Abstract 7249: TME hypoxia drives immunopeptidomic deficiencies in non-small cell lung cancer by blocking translation of interferon stimulated genes within the MHC I pathway

Matthew G. Smith; Alexis Ramos; Heena Panchal; Adam Mailloux

doi:10.1158/1538-7445.AM2025-7249

Loss or reduction in the expression of interferon stimulated genes (ISGs) which augment the MHC class I presentation pathway (MHC1PP) predict poor responsiveness to immunotherapy as these genes are required for tumor rejection mediated through CD8+ T cells. Understanding how these MHC1PP components are regulated is critical for the design of strategies to enhance immunotherapy responses in solid tumors, particularly those deemed "cold" and poorly immunogenic through low-level expression of the MHC1PP. Here, we present evidence that tumor hypoxia, a hallmark of solid cancers, blocks the translation of ISGs comprising the MHC1PP. We first established that tumor hypoxia in vitro (2% O2) blocks IFN-γ induction of MHC1PP components including the immunoproteasome (IP) proteolytic subunits and ER chaperones and transporter proteins involved in generating a peptide-MHC I complex (p-MHC I) via Western blot analysis of human (A549) and murine (LLC) non-small cell lung cancer (NSCLC) lines cultured at normoxia (20% O2) versus hypoxia. We then assessed the immunopeptidome of A549 cells through immunoprecipitation of intact p-MHC I and analysis of presented antigen via LC-MS based immunopeptidomic workflows. We showed that under normoxia, IFN-γ induction of the MHC1PP results in significantly enhanced antigen diversity and a three-fold increase in total unique tumor antigens, while hypoxia completely abolishes this augmentation of the immunopeptidomic landscape. Mechanistic studies indicate that while MHC1PP components are blocked at the protein level, mRNA transcript levels are largely unaffected as determined by RT-PCR of A549 cultures. 5-methylcytosine (m5C) ELISA experiments showed that hypoxia significantly increases the abundance of m5C modifications on RNA and reversing this accumulation through RNA but not DNA specific methyltransferase inhibitors corresponds with restored protein expression of MHC1PP components in hypoxic A459 cells, suggesting a potential epitranscriptomic mechanism of translation regulation. In vitro, restoring oxygen to A549 cells rapidly reverses the blocked induction of MHC1PP components. To corroborate this in vivo, subcutaneous LLC tumors were grown on C57Bl/6 mice subjected to whole body hyperbaric oxygen therapy (HBOT) versus controls. Compared to controls, HBOT treated mice had significantly smaller tumors, showed prolonged survival, and exhibited greater CD8+ tumor infiltrating lymphocyte (TIL) activation status. Overall, these studies indicate that tumor hypoxia regulates the translation of ISGs comprising the MHC1PP, potentially driving or contributing to immunologically “cold” TME in solid cancers such as NSCLC, and restoring tumor oxygen in vivo is a potential strategy to augment tumor immunogenicity and improve responses to additional treatment modalities.

Abstract 7249: TME hypoxia drives immunopeptidomic deficiencies in non-small cell lung cancer by blocking translation of interferon stimulated genes within the MHC I pathway

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