Project Grant F31CA301909

UNCOVERING THE ROLES OF SYMMETRIC DIMETHYLATION OF ARGININE ON ZNF326 - PROJECT SUMMARY DRIVER MUTATIONS ARE CRITICAL IN THE DEVELOPMENT OF CANCERS AND OCCUR THROUGH THE DELETION OF TUMOR SUPPRESSOR GENES OR THE ACTIVATION OF ONCOGENES. CDKN2A, OR CYCLIN-DEPENDENT KINASE INHIBITOR 2A, IS A GENE THAT FALLS INTO THE FIRST CATEGORY, AND WHEN THE CDKN2A TUMOR SUPPRESSOR GENE IS DELETED, ANOTHER GENE ENCODING MTAP, OR METHYLTHIOADENOSINE PHOSPHORYLASE, IS DELETED AS A PASSENGER DELETION. THIS CO-DELETION IS SEEN IN 15% OF ALL SOLID HUMAN TUMORS, INCLUDING SEVERAL AGGRESSIVE CANCERS THAT LACK EFFECTIVE THERAPY SUCH AS PANCREATIC CANCER. CANCER CELLS WITH THE CDKN2A/MTAP CO-DELETION HAVE A HIGHER SUSCEPTIBILITY TO THE INHIBITION OF PROTEIN ARGININE METHYLTRANSFERASE 5 (PRMT5) COMPARED TO HEALTHY CELLS, AND THIS SYNTHETIC LETHALITY HAS LED TO EXCITING THERAPEUTIC OPPORTUNITIES FOR THE TREATMENT OF THIS SUBSET OF CANCERS. ALTHOUGH PRMT5 INHIBITORS ARE BEING DEVELOPED AND TESTED IN CLINICAL TRIALS, THE EXACT ROLE OF PRMT5 IN THE CELL IS NOT FULLY KNOWN. ADDITIONALLY, THE WAY BY WHICH PRMT5 BINDS ITS SUBSTRATES HAS NOT BEEN FULLY ELUCIDATED. FURTHER UNCOVERING THE BINDING MECHANISMS OF PRMT5 COULD LEAD TO IDENTIFICATION OF DRUGGABLE SITES DIRECTLY ON THE ENZYME, AND BETTER UNDERSTANDING OF THE ROLE PRMT5 METHYLATION PLAYS ON ITS SUBSTRATES COULD LEAD TO THE IDENTIFICATION OF SUBSTRATE TARGETS THAT CAN BE INHIBITED TO HAMPER PRMT5 ACTIVITY INDIRECTLY. THE GOAL OF THIS PROJECT IS TO DETERMINE THE CONSEQUENCE OF PRMT5 ACTIVITY ON ITS SUBSTRATES (AIM 1), TO UNCOVER NOVEL BINDING MECHANISMS TO PRMT5 (AIM 2), AND TO UNDERSTAND THE ROLE THAT ZNF326 PLAYS IN PANCREATIC DUCTAL ADENOCARCINOMA (AIM 3). THE PROJECT WILL STUDY PRMT5 ACTIVITY THROUGH ONE OF ITS SUBSTRATES, ZNF326, WHICH IS REPRESENTATIVE OF PRMT5 SUBSTRATES DUE TO ITS PROPERTIES AS AN RNA-BINDING PROTEIN. IN AIM 1, A CO- IMMUNOPRECIPITATION (CO-IP) MASS-SPECTROMETRY EXPERIMENT WILL BE PERFORMED TO TEST WHETHER PRMT5 ACTIVITY IMPACTS THE PROTEIN-PROTEIN INTERACTIONS OF ZNF326. I HYPOTHESIZE THAT METHYLATION BY PRMT5 WILL IMPAIR THE ABILITY OF ZNF326 TO FORM NECESSARY INTERACTIONS WITH OTHER PROTEINS. IN AIM 2, A CO-IP EXPERIMENT USING TRUNCATED MUTANTS OF ZNF326 WILL HELP UNCOVER THE SEGMENT OF ZNF326 NEEDED FOR BINDING TO PRMT5, AND IN- VIVO STUDIES WILL BE PERFORMED TO FURTHER TEST THIS INTERACTION. ZNF326 IS UNDERSTUDIED IN CANCERS, BUT EXISTING STUDIES FIND THAT IT MAY BE INVOLVED IN CARCINOGENESIS. AIM 3 WILL FOCUS ON TESTING THE ROLE OF THIS TRANSCRIPTION FACTOR IN PANCREATIC CANCER, AND HOW IT MAY BE RESPONDING IN CELLS TO INHIBITION OF OTHER SPLICING FACTORS AND PRMT5. THE EFFECTS OF PRMT5 METHYLATION ON ZNF326 ACTIVITY IN PANCREATIC CANCER WILL BE ASSESSED VIA RNA- SEQ. RESULTS OF THIS PROJECT WILL ENHANCE OUR UNDERSTANDING OF THE SYMMETRIC DIMETHYLARGININE (SDMA) MARK THAT PRMT5 PLACES ON ITS SUBSTRATES, WHICH WILL HELP US UNDERSTAND THE ROLE OF THIS POST TRANSLATIONAL MODIFICATION IN CANCERS AND ILLUMINATE NOVEL AVENUES BY WHICH THIS ENZYME CAN BE TARGETED FOR THE TREATMENT OF CDKN2A/MTAP DEFICIENT CANCERS.