Abstract Background Activating KRAS mutations, stromal desmoplasia, and immune exclusion are fundamental to therapeutic resistance in PDAC. We have previously shown an inverse correlation of reciprocally-activated MEK1 and STAT3 signaling, combined inhibition of which remodels the tumor stroma resulting in enhanced effector immune cell infiltration and improved survival in an aggressive genetically engineered mouse model (GEM) of PDAC. Based on these results, we sought to determine the effects of cancer-associated fibroblasts (CAF) specific silencing of MEK1 and STAT3 on the immune microenvironment and PDAC tumor growth. Methods CRISPR/Cas9 ablation of MEK1 and STAT3 was performed in CAFs derived from LSL-KrasG12D/+; Trp53fl/+; Pdx1-Cre (KPC) GEM. RNA sequencing identified transcriptomics changes in MEK1 KO / STAT3KO CAFs vs. wildtype KPC-CAFs. The effect of CAF-specific deletion of MEK1/STAT3 in modulating the immune microenvironment was assessed using multiplex immunophenotyping in orthotopic tumor implantation models of co-cultured KPC tumor cells with MEK1KO / STAT3KO CAFs. Results CAF-specific MEK1/STAT3 ablation resulted in significant retardation of growth kinetics of 3D spheroids, as well as migration and invasion, in KPC tumor cell: CAF co-culture systems. Interestingly, the transcriptome of MEK1KO/ STAT3KO CAFs was strongly enriched for antigen-presenting machinery, IL-12, and IFN signaling pathways, while revealing downregulation of Wnt/β-catenin and collagen pathways. Multiplex cytokine arrays confirmed dramatically increased IL-12p70, IL-2, CXCL10 secretion, and decreased IL-10 secretion in MEK1KO / STAT3KO vs. wildtype. Flow cytometric analysis confirmed increased expression of class I and class II MHC, but not co stimulatory molecules, expression in these double knock out CAFs. Orthotopic implantation of MEK1KO / STAT3KO vs. wildtype CAFs with KPC tumor cells in C57BL/6 pancreata revealed significantly increased infiltration of activated effector degranulating CD8+ T-cells (CD62LlowCD44highCD107low) with reduced exhaustion phenotype (PD1highCD107low)in MEK1KO /STAT3KO vs. wild type CAF tumor-bearing mice. Conclusion CAF-specific ablation of MEK1 and STAT3 generates features suggestive of augmented antigen-presenting capacity and results in adaptive immune invigoration in the PDAC tumor microenvironment. Further mechanistic studies exploring this novel CAF-specific phenotype is warranted. Citation Format: Siddharth Mehra, Xizi Dai, Anna Bianchi, Austin Dosch, Iago de Castro Silva, Prateek Sharma, Samara Singh, Supriya Srinivasan, Nagaraj Nagathihalli, Jashodeep Datta, Nipun Merchant. Targeting of cancer associated fibroblast-specific MEK1 and STAT3 to overcome immunosuppressive microenvironment in Pancreatic Ductal Adenocarcinoma (PDAC) [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-056.