Abstract
Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) remains an aggressive malignancy, and its unique genetic makeup and immunosuppressive tumor microenvironment (TME) produce a lack of response to current therapies. Previously, we have identified Cyclic AMP Response Element Binding protein 1 (CREB) as an oncogenic transcriptional factor that promotes disease aggressiveness, poor survival, and immune suppression. Based on these, we sought to determine the role of tumor intrinsic CREB in promoting immunosuppressive TME in PDAC. Methods: We have generated a genetically engineered mouse model (GEMM) of pancreas-specific CREB deletion (CREBfl/fl) in LSL-KrasG12D/+; Trp53 R172H/+; Pdx1Cre/+ (KPC) mice that phenocopy human PDAC disease. CRISPR/CAS9-based genomic editing was utilized to ablate CREB (CREBKO) in KPC tumor cells. RNA-sequencing analysis was performed in KPC CREB wild type (CREBWT) vs. CREBKO tumor cells to identify CREB-mediated transcriptomic changes. Chromatin immunoprecipitation (ChIP-qPCR) analysis was performed in KPC tumor cells. Syngeneic orthotopic tumor implantation of these cells was performed in the pancreata of mice. Immunophenotyping was accomplished to assess changes in the immune subsets with CREB deletion invivo. Additionally, these tissues were also processed for single-cell RNA (scRNA) transcriptomics analysis to evaluate changes on different cellular constituents. Results: Pancreas-specific CREB deletion in the KPC GEMM led to a significant reduction in the primary tumor burden, liver metastases, and improved overall survival compared to wild-type KPC. In assessing the immune repercussions of CREB deletion in pancreatic tumors, we observed a decreased infiltration of tumor-promoting CD11b+ F4/80+ CD206+ [M2-like tumor-associated macrophages (TAMs)] and a concomitant increase in the antigen-presenting M1-like macrophages (F4/80+MHC-IIhighCD86high). Additionally, scRNA sequencing analysis within the macrophage compartment in CREBKO tumors revealed significant enrichment of M1 hallmark signaling pathways. Also, CREB ablation in these tumors further facilitated increased infiltration of activated effector memory CD8+ T cells and resulted in enhanced adaptive immune response within the PDAC TME. Mechanistically, RNA transcriptomic-based analysis of CREBKO tumor cells revealed, Leukemia inhibitory factor (LIF) as one of the downstream targets of CREB. ChIP qPCR analysis after CREB1 pulldown confirmed its occupancy on LIF promoter regulatory region. Incubation of macrophages with CREBWT conditioned media in the presence of LIF neutralizing antibody or blocking its receptor expression using EC359 pushed these macrophages towards an M1-like phenotype, confirming its role as a mediator of tumor cell macrophage crosstalk. Conclusion: These findings broaden our understanding of the tumor cell-intrinsic role of CREB and provide new insights into its molecular underpinnings in fostering immunosuppressive profile by promoting skewness of TAMs towards M2 phenotype in PDAC. Citation Format: Siddharth Mehra, Vanessa T. Garrido, Samara Singh, Iago De Castro Silva, Anna Bianchi, Luis A. Nivelo, Nilesh U. Deshpande, Austin R. Dosch, Zhou Zhiqun, Supriya Srinivasan, Christine I. Rafie, Ifeanyichukwu C. Ogobuiro, Xi Chen, Alejandro Villarino, Jashodeep Datta, Nipun B. Merchant, Nagaraj Nagathihalli. CREB-LIF axis drives immune suppression by promoting macrophage polarization in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C040.