Abstract 656: Suppression of pyruvate carboxylase drives tumor microenvironment immunosuppression
Authors: Coleman Michael F. , Cotul Eylem Kulkoyluoglu , Pfeil Alexander J. , Devericks Emily N. , Safdar Muhammad H. , Chen Hao , Kiesel Violet A. , Teegarden Dorothy , Hursting Stephen D. , Wendt Michael K. 
Published: April 2023

Abstract
Metabolic reprogramming and immune evasion are established hallmarks of the tumor microenvironment (TME). Tumor metabolic dysregulation is a key mediator of tumor immune evasion. High levels of lactate produced by the tumor potently suppress antitumor immunity in the TME. Pyruvate carboxylase (PC), the enzyme responsible for the anaplerotic conversion of pyruvate to oxaloacetate, is required for lung metastasis in triple negative breast cancer (TNBC). Moreover, PC may be dispensable in some cells within the TME, and loss of PC expression is associated with immunosuppression. Here we test whether PC suppression in two murine models of TNBC alters tumor metabolism and promotes immunosuppression. C57BL6/J mice were orthotopically injected with E0771 or M-Wnt tumor cells bearing shRNA either targeting PC or a scramble control (n=3-6/group for each experiment). Tumor growth was monitored by digital calipers. RNA isolated from tumors was used for transcriptomic profiling using Affymetrix Clariom D microarray. Gene set enrichment analysis was used to identify pathways and processes enriched in each condition, as well as to define a hypoxia signature associated with PC expression. Immunohistochemistry was performed using an anti-CD3 antibody to identify T cell infiltration. Untargeted metabolomics, extracellular flux analysis and high resolution respirometry were used to assay metabolic effects of PC suppression. We found that suppression of PC in multiple animal models promotes primary tumor growth (~2 fold increase in size) and alters metabolic, transcriptomic and immunohistochemical markers of tumor immunosurveillance. Specifically, suppression of PC drove reduction of OCR, which was restored when exogenous malate was provided to ensure anaplerosis was non-limiting. Simultaneously, lactate production was elevated by PC suppression, as was in vitro sensitivity to inhibition of lactate metabolism. Critically, we identified that loss of immunosurrveilance following PC suppression could be restored by inhibition of lactate transport. Finally, we demonstrated that PC expression is markedly lower in tumors of patients whose transcriptomic profile indicated higher levels of hypoxia, and confirmed in vitro that hypoxia can directly suppress PC. Taken together, these data demonstrate hypoxia-driven PC suppression may be a key mechanism through which primary tumors limit antitumor immunity in part via enhanced lactate production. Thus, these data highlight that PC-directed tumor metabolism is a nexus of tumor progression and antitumor immunity. Ongoing work will examine the immune profile of the TME from PC suppressed tumors, and identify if PC overexpression is sufficient to enhance antitumor immunity.
Citation Format: Michael F. Coleman, Eylem Kulkoyluoglu Cotul, Alexander J. Pfeil, Emily N. Devericks, Muhammad H. Safdar, Hao Chen, Violet A. Kiesel, Dorothy Teegarden, Stephen D. Hursting, Michael K. Wendt. Suppression of pyruvate carboxylase drives tumor microenvironment immunosuppression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 656.