CSIG-34. TYROSINE PHOSPHORYLATION OF NON-CANONICAL NF-ΚB P52 PROMOTES GLIOMA GROWTH AND CHEMORESISTANCE IN GBM

Abstract p52 is a nuclear factor-κB (NF-κB) transcription factor that comprises the non-canonical NF-κB pathway. The impact and mechanism of non-canonical NF-κB signaling remains understudied in glioblastoma (GBM). Subunit post-translational modification is a known mechanism of NF-κB activation, however phosphorylation of p52 has never been demonstrated. Elevated tyrosine kinase activity is a known-feature of GBM and portends worse survival. Further, p52 has been shown to upregulate factors that likely promote invasion of GBM, thereby reducing survival. Activation of p52 may serve as a link between increased kinase activity and poor survival in GBM. p52-knockout cell lines were generated using the CRISPR-Cas9 system and assessed for growth and survival differences in vitro and in syngeneic murine glioma models. Public glioma datasets and an institutional cohort were surveyed to corroborate p52-dependent survival differences in GBM. To determine whether p52 is post-translationally modified, we performed tandem mass spectrometry on p52 in GBM cells. Interacting factors were also analyzed and tested for site-specific modification of p52 via co-IP studies. We then generated PTM-null, knock-in glioma cell lines using CRISPR-CAS9 editing to demonstrate the functional significance of p52 PTMs, which were assessed using assays of relative cell growth, stemness, chemosensitivity and survival. Finally, we performed unbiased analysis of the p52 PTM-dependent transcriptome via RNA-seq. p52 loss significantly reduced glioma growth rate in vitro and improves murine survival. Retrospective analysis of GBM in patients suggests that p52 levels are negatively prognostic. Mass spectrometry identified multiple p52 PTMs with tyrosine phosphorylation sites as top hits. Identified phospho-tyrosine residues specifically interact with SRC-family kinases. PTM site editing significantly reduces glioma cell growth and stemness while enhancing chemosensitivity, reflecting broad transcriptome level changes. In sum, p52 activity enhances GBM growth and chemoresistance, which is partially modulated by tyrosine-specific phosphorylation. p52 warrants investigation as a therapeutic target in GBM, possibly in combination with kinase inhibitors and standard alkylating chemotherapy.