Supplemental Legends

Supplemental Legends

Supplementary Table 1. (Top) IC50 (nM), fold changes (FC) and (Bottom) % viability at 10uM drug treatment compared to DMSO control for BRAF mutant cell lines, A375, MALME-3M, 451Lu and IGR1 (expresses RAC1 P29S) are shown.

Supplementary Table 2. % viability at 1uM drug treatment compared to DMSO control for the NRAS Q61K mutant cell lines, CP66, HMVII, and WM3060 (expresses RAC1 P29S) are shown.

Supplementary Table 3. IC50s (nM) and fold changes (FC) comparing IC50s of RAC1 wild-type and P29S mutant to GFP are shown for indicated drug, cell lines and expression plasmid.

Supplementary Table 4. STR profiles for cell lines used in this study.

Supplementary Figure 1: Identification of RAC1 P29S mutant melanoma cell lines. A and B, Left panels) Mass spectrometry Sequenom results for IGR1 and WM3060 showing peaks for presence of both cytosine (C) and thymine (T) at position 85 (the latter encoding for amino acid S29), which is absent in 451Lu. A and B, Right panels) Sanger sequencing results are shown confirming presence of T at position 85 for IGR1 and WM3060, but only C for 451Lu. UEP: unextended primer.

Supplementary Figure 2: RAC1 P29S mutant melanoma cell lines have higher levels of activated RAC1. A) Active RAC1 protein levels measured by PAK p21 binding domain (PBD) pull downs in the presence of GDP or a nonhydrolysable GTP analog (GTPƔ) from NRAS Q61K mutant melanoma cell lines CP66, HMVII, and WM3060, which possess the c.C85T SNV encoding for the P29S amino acid change. B) Log-transformed dose response curves (%viability compared to DMSO treated control) for CP66, HMVII, and WM3060 treated with MEK inhibitors B) trametinib (GSK11202120) and C) PD325901 for 72h with doses up to 10 000, and serial dilutions of 1 000, 333.33, 111.11, 37.04, 12.35, 4.12, 1.37, 0.46, 0.15 nM. X axis is presented as log transformed drug doses in pM concentrations. Error bars represent standard error means.

Supplementary Figure 3: Overexpression of RAC1 P29S increases cell viability of 451Lu cells to RAF and MEK inhibitor treatment. A) Immunoblot of 451Lu cells stably expressing GFP, RAC1 wild-type (WT) and P29S mutant from pHAGE-EF1α plasmids showing active GTP-loaded RAC1 from PBD pull down with corresponding input lysates. Log-transformed dose response curves (%viability compared to DMSO treated control) for 451Lu isogenic cell lines treated with RAF inhibitors B) vemurafenib (PLX4032) C) dabrafenib (GSK21118436) and MEK inhibitors D) trametinib (GSK11202120) and E) PD325901 (bottom panels) for 96h with doses up to 10 000 and serial dilutions of 1 000, 333.33, 111.11, 37.04, 12.35, 4.12, 1.37, 0.46, 0.15 nM. X axis is presented as log transformed drug doses in pM concentrations. Error bars represent standard error means.

Supplementary Figure 4: Overexpression of RAC1 P29S increases cell viability of A375 to RAF and MEK inhibitor treatment. A) Immunoblot of A375 cells stably expressing GFP, RAC1 wild-type (WT) and P29S mutant from pHAGE-EF1α plasmids showing active GTP-loaded RAC1 from PBD pull down with corresponding input lysates. Log-transformed dose response curves (%viability compared to DMSO treated control) for A375 isogenic cell lines treated with RAF inhibitors B) vemurafenib (PLX4032) C) dabrafenib (GSK21118436) and MEK inhibitors D) trametinib (GSK11202120) and E) PD325901 for 72h with doses up to 10 000, and serial dilutions of 1 000, 333.33, 111.11, 37.04, 12.35, 4.12, 1.37, 0.46, 0.15 nM. X axis is presented as log transformed drug doses in pM concentrations. Error bars represent standard error means.

Supplementary Figure 5: Overexpression of RAC1 P29S increases cell viability of MALME-3M to RAF inhibitor treatment. A) Immunoblot of MALME-3M cells stably expressing RAC1 wild-type (WT) and P29S mutant from pHAGE-EF1α plasmids showing active GTP-loaded RAC1 from PBD pull down with corresponding input lysates. Log-transformed dose response curves (%viability compared to DMSO treated control) for MALME-3M isogenic cell lines treated with RAF inhibitors B) vemurafenib (PLX4032) and C) dabrafenib (GSK21118436) for 72h with doses up to 10 000, and serial dilutions of 1 000, 333.33, 111.11, 37.04, 12.35, 4.12, 1.37, 0.46, 0.15 nM. X axis is presented as log transformed drug doses in pM concentrations. Error bars represent standard error means. D) Immunoblots of cleaved PARP and β-Actin from lysates of MALME-3M P29S overexpressing cells compared to parental cell line treated with 0.6 and 1.25 μM of dabrafenib (GSK2118436) for 48h. E) IC50 (nM) are indicated for the MALME-3M cell line in response to listed drugs with indicated fold changes (FC) comparisons.

Supplementary Figure 6: RAC1 P29S knockdown increases sensitivity of IGR1 melanoma cell lines to MAPK inhibition. A) Immmunoblot of RAC1 from lysates of stably expressing doxycycline (DOX)-inducible RAC1 shRNA IGR1 cells following 72h of DOX treatment. B) Log-transformed dose response curves (%viable compared to DMSO treated control) for indicated IGR1 cell lines treated with B) dabrafenib (GSK21118436) C) vemurafenib (PLX4032) D) PD325901 for 72h with doses of 2, 0.8, 0.32, 0.128, 0.0512, 0.02048 μM of drug. D) % Cell viability IGR1 cell lines (mean +/- SD (standard deviation) at doses of 2 μM for indicated drugs. DOX-induced cells were in culture for a total of 96h with DOX containing media. X axis is presented as log transformed drug doses in nM (0=DMSO treated control). Error bars represent standard deviations. LUC=luciferase, GFP= Green Fluorescent Protein.

Supplementary Figure 7. RAC1 P29S decreases effect of RAF inhibitor treatment on tumor growth in vivo. A) Tumor volumes measurements in the presence of PLX4720 drug are shown up until day 19. B) Photographs of mice tumors taken between days 19 to 21 days post-injection.