Project Grant F30CA310119

TARGETING THE MAIN EFFECTOR OF THE WNT PATHWAY WITH PROTACS - PROJECT SUMMARY COLORECTAL CANCER (CRC) IS THE SECOND LEADING CAUSE OF CANCER-RELATED DEATHS WORLDWIDE, WITH MORTALITY PROJECTED TO NEARLY DOUBLE BY 2040. OVER 90% OF CRC CASES EXHIBIT MUTATIONS THAT ABERRANTLY ACTIVATE WNT SIGNALING, STABILIZING CYTOPLASMIC B-CATENIN, A TRANSCRIPTIONAL CO-ACTIVATOR THAT DRIVES EXPRESSION OF GENES PROMOTING CELL PROLIFERATION AND GROWTH. NO FDA-APPROVED DRUGS CURRENTLY TARGET THE WNT PATHWAY IN THE SETTING OF CANCER. DIRECTLY INHIBITING B-CATENIN IS CHALLENGING DUE TO ITS LACK OF ENZYMATIC SMALL-MOLECULE BINDING SITES AND ITS CRITICAL ROLE IN FORMING ADHERENS JUNCTIONS AT THE MEMBRANE, RAISING CONCERNS ABOUT ON-TARGET TOXICITY. PROTEOLYSIS TARGETING CHIMERAS (PROTACS) OFFER A PROMISING SOLUTION BY INDUCING DEGRADATION OF TARGET PROTEINS VIA THE UBIQUITIN-PROTEASOME PATHWAY. UNLIKE TRADITIONAL INHIBITORS, PROTACS CAN EFFECTIVELY TARGET PROTEINS WITHOUT SMALL-MOLECULE BINDING POCKETS AND SELECTIVELY DEGRADE CYTOPLASMIC PROTEINS, MAKING THEM IDEAL FOR ADDRESSING B-CATENIN. RECENTLY, THE FESIK LAB AT VANDERBILT IDENTIFIED A NOVEL B-CATENIN BINDING SITE, ENABLING THE DEVELOPMENT OF POTENT, DRUG-LIKE B-CATENIN PROTACS. MY PRELIMINARY STUDIES WITH ONE SUCH PROTAC, VU9132, DEMONSTRATE THAT IT: 1) SELECTIVELY DEGRADES CYTOPLASMIC B-CATENIN (WNT POOL) WHILE SPARING MEMBRANE- ASSOCIATED, CADHERIN-BOUND B-CATENIN (TOXIC POOL), AND 2) INHIBITS WNT TARGET GENE TRANSCRIPTION IN HEK293T AND SW480 (CRC) CELLS. IN COLLABORATION WITH THE VIVIAN WEISS LAB AT VANDERBILT UNIVERSITY MEDICAL CENTER, WE FURTHER SHOWED THAT VU9132 PROTACS REDUCE DLD1 CRC CELL GROWTH. BASED ON THESE PRELIMINARY FINDINGS, I HYPOTHESIZE THAT B-CATENIN PROTACS WILL SELECTIVELY DEGRADE CYTOPLASMIC B-CATENIN AND SUPPRESS CRC GROWTH ACROSS A WIDE LANDSCAPE OF CRC MUTATIONS THROUGH A WNT DEPENDENT MECHANISM. IN AIM 1, I WILL DETERMINE WHETHER E-CADHERIN BINDING PREVENTS PROTAC DEGRADATION OF MEMBRANE BOUND B-CATENIN REGARDLESS OF CELL LOCATION VIA OVEREXPRESSION OF FULL LENGTH E-CADHERIN AND THE E-CADHERIN INTRACELLULAR DOMAIN FOLLOWED BY QUANTIFICATION OF B-CATENIN POST PROTAC TREATMENT. IN ADDITION, I WILL FURTHER CHARACTERIZE THIS SELECTIVITY OF B- CATENIN PROTACS USING 1) ENGINEERED CELL LINES THAT ALLOW FOR THE QUANTIFICATION OF CYTOPLASMIC AND CADHERIN- BOUND B-CATENIN THROUGH LUMINESCENCE AND 2) BIOCHEMICAL FRACTIONATION ACROSS A PANEL OF SIX CRC CELL LINES WITH VARYING WNT AND NON-WNT MUTATIONS. IN AIM 2, I WILL EVALUATE THE ABILITY OF B-CATENIN PROTACS TO INHIBIT CRC GROWTH USING CRC MONOLAYERS, SPHEROIDS, AND PATIENT-DERIVED ORGANOIDS. WNT TRANSCRIPTIONAL ACTIVITY WILL BE MEASURED BY QPCR, AND TUMOR GROWTH WILL BE ASSESSED VIA CELL VIABILITY, PROLIFERATION, AND APOPTOSIS ASSAYS. THIS STUDY WILL BE THE FIRST TO CHARACTERIZE THE ABILITY FOR B-CATENIN DEGRADERS TO SELECTIVELY TARGET CYTOPLASMIC B-CATENIN OVER MEMBRANE-BOUND, CADHERIN-ASSOCIATED B-CATENIN - A CRITICAL STEP TOWARD VALIDATING B-CATENIN-TARGETING DRUGS AS VIABLE THERAPEUTICS. SUCCESSFUL COMPLETION OF THESE AIMS WILL ALSO HIGHLIGHT THE POTENTIAL OF B-CATENIN PROTACS TO INHIBIT WNT SIGNALING AND CRC TUMOR GROWTH AT CLINICALLY RELEVANT DOSES.