CS-23 * DEFINING THE NETWORKS AND INTERSECTION OF EGFR AMPLIFICATION AND NFKBIA DELETION IN HUMAN GLIOMA

Abstract

Nuclear factor KappaB (NF-κB) is a critical transcriptional regulator in glioblastoma (GBM) and is normally inhibited by IKBα, the product of NFKBIA. The relative mutual exclusivity of EGFR amplification and NFKBIA deletion suggested a redundancy of fundamental signaling pathways mediated by EGFR and NF-κB. Our bioinformatics analysis of TCGA GBM data showed that only 11.2% cases with EGFR amplification had hemizygous deletion for NFKBIA gene and only 0.2% had homozygous deletion, whereas non-amplified tumors had 18.5% hemizygous deletion and 0.4% homozygous deletion. Using TCGA GBM data, we identified common genes that were upregulated or downregulated in a similar fashion in both EGFR amplified and NFKBIA deleted GBM samples. Ingenuity Pathway Analysis (IPA) of common genes implicated TNF, p53, NF-κB and c-MYC as shared signaling pathways, suggesting they could be final common pathways downstream of EGFR amplification and NFKBIA deletion and explain relative exclusivity. Therefore, we examined the relationship of EGFR status on NFKBIA expression and the impact of aberrant NFKBIA expression on glioma viability. We found that stable over-expression of wt-EGFR in U87MG cells is associated with reduced NFKBIA compared to cells expressing basal levels of EGFR. When we transiently silenced NFKBIA in these cells, we noted increased level of PARP mediated apoptosis, suggesting that complete loss of NFKBIA in EGFR amplified cells is less compatible with viability. Additionally, overexpression of NFKBIA led induction of apoptosis in stably transformed human astrocytes as determined by FACS analysis of AnnexinV. Therefore our results demonstrate a complex interplay of EGFR and NFKBIA expression, which ultimately dictates downstream NF-κB signaling. These findings are consistent with our insilico analysis of TCGA GBMs, which demonstrate relative exclusivity of NFKBIA loss and EGFR amplification, yet aberrant NF-κB signaling across all GBM subclasses, independent of EGFR status. Our ultimate goal is to therapeutically manipulate NFKBIA downstream signaling networks.