In vivo treatment of zebrafish melanomas reveals different sensitivities to BRAF inhibition between BRAF/tp53 and BRAF/tp53/spred1 tumors. (A) Distribution of SPRED1 mutant alleles in cultures of BRAF-driven A375 human melanoma cells transiently transfected with a vector expressing Cas9 and either of three gRNAs targeting SPRED1 after five passages in 100 nM dabrafenib, as measured by deep sequencing of the CRISPR target loci. Indels are indicated as the number of base pairs altered followed by the type of indel: insertion (I) or deletion (D). (B) Most common mutant alleles found in the cells described in A. (C) Schematic representation of the procedure to treat adult zebrafish bearing primary tumors. Fish were immerged in 50 ml water containing the drug for 12 h (overnight). Treatment was repeated daily for 14 d. ts, tumor suppressor. (D) Western blot analysis of MAPK pathway activity in primary zebrafish tumors treated with DMSO (control) or 5 µM dabrafenib for 3 d following the protocol described in C. BRAF, BRAF^V600E. Representative of two independent experiments. (E) Quantification of tumor size after 7 d of dabrafenib treatment relative to tumor size before treatment in zebrafish bearing BRAF/tp53 (n = 13) or BRAF/tp53/spred1 (n = 9) tumors. Pooled data of three independent experiments. *, P = 0.03, two-tailed t test. (F) Diagram summarizing the effect of SPRED1 loss in BRAF-driven melanoma. Under treatment with the BRAF^V600E inhibitor dabrafenib, SPRED1 loss annihilates the inhibitory activity of NF1 on Ras, which results in the activation of the RAF/MEK/ERK cascade that fuels cell survival and proliferation.