Project Grant F31CA294913

DECODING ADAPTIVE FUNCTIONS OF STING IN CANCER PROGRESSION - PROJECT SUMMARY CANCER METASTASIS IS A COMPLEX, MULTISTEP PROCESS THAT REQUIRES CELLS TO ADAPT AND SURVIVE IN STRESSFUL CONDITIONS. THESE ADAPTIVE RESPONSES ARE CARRIED OUT BY PROTEINS WITHIN THE CELL. MANY PROTEINS CAN PLAY DIFFERENT ROLES BASED ON THEIR CONTEXT, AS HAS BEEN DEMONSTRATED FOR STIMULATOR OF INTERFERON GENES (STING). STING HAS A WELL CHARACTERIZED ROLE IN INNATE IMMUNITY AND HAS BEEN PROPOSED TO ACTIVATE AN ANTI-TUMOR IMMUNE RESPONSE EARLY IN DISEASE PROGRESSION. HOWEVER, RECENT WORK SHOWS THAT ITS CHRONIC ACTIVATION IN CANCER CELLS CAN DRIVE METASTASIS. IN ADDITION TO ITS ROLE IN THE TYPE 1 INTERFERON RESPONSE, STING ALSO HAS PUTATIVE ROLES IN ER STRESS RESPONSE SIGNALING AND THE NF-KB PATHWAY. AN EXPLANATION FOR HOW STING PLAYS THESE ROLES IS PLEIOTROPY, MEANING ONE GENE CAN CAUSE MULTIPLE, UNRELATED PHENOTYPES. IT IS UNCLEAR HOW THESE ROLES ARE MEDIATED AND HOW THESE ROLES CHANGE IN DIFFERENT CONTEXTS SUCH AS DURING CANCER PROGRESSION. MY CENTRAL HYPOTHESIS IS THAT STING ADOPTS ADAPTIVE FUNCTIONS MEDIATED THROUGH BINDING PARTNERS THAT INTERACT WITH DISTINCT FUNCTIONAL DOMAINS. THE 2 SPECIFIC AIMS OF MY PROJECT ARE (1) TO MAP THE EMERGENT DOWNSTREAM FUNCTIONS OF STING AND DETERMINE IF THESE ARE MEDIATED BY BINDING PARTNERS, AND (2) PROBE ONE PRO-METASTATIC STING VARIANT (K337X) TO UNDERSTAND HOW BINDING PARTNERS THAT STABILIZE OR DEGRADE STING INFLUENCE ITS FUNCTION. TO QUANTIFY RESIDUE-SPECIFIC FUNCTIONS OF STING IN A HIGH-THROUGHPUT MANNER, MY LAB HAS GENERATED A SATURATION MUTAGENESIS LIBRARY OF STING WITH EACH VARIANT DISTINGUISHED BY A DNA ENCODED BARCODED. WE HAVE PERFORMED SCRNA-SEQ SCREENS ON THE VARIANT LIBRARY WITH THE NATURAL AGONIST OF STING (CGAMP) TO QUANTIFY DOWNSTREAM TRANSCRIPTIONAL EFFECTS WITH EACH VARIANT. I AM NOW COUPLING SELECT VARIANTS IDENTIFIED IN THE SCREEN WITH FUNCTIONAL FLUORESCENT REPORTERS TO CLASSIFY AND VALIDATE VARIANTS ABLE TO PERFORM DOWNSTREAM INTERFERON, ER STRESS, AND NF- KB FUNCTIONS. I WILL THEN USE PROTEOMICS TO IDENTIFY IF BINDING PARTNERS ARE RESPONSIBLE FOR DIFFERENCES IN VARIANT FUNCTION. WE HAVE ALSO PERFORMED 2 METASTASIS ASSAY SCREENS WITH THE LIBRARY AND IDENTIFIED ONE STRIKING PRO- METASTATIC VARIANT, K337X. I AM FOLLOWING UP ON 2 SPECIFIC BINDING PARTNERS THAT ARE KNOWN TO EITHER STABILIZE OR PROMOTE DEGRADATION OF STING TO TEST IF THE CONTEXT OF STING, MEANING BINDING PARTNERS AVAILABLE, RATHER THAN SPECIFIC CLINICAL MUTATIONS COULD BE RESPONSIBLE FOR ITS EMERGENT FUNCTIONS. I WILL STUDY THESE FUNCTIONS OF STING IN 2 MOUSE MODELS OF CHROMOSOMALLY UNSTABLE TRIPLE NEGATIVE BREAST CANCER CELL LINES. THE LONG-TERM GOAL OF THIS WORK IS TO BETTER UNDERSTAND THE MEDIATORS OF STING'S FUNCTIONS SO THAT ITS DYSREGULATION IN CANCER CAN BE BETTER UNDERSTOOD AND MORE EFFECTIVELY TARGETED WITH THERAPEUTICS.