. As NFAs have the capacity to adduct protein Cys residues, and preliminary data also support an NFA-mediated inhibition of another DNA DSB repair pathway, known to be upregulated after Rad51 inhibition, other possible NFA protein targets in DNA DSB repair, will be examined by click-chemistry based HPLC-MS/MS proteomic analysis of NFA targets. Secondly, a TNBC patient-derived xenograft breast cancer model in combination with a genetically engineered TNBC mouse model, ...