Resistance to Hedgehog inhibitor through Smoothened receptor mutation in basal cell carcinoma.

The inappropriate activation of Hedgehog (Hh) pathway is involved in the pathogenesis of skin basal cell carcinoma (BCC). Loss of function of the transmembrane receptor Patched (PTCH1), or gain of function of the Smoothened (SMO), Sonic Hh (SHH), and GLI family transcription factors can be ligand-independent oncogenic drivers of this disease. The activity of Vismodegib, a small molecule inhibitor of the Hh pathway that binds and inhibits SMO, is currently being evaluated in clinical trials against advanced BCC. Here, we report two cases of BCC, the first showing primary resistance to Vismodeginb treatment and the second characterized by a dramatic response to Vismodegib treatment (150 mg/day) followed by insurgence of secondary resistance. Formalin-fixed specimens of pre-treatment primary tumors and, for the second case, recurrence arisen during Vismodegib regimen were investigated for the mutational status of the serpentine receptor SMO by sequencing (3500DX Genetic Analyzer). In the case of primary resistance, SMO sequencing revealed the presence of the missense point mutation G497W while in the case of secondary resistance the D473Y mutant isoform of SMO receptor was found in the recurrence specimen. This substitution was not detected in the corresponding primary tumor specimen. In silico simulations of the wild type and mutated SMO receptor proteins in complex with Vismodegib showed that two different mechanisms are at work leading to primary and secondary inhibitor resistance, respectively. In the case of G497W (primary resistance), this residue is in a distal position with respect to the receptor drug binding site; however, the mutant residue reflects in a rearrangement of the corresponding loop ultimately leading in a partial obstruction to inhibitor entry. In the case of D473Y (secondary resistance), this residue is involved with other two residues (R400, H470) in a peculiar hydrogen bonds network, and hence appears to play a crucial role in stabilizing Vismodegib binding to the receptor. The mutation D473Y alters this equilibrium leading to a substantial decrease in receptor affinity for the inhibitor. Accordingly, the G497W and the D473Y mutations in SMO could represent two paradigm of primary and acquired resistance to Vismodegib in BCC, driven by different mechanism of SMO/Vismodegib interactions. Further ongoing elucidation of such mechanisms will help in developing therapeutic strategies able to overcome both type of Hh inhibitors resistance