Smoothened mutation confers resistance to a Hedgehog pathway inhibitor in medulloblastoma

Abstract

The Hedgehog (Hh) signaling pathway is inappropriately activated in certain human cancers, including medulloblastoma, an aggressive brain tumor. GDC-0449, a drug that inhibits Hh signaling by targeting the serpentine receptor Smoothened (SMO), has produced promising anti-tumor responses in early clinical studies of cancers driven by mutations in this pathway. To evaluate the mechanism of resistance in a medulloblastoma patient who had relapsed after an initial response to GDC-0449, we determined the mutational status of Hh signaling genes in the tumor after disease progression. We identified an amino acid substitution at a conserved aspartic acid residue of SMO that had no effect on Hh signaling but disrupted the ability of GDC-0449 to bind SMO and suppress this pathway. A mutation altering the same amino acid also arose in a GDC-0449-resistant mouse model of medulloblastoma. These findings show that acquired mutations in a serpentine receptor with features of a G protein-coupled receptor can serve as a mechanism of drug resistance in human cancer.

Fig 1

Identification of a SMO mutation in tumor samples from a medulloblastoma patient who relapsed after an initial response to GDC-0449. (A) Nucleotide sequence tracings showing a heterozygous mutation in SMO causing a Asp>His change at amino acid 473 (asterisk). This mutation was present in a metastatic biopsy taken at relapse but was not present in the primary tumor before GDC-0449 treatment. (B) The GPCR architecture of SMO maps the location of the D473H mutation to the C-terminal end of TM6. Looking down at the extracellular face of the GPCR helix bundle (color-ramped from TM1 in blue to TM7 in red, with ectoloops left out for clarity), a molecular model of SMO built upon the rhodopsin [Protein Data Bank (PDB) number 2Z73] and β1-adrenergic receptor template (PDB number 2VT4) with MODELLER (18) shows the position of the Asp-473 residue facing the central binding cavity.

Fig 2

The SMO-D473H mutation confers resistance to the Hh pathway inhibitor GDC-0449. (A) GLI-luciferase reporter activity after transfection of SMO variants in the presence (gray bars) or absence (black bars) of PTCH1 DNA (20 ng). SMO-M2 represents a previously identified activating mutation. (B) GLI-luciferase reporter activity in C3H10T1/2 cells transfected with SMO-WT (closed circles) or SMO-D473H (open circles) after treatment with various doses of GDC-0449. Reporter activity is normalized to untreated cultures. (C) Binding of ¹⁴C-labeled GDC-0449 (5 nM) to human embryonic kidney–293 cells transfected with SMO variants in the presence or absence of unlabeled GDC-0449 (5 μM), to demonstrate specificity. Data in all experiments represent mean ± SD.

Fig 3

Acquired resistance to GDC-0449 through Smo mutation in a genetically engineered mouse model of medulloblastoma. (A) Medulloblastoma allografts from Ptch1^+/−;p53^−/− mice were made GDC-0449–resistant through intermittent daily dosing with 75 mg/kg GDC-0449. Treatment days are represented by triangles, and tumors were excised once they failed to respond to twice-daily dosing with GDC-0449. (B) Nucleotide sequence tracings from parental and a GDC-0449–resistant (SG274) medulloblastoma allografts showing a heterozygous mutation resulting in a D>G change at amino acid 477 of Smo (homologous to position 473 of human SMO). (C) GLI-luciferase reporter activity in C3H10T1/2 cells transfected with SMO-WT (closed circles) or SMO-D473G (open circles) after treatment with various doses of GDC-0449. (D) Quantitation of Gli1 mRNA levels by quantitative reverse transcription polymerase chain reaction from multiple (n = 3) tumors collected 6 hours after treatment with vehicle control (open bars) or 75 mg/kg GDC-0449 (closed bars) from parental and SG274 tumor-bearing mice. Data indicate mean ± SD. *P < 0.05 (t test).