Human tumor genomics and zebrafish modeling identify SPRED1 loss as a driver of mucosal melanoma

Abstract

Melanomas originating from mucosal surfaces have low mutation burden, genomic instability, and poor prognosis. To identify potential driver genes, we sequenced hundreds of cancer-related genes in 43 human mucosal melanomas, cataloging point mutations, amplifications, and deletions. The SPRED1 gene, which encodes a negative regulator of mitogen-activated protein kinase (MAPK) signaling, was inactivated in 37% of the tumors. Four distinct genotypes were associated with SPRED1 loss. Using a rapid, tissue-specific CRISPR technique to model these genotypes in zebrafish, we found that SPRED1 functions as a tumor suppressor, particularly in the context of KIT mutations. SPRED1 knockdown caused MAPK activation, increased cell proliferation, and conferred resistance to drugs inhibiting KIT tyrosine kinase activity. These findings provide a rationale for MAPK inhibition in SPRED1-deficient melanomas and introduce a zebrafish modeling approach that can be used more generally to dissect genetic interactions in cancer.

Fig. 1.. Genetic alterations in 43 mucosal melanomas.

(A) Tiling plot summarizing pathogenic mutations, copy number alterations (CNAs), and structural variants in various genes (listed on left). Mucosal melanoma (MM) samples are arranged in columns. Gene alteration frequencies (including point mutations and CNAs) are shown as percentages. The bar graph on top shows the genome instability index (GII), which is the fraction of the genome affected by CNAs, for each sample. HIPPO, Hippo signaling pathway. (B) Mutation frequencies (which include pathogenic amplifications and deep deletions) from the TCGA skin cutaneous melanoma (SKCM) cohort are shown for comparison. The bar graph shows P values from Fisher’s exact tests for significance of increased (purple) or decreased (green) mutation frequencies compared with the TCGA SKCM cohort. The dashed line marks the 5% false detection rate (FDR) threshold. *TERT: 20% (4/20) activating promoter mutation, 7% (3/43) gene amplification, and 5% (1/20) promoter structural rearrangement. **TERTpromoter mutation frequency in cutaneous melanomas (29). Mucosal M, mucosal melanoma. (C) GISTIC2 analysis identifies significant recurrent amplifications (red, top) and deletions (blue, bottom). Green lines indicate the q value thresholds for significance. Chr, chromosome.

Fig. 2.. Focused deletions at chromosome band 15q14 nominate SPRED1 as a frequently lost tumor suppressor gene.

(A) Heat maps show SPRED1 deep deletion in nine tumors. Log₂-transformed copy number ratios are represented on a color scale. (B) SPRED1 immunohistochemistry (IHC) results for 26 tumors with available material. All samples with biallelic inactivation of SPRED1 loss were negative for SPRED1. *In one sample, weak SPRED1 staining was present in 25% of tumor cells, suggesting the presence of a subclonal tumor population that retains SPRED1. (C) Shown are mucosal melanoma and TCGA skin cutaneous melanoma cases that harbor SPRED1 biallelic loss-of-function mutations and/or exhibit loss of SPRED1 protein by IHC (for mucosal melanomas only), along with other MAPK pathway–activating mutations. In all cases where point mutations occur with gain or amplifications, the amplification occurs on the mutated allele. An asterisk indicates nonsense mutation. fs, frameshift; WT, wild type. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; G, Gly; I, Ile; K, Lys; L, Leu; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; and W, Trp.

Fig. 3.. Melanoma modeling in zebrafish establishes genetic cooperation between SPRED1 loss and KIT mutations.

(A) Schematic representation of the MiniCoopR vector (left) and the CRISPR MiniCoopR vector (right), enabling melanocyte-specific gene expression or inactivation, respectively. Pmitfa, mitfa promoter; pA, polyadenylation signal; ts, tumor suppressor. Injection of MiniCoopR or CRISPR MiniCoopR vectors into casper (mitfa^−/−;roy^−/−) embryos rescues melanocyte formation in a mosaic fashion in adult zebrafish. Combinations of vectors expressing the oncogenes KIT^K642E,BRAF^V600E, or NRAS^Q61R and targeting tp53 initiate melanoma. (B to E) Melanoma-free survival curves of casper zebrafish injected with the indicated combinations of vectors expressing KIT^K642E, BRAF^V600E, or NRAS^Q61R and targeting tp53, pten, and/or spred1. P values were calculated by log-rank test. ns, not significant.

Fig. 4.. Loss of SPRED1 confers resistance to KIT inhibition in KIT-mutant melanoma by sustaining MAPK signaling and cell proliferation.

(A) Proliferation of the KIT^L576P-driven human melanoma cell line WM3211 stably expressing a control shRNA (shctrl) or a shRNA directed against SPRED1 (shSPRED1) measured after 4 days in culture and normalized to the control shRNA. Data are means ± SD of three independent experiments, and P = 0.03 by paired Student’s t test. (B) Western blot analysis of MAPK pathway activity in the cells described in (A) treated with the indicated concentrations of dasatinib (Dasa) for 6 hours. C, shctrl; Sh, shSPRED1; p-ERK, phosphorylated ERK. Actin was used as a loading control. (C and D) Viability of the cells described in (A) treated with increasing concentrations of the KIT inhibitor dasatinib (C) or the MEK inhibitor trametinib (D) for 4 days. Data are means ± SD of three independent experiments, and *P < 0.05 by paired Student’s t test. (E) Evolution of frameshift variant allele fraction (VAF) in cultured WM3211 human melanoma cells transiently transfected with two independent vectors (S1 and S2) targeting SPRED1 by CRISPR and treated with either dimethyl sulfoxide (DMSO) or 500 nM dasatinib over five passages. Data are from one representative experiment. (F) Quantification of tumor size in zebrafish injected with combinations of vectors expressing KIT^K642E and targeting either tp53 or both tp53 and spred1, after 14 days of 1 mM dasatinib treatment, relative to tumor size before treatment. P < 0.001 by two-tailed Student’s t test.