Abstract 2997: Tumor hypoxia blocks the induction of antigen processing machinery
Authors: Ramos Alexis Rebecca , Smith Matthew , Panchal Heena , Mailloux Adam 
Published: April 2023

Abstract
Antigen presentation is critical for adaptive immune response to cancer but is often subverted to evade immune response. While hypoxia is a pervasive feature of solid tumors, it’s role in tumor antigen processing remains understudied. To assess the effects of reduced oxygen on antigen processing machinery, we used interferon (IFN)-γ to induce the expression of the immunoproteasome (IP), a proteasome isoform critical for the tumor antigen processing, in A549 human lung cancer cells cultured under 20% O2, or 2% O2. Treatment with IFN-γ induced protein expression of IP subunits LMP2, LMP7, and MECL-1 in a dose-dependent manner under 20% O2 as assessed by western blotting. In contrast, IFN-γ failed to induce protein expression of these subunits under hypoxic conditions. We observed similar blockades of other antigen processing pathway components in response to IFN-γ under hypoxic conditions including TAP, tapasin, and calreticulin, as well as secondary mediators of IFN-γ-stimulated genes including several members of the interferon regulatory factor (IRF) family. Surprisingly, under identical conditions, induction of IP subunit mRNAs appear intact under hypoxia as assessed by RT-PCR, with similar levels of LMP2, LMP7, and MECL-1 upregulated by IFN-γ regardless of oxygen level, suggesting hypoxic IP blockade occurs post-transcriptionally or post-translationally. In line with this notion, primary mediators of IFN-γ signaling, including phosphorylated (Tyr701) STAT1 levels were unaffected by oxygen levels. To investigate the mechanism behind this phenomenon, we created hypoxia-inducible factor (HIF)-1a and/or HIF-2a knockout A549 cell lines or used cobalt chloride (CoCl2), a hydroxylase inhibitor, to directly interrogate the hypoxia signaling pathway. Blockade of IP subunit induction in response to IFN-γ was not observed after CoCl2 treatment, nor did HIF-1a and/or HIF-2a genetic deletion rescue IP subunit blockade in A549 cells cultured under hypoxia, indicating that hypoxia-induced IP blockade is independent of the HIF pathway. To test if this phenomenon is regulated by epigenic means, we treated cells with 5-Azacytidine (5-aza), a cystine analog nucleoside, and observed a re-establishment of IP subunit protein expression under hypoxia in response to IFN-γ. Given that 5-aza preferentially integrates RNA over DNA, and that IP subunit mRNA levels appear unaffected by hypoxia, suggests that hypoxia-induced IP blockade may be due to hypoxia-specific cytosine modifications within mRNA transcripts. Current studies include efforts to map mRNA methylation patterns and interrogate mRNA methylation transferases under hypoxic conditions. These studies introduce a novel link between hypoxia and reduced cancer cell antigen processing machinery and may help identify druggable epigenetic targets that can be used to enhance the immunogenicity of hypoxic tumors.
Citation Format: Alexis Rebecca Ramos, Matthew Smith, Heena Panchal, Adam Mailloux. Tumor hypoxia blocks the induction of antigen processing machinery [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 2997.