Project Summary Basal cell carcinoma (BCC), caused by the inappropriate activation of the hedgehog (Hh) pathway, represents a common skin cancer that, while treated surgically, can also exhibit rapid invasion and metastasis. While Smoothened inhibitors (Smoi) show efficacy, the majority of advanced BCCs acquire resistance. 5ARO54780 showed that increased expression in BCCs of the polarity kinase atypical protein kinase C (PRKCi) amplifies Gli activity and confers resistance, although the mechanism remains unknown. PRKCi cooperates with histone deacetylase 1 (HDAC1) to promote Gli deacetylation and chromatin association. A vicinal proteomics approach led to the surprising identification that isoforms of the Lamina-Associated Polypeptide 2 (LAP2) bind to the Gli zinc finger domain. Nuclear envelope tethered LAP2b and an associated acetyl-lysine reader promote Gli accumulation at a site termed the paused nuclear complex that protects Gli from degradation or nuclear export. By contrast, nucleoplasmic LAP2a binds both HDAC1 and Gli allowing it to stably accumulate on chromatin. In turn, PRKCi prevents LAP2a-HDAC1-GLI degradation while promoting switching from LAP2b to LAP2a. Now in its 10th year, 5ARO54780 will test the overarching hypothesis that PRKCi-dependent Gli deacetylation directs association with distinct LAP2 isoforms that amplify pathway activity in resistant BCCs. We will: 1) Elucidate the structure and function of the Gli-LAP2b paused nuclear complex as we identify the LAP2b functional domains required for Gli activity, identify components of the Gli-LAP2b acetyl-lysine reader, and dissect how LAP2 regulates Gli localization and mobility; 2) Elucidate the structure and function of the GLI-LAP2a activation complex as we determine whether LAP2a requires Gli for colocalization, define the non-base contact surface of LAP2-Gli1, and elucidate how PRKCi regulates LAP2a-HDAC1-Gli stability; 3) Establish resistance pathway efficacy and epistasis as we determine the prevalence of the BCC resistance pathways and determine efficacy of the LAP2-LLD in human and mouse BCC explants. Completion of the project will deepen our mechanistic insights into transcription factor trafficking and regulation in the nucleus, help optimize rational BCC therapy, and identify new therapeutic targets.