Abstract B050: Mitochondrial NF-kB–inducing kinase orchestrates metabolic fitness, stress responses and immune signaling in glioma

Abstract Background: While NF-kB-inducing-kinase (NIK, MAP3K14) is primarily recognized for its key functions in immunity and inflammation, recent studies suggest a critical role for NIK as a bioenergetic rheostat that links mitochondrial stress and metabolic adaptation in glioma progression. We previously demonstrated that a discrete pool of NIK localizes to mitochondria in glioma cells where it promotes oxidative metabolism, metabolic fitness, cell survival, invasion and proliferation. Indeed, NIK knockout (NIKKO) glioma cells exhibit substantially reduced oxygen consumption and impaired overall metabolic capacity with a compensatory shift toward glycolysis, indicating that glioma cells co-opt mitochondrial NIK to support tumor progression. Notably, these metabolic functions are independent of downstream NF-kB signaling and the mechanism by which NIK rewires cancer cell metabolism remains unknown, thereby limiting the ability to therapeutically exploit this pathway. Methods: Whole-cell proteomic analysis was used to define metabolic dysfunction in NIKKO glioma cells. Immunoprecipitation–mass spectrometry and proximity ligation assays with confocal microscopy were performed to identify mitochondrial NIK-interacting proteins. Site-directed mutagenesis and structure–function analyses were used to map and characterize putative NIK GxxxG and WN motifs, conserved sequences shown to mediate mitochondrial inner membrane protein-protein interactions. The effect of NIK mutations (GxxxG to LxxxL and WN to AA) on mitochondrial function and NF-kB signaling were evaluated using metabolic, biochemical, and immunoblot assays. Results: The top dysregulated pathways in NIKKO glioma cells include impaired mitochondrial respiration, stress response and quality control, as well as heightened innate/adaptive immune signaling and antigen presentation. NIK was observed to interact with several mitochondrial proteins, including TIM50 and TIM8A. Mutating conserved GxxxG and WN motifs identified in NIK abrogated mitochondrial respiration and metabolic fitness in glioma cells. Immunoblot analysis revealed no impairment of activation of either the canonical or non-canonical NF-κB pathway in NIK-mutants compared to wild-type NIK cells, suggesting a novel independent role for NIK at mitochondria. Conclusions: Our findings identify NIK as a putative mitochondrial scaffold, revealing a previously unrecognized function for NIK in integrating bioenergetic state, stress signaling programs and innate immune antigen-presentation pathways in glioma. These data position mitochondrial NIK as a tractable therapeutic target to reprogram glioma cell metabolism and enhance potential responsiveness to immunotherapy. Moreover, by directly linking mitochondrial function in tumor cells to immune activation pathways, mitochondrial NIK emerges as a key regulator of the neuro-immune landscape that shapes glioma progression and treatment response. Citation Format: Victoria Bunting, Raquel Sitcheran. Mitochondrial NF-kB–inducing kinase orchestrates metabolic fitness, stress responses and immune signaling in glioma abstract. In: Proceedings of the AACR Special Conference in Cancer Research: Brain Cancer; 2026 Mar 23-25; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2026;86 (6Suppl): Abstract nr B050.