Smoothened (SMO) receptor mutations dictate resistance to vismodegib in basal cell carcinoma

Abstract

Basal cell carcinomas (BCCs) and a subset of medulloblastomas are characterized by loss-of-function mutations in the tumor suppressor gene, PTCH1. PTCH1 normally functions by repressing the activity of the Smoothened (SMO) receptor. Inactivating PTCH1 mutations result in constitutive Hedgehog pathway activity through uncontrolled SMO signaling. Targeting this pathway with vismodegib, a novel SMO inhibitor, results in impressive tumor regression in patients harboring genetic defects in this pathway. However, a secondary mutation in SMO has been reported in medulloblastoma patients following relapse on vismodegib to date. This mutation preserves pathway activity, but appears to confer resistance by interfering with drug binding. Here we report for the first time on the molecular mechanisms of resistance to vismodegib in two BCC cases. The first case, showing progression after 2 months of continuous vismodegib (primary resistance), exhibited the new SMO G497W mutation. The second case, showing a complete clinical response after 5 months of treatment and a subsequent progression after 11 months on vismodegib (secondary resistance), exhibited a PTCH1 nonsense mutation in both the pre- and the post-treatment specimens, and the SMO D473Y mutation in the post-treatment specimens only. In silico analysis demonstrated that SMO(G497W) undergoes a conformational rearrangement resulting in a partial obstruction of the protein drug entry site, whereas the SMO D473Y mutation induces a direct effect on the binding site geometry leading to a total disruption of a stabilizing hydrogen bond network. Thus, the G497W and D473Y SMO mutations may represent two different mechanisms leading to primary and secondary resistance to vismodegib, respectively.

Keywords: Basal cell carcinoma; Hedgehog pathway; PTCH1; Primary resistance; SMO; Secondary resistance; Vismodegib.

Figure 1

Pre‐treatment BCC liver metastasis of the first case (patient 1, primary resistance) showed PTCH1 wild type gene (A) and the SMO G49W mutation (C). Pre‐treatment primary tumor and BCC recurrence of the second case (patient 2, acquired resistance) carried the nonsense Q84Stop PTCH1 mutation (B) while the SMO D473Y mutation was observed only in the recurrence BCC sample (D).

Figure 2

Cross section of the 3D structure of the SMO receptor embedded in a 1‐hexadecanoyl‐2‐[(9Z)‐octadecenoyl]‐sn‐glycero‐3‐phosphocholine (POPC)/1‐palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐phosphoethanolamine (POPE) (2:2) membrane model. Water is shown as a light cyan surface, while Na+ and Cl− ions are visible as green and purple spheres, respectively. Lipids are portrayed as ball‐and‐sticks, the polar heads of POPC and POPE depicted in white and cyan, respectively, while the corresponding hydrophobic tails are colored green and salmon, respectively. The membrane solvent accessible surface area is highlighted in transparent forest green. The SMO receptor protein is shown as a red ribbon, the inhibitor binding region being evidenced by a yellow sphere.

Figure 3

(A) Zoomed view of the SMOG497W binding site in complex with vismodegib. The receptor is shown as a secondary‐structure colored ribbon (orange, α‐helices; purple, β‐sheets; gray, coils). Vismodegib is portrayed as atom‐colored sticks‐and‐balls (red, O; blue, N; green, Cl; S, sulfur; gray, C). Residue W497 is evidenced as dark red sticks. Hydrogen atoms, water molecules, ions and counterions are omitted for clarity. (B) SMD snapshots of vismodegib entering the receptor binding pocket. Vismodegib is highlighted by its green/red van der Waals surface. Hydrogen atoms, ions, counterions and water molecules are omitted for clarity. (C) Rupture force vs. time during the entry process of vismodegib within the WT (green) and SMOG497W (red) binding site.

Figure 4

(A) Zoomed views of the wild type (WT) and (B) SMOD473Y mutant binding sites in complex with vismodegib. In both panels, the receptor secondary structure is outlined as a semi‐transparent ribbon (orange, α‐helices; purple, β‐sheets; gray, coils). Vismodegib is portrayed as atom‐colored sticks‐and‐balls (red, O; blue, N; green, Cl; S, sulfur; gray, C). The triad of residues involved in the hydrogen‐bond network are highlighted colored sticks: R300, dark magenta; H470, olive drab; D/Y473, dark cyan. Y394 is also shown as dark red sticks. H‐bonds are evidenced as black lines. (C) Comparison of vismodegib binding energy contributions from WT (green) and D473Y (red) SMO residues. (D) Comparison between hydrogen bond network stabilization energies for SMO residues belonging to the WT (green) and SMOD473Y triad residues in the relevant vismodegib complexes. X denotes either D or Y residue at position 473.