Abstract PO-095: A cancer cell-intrinsic GOT2-PPARδ axis suppresses antitumor immunity

Abstract

Abstract Glutamic-oxaloacetic transaminase 2 (GOT2) encodes a product with dual functions: primarily as a metabolic enzyme in mitochondria but also as a putative fatty acid (FA) binding protein. The purpose of this study is to investigate GOT2 function and FA-binding in PDA carcinogenesis. Depletion of GOT2 expression with CRISPR-Cas9 or shRNA in human and murine PDA cell lines shows no proliferation defects in vitro. However, GOT2-depleted cells orthotopically implanted in immunocompetent hosts fail to form large tumors. Analysis of cancer cell proliferation shows no difference between control and knockout (KO) tumors, indicating proliferation is not affected by GOT2 in vivo. Genes negatively correlated with GOT2 expression in human PDA reveal ontology clusters associated with adaptive immune response, which suggests an immune-modulatory role of GOT2. Quantitation of immune markers using conventional and multiplex immunohistochemistry confirmed enhanced immune cell infiltration in KO tumors. Inhibition of T cells with blocking antibodies rescued the growth of GOT2 KO tumors, further validating the role of GOT2 in mediating adaptive tumor immunity. In addition to its known mitochondrial and plasma membrane localization, GOT2 expression was observed in the nuclei of PDA cells. It was hypothesized that nuclear GOT2 can deliver FA-ligand to activate PPARs (peroxisome proliferator activator receptor), a class of ligand-activated transcription factors with tumor-promoting properties; specifically, the ubiquitously expressed PPARδ. Analysis of human PDA RNA-seq data shows a significant positive correlation between GOT2 and PPARδ target genes. GOT2-dependent PPARδ activation in PDA cells was confirmed in vitro. Computational modeling of the crystal structure of GOT2 revealed a suitable arachidonic acid (AA) docking site, a known PPARδ ligand. Lipid binding assays of purified protein confirmed direct GOT2-AA binding. The FA docking site of GOT2 was mutated and KO cells were reconstituted with wild type and mutant GOT2. GOT2 mutant cells, compared to wild-type GOT2 cells, showed reduced PPARδ activity, nuclear localization, and interaction with PPARδ. In vivo, mutant GOT2 increased immune cell infiltration. Lastly, the rescue of PPARδ activity in GOT2 KO tumors restores the formation of large tumors with similar immune microenvironments to control tumors. We conclude that the enzymatic function of GOT2 is dispensable for PDA proliferation. However, GOT2 direct FA binding enhances activation of PPARδ to promote an immune-suppressed microenvironment. This non-canonical function of GOT2 can be further explored to elucidate mechanisms of immune evasion in PDA and aid in the development of efficient immunotherapies to improve disease outcomes. Citation Format: Jaime Abrego, Hannah Sanford-Crane, Chet Oon, Xu Xiao, Courtney Betts, Duanchen Sun, Shanthi Nagarajan, Zheng Xia, Lisa Coussens, Peter Tontonoz, Mara Sherman. A cancer cell-intrinsic GOT2-PPARδ axis suppresses antitumor immunity [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-095.