Project Grant F31CA318249

ROLE OF P2X4 RECEPTORS IN GOLGI PH REGULATION IN PANCREATIC CANCER - PROJECT SUMMARY PANCREATIC DUCTAL ADENOCARCINOMA (PDAC) IS AMONG THE MOST LETHAL CANCERS, IN PART BECAUSE IT CO- OPTS CORE CELLULAR PROCESSES TO SUPPORT ITS GROWTH AND EVADE INTERVENTION. ONE SUCH ADAPTATION IS THE ABILITY OF PDAC CELLS TO MAINTAIN AN ALKALINE INTRACELLULAR PH (PHI), WHICH SUPPORTS PROLIFERATION, SURVIVAL, AND METASTASIS DESPITE THE ACID LOAD GENERATED BY HIGH METABOLIC ACTIVITY. WHILE MUCH ATTENTION HAS BEEN GIVEN TO PLASMA MEMBRANE TRANSPORTERS THAT EXTRUDE PROTONS, LESS IS KNOWN ABOUT HOW INTERNAL ORGANELLES CONTRIBUTE TO PH HOMEOSTASIS. EMERGING EVIDENCE SUGGESTS THAT ACIDIC COMPARTMENTS LIKE LYSOSOMES AND THE GOLGI PLAY A CENTRAL ROLE BY BUFFERING INTRACELLULAR PROTONS. TO UNCOVER CANCER- SPECIFIC MECHANISMS OF PHI REGULATION, WE PERFORMED A HIGH-THROUGHPUT CHEMICAL SCREEN USING A RATIOMETRIC GOLGI-TARGETED PH PROBE. THIS LED TO THE IDENTIFICATION AN INHIBITOR OF P2X4, WHICH IS AN ATP- GATED ION CHANNEL EXPRESSED IN PDAC. WE FOUND THAT PHARMACOLOGICAL INHIBITION OR GENETIC DEPLETION OF P2X4 DISRUPTS GOLGI ACIDIFICATION, DECREASES PHI, AND SELECTIVELY REDUCES VIABILITY IN PDAC CELLS WHILE SPARING NORMAL CELLS. THESE FINDINGS POINT TO A NOVEL ROLE FOR P2X4 IN MAINTAINING INTRACELLULAR PH THROUGH GOLGI FUNCTION, AN UNEXPECTED VULNERABILITY THAT COULD BE LEVERAGED FOR THERAPEUTIC GAIN. WE HYPOTHESIZE THAT P2X4-MEDIATED CATION FLUX IS REQUIRED TO MAINTAIN GOLGI ACIDIFICATION, AND TARGETING THIS MECHANISM DISRUPTS PHI HOMEOSTASIS AND TUMOR CELL FITNESS. IN AIM 1, WE WILL DEFINE THE MECHANISM BY WHICH P2X4 REGULATES GOLGI PH IN PDAC. WE WILL ALSO DETERMINE WHETHER P2X4 REGULATES THE PH OF OTHER INTRACELLULAR ORGANELLES SUCH AS LYSOSOMES. IN AIM 2, WE WILL EVALUATE THE THERAPEUTIC POTENTIAL OF P2X4 INHIBITION IN PRECLINICAL PDAC MOUSE MODELS. WE WILL ALSO EVALUATE THE POTENTIAL SYNERGY OF P2X4 INHIBITORS WITH STANDARD-OF-CARE CHEMOTHERAPIES AND MECHANISM-SPECIFIC COMPOUNDS IN VIVO. BY UNCOVERING A PREVIOUSLY UNRECOGNIZED ROLE FOR P2X4 IN PH REGULATION AND LINKING IT TO CANCER CELL SURVIVAL, THIS WORK COULD OPEN NEW AVENUES FOR THERAPEUTIC INTERVENTION IN A DISEASE WITH FEW EFFECTIVE TREATMENT OPTIONS.