Smoothened-activating lipids drive resistance to CDK4/6 inhibition in Hedgehog-associated medulloblastoma cells and preclinical models.

Medulloblastoma is an aggressive pediatric brain tumor that can be driven by misactivation of the Hedgehog (HH) pathway. CDK6 is a critical effector of oncogenic HH signaling, but attempts to target the HH pathway in medulloblastoma have been encumbered by resistance to single-agent molecular therapy. We identified mechanisms of resistance to CDK6 inhibition in HH-associated medulloblastoma by performing orthogonal CRISPR and CRISPR interference screens in medulloblastoma cells treated with a CDK4/6 inhibitor and RNA-Seq of a mouse model of HH-associated medulloblastoma with genetic deletion of Cdk6. Our concordant in vitro and in vivo data revealed that decreased ribosomal protein expression underlies resistance to CDK6 inhibition in HH-associated medulloblastoma, leading to ER stress and activation of the unfolded protein response (UPR). These pathways increased the activity of enzymes producing Smoothened-activating (SMO-activating) sterol lipids that sustained oncogenic HH signaling in medulloblastoma despite cell-cycle attenuation. We consistently demonstrated that concurrent genetic deletion or pharmacological inhibition of CDK6 and HSD11ß2, an enzyme producing SMO-activating lipids, additively blocked cancer growth in multiple mouse genetic models of HH-associated medulloblastoma. Our data reveal what we believe to be a novel pathway of resistance to CDK4/6 inhibition as well as a novel combination therapy to treat the most common malignant brain tumor in children.

Sterol and oxysterol synthases near the ciliary base activate the Hedgehog pathway.

Vertebrate Hedgehog signals are transduced through the primary cilium, a specialized lipid microdomain that is required for Smoothened activation. Cilia-associated sterol and oxysterol lipids bind to Smoothened to activate the Hedgehog pathway, but how ciliary lipids are regulated is incompletely understood. Here we identified DHCR7, an enzyme that produces cholesterol, activates the Hedgehog pathway, and localizes near the ciliary base. We found that Hedgehog stimulation negatively regulates DHCR7 activity and removes DHCR7 from the ciliary microenvironment, suggesting that DHCR7 primes cilia for Hedgehog pathway activation. In contrast, we found that Hedgehog stimulation positively regulates the oxysterol synthase CYP7A1, which accumulates near the ciliary base and produces oxysterols that promote Hedgehog signaling in response to pathway activation. Our results reveal that enzymes involved in lipid biosynthesis in the ciliary microenvironment promote Hedgehog signaling, shedding light on how ciliary lipids are established and regulated to transduce Hedgehog signals.