Project Grant R37CA286908

TARGETING BASE DAMAGE REPAIR IN BRCA-MUTANT CANCERS - PROJECT SUMMARY/ABSTRACT POLY (ADP-RIBOSE) POLYMERASE INHIBITORS (PARPI) ARE A MAINSTAY FOR THE CHEMOTHERAPEUTIC REGIMEN OF BRCA MUTANT OVARIAN AND BREAST CANCERS. DESPITE INITIAL POSITIVE RESPONSES, LONG -TERM CLINICAL SUCCESS WITH PARPI THERAPY IS LIMITED OWING TO THE INEVITABLE EMERGENCE OF RESISTANCE AND THE SIDE EFFECTS ASSOCIATED WITH THE CURRENT DOSAGE. WE AND OTHERS RECENTLY REPORTED THAT LOSS OF A NUCLEOSOME SLIDING ENZYME, AMPLIFIED IN LIVER CANCER 1, (ALC1), HYPERSENSITIZES BRCA MUTANT CANCER CELLS TO PARPI. NOTABLY, ALC1 LOSS PERMITS KILLING OF BRCA MUTANT CANCER CELLS AT SUB-NANOMOLAR PARPI DOSAGE AND RESTORES PARPI SENSITIVITY ACROSS VARIOUS ENGINEERED MODELS OF CHEMORESISTANCE. BASED ON THESE OBSERVATIONS, OUR OVERARCHING GOAL IS TO EMPLOY ALC1-DEFICIENT BRCA MUTANT CANCER CELLS TO DEFINE THE CELLULAR AND BIOCHEMICAL MECHANISMS THAT CAN BE EXPLOITED TO CIRCUMVENT CLINICAL HURDLES ASSOCIATED WITH PARPI. OUR PRELIMINARY DATA HIGHLIGHT A ROLE OF ALC1 MEDIATED NUCLEOSOME SLIDING IN PROMOTING THE REPAIR OF BASE DAMAGE LESIONS CALLED ABASIC SITES. HOWEVER, IT IS UNCLEAR HOW ALC1 LOSS GENERATES ABASIC SITES AND HOW THIS CONTRIBUTES TO PARPI HYPERSENSITIVITY IN BRCA MUTANT CANCER CELLS. THE PROPOSAL ADDRESSES THIS KNOWLEDGE GAP VIA THE FOLLOWING SPECIFIC AIMS. IN SPECIFIC AIM 1, WE WILL INTEGRATE IN VITRO RECONSTITUTION, GENETIC COMPLEMENTATION ANALYSIS AND DNA REPAIR AND REPLICATION ASSAYS TO DEFINE THE MECHANISM(S) THAT LEAD TO INCREASED ABASIC SITES ON THE CHROMATIN UPON THE LOSS OF ALC1. THESE EXPERIMENTS WILL PROVIDE NEW MECHANISTIC INSIGHTS INTO HOW PERTURBING CHROMATIN REMODELING INVOLVED IN BASE DAMAGE REPAIR CAN BE LEVERAGED FOR AUGMENTING PARPI SENSITIVITY IN BRCA MUTANT CANCERS. IN AIM 2, WE WILL USE SINGLE-MOLECULE REPLICATION TRACT LABELING ASSAYS, ELECTRON MICROSCOPY AND CRISPR-BASED GENETIC EDITING OF PATIENT-DERIVED PRIMARY CELLS TO DETERMINE HOW ABASIC SITES RESULTS IN REMODELING OF REPLICATION FORKS AND GENERATION OF LESIONS THAT ACCENTUATE PARPI SENSITIVITY. THESE EXPERIMENTS WILL UNCOVER HOW THE COMMUNICATION BETWEEN BASE DAMAGE REPAIR AND REPLICATION FORKS CAN BE EXPLOITED TO ENHANCE THE THERAPEUTIC POTENTIAL OF PARPI. OUR STUDIES WILL PROVIDE THE FOUNDATION TO DEVELOP NEW APPROACHES TO IMPROVE THE EFFICACY AND TOXICITY PROFILE OF CLINICALLY USED PARPI WHILE SIMULTANEOUSLY HIGHLIGHTING NEW BIOMARKERS THAT CAN EFFECTIVELY PREDICT PARPI RESPONSES IN BRCA MUTANT PATIENTS.