Abstract 7250: Extracellular acidity in the TME interferes with the IFN-γ induction of the immunoproteasome

Abstract The immunoproteasome (IP) is a component of the MHC class I presentation pathway (MHC1PP) that is induced by IFN-γ, functions to augment the immunogenicity of cancer cells by generating optimal tumor antigens from polypeptide precursors, and is correlated with favorable responses to immunotherapy. Understanding tumor intrinsic factors that regulate the IP in cancer is essential for addressing immunologically cold tumors which respond poorly to immunotherapy. Extracellular acidity (pH 6.5) is a hallmark of solid tumors and despite being shown to impair CD8+ anti-tumor immunity, remains understudied as a driver of immune-evasion in cancer. Here, we present data that extracellular acidity in vitro can restrict the induction of the catalytically active subunits of the IP, in particular 20s proteasome subunit beta type 9 (β1i) and 20s proteasome subunit beta type 10 (β2i). Various human and murine cancer cell lines (Human: pancreatic ductal adenocarcinoma PANC1, hepatocellular carcinoma HEPG2, non small cell carcinoma A549; Murine: non small cell carcinoma LLC) were cultured in control media (DMEM pH 7.4) versus acidic media (DMEM pH 6.5) and stimulated with IFN-γ to induce the IP. Cell lysates were assessed by Western blot and demonstrated a sharp reduction in the protein induction of β1i and β2i, which are both required for the formation of the mature IP, under acidic conditions compared to controls. RT-PCR analysis of A549 cells indicates that these proteins are blocked transcriptionally as well as translationally. Mechanistically, we demonstrate via Western blot that activation of signal transducer and activator of transcription 1 (STAT1), the critical transcription factor required for IFN-γ induction of the IP, is delayed in acidic conditions compared to controls. We further show that signal transducer and activator of transcription 3 (STAT3), a pro-tumorigenic transcription factor and antagonist of STAT1 transcriptional activity, is activated under acidic conditions in A549 cells and furthermore IFN-γ enhances this STAT3 activation but only under acidic conditions. Functionally, we show via flow cytometry that downstream of the blockade in IP subunit induction that occurs under acidic conditions, surface MHC I expression is diminished. Overall, these studies indicate extracellular acidity in the TME restricts the induction of IP components potentially through a STAT3 dependent mechanism, suggesting that targeting STAT3 activation in acidic tumors may improve tumor immunogenicity and responses to immunotherapy. Citation Format: Matthew G. Smith, Alexis Ramos, Heena Panchal, Adam Mailloux. Extracellular acidity in the TME interferes with the IFN-γ induction of the immunoproteasome [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 7250.