Hedgehog (Hh) signaling has emerged as a key growth pathway in human carcinogenesis and inappropriate Hh target gene activation drives the growth of tumors such as skin basal cell carcinomas (BCCs). Experimental support from 5ARO54780 facilitated approval by the FDA of the first therapy aimed at the Smoothened (Smo) receptor. While naïve tumors responded, resistance appears to be common, reiterating the need for developing new therapies targeting the Gli transcription factors downstream of Smo. 5ARO54780 in the prior funding period focused on factors that regulate Gli activity and showed the importance of the primary cilium and the basal body-associated Missing-in- Metastasis. Moreover, a proteomics screen to identify druggable components isolated the oncogenic polarity kinase aPKC-ι/λ and demonstrated that aPKC-ι/λ plays a key role in hedgehog signaling, BCC tumor growth, and Smo inhibitor resistance. In tumors, aPKC-ι/λ phosphorylates and activates Gli1 within the regulatory region of the DNA binding domain at residue Gli T304. By contrast, phosphorylation within the linker region of the DNA binding domain appears to inhibit function, revealing a gap in our understanding how DNA binding domain modifications affect activity. Studies in the next funding period will test the hypothesis that aPKC-ι/λ-mediated DNA binding domain phosphorylation regulates Gli activity and Smo inhibitor resistance by: Determining the consequences of Gli DNA binding domain phosphorylation; Determining how aPKC phosphorylation affects the Gli- DNA interaction; and by 3) Determining the functional consequences of resistant human BCC tumor variants on BCC growth. Completion of the above aims will provide needed information about the pivotal Gli transcription factor and provide insights to help diagnose and treat naïve and resistant hedgehog-dependent tumors.