Abstract 6403: In vivo CRISPR/Cas9 screening identifies fatty acid synthase (FASN) as a target to enhance anti-PD-1 therapy in pancreatic cancer

Purpose: PDAC exhibits significant resistance to anti-PD-1 immunotherapy, limiting its efficacy for the majority of patients. This study aimed to identify novel genetic regulators of immunotherapy resistance in PDAC using in vivo CRISPR/Cas9 screening. We sought to uncover actionable targets that could enhance the efficacy of anti-PD-1 therapy, while also elucidating the underlying mechanisms driving resistance. Methods: A personalized sgRNA library was constructed by integrating bioinformatic analyses, including correlation with PD-1/PD-L1/MHC-I expression and immune scores derived from ESTIMATE, Library infected KPC cells were orthotopically implanted mice treated with anti-PD-1. Single-cell RNA sequencing (GEM-X technology) and flow cytometry were performed to evaluate immune microenvironment changes. The OVA-I model was employed to assess tumor cell-mediated activation of naïve CD8+ T cells and the cytotoxicity of activated CD8+ T cells in vitro. Different pancreatic tumor models were used to evaluate the therapeutic efficacy of FASN inhibition combined with anti-PD-1 therapy. Results: FASN was identified as one of the most depleted genes in the screen and the only top candidate with an inhibitor currently undergoing clinical trials. Knockout of FASN in KPC cell lines reduced tumor cell proliferation across multiple models and enhanced MHC-I expression, increasing antigen presentation. OT-I model demonstrated that FASN knockout improves the activation of naïve CD8+ T cells and enhances CD8+ T cells cytotoxicity against tumor cells. Single-cell RNA sequencing of 41,000 CD45+ immune cells from tumors microenvironment revealed that FASN knockout had minimal impact on total T cell populations but led to a significant increase in cytotoxic T cells. Macrophage subpopulation analysis showed a decrease in precursor macrophages (monocyte-derived macrophages) and M2-like macrophages in the FASN knockout group. Combined transcriptomic analysis in tumor cell line indicated that FASN knockout suppressed CCL2 secretion, reducing monocyte recruitment and subsequent macrophage differentiation in the tumor microenvironment. In multiple in vivo models, FASN knockout or inhibition combined with anti-PD-1 therapy significantly delayed tumor growth and extended overall survival in treated mice. Conclusions: FASN inhibition enhances the efficacy of aPD-1 in PDAC through dual mechanisms. Tumor-intrinsic effects include reduced proliferation and increased antigen presentation, while microenvironmental effects involve T cell sensitization, reduced monocyte recruitment, and macrophage reprogramming. This partially shifts the immunosuppressive "cold" tumor microenvironment to a more inflamed "hot" state. These findings highlight the potential of combining FASN inhibitors with anti-PD-1 therapy to improve immunotherapy outcomes in PDAC. Citation Format: Feng Guo, Chia-Chin Wu, David Eisenbarth, Tadahito Yasuda, Fei Gao, Hyejin Oh, Y Alan Wang. In vivo CRISPR/Cas9 screening identifies fatty acid synthase (FASN) as a target to enhance anti-PD-1 therapy in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6403.