Abstract The PI3K-AKT pathway is the most frequently aberrantly activated pathway in breast cancer (BC), with alterations in genes encoding the pathway components (e.g. RTKs, HER2, PTEN, PIK3C and AKT) occurring in ~80% of cases. However, inhibition of this pathway is either not effective or often results in development of resistance in BC. TRAF2 is a key effector protein downstream of the TNF receptor superfamily, and activates NF-κB to promote BC cell survival. We mapped two phosphorylation sites (Ser-11 and Ser-274) in TRAF2, and identified TBK1/IKKϵ and AKT as the kinases that directly phosphorylate TRAF2 at Ser-11 and Ser-274, respectively. TBK1 and IKKϵ are serine/threonine kinases activated by inflammatory stimuli and the oncogenic RTK-Ras-RALB pathway, and play a critical role in BC transformation and survival. IKKϵ and TRAF2 are overexpressed in a significant portion of BC, and their overexpression is associated with poor prognosis in patients with invasive breast carcinoma. We found that inhibition of AKT leads to increased TBK1 activation and TRAF2 Ser-11 phosphorylation in triple negative BC (TNBC) cells. Importantly, combination of AKT and TBK1/IKKϵ inhibitors synergistically induced apoptosis in all TNBC cell lines tested in vitro, suggesting that inhibition of PI3K-AKT derepresses the pro-survival TBK1-TRAF2 pathway, which in turn confers resistance to AKT inhibition in TNBC. We believe that any positive outcomes from our ongoing xenograft experiments will have an immediate translational impact in TNBC treatment. Citation Format: Hasem Habelhah, Laiqun Zhang. TRAF2 Ser-11 phosphorylation confers resistance to AKT inhibitors in breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4024.