Abstract 4128: Mapping the hypoxic regulation of IFNγ and TNFα-mediated kinase pathways in melanoma
Authors: Simar Pal Singh, Alex Miranda Rodriguez, Russell W. Jenkins
Journal: Cancer Research (2025)

Abstract Introduction: Hypoxia can promote immunologic, transcriptomic, and metabolic alterations in the tumor microenvironment that contribute to immune evasion. Activation of hypoxia signaling pathways has been linked to cancer cell-intrinsic immune evasion and resistance. Hypoxic tumor cells show less susceptibility to direct killing by cytotoxic T lymphocytes and NK cells through several mechanisms, including upregulation of immune checkpoint ligands. IFNγ and TNFα are immune cell-derived effector cytokines critical for tumor immunity. However, the effect of these cytokines in the hypoxic tumor microenvironment is less well studied. Since, kinase signaling pathways are directly activated downstream of cytokine receptor, we explored the functional activity of kinases downstream of IFNγ and TNFα. Methodology: Murine B16F10 melanoma cells were treated with 40 ng/ml of IFN-gamma and TNF-alpha under normoxia (21 % oxygen) and hypoxia (1% oxygen) conditions for 6 or 24 hours. Following treatment, cells were lysed with M-PER Mammalian Extraction Buffer supplemented with Halt Phosphatase Inhibitor Cocktail and Halt Protease Inhibitor Cocktail EDTA free and protein quantification was determined using Pierce Coomassie Plus (Bradford) Assay Kit. Tyrosine and Serine/threonine kinase activity profiles were generated using PamChip® comprising 340 different phosphosites that are known downstream substrates of kinases. Peptide phosphorylation and Upstream Kinase Analysis was performed with PamGene’s tercen analysis pipeline. Results: We found a global reduction of the cytokines induced kinome activation when cells were treated under hypoxia for 6h. However, after 24h a group of kinases associated mainly with the MAPK, RAS and Calcium signaling were increasingly activated in the hypoxia condition. A dynamic activation of the PI3K/AKT pathway was observed when comparing cells exposed to cytokines in normoxia or hypoxia conditions. At 6 h after IFNγ and TNFα treatment under normoxia, we observed an increased PI3K/AKT activity that decreased and became undetectable after 24h. However, when cells were exposed to the cytokines under hypoxia the reduced PI3K/AKT kinase activity at 6 h is reverted after 24h of treatment. Interestingly, the downstream mTOR pathway is predicted to have lower activity in the hypoxia conditions across both time points evaluated. Conclusions: In the present work we unveil strong diversity on the kinome of melanoma cells when exposed to the inflammatory cytokines IFNγ and TNFα under different oxygen levels. These results provide deeper insight into the mechanisms by which hypoxia contributes to immune evasion and response to cytokines. Our data can help inform future therapeutic strategies targeting these pathways to enhance immune-mediated tumor suppression. Citation Format: Simar Pal Singh, Alex Miranda Rodriguez, Russell W. Jenkins. Mapping the hypoxic regulation of IFNγ and TNFα-mediated kinase pathways in melanoma [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 4128.