Extended Data Fig. 2: ARAF p.G387 mutations confer mutational specific belvarafenib resistance.
From: ARAF mutations confer resistance to the RAF inhibitor belvarafenib in melanoma
a, Cellular viability of IPC-298 belvarafenib-resistant clones (round 2) treated with belvarafenib for 3 days. Data are mean ± s.e.m., n = 3 replicates. b, Clonogenic assay of IPC-298 parental, BRC9 and BRC9-WO cells treated with increasing concentrations of belvarafenib. Cells were cultured for 8 days then stained with crystal violet. c, Cell growth in IPC-298 parental or BRC9 cells treated with DMSO or 10 μM belvarafenib. d, MAPK signalling in parental, BRC9 or BRC9-WO cells after 24 h treatment with 10 μM belvarafenib. e, Relative ARAF expression in BRC cells compared to parental IPC-298 cells. Data are mean ± s.e.m. (ΔΔCt), n = 4 replicates. ***P < 0.0001, one-way ANOVA, followed by Dunnett’s multiple-comparisons test. f, Cellular viability of BRC or parental cells treated with vemurafenib for 3 days, n = 5 BRCs. Data are mean ± s.e.m., n = 3 replicates. g, IGV view of exome sequencing reads from BRCs around ARAF p.Gly387 (c.1168G>C or c.1169G>A). Wild-type G in orange, mutant C allele in blue, mutant A allele in green. h, Allelic frequency of ARAF p.G387 in a panel of BRCs and BRC9-WO. i, IGV view of exome sequencing reads from IPC-298 parental, BRC9 and BRC9-WO cells around ARAF p.Gly387 (c.1169G>A). j, Clonality of belvarafenib-resistant cells assessed by high-complexity genomic barcoding using Cellecta CloneTracker 50M library. k, Sanger sequencing reads of ARAF p.Gly387 of BRC1-1 and BRC6-3. l, Cellular viability of IPC-298 belvarafenib-resistant clones (Cellecta CloneTracker) treated with belvarafenib for 3 days. Data are mean ± s.e.m., n = 3 replicates. m, n, Deep sequencing nucleotide reads of ARAF p.Gly387 in BRC9, IPC-298 parental, and MelJuso. o, Cellular growth of IPC-298 or BRC9 doxycycline-inducible shRNA knockdown cells against ARAF, BRAF or CRAF for 128 h in the presence of doxycycline treated with DMSO. p, MAPK signalling and RAF protein levels after shRNA knockdown in the presence of doxycycline for 24 h (pre-seeding levels for o, q). q, Cellular growth of doxycycline-inducible IPC-298 or BRC9 cells after shRNA knockdown of ARAF, BRAF or CRAF for 128 h in the presence of doxycycline treated 10 μM belvarafenib. r, MAPK signalling and RAF protein levels in doxycycline-inducible IPC-298 or BRC9 cells after shRNA knockdown of ARAF and treatment with serial titrations of belvarafenib for 24 h in the presence of doxycycline. s, ARAF protein levels after shRNA knockdown, as shown in Fig. 2e.
