Elucidating the functional role and therapeutic potential of GPCR kinase 2 in primary and therapy-resistant basal cell carcinoma

Inappropriate activation of the Hedgehog (HH) pathway drives basal cell carcinoma (BCC) of the skin, the most common cancer in the United States with greater than 4 million cases annually. Existing drugs target oncogenic HH signaling by inhibiting the key pathway regulator SMOOTHENED (SMO). Tumors initially respond to SMO inhibitors, but these molecules ultimately lose effectiveness, largely due to secondary mutations within SMO that render it drug-resistant. To elicit expression of oncogenic target genes involved in tumorigenesis, SMO signals intracellularly to GLI transcription factors, but for decades the underlying signaling mechanism remained obscure. We recently identified a key aspect of SMO-GLI communication that enables us to propose new therapeutic strategies to minimize resistance issues. Working in cultured cells and embryos, we discovered that the active, agonist-bound form of SMO undergoes phosphorylation by the kinase GRK2, enabling SMO to directly bind the PKA catalytic subunit, inhibit its enzymatic activity, and thereby prevent PKA from phosphorylating and inactivating GLI. We hypothesize that this novel SMO-GRK2-PKA signaling pathway transmits oncogenic HH signals from the cell surface to the nucleus during BCC tumorigenesis. As such, GRK2 represents a promising therapeutic target for both primary and SMO inhibitor-resistant BCC. The goal of our MPI R01 grant is to define the role of GRK2 in mediating oncogenic SMO-GLI communication in BCC, elucidate the underlying mechanism of phosphorylation, and to evaluate GRK2 inhibition as a BCC therapeutic strategy. Using cultured cell and in vivo BCC models, we will determine whether SMO undergoes GRK2 phosphorylation in primary cilia and probe the functional significance of these phosphorylation events to oncogenic GLI activation and BCC tumorigenesis. We will also block GRK2 activity using either genetic or pharmacological approaches, the latter capitalizing on selective, high-affinity, bioavailable GRK2 inhibitors that have already been developed and utilized in cardiovascular disease models. Lastly, we will uncover the biochemical and cell biological mechanism by which GRK2 recognizes the SMO active conformation at the cell membrane, which is its essential role in HH signal transduction, and capture a cryoEM structure of SMO-GRK2 or SMO-GRK2-Gβγ complex to understand this process at atomic resolution. To carry out our studies, we have assembled a team of experts in biochemical / cell biological mechanisms of HH signal transduction (Myers), BCC tumorigenesis (Atwood), and GRK2 structure / pharmacology (Tesmer). We expect these studies to define an essential and unique role for GRK2 phosphorylation of SMO in transmitting oncogenic HH signals from the cell surface to the nucleus, and for GRK2 inhibition, either alone or as a combination therapy with FDA-approved SMO inhibitors, to block tumorigenesis and forestall resistance more effectively than existing standard methods of care. Overall, our work will provide valuable insights into the mechanism of BCC tumorigenesis and validate a novel BCC therapeutic strategy, with the potential to transform treatment paradigms for many malignancies driven by ectopic SMO or GRK2 activity.