Smoothened variants explain the majority of drug resistance in basal cell carcinoma

Abstract

Advanced basal cell carcinomas (BCCs) frequently acquire resistance to Smoothened (SMO) inhibitors through unknown mechanisms. Here we identify SMO mutations in 50% (22 of 44) of resistant BCCs and show that these mutations maintain Hedgehog signaling in the presence of SMO inhibitors. Alterations include four ligand binding pocket mutations defining sites of inhibitor binding and four variants conferring constitutive activity and inhibitor resistance, illuminating pivotal residues that ensure receptor autoinhibition. In the presence of a SMO inhibitor, tumor cells containing either class of SMO mutants effectively outcompete cells containing the wild-type SMO. Finally, we show that both classes of SMO variants respond to aPKC-ι/λ or GLI2 inhibitors that operate downstream of SMO, setting the stage for the clinical use of GLI antagonists.

Figure 1. Hedgehog signaling is upregulated in resistant BCCs

(A) Clinical photographs (scale bar=1 in) and histology (scale bar=100 µm) depicting the time course of a sensitive and a resistant BCC in the same patient during vismodegib therapy. (B) Pathway-driven gene set enrichment analysis (DAVID) in resistant BCCs as compared with sensitive BCCs and normal skin. (C) A box-plot representation comparing the log₂ RPKM for GLI1 in resistant BCCs, sensitive BCCs, and normal skin (p=0.0001). Box represents first and third quartiles, with whiskers representing range; center line: median; diamond: mean; circle: outliers (D) Quantification of GLI1 immunofluorescence pixel intensity in Keratin 14 (K14)-positive regions (n=10). Error bars, s.e.m. (E) Representative immunofluorescence staining against GLI1 and K14, as well as DAPI counterstain. Adjacent sections stained with hematoxylin and eosin. Scale bar=100 µm.

Figure 2. Resistant BCCs harbor recurrent SMO mutations

(A) Schematic overview of the tumor biopsy and adjacent normal skin collection followed by whole-exome or genome sequencing and analysis. (B) List of SMO, PTCH1, and TP53 mutations identified for each resistant BCC samples subjected to exome sequencing. (C). Spectrum of HH pathway genes with genetic alterations seen in exome sequencing of resistant tumor-normal pairs. The genes are listed on the left hand side and the tumor samples are across the bottom. The fraction of samples with HH pathway mutations is listed in the bar graph to the right. (D) Bar graph showing recurrent, LBP, and COSMIC database SMO mutations in resistant BCCs compared with untreated samples. (E) Schematic showing SMO mutations in resistant BCCs as compared with untreated BCCs. SMO mutations are listed on the left hand side of each row and each column represents a unique sample with patient number and other relevant information listed at the bottom. The mutations are color-coded. Red color: a mutation in an amino acid located in the SMO-LBP. Blue color: a mutation also reported as somatically mutated in cancer in the COSMIC database. Green color: a recurrent mutation in neither the LBP nor the COSMIC database.

Figure 3. Variation in the responsiveness of SMO ligand binding pocket mutations

(A) SMO variants expressed in Smo^−/− MEFs and treated with SHH-N conditioned media (CM) with or without 100 nM vismodegib. Western blot shows the expression of SMO WT and SMO variants. (B) Side view (left) and top down view (right) of the position of the SMO variants within the SMO crystal structure showing their arrangement relative to an inhibitor (Wang et al., 2013). (C) Response of indicated SMO mutants with different concentrations of vismodegib. IC₅₀ are shown in brackets. (D) HH pathway activity in Smo^−/− MEFs expressing the indicated SMO and treated with SHH-N conditioned media (CM) with or without 10, 20, or 80 nM vismodegib. All error bars, s.e.m.

Figure 4. Two distinct mechanisms of SMO-mediated resistance in BCCs

(A) Position of the SMO variants within the SMO crystal structure showing their arrangement relative to an inhibitor (Wang et al., 2013) in TM3 (V321), TM5 (F460), TM6 (L412F), and TM7 (W535L). (B) Side view (left) and top down view (right) of the overlay of pivot regions of B₂ adrenergic receptor (grey) with those of SMO (green). Black numbers represent prolines in the B₂ adrenergic receptor structure around which the lower receptor pivots. (C) Baseline HH pathway activity in Smo^−/− MEFs under serum-starvation conditions expressing SMO WT or indicated SMO-CA variants. Western blot of expression of SMO WT compared to SMO variants. (D) HH pathway activity in Smo^−/− MEFs expressing the indicated SMO treated with SHH-N CM with or without 100 nM vismodegib. (E) Response of indicated SMO with different concentrations of vismodegib. IC₅₀ are shown in brackets. (F) Coexpression of SMO-CA variants and PTCH1 or GFP in a GLI-luciferase reporter assay. All error bars, s.e.m.

Figure 5. SMO mutations drive tumor evolution and drug resistance

(A) Bar graph showing the allele fraction (red) of the indicated SMO-LBP or CA mutants in pre-treated (Pre) or treated and resistant (Post) BCCs. (B) A schema showing sequencing of two resistant clones arising from the same sporadic BCC under vismodegib selection. (C) Frequencies of BCC having functional SMO mutations shown to either impart constitutive activity or confer resistance to vismodegib. (D, E) Representative fluorescent images (D, scale bar 100 µm) and quantitation (E) of competition assay with stable ASZ001 BCC cell lines coexpressing SMO WT and mCherry or SMO variants and GFP with or without vismodegib. (F) HH pathway activity in Smo^−/− MEFs expressing the indicated SMO variant and treated with SHH-N CM with or without 32 nM vismodegib, 20 µM PSI, or 8 µM ATO. All error bars, s.e.m.