The Hedgehog (Hh) family of lipid-modified signaling proteins directs embryonic development and tissue homeostasis, and dysregulated Hh signaling drives familial and sporadic cancers. Hh ligands bind to and inhibit the tumor suppressor Patched (PTCH) and allow the oncoprotein Smoothened (SMO) to accumulate in cilia, which in turn activates the GLI family of transcription factors. Recent work has demonstrated that oxysterol lipids bind and modulate SMO activity. This thesis explores the central theme that lipids regulate the Hh pathway which in turn regulates lipid metabolism. First, I review the myriad of sterols that activate or inhibit the Hedgehog pathway. I discuss the possibility utility of Smo ligands or inhibitors in sterol metabolism as cancer therapeutics. Next, I define the Hh gene expression program using RNA sequencing of cultured cells treated with ciliary ligands, human BCCs, and Hh-associated medulloblastomas from humans and mice. These results reveal Hh target genes such as the oxysterol synthase Hsd111 and the adipokine Retnla regulate lipid metabolism to drive cell fate decisions in response to Hh pathway activation. Lastly, I demonstrate that Hedgehog signaling can be co-opted to drive resistance to pharmacological blockade of CDK6, an important single-agent molecular therapy in cancer therapeutics. Through CRISPR screens and RNA-sequencing of a mouse model of HH-associated medulloblastoma with genetic deletion of Cdk6, I demonstrate that decreased ribosomal protein expression underlies resistance to CDK6 inhibition in HH-associated medulloblastoma, leading to endoplasmic reticular (ER) stress and activation of the unfolded protein response (UPR). I then demonstrate concurrent genetic deletion or pharmacological inhibition of CDK6 and HSD11ß2, an enzyme producing Smoothened-activating lipids, additively blocked cancer growth in multiple mouse genetic models of HH-associated medulloblastoma. In sum, this research program demonstrates that lipid signaling is an avenue to target Hh-dependent cancers and I demonstrate a proof-of-principle therapeutic to target lipid signaling in Hh signaling
