Project Grant F31CA298263

DETERMINING THE ROLE OF NUCLEAR INTEGRITY LOSS SURVEILLANCE IN PARPI-INDUCEDCELL DEATH IN BRCA1-DEFICIENT CANCER - PROJECT SUMMARY/ABSTRACT POLY (ADP-RIBOSE) POLYMERASE INHIBITORS (PARPI) ARE SYNTHETICALLY LETHAL IN CELLS WITH HOMOLOGOUS RECOMBINATION (HR) DEFECTS, A PHENOTYPE OF CERTAIN CANCERS. BRCA1 IS ONE OF THE MOST COMMONLY MUTATED GENES IN HEREDITARY, HR-DEFICIENT CANCERS. UNFORTUNATELY, PATIENTS WITH HR-DEFICIENT CANCERS COMMONLY ACQUIRE RESISTANCE TO PARPI, AND THE MECHANISMS OF PARPI-INDUCED CYTOTOXICITY ARE UNDEREXPLORED. UNDERSTANDING THESE PATHWAYS COULD PROVIDE INSIGHT INTO HOW PATIENTS ACQUIRE PARPI RESISTANCE. THE PARPI CYTOTOXIC RESPONSE REQUIRES CELLS TO TRANSIT MITOSIS, AND CELLS THAT PERSIST THROUGH PARPI TREATMENT (PERSISTER CELLS) ARREST FROM THE CELL CYCLE. THUS, DETERMINING THIS MECHANISM OF CELL CYCLE COMMITMENT IS CENTRAL TO UNDERSTANDING PARPI-INDUCED CYTOTOXICITY AS CELLS FACE TWO POTENTIAL FATES IN RESPONSE TO PARPI: UNDERGO MITOSIS AND TRIGGER CELL DEATH OR ARREST FROM THE CELL CYCLE AND PERSIST. MY PRELIMINARY DATA SUGGESTS THAT ARRESTED PERSISTER CELLS HAVE DECREASED LEVELS OF LAMIN B1 (LMNB1, A NUCLEAR LAMINA PROTEIN): LOSS OF WHICH IS NECESSARY AND SUFFICIENT TO INDUCE CELL CYCLE ARREST IN OTHER CONTEXTS. THUS, LMNB1 DOWNREGULATION MAY CONTRIBUTE TO THE CELL CYCLE ARREST MECHANISM IN THESE PERSISTER CELLS. FOR PARPI-TREATED CELLS THAT UNDERGO MITOSIS-DEPENDENT CELL DEATH, THE PATHWAYS GOVERNING THIS CYTOTOXICITY ARE UNCLEAR. PARPI HAVE BEEN SHOWN TO SYNERGIZE WITH OTHER DRUGS TO INDUCE VARIOUS FORMS OF REGULATED CELL DEATH (RCD) IN DIFFERENT CELL LINES, SUGGESTING THAT MULTIPLE FORMS OF RCD COULD BE TRIGGERED IN BRCA1-DEFICIENT CELLS. A SYSTEMATIC INVESTIGATION IS NEEDED TO DETERMINE THE RCD MECHANISMS INVOLVED IN THE PARPI RESPONSE AND THE UPSTREAM REGULATORY PATHWAYS THAT INITIATE THEM. ONE HYPOTHESIS OF A CONTRIBUTING REGULATORY PATHWAY IS CYCLIC GMP-AMP SYNTHASE (CGAS) / STIMULATOR OF INTERFERON GENES (STING) SIGNALING, WHICH CANONICALLY INDUCES INNATE IMMUNE SIGNALING IN RESPONSE TO CYTOSOLIC DNA. CGAS/STING SIGNALING IS INCREASED IN HR-DEFICIENT, PARPI-TREATED CELLS. HIGHER INSTANCES OF MITOTIC ERRORS, INCLUDING PERSISTENT DNA BRIDGES, ARE ALSO OBSERVED IN RESPONSE TO PARPI TREATMENT. AS THESE STRUCTURES ARE PRONE TO RUPTURE, LEADING TO LOSS OF NUCLEAR ENVELOPE INTEGRITY, WE HYPOTHESIZE THAT THEY COULD BE SURVEILLED BY CGAS TO SIGNAL RCD IN THE PARPI RESPONSE. THE OBJECTIVE OF THIS PROPOSAL IS TO DETERMINE THE MECHANISMS THAT CONFER VULNERABILITY OF BRCA1-DEFICIENT CELLS TO PARPI-INDUCED CYTOTOXICITY. USING BRCA1-DEFICIENT BREAST AND OVARIAN CANCER CELL LINES AS MODELS, IN AIM 1 I WILL DETERMINE MECHANISM OF CELL CYCLE EVASION THAT ALLOWS CELLS TO PERSIST THROUGH PARPI TREATMENT, SPECIFICALLY INVESTIGATING LMNB1 DOWNREGULATION AS A CONTRIBUTING PATHWAY. IN AIM 2 I WILL CONDUCT AN ARRAYED CRISPR SCREEN TARGETING RCD DRIVER GENES AND OTHER REGULATORY PATHWAYS TO DETERMINE THE MECHANISMS OF PARPI-INDUCED CYTOTOXICITY IN CYCLING CELLS, INCLUDING INVESTIGATING A ROLE FOR CGAS/STING SIGNALING. OVERALL, THIS PROPOSAL WILL ENHANCE OUR UNDERSTANDING OF HOW CELLS EVADE OR SUCCUMB TO PARPI-INDUCED CELL DEATH. THIS WORK COULD INFORM COMBINATION THERAPIES WITH PARPI TO COMBAT PARPI RESISTANCE IN PATIENTS WITH BRCA1-DEFICIENT CANCERS.