Members of the nuclear-factor-κB (NF-κB) transcription factor family play a central role in regulating many physiological processes including innate and adaptive immunity. In addition, accumulating evidence suggests that inappropriate activation of NF-κB occurs in many types of human cancers. Indeed, genome characterization studies have uncovered genetic alterations involving many components of the NF-κB signaling cascade in both hematopoietic and epithelial cancers, and accumulating evidence indicates that NF-κB signaling contributes to resistance to targeted cancer therapy. Using integrated genomic approaches, we identified the two closely related, non-canonical inhibitors of κB kinase, IKKϵ (IKBKE, IKKi) and TBK1, as an amplified breast cancer oncogene and an essential gene in KRAS-driven human cancers, respectively. During the past funding period, we have used biochemical, genetic and molecular biological approaches to solve the structure of these two IKKs and identify substrates of these two IKKs critical for cell transformation. We have also identified K63-linked ubiquitination as one mechanism of regulation of these IKKs, essential for their tumorigenic function. These observations provide new insights into the function of these IKKs in both innate and oncogenic contexts. Based on these observations, this proposal focuses on delineate the mechanism(s) by which these immune regulators contribute to malignant transformation and to credential IKKϵ and TBK1 inhibitors. We will perform mechanistic studies critical to understanding how perturbing IKKϵ function affects tumor maintenance and will develop small molecule inhibitors of IKKϵ. Specifically, biochemical, genetic, molecular biological and pharmacologic approaches will be applied to interrogate the regulation of IKKϵ in both immune and oncogenic contexts, to identify effector pathways that mediate IKKϵ-induced cell transformation and to validate IKKϵ inhibitors preclinically. Investigating the regulation and function of IKKϵ in breast cancer development will not only enhance our mechanistic understanding of this non-canonical IKK regulator but will also clarify the role of NF-κB signaling in the development of human epithelial cancers. In addition, these studies will provide a foundation for strategies to target this kinase oncogene therapeutically.