Identification of recurrent SMO and BRAF mutations in ameloblastomas

Abstract

Here we report the discovery of oncogenic mutations in the Hedgehog and mitogen-activated protein kinase (MAPK) pathways in over 80% of ameloblastomas, locally destructive odontogenic tumors of the jaw, by genomic analysis of archival material. Mutations in SMO (encoding Smoothened, SMO) are common in ameloblastomas of the maxilla, whereas BRAF mutations are predominant in tumors of the mandible. We show that a frequently occurring SMO alteration encoding p.Leu412Phe is an activating mutation and that its effect on Hedgehog-pathway activity can be inhibited by arsenic trioxide (ATO), an anti-leukemia drug approved by the US Food and Drug Administration (FDA) that is currently in clinical trials for its Hedgehog-inhibitory activity. In a similar manner, ameloblastoma cells harboring an activating BRAF mutation encoding p.Val600Glu are sensitive to the BRAF inhibitor vemurafenib. Our findings establish a new paradigm for the diagnostic classification and treatment of ameloblastomas.

Figure 1

Mutation frequency, distribution and relationship with pathological features. (a) Mutation status for four genes is indicated, and the overall percentage of mutant cases is given in parentheses. Information on histology and anatomical site is included below for each case. Each column represents a single case. Colors correspond to a specific mutation, histology or anatomical site. Details on additional clinical parameters are included in supplementary table 1. (b) Illustration of the distribution of tumors with the identified mutations.

Figure 2

SMO Leu412Phe activity and inhibition. (a) Relative expression of a Hedgehog-sensitive Gli-driven luciferase reporter in Smo^−/− MEFs expressing GFP (negative control), wild-type human SMO (WT) or the Leu412Phe or Trp535Leu SMO mutants following stimulation with control medium or medium containing the Shh N-terminal domain (ShhN). Note the significant basal induction of Hedgehog-pathway activity by Leu412Phe and Trp535Leu. **P < 0.01, two-sided Student’s t test. Results are representative of five independent trials. (b) Effect of treatment with vismodegib (200 nM), itraconazole (2 μM), KAAD-cyclopamine (300 nM) and ATO (8 μM), showing significant reduction in Hedgehog activity in cells expressing SMO Leu412Phe and SMO Trp535Leu with KAAD-cyclopamine and ATO treatment. ***P < 1 × 10⁻⁵, ****P < 1 × 10⁻⁶, two-sided Student’s t test. Results are representative of two independent trials. Data in a,b are from three independent transfections (three biological replicates), and error bars represent s.d. (c) Crystal structure of human SMO (Protein Data Bank (PDB) 4JKV) bound to the LY2940680 inhibitor (yellow) with amino acids 412 and 535 highlighted (red) to show transmembrane domain positioning.

Figure 3

SMO Leu412Phe enhances ameloblast-lineage cell proliferation. (a) Overexpression of wild-type SMO, SMO Trp535Leu, SMO Leu412Phe or empty vector control in mouse ameloblast-lineage (ALC) cells, shown by protein blot (antibody to Myc tag). GAPDH serves as a loading control. (b) Relative cell proliferation (in optical density (OD) units) evaluated by WST-1 assay (Roche) 24, 48 and 72 h after plating equal numbers of cells. Overexpression of SMO constructs significantly enhances cell proliferation compared to empty vector control; SMO Leu412Phe also enhances proliferation in comparison to wild-type SMO (two-sided Student’s t test, P values indicated). SMO expression was engineered by lentiviral transduction, and stable cell pools (with approximately 1 × 10⁵ independent integrations) were assayed. Cell proliferation was evaluated by three independent cell platings; error bars, s.d. Results presented are representative of three independent trials.

Figure 4

An ameloblastoma cell line harboring BRAF p.Val600Glu is sensitive to the BRAF inhibitor vemurafenib. (a) PCR amplification with Sanger sequencing identifies a BRAF mutation encoding p.Val600Glu in the AM-1 ameloblastoma cell line. (b) Expression of RAF Val600Glu in AM-1 cells was confirmed by protein blot using Val600Glu-specific antibody. COLO320 (wild-type BRAF) and COLO205 (BRAF Val600Glu) colorectal cancer cell lines served as controls. (c) Vemurafenib inhibits AM-1 cell proliferation/viability (fractional viability normalized to vehicle control) with an IC₅₀ of 0.19 μM. Respective IC₅₀ values for the control BRAF-mutant COLO205 and BRAF–wild type COLO320 cell lines are indicated. Data are representative of three independent cell platings; error bars, s.d. Results presented are representative of two independent trials.