Extended Data Fig. 1: Belvarafenib effectively inhibits NRAS-mutant melanoma cells.

a, Cell lysate treated with 10 μM belvarafenib or DMSO for 1 h before performing thermal-shift CETSA assay. b, Inhibition of pMEK by belvarafenib after treatment for 24 h in A549 cells engineered to express a single RAF isoform. Ratio of phosphorylated and total MEK plotted after treatment. Data are mean ± s.e.m., n = 2 replicates. c, Representation of RAF inhibitor binding for vemurafenib (left) or belvarafenib (right). d, e, Inhibition of pMEK by belvarafenib or vemurafenib after 24 h in Hec-1-A BRAF^null cells transiently transfected with BRAF(V600E) or BRAF(V600E/E568K). Ratio of phosphorylated and total MEK plotted. Data are mean ± s.e.m., n = 2 replicates. f–h, MAPK signalling in SK-MEL-28 (f), SK-MEL-2 (g), or human melanocytes, HEMn-LP (h) after treatment with serial titration of vemurafenib or belvarafenib for 24 h. i, Sensitivity of melanoma cell lines to vemurafenib or belvarafenib. In vitro IC₅₀ screening data for a panel of 25 melanoma cell lines. j, Cell viability of a panel of melanoma cell lines treated with belvarafenib (left) or cobimetinib (right) for 3 days. Data are mean ± s.e.m., n = 2 replicates. k, Clonogenic assay of panel of BRAF^V600E, NRAS, and BRAF non-canonical mutant melanoma cells treated with vemurafenib or belvarafenib. Cells were cultured for 8 days then stained with crystal violet. l, Cell viability of Ba/F3 cells expressing RAS-mutations treated with belvarafenib (left) or vemurafenib (right) for 3 days. Data are mean ± s.e.m., n = 6 replicates. m, Confirmation of RAS overexpression in Ba/F3 cells by western blot. Tubulin was stained on a separate gel as a sample processing control. n, Mice with established A431 tumours treated with vemurafenib, dabrafenib or belvarafenib. Data are mean ± s.e.m., n = 6 mice per group. *P = 0.0374 (Belva 30 versus vehicle), **P = 0.0003 (Belva versus vem), one-way ANOVA, followed by Dunnett’s multiple-comparisons test.

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