P02.17.B CREB REGULATES PRO-TUMOR MACROPHAGE POLARIZATION IN GBM AND CAN BE TARGETED TO REVERSE IMMUNOSUPPRESSION

Abstract BACKGROUND The cAMP Responsive Element Binding (CREB) protein is a kinase-inducible transcription factor with prominent roles in the brain, immune cells and tumor cells. We investigated CREB activation in immune cells, in glioblastoma (GBM). As tumor associated macrophages (TAM) are key regulators of tumor immunosuppression, we hypothesized that CREB is an important regulator of macrophage polarization in GBM. MATERIAL AND METHODS We characterized CREB phosphorylation in pro-tumor immunosuppressive and anti-tumor inflammatory TAMs in the GBM tumor microenvironment, using spatially resolved multiplex immunohistochemistry. To test if macrophages respond to GBM cell secreted factors and that these factors regulate TAMs, we performed in vitro experiments using macrophage-differentiated peripheral blood mononuclear cells (PBMC) and stimulated these with GBM cell conditioned media (CM), to measure changes in pCREB expression by western blotting (WB). To test whether CREB directly regulates pro-tumor immunosuppression, we used a CREB inhibitor. To identify CREB target genes in GBM TAMs, we used RNA-seq and Olink proteomics in TAMs, which were either GBM-CM stimulated or not, in the presence or absence of the CREB inhibitor. RESULTS Spatial data revealed that circulating bone marrow derived macrophages (BMDM) arrive at the tumor, expressing inflammatory biomarkers and are rapidly polarized toward an immunosuppressive state when they encounter the GBM tumor parenchyma. TAM immunosuppression correlated with CREB activity, where almost all CD163+ TAMs were also pCREB+, while CREB163- TAMs were pCREB-. In vitro data showed that GBM cell secreted factors provide the necessary signals for CREB activation and pro-tumor TAM polarization. Use of cell signaling inhibitors identified that the cAMP pathway regulates CREB activation in GBM TAMs. Differential expression analysis identified key CREB target genes which regulate TAM immunosuppression. CONCLUSION GBM cell-derived factors are sufficient for pro-tumor M2-like macrophage polarization, which is regulated by a cAMP-CREB signaling-transactivation axis. This study also defines a set of CREB regulated target genes encoding for cytokines and chemokines expressed by GBM-TAMs. Importantly, the expression of these immunosuppressive factors in TAMs can be inhibited by pharmacological targeting of CREB, resulting in reversal of pro-tumor M2-like TAM polarization.