DNA double strand breaks (DSBs) are the most lethal type of DNA damage. Defects in DSB repair by homologousrecombination-directed(HDR)DNArepairsensitizescancercellstoinhibitorsofpoly(ADPribose) polymerase(PARP),anenzymefacilitatingsinglestrandbaserepair(SSB).Inmetastatictriplenegativebreast cancer (TNBC) patients carrying HDR-inactivating germline mutations in the HDR genes BRCA1 and BRCA2 (gBRCAm),theEMBRACAtrialofthePARPinhibitor(PARPi)olapapribhasshownlife-prolongingeffects.Thus, the olapaprib is now a FDA-approved monotherapy for gBRCAm TNBC patients. These findings are highly significant,astheyendorsetheconceptthatgeneticdefectsinHDRpavethewaytocancercellkillingbyPARPi thatpreventsinglestrandDNArepair.Only15%ofallTNBCpatientsaregBRCAmcarriers,withtheremaining 85% of gBRCAm-negative TNBC showing inconsistent responses to PARPi despite BRCA-like phenotypes (BRCAness).ThissignifiestheResearchPlanpresentedhereinasitproposestoinduceHDR-deficiencythrough Rad51inhibition,whichtheninturnamplifiesPARPiefficacy.WehaveidentifiedanovelclassofHDR-inhibitors thatarefattyacids(NFA)nitroalkenes,whicharewelltoleratedandreadilydeployableinhumans.Ourresearch 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 alkylationbytheNFAsandtheimpactonHDR.Inaddition,anefficaciousNFAregioisomersdesignedfromX- raystructure-basedmodelingstudies,thatalreadyshowedincreasedTNBCcellkilling,willbefurtherevaluated for extents of Rad51 targeting, inhibition of HDR repair in combination with PARPi and net effects on TNBC killinginaTNBCcelllinepanel.AsNFAshavethecapacitytoadductproteinCysresidues,andpreliminarydata 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 incombination with a genetically engineered TNBC mouse model, will facilitate more clinically relevanteffectsofNFAs,incombinationwithPARPi,exandinvivo.TheResearchPlanwillrevealanoveldrug strategy for TNBC therapy, where the inhibition of Rad51-mediated DNA repair by NFAs renders TNBC cells moresensitivetoPARPinhibitionthusincreasingTNBCcellkilling.
