Abstract Triple negative breast cancer (TNBC) is a heterogeneous group of clinically aggressive diseases that account for approximately 15-20% of invasive breast cancers. TNBC patients have high risk of recurrence and metastasis, and current treatment options remain limited. There is strong evidence for the involvement of Notch signaling in TNBC. Notch1 is highly expressed in Basal-like 1 (BL1) and especially Mesenchymal-Stem-Like (MSL) TNBCs. Expression of Notch1 protein correlates with pAKT and nuclear NF-κB in TNBC specimens. Expression of Notch1 and its ligand Jagged1 correlate with poor prognosis. NF-κB also plays key roles in promoting cancer cell survival in TNBC. Notch-NF-κB cross-talk through several mechanisms has been demonstrated in T-cell acute leukemia, cervical cancer and Luminal A breast cancer cells, but not TNBC. Here, we demonstrate that Notch1 promotes cell survival in MDA-MB-231 cells, representative of MSL TNBC, in part by activating NF-κB. Notch activation by Jagged1-expressing stromal cells enhances transcription of the anti-apoptotic gene cIAP-2 (BIRC3), a known NF-κB target. This event is dependent on recruitment to the cIAP-2 promoter of NF-κB subunits, IKKα and Notch1. However, Notch1 is recruited to the chromatin later than NF-κB subunits and IKKα, suggesting that early, non-nuclear events contribute to this process. NF-κB activation correlates with AKT activation as revealed by S473 phosphorylation. Pharmacological inhibition of AKT or expression of dominant-negative AKT decreases NF-κB activity. Inhibition of Notch activity through GSIs (γ-secretase inhibitors) leads to decreased AKT phosphorylation. Our results indicate that AKT activation is independent of canonical nuclear Notch1 partner CBF-1. Short term exposure of MDA-MB-231 cells (MSL, PTEN wild-type), but not MDA-MB-468 cells (BL1, PTEN-null) to recombinant Jagged1 leads to AKT phosphorylation within 1 hour. This is suppressed by dual mTORC1/2 inhibitors, PI3K inhibitors and IKKα inhibitors but not Everolimus (mTORC1-selective inhibitor). Pharmacological inhibition of AKT, or mTORC1/2 or IKKα decreases AKT phosphorylation and cell proliferation. These observations support a model where canonical and non-canonical mechanisms downstream of Notch1 trigger AKT phosphorylation and NF-κB activation in PTEN wild type TNBC cells. Rapid AKT phosphorylation downstream of Notch1 requires mTORC2, PI3K and IKKα, and contributes to NF-κB activation. Notch1 has been shown to activate mTORC2 in T-cells, while IKKα has been reported to activate mTORC2 in other models. Our data indicate that this pathway may be active in some TNBC subtypes as well. Our data indicate that therapeutic regimens targeting this pathway deserve further investigation in PTEN-wild type but not PTEN-mutant TNBCs. Citation Format: Fokhrul Hossain, Yin Peng, Antonio Pannuti, Kandis Backus, Todd Eliot Golde, Barbara Osborne, Lucio Miele. A novel non-canonical Notch-AKT- NF-κB pathways in triple negative breast cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4616.