Genotypic Interactions in Brain Cancer Heterogeneity

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Project Summary Minimal improvement in the 12-15-month median survival of patients with IDHwt glioblastoma (GBM) has occurred despite advances in neurosurgery, radiation therapy, and clinical trials for novel therapeutics. A central issue confounding successful treatment is the heterogeneous nature of this aggressive tumor. Multi-omics analyses have provided granularity into GBM cellular composition, illustrating that these malignancies can be classified into three molecular subtypes - classical (CL), mesenchymal (MES), and proneural (PN) - with individual tumors typically harboring mixtures of all three subtypes. As a result, multiple spatially distinct, heterotypic populations exist within GBM, making any lesion- or pathway-specific therapy less effective. While considerable effort has been placed on understanding cell intrinsic mechanisms conferring therapeutic resistance, much less is known about interactions between heterotypic GBM cells that contribute to its recalcitrant nature. A hallmark mutation present in 60% of GBM cases is amplification and mutation of the epidermal growth factor receptor (EGFR). The most common EGFR alteration, EGFRvIII, results from structural deletion within its extracellular domain (ECD) yielding a constitutively active receptor which conveys tumor enhancing and therapy resisting functions. Furthermore, EGFRvIII-expressing cells can transmit these properties to amplified EGFRwt cells through pro-survival, paracrine factors,1 akin to TNFα inflammatory signaling. Like TNFα, EGFRvIII activity, specifically in the context of PTEN inactivation, results in NF-κB RelA/p65 nuclear localization, association with members of the acetylated lysine-binding BET (bromodomain and extra terminal domain) family of enhancer proteins, and activation of inflammatory transcription.1 Given the requirement of RelA K310 acetylation (acK310-RelA) for BET bromodomain interactions2 and central role of NF-κB in driving a PN/CL to MES phenotype transition (MESt),3 we postulate that RelA K310 acetylation acts as a regulatory switch controlling MESt and therapy resistance associated with tumors enriched for MES gene signatures.4 The overall goal of this renewal project is to dissect and target mechanisms whereby GBM PN or EGFR-driven CL tumors transition to MES gene expression (collectively referred to here as MES transition (MESt)) and acquire therapeutic resistance through activation of acK310-RelA/BET-mediated inflammatory gene expression. The following lines of experimentation will be employed: 1) biochemical interrogation of the PTEN signaling pathway to determine effectors mediating RelA K310 acetylation and associated MESt, tumor abundance of microglia and macrophages, and resistance to DNA damage; 2) functional analysis of BET family members, BRD2, 3 and 4, through inducible protein degradation, gene editing, bromodomain (BD1, BD2) pharmacological targeting, and assessment of transcription coupled with acK310-RelA/BET enhancer landscape rewiring; and 3) pharmacological interrogation of the PTEN/RelA/BET axis as a means to overcome resistance to salvage radiation therapy in recurrent GBM patients. 1