DDDR-17. CHEMOTHERAPEUTIC STRESS INDUCES NONCANONICAL HIF1Α SIGNALING UNDER NORMOXIA IN GLIOBLASTOMA DRIVING THERAPEUTIC RESISTANCE

Abstract

Abstract Glioblastoma (GBM) is an aggressive brain tumor with no cure. Standard treatment includes adjuvant, concomitant temozolomide (TMZ) and radiation, but most patients do not survive more than 15 months. Tumor adaptations to temozolomide (TMZ) contribute to glioblastoma (GBM) chemoresistance and recurrence, and hypoxia inducible factor subunit alpha (HIF1α) and downstream effectors are implicated in attenuated therapeutic responses. We have previously shown that TMZ-dependent hijacking of HIF1α signaling under normoxia is independent from physiological hypoxia in the tumor core. Our results further showed that TMZ promotes HIF1α accumulation enhancing stem-like characteristics of GBM cells driving therapy-resistant populations involved in recurrence. However, noncanonical HIF1α signaling mechanisms are not well understood preventing the success of HIF1α inhibitors in GBM thus far. Here, we investigate how TMZ induces HIF1α in an oxygen-independent manner by characterizing the HIF1a binding profile post-therapy. We cultured patient-derived xenograft (PDX) models of GBM in vitro and found HIF1α expression and hypoxia response element (HRE) activity peak 96h post-therapy using western blot and luciferase assays. Our lab also performed a whole-genome CRISPR-Cas9 knockout screen in GBM cells over 28 days to determine genetic drivers of GBM aggression. HIF1α was found to be particularly enriched in this screen, further supporting the role of chronic hypoxia signaling for GBM proliferation (p-value < 0.05). We show TMZ therapy prevents proteasome-dependent degradation through reduced binding of PHD2 and VHL allowing nuclear translocation of HIF1a through western blot and immunocytochemistry. Thus, we performed immunoprecipitation of HIF1α 96h post-therapy for proteomic characterization to establish how TMZ uniquely regulates HIF1α stabilization in the absence of oxygen tension, and these results will be presented. We will subsequently characterize the regulatory role of TMZ-dependent post-translational modifications to HIF1α. Elucidating the molecular mechanisms of therapy-mediated HIF1α regulation can produce better therapeutic targets and increase TMZ efficacy.