Project Grant R01CA282027

BHLHE40 REGULATION OF T CELL FUNCTION DURING CANCER IMMUNOTHERAPY - PROJECT SUMMARY/ABSTRACT FOR CANCER IMMUNOTHERAPIES SUCH AS IMMUNE CHECKPOINT THERAPY (ICT), SUCCESS DEPENDS ON SUSTAINED ACTIVATION OF INTRATUMORAL EFFECTOR T CELLS RECOGNIZING TUMOR ANTIGENS. WHILE MANY OF THE MOLECULAR CHANGES REQUIRED FOR EFFECTOR T CELLS TO CONTROL CANCER ARE KNOWN, MANY KEY EPIGENETIC AND TRANSCRIPTIONAL FEATURES REQUIRED FOR ICT-INDUCED ANTI-TUMOR IMMUNITY ARE UNKNOWN. WE RECENTLY FOUND IN PRECLINICAL SARCOMA AND MELANOMA MODELS THAT ANTI-PD-1 OR ANTI-CTLA-4 ICT INDUCES STRONG UPREGULATION OF THE TRANSCRIPTION FACTOR, BHLHE40, IN TUMOR ANTIGEN-SPECIFIC CD8 AND CD4 T CELLS, WHICH ARE CRUCIAL FOR ICT-INDUCED TUMOR REJECTION AND ELIMINATION. WE FURTHER DISCOVERED CONDITIONAL DELETION OF BHLHE40 IN CD4+ T REGULATORY CELLS (TREGS), CD4 T CELLS, AND CD8 T CELLS RENDERED MICE TREATED WITH ICT INCAPABLE OF REJECTING ICT-SENSITIVE TUMORS. BHLHE40- DEFICIENT CD4 AND CD8 T CELLS EXHIBITED NOTABLE REDUCTIONS IN ICT-DRIVEN INTERFERON GAMMA (IFN-G) PRODUCTION ASSOCIATED WITH DEFECTS IN ICT-INDUCED REMODELING OF INTRATUMORAL MACROPHAGES. GENE SET ENRICHMENT ANALYSIS INDICATED DYSREGULATED METABOLISM WITHIN CERTAIN SUBPOPULATIONS OF CD4+ AND CD8+ T CELLS IN THE ABSENCE OF BHLHE40. HOWEVER, THESE ANALYSES WERE DONE IN MICE LACKING BHLHE40 IN TREGS AND CONVENTIONAL CD4 AND CD8 T CELLS. THEREFORE, IT IS IMPORTANT TO DELINEATE HOW BHLHE40 CONTRIBUTES TO NOT ONLY CD8 BUT ALSO CD4 T CELL (WHICH ARE NOW ACKNOWLEDGED TO BE CRITICAL FOR EFFECTIVE ANTI-TUMOR IMMUNITY) EFFECTOR FUNCTION. WE WILL TEST THE CENTRAL HYPOTHESIS THAT BHLHE40 IS A PIVOTAL TRANSCRIPTIONAL REGULATOR OF BOTH CD4 AND CD8 T CELL ANTI-TUMOR EFFECTOR FUNCTION DURING ANTI-PD-1/ANTI-CTLA-4 ICT AND TUMOR-SPECIFIC CANCER VACCINE THERAPIES. IN AIM 1, WE WILL USE CONVENTIONAL CD4 OR CD8 T CELL-SPECIFIC BHLHE40 KNOCKOUT (KO) MICE, NEWLY GENERATED MOUSE MELANOMA MODELS THAT EXPRESS DEFINED NEOANTIGENS, AND MOUSE SARCOMA LINES TO DETERMINE HOW CELL- SPECIFIC DELETION OF BHLHE40 IMPACTS RESPONSE TO ANTI-PD-1 AND/OR ANTI-CTLA-4 ICT OR TUMOR-SPECIFIC NEOANTIGEN CANCER VACCINE TREATMENT. WE WILL THEN ANALYZE TUMORS FROM CD4 OR CD8 T CELL SPECIFIC BHLHE40 KO MICE AND BHLHE40 REPORTER MICE BY SINGLE CELL RNA SEQUENCING (SCRNASEQ), MASS CYTOMETRY (CYTOF), AND CODEX MULTIPLEX IMAGING, TO ASSESS HOW LOSS OF BHLHE40 IN CD4 OR CD8 T CELLS ALTERS THE IMMUNE TUMOR MICROENVIRONMENT. IN AIM 2, WE WILL DISSECT THE MECHANISM BY WHICH BHLHE40 REGULATES CD4 AND CD8 T CELL FUNCTION IN THE CONTEXT OF DIFFERENT IMMUNOTHERAPIES BY SEVERAL APPROACHES SUCH AS FUNCTIONAL ASSAYS, SINGLE- CELL SEQUENCING ASSAY FOR TRANSPOSASE-ACCESSIBLE CHROMATIN (SCATACSEQ), CHROMATIN IMMUNOPRECIPITATION SEQUENCING (CHIPSEQ), AND LUCIFERASE REPORTER ASSAYS. IN AIM 3, WE WILL RELATE OUR FINDINGS IN MICE TO HUMANS BY FIRST MANIPULATING THE EXPRESSION OF BHLHE40 IN HUMAN CD4 AND CD8 T CELLS TO DETERMINE HOW BHLHE40 REGULATES THEIR EFFECTOR FUNCTION AND THEN ANALYZING PRE- AND POST-ICT TREATMENT PATIENT SAMPLES. THESE STUDIES WILL CONTRIBUTE TO OUR UNDERSTANDING OF HOW T CELLS RECOGNIZING TUMOR ANTIGENS MAINTAIN EFFECTOR FUNCTIONS DURING ICT AND THERAPEUTIC NEOANTIGEN CANCER VACCINES AND COULD IDENTIFY BHLHE40 AS A NOVEL THERAPEUTIC TARGET.