Abstract Nuclear factor Kappa B (NF-κB)- a nuclear transcription factor that is retained in the cytoplasm by IKBα (encoded by NFKBIA)- is implicated as a mediator of aggressive behavior in all glioblastoma (GBM) subclasses. We are interested in elucidating molecular mechanisms that explain the relative mutual exclusivity of EGFR amplification and NFKBIA deletion in GBMs. Bioinformatics analysis of TCGA GBM data showed that NFKBIA hemizygous deletions were significantly depleted in samples with EGFR amplification (one-tailed p value of Fisher Exact test, P = 5.97e-4). Moreover, we showed by immunohistochemistry that IKBα was more highly expressed in EGFR-amplified than non-amplified GBMs. We use Ingenuity Pathway Analysis (IPA) to identify gene sets common to both EGFR amplification and NFKBIA hemizygous deletion to identity shared pathways that might explain mutual exclusivity and identified TNF, p53, PI3K, and NF-κB proliferation and apoptosis signaling pathways. We next genetically modeled glioma cell lines and GBM neurosphere cultures by transient transfection/stable viral transduction to recapitulate EGFR/NFKBIA combinations. qPCR of DNA and mRNA for EGFR and NFKBIA verified genotypes. Among the top hits predicted by our in silico studies, we identified sparcl1 and hgpx4 by RT-qPCR and immunoblots as concordantly upregulated in the EGFRamp/NFKBIA+/+ and EGFRnon-amp/NFKBIA+/- cell lines/neurospheres. We also showed differential DNA binding of NF-κB (by electro-mobility shift assay); target gene expression (eg. bcl-2, Il-6); and expression of upstream regulators of NF-κB pathways (viz. pAKT (S436) and IKKβ (Y466)) in GBM neurospheres with distinct EGFR/NFKBIA status. Moreover in U87MG cells (NFKBIA+/−), transiently silencing and overexpressing NFKBIA in different EGFR backgrounds (non-amplified and amplified) led to activation of PARP-dependent but caspase-3-independent apoptotic pathways. Interestingly, in U87MG cells and HPV E6/E7 transformed astrocytes, Annexin V/PI based FACS experiments indicated that NFKBIA overexpression suppressed necrosis. These data demonstrate a complex interplay of EGFR and IKBα/NF-κB in GBMs and identify shared pathways that might explain the relative mutual exclusivity of EGFR amplification and NFKBIA deletion. Citation Format: Alok Mishra, Jun Kong, Daniel J. Brat. Signal transduction in EGFR-amplified and NFKBIA-deleted human gliomas. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-029. doi:10.1158/1538-7445.AM2015-LB-029