BSBM-20 INVESTIGATING THE ROLE OF OXIDATIVE PHOSPHORYLATION (OXPHOS) IN CD4+ T CELL ACTIVATION AND RESPONSE TO ANTI-PD1/ANTI-LAG3 IMMUNOTHERAPY IN MELANOMA BRAIN METASTASIS (MBM)

Abstract

Abstract Brain metastasis is a frequent and deadly complication of metastatic melanoma. While extracranial metastases (ECMs) are often treatable, MBMs remain a critical treatment (tx) challenge to overcome. We previously showed that MBMs are characterized by increased utilization of OXPHOS compared to ECMs, and OXPHOS inhibition increased expression of immune activation transcriptional signatures in MBMs. Thus, we tested the role of tumor OXPHOS on the immune microenvironment and checkpoint immunotherapy response in a syngeneic mouse model with concurrent intracranial (ICr) and subcutaneous (SQ) tumors. Selective OXPHOS inhibition was achieved via CRISPR-KO of Ndufs4 (mitochondrial complex I) in B16-F10 cells and confirmed via Seahorse Analyzer MitoStress Test. Multiplex cytokine analysis of culture supernatant revealed that NDUFS4 KO increased anti-tumor cytokines IFNγ (p=0.016) and IL-2 (p=0.023) and decreased pro-tumor chemokine CXCL2 (p<0.001). Next, we implanted concurrent ICr and SQ B16-F10 tumors in C57BL/6J mice. Flow cytometry demonstrated decreased CD4+ and CD8+ T cell infiltration of ICr versus SQ tumors in both B16-F10 and NDUFS4 KO tumors (all p<0.001). NDUFS4 KO increased total CD4+ T cells (p=0.041) and activated CD4+ T cells (p<0.001) in ICr tumors. Finally, we tested the efficacy of tx with combination anti-PD1/anti-LAG3 (200μg/100μg intraperitoneal 3x/week) versus isotype controls (Ctrl) in these mice. Compared to Wildtype (WT) tumors with Ctrl tx, we observed improved overall survival (OS) in mice with NDUFS4 KO tumors + Ctrl (HR 0.117; p=0.015), WT tumors + anti-PD1/anti-LAG3 (HR 0.071; p=0.005), and NDUFS4 KO tumors + anti-PD1/anti-LAG3 (HR 0.049; p=0.002). For anti-PD1/anti-LAG3 tx, there was a trend for improved OS with NDUFS4 KO versus WT tumors (HR 0.317; p=0.130). Together, these data implicate tumor OXPHOS in CD4+ T cell immunosuppression and a potential target to enhance response to checkpoint immunotherapy in MBMs.