Project Summary Advanced basal cell carcinomas are caused by the inappropriate activation of the hedgehog pathway, and rapidly acquire resistance to Smoothened inhibitors. Previous work by ARO46786 developed a bioinformatic pipeline with cell line and animal model validation to identify tumor mutations in canonical pathway members such as Smoothened, increased expression of the Gli kinase atypical protein kinase C, and tumor pathway switching to another epidermal cancer, squamous cell carcinoma. Despite these findings, many of the resistant BCCs lacked identified resistance mechanisms, suggesting the existence of additional resistance pathways. Using multi- dimensional genomics approaches and a new murine model of resistant BCCs, we have uncovered two additional novel resistance pathways whose biology will be explored in the next funding cycle: 1) Hh pathway repressor mutations of the primary cilium, where the normal mechanisms that the centrosome-based organelle uses to dampen Gli activity are lost. We will study one of the most commonly mutated genes protocadherin15 by dissecting how protocadherin15 regulates hedgehog signaling and ciliogenesis and determining the fitness of protocadherin15 mutations in vivo; 2) Cytoskeletal-associated transcription, where signals from the local environment enhance Gli activity through the activation of the serum response factor / myocardin-related transcription factor complex that binds to Gli. We will dissect the functional interaction between Gli and the serum response factor / myocardin-related transcription factor complex and elucidate the upstream environmental activators that confer resistance. Completion of these aims will fill in major gaps in our understanding of tumor evolution and help develop new therapeutics for advanced BCCs and other cancers.