Further information and reasonable requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Victoria Sanz-Moreno ( v.sanz-moreno@qmul.ac.uk ). All unique/stable reagents generated in this study are available from the Lead Contact with a completed Materials Transfer Agreement.

Animals used in this study were from Charles River UK: 5-week-old female nude CD-1 nu/nu mice; 5-8-week old NOD/SCID/IL-2Rγ-/- (NSG) mice (male and female); 5-7-week-old female C57BL/6J mice. Tumors were allowed to establish, sizes (average 60-100 mm) were matched and then mice were randomly allocated to groups of 6-8 animals. No blinding was used in the treatment schedules for these experiments since the different treatments were identified by ear notching/mark on tail. Based on previous studies in the literature () and our own experience, groups of 6-8 animals were used to have sufficient animals per group to provide statistically significant data while keeping the number of animals used to a minimum. Tumor size was determined by caliper measurements of tumor length, width and depth and tumor volume was calculated as volume = 0.5236 x length x width x depth (mm). Note that this formula calculates smaller tumors (approximately 2-fold smaller) compared to those calculated using the formula volume = 0.5236 x length x width(mm).

All animals were maintained under specific pathogen-free conditions and handled in accordance with the Institutional Committees on Animal Welfare of the UK Home Office (The Home Office Animals Scientific Procedures Act, 1986). All animal experiments were approved by the Ethical Review Process Committees at Barts Cancer Institute, King's College London and The Francis Crick Institute, in accordance with the Animals (Scientific Procedures) Act 1986 and according to the guidelines of the Committee of the National Cancer Research Institute.

A375, A375/PLX/R, Colo829, Colo829/PLX/R, SKMEL5 cells and Patient-derived cell lines were confirmed by STR profiling at CRUK Manchester Institute; A375M2, WM983A, WM983B at King’s College London; WM88 and WM793B cells were purchased from Coriell Institute in June 2014.

Brafmouse melanoma cell lines 5555, 4434, 4599 and 690cl2 (from Richard Marais) were established from the following C57BL/6 mouse models: Braf;Tyr::CreERT2;p16(5555, 4434); Braf;Tyr::CreERT2(4599); Pten-null Braf;Tyr::CreERT2;p16;Pten-/- (690cl2) (). NRAS mutant cell lines used: D04 was from Kevin Harrington (The Institute of Cancer Research); MM485 was obtained from the Wellcome Trust Functional Genomics Cell Bank (UK). HEK293T cells were from Jeremy Carlton (The Francis Crick Institute).

Patient-derived melanoma cell lines (#2, #35, #62T3, #58, #33) were a very kind gift from Richard Marais and were grown in RPMI. Patient #2, #35, #62T3 were grown in complete RPMI supplemented with 1 μM PLX4720. Patient #2 cell line was established from a patient with stage IV BRAF mutant melanoma with primary resistance to vemurafenib and ipilimumab. Patient #/35 cell line was established from a lymph node metastasis after treatment with vemurafenib for 3 months. Patient #62T3 cell line was established from a resected tumor upon disease progression following vemurafenib treatment (acquired resistance) and immunotherapy (refractory to ipilimumab and subsequent pembrolizumab). Patient #58 cell line (wild-type for BRAF/NRAS) was established from a metastasis from a patient that never responded to ipilimumab treatment (3 months). Patient #33 cell line (BRAF K601E ) was established from a metastasis from a patient that never responded to ipilimumab treatment (1 month). Patient #58 and #33 had also been treated with dacarbazine (DTIC) before ipilimumab. Patient #26 cell lines were established before and after nivolumab treatment.

PLX4720-resistant (A375/PLX/R, Colo829/PLX/R) () and dabrafenib+trametinib-resistant (A375/D+T/R) cell lines were a kind gift from Richard Marais (Cancer Research UK Manchester Institute). Resistant cells were generated after exposure of parental A375 and Colo829 to increasing concentrations of drugs (up to 1 μM PLX4720; 1 μM dabrafenib plus 10 nM trametinib) until cells resumed growth. Cells were grown in complete DMEM (A375-derivatives) or complete RPMI (Colo829-derivatives) supplemented with 1 μM PLX4720 (A375/PLX/R and Colo829/PLX/R cells); 1 μM dabrafenib plus 10 nM trametinib (A375/D+T/R) or equivalent volume of DMSO (parental A375 and Colo829 cells).

Cell lines used are listed in the Key Resources Table . Cell lines were cultured under standard conditions in complete medium (DMEM or RPMI medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (all from Gibco)). Cell lines were tested to be free from mycoplasma contamination. All melanoma cell lines used were BRAFunless otherwise stated. A375, Colo829 and SKMEL5 cells were from ATCC. WM88, WM983A, WM983B, WM793B were purchased from Coriell Institute. A375M2 were from Dr Richard Hynes (HHMI, MIT, USA). LOX-IMVI cell line was a gift from Prof Øystein Fodstad (Oslo University Hospital). SKMEL5, WM983A, WM983B, WM793B, LOX-IMVI were grown in complete RPMI, WM88 was grown in complete DMEM. PLX4720-resistant WM983A, WM983B and WM88 cells were derived after exposure to PLX4720 for 2-3 months (1 μM PLX4720 for WM983A and WM983B; 0.5 μM PLX4720 for WM88), controls were treated with equivalent volume of DMSO.

Human melanoma samples were a kind gift from Paul Lorigan (University of Manchester). Tumor samples were collected under the Manchester Cancer Research Centre (MCRC) Biobank ethics application #07/H1003/161+5 with full informed consent from the patients. The work presented in this manuscript was approved by MCRC Biobank Access Committee application 13_RIMA_01. Patient sample information is in Table S6

Method Details

Chemicals Chemicals used in this study (stocks resuspended in DMSO unless otherwise stated): BRAFi PLX4720 and PLX4032 (Selleck), BRAFi Dabrafenib (GSK2118436, ChemieTek), MEKi Trametinib (GSK1120212, Selleck), MEKi PD184352 (Selleck), MEKi AZD6244 (Selleck), ERKi SCH772984 (Selleck), ROCKi GSK269962A (Axon Medchem), ROCKi H1152 (resuspended in water; Calbiochem), AGC kinase inhibitor and ROCKi AT13148 (Selleck), myosin II inhibitor blebbistatin (in 95% DMSO; Calbiochem). Concentrations used unless otherwise stated in other STAR Methods sections: 5 μM ROCKi GSK269962A, 5 μM ROCKi H1152, 5 μM ROCKi AT13148, 25 μM myosin II inhibitor blebbistatin, 5 μM BRAFi PLX4720. “Analysis of cell morphology” section lists the inhibitors and concentrations used for those experiments.

Antibodies Antibodies and concentrations used: pThr18/Ser19-MLC2 (#3674; 1:750, immunoblot), pSer19-MLC2 (#3671; 1:50, immunohistochemistry; 1:200, immunofluorescence), MLC2 (#3672; 1:750), pT202/Y204-p44/42 (ERK1/2) (#4370; 1:1,000), pY705-STAT3 (#9145; 1:750), PD-L1 (clone E1L3N, #13684, 1:200) from Cell Signaling Technology; STAT3 (sc-482; 1:500), ERK2 (sc-154; 1:1,000), MCL-1 (sc-819; 1:1,000), GFP (sc-8334; 1:1,000) from Santa Cruz Biotechnology; GAPDH (MAB374; 1:10,000) from Millipore; P-H2A.X (S139) (ab2893;1:1000), CD206 (ab64693; 1:1,000), CD3 (anti-mouse, ab134096; 1:500), CD4 (anti-mouse, clone I3T4, ab183685; 1:300), FoxP3 (anti-human, clone 236A/E7, ab20034; 1:200) from Abcam; F4/80 (anti-mouse, clone BM8, MF48000, 1:1000), CD8a (anti-mouse, clone Ly2, 14-0808-82; 1:200), FoxP3 (anti-mouse, clone FJK-16s, 14-5773-82; 1:200) from Invitrogen; CD4 (anti-human, clone 11E9, NCL-L-CD4-368; 1:300) from Novocastra.

Long-Term Survival Long-term survival was performed on tissue culture plastic dishes unless otherwise specified. Cells were seeded in 6-well plates (10,000 cells/well) and treated for 5-14 days, re-adding drugs in fresh media every 2-3 days (daily for blebbistatin). Then cells were fixed with 1% formaldehyde and stained with 0.25% crystal violet. Plates were scanned and images analyzed using ImageJ software. For experiments with inhibitors, percentage of the well covered by crystal violet-stained cells was calculated and shown relative to control cells. For dose-response experiments, cells were seeded in 12-well or 96-well plates and survival was analyzed after 3-5 days treatment with indicated drugs using crystal violet. Crystal violet was solubilized with 10% acetic acid and absorbance was measured at 590 nm. In dose-response experiments, BRAFi-resistant cells were cultured in the presence of BRAFi throughout the experiment unless otherwise stated. In Figure 5 C, 4434- and 5555-derivatives were treated with 0.1 μM ROCKi. Di Veroli et al., 2016 Di Veroli G.Y.

Fornari C.

Wang D.

Mollard S.

Bramhall J.L.

Richards F.M.

Jodrell D.I. Combenefit: an interactive platform for the analysis and visualization of drug combinations. For synergy experiments, 1,000 A375 cells were seeded in 96-well plates, cultured overnight and next day treated in quintuplicates with ROCKi GSK269962A or BRAFi PLX4720, either alone or in several combinations in complete medium. Three days later, plates were fixed, stained with crystal violet and solubilized and quantified as above. Values were normalized to vehicle controls and analyzed with Combenefit software (Loewe model) (). Average of 4 independent experiments is shown.

Long Term Survival on Collagen I Matrices Orgaz et al., 2014b Orgaz J.L.

Pandya P.

Dalmeida R.

Karagiannis P.

Sanchez-Laorden B.

Viros A.

Albrengues J.

Nestle F.O.

Ridley A.J.

Gaggioli C.

et al. Diverse matrix metalloproteinase functions regulate cancer amoeboid migration. Bankhead et al., 2017 Bankhead P.

Loughrey M.B.

Fernandez J.A.

Dombrowski Y.

McArt D.G.

Dunne P.D.

McQuaid S.

Gray R.T.

Murray L.J.

Coleman H.G.

et al. QuPath: open source software for digital pathology image analysis. 2 were normalized to each untreated control as percentage of area covered by cells. For Patient #2 cells, spheroid-forming ability was quantified as the sum of areas occupied by spheroids from phase-contrast images using ImageJ. Cells were grown on collagen I matrices as described (). Briefly, bovine collagen I (PureCol, #5005-B; Advanced BioMatrix) thick gels were polymerized at 1.7 mg/ml in 24-well plates. Cells were seeded at 10,000 cell/well and treatments started 16 hr later for 5-14 days. In experiments using A375-derivatives, cells were treated with 1 μM ROCKi, 1 μM BRAFi or both. Patient-derived cell lines were treated with 5 μM ROCKi. Fresh complete media with drugs was added every 2-3 days. At the end of the experiment collagen I gels were fixed with 4% formaldehyde and phase-contrast images were taken. Percentage of area covered by cells was quantified using QuPath software Version 0.1.2 and a SLIC superpixel image segmentation (Gaussian sigma value 5 pixels, superpixel spacing 20 pixels) (). Software was trained to identify cells and background (surrounding collagen). Detection measurements were then exported to Excel and values for area/pixelwere normalized to each untreated control as percentage of area covered by cells. For Patient #2 cells, spheroid-forming ability was quantified as the sum of areas occupied by spheroids from phase-contrast images using ImageJ.

MTT Assay Cells were seeded in 96-well plates (2,000 cells/well). Drugs were added every 2 days. Three days after seeding, plates were incubated with MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; Millipore) following the manufacturer’s instructions and absorbance measured at 572 nm. Background at 630 nm was subtracted and data represented as relative viability.

Cell Cycle Analysis For DNA cell cycle analysis, floating and adherent cells were fixed in 70% ethanol at -20°C, washed in phosphate-buffered saline and treated with 40 μg/ml propidium iodide (PI) (Biolegend) and 100 μg/ml ribonuclease (Sigma) for 25 min at 37°C. Staining was detected using a FACS BD Canto II (BD Biosciences) and analyzed and plotted using FlowJo (FlowJo LLC). The starting gating of the whole cell population, excluding any debris, was performed with FSC-A/SSC-A. Using this as a parental gate, doublets were excluded using PerCP-Cy5.5-A/ PerCP-Cy5.5-W (PI). The gated singlets were represented as histograms for PerCP-Cy5.5-A to show the peaks for the cell cycle phases.

AnnexinV/Propidium Iodide FACS Floating and adherent cells were collected, spun down, and labelled with FITC Annexin V Apoptosis Detection Kit with PI (#640914, Biolegend UK Ltd), following the manufacturer’s instructions. Staining was detected using a FACS BD Canto II and analyzed and plotted using FlowJo. The starting gating of the whole cell population, excluding any debris, was performed with FSC-A/SSC-A. This was followed by a double exclusion of doublets using first FSC-H/FSC-W and then SSC-H/SSC-W. The gated singlets were then gated as ‘quad gates’ using FITC-A (AnnexinV) versus PerCP-Cy5.5-A (PI) and represented as FACS dot plots. Graphs show percentage of dead cells as the sum of percentage of early apoptotic (annexin Vhigh, propidium iodidelow) and percentage of late apoptotic/necrotic cells (annexin Vhigh, propidium iodidehigh).

ROS Detection Cells were treated with 1 μM ROCKi for 24 hr (A375 pair) or 48 hr (WM983A pair). Then cells were collected and ROS levels were detected using CellROX Green Flow Cytometry Assay Kit (C10492, Life Technologies), according to the manufacturer’s instructions. FACS and gating strategy were as described in Cell Cycle section.

Time Lapse Microscopy 2 using a 10X/0.3 NA Plan Fluor ELWD objective lens on a fully motorized (Prior Scientific) multi-field Nikon TE2000 microscope with an ORCA camera (Hamamatsu) controlled by Micro-Manager ( Multi-site bright-field microscopy of cells in 24-well plates was performed in a humidified chamber at 37°C and 5% COusing a 10X/0.3 NA Plan Fluor ELWD objective lens on a fully motorized (Prior Scientific) multi-field Nikon TE2000 microscope with an ORCA camera (Hamamatsu) controlled by Micro-Manager ( https://micro-manager.org/ ) and ImageJ. Sixteen hr after seeding, cells were treated for 72 hr with ROCKi, BRAFi or both in the presence of 1.5 μM PI to identify dead cells. Total number of cells, percentage of multinucleated (alive, dead) and total dead cells were quantified for 72 hr.

RNAi One hundred thousand cells were plated per 35-mm dish and transfected the next day with 26 nM siRNA oligonucleotides, using Optimem-I and Lipofectamine 2000 (Invitrogen). Forty eight hr after transfection cells were harvested and equal numbers re-seeded on 35-mm wells. Cells were transfected again 2 days later and plates were fixed and stained with crystal violet 2-4 days after the second transfection. Crystal violet was solubilized and absorbance at 590 nm measured as above. Cells were grown in the presence of 1 μM PLX4720 during the whole experiment. All siRNA sequences were On-Targetplus (OT) from Dharmacon (Lafayette, USA) and are listed in Table S7

MLC2 Rescue Experiments Calvo et al., 2013 Calvo F.

Ege N.

Grande-Garcia A.

Hooper S.

Jenkins R.P.

Chaudhry S.I.

Harrington K.

Williamson P.

Moeendarbary E.

Charras G.

Sahai E. Mechanotransduction and YAP-dependent matrix remodelling is required for the generation and maintenance of cancer-associated fibroblasts. +) was quantified within transfected (GFP+) cells. One hundred thousand cells were plated per 35-mm dish and transfected the next day with Lipofectamine 2000 and 1 μg plasmid encoding GFP (as control), wild-type rat MLC2 (MYL12B) fused with GFP or inactive phospho-mutant TASA-MLC2 fused with GFP (T18A, S19A) () (plasmids were a gift from Fernando Calvo). Next day cells were transfected with 26 nM siRNA oligonucleotides against MYL12B. Cell death was assessed 2-3 days after siRNA transfection by PI (1.5 μM) incorporation by FACS. Percentage of dead cells (PI) was quantified within transfected (GFP) cells.

MLC2 Stable Overexpression Takaki et al., 2017 Takaki T.

Montagner M.

Serres M.P.

Le Berre M.

Russell M.

Collinson L.

Szuhai K.

Howell M.

Boulton S.J.

Sahai E.

Petronczki M. Actomyosin drives cancer cell nuclear dysmorphia and threatens genome stability. Lentivectors encoding EGFP-fused rat MLC2-derivatives (wild-type, phospho-mimetic TDSD (T18D, S19D) and inactive phospho-mutant TASA (T18A, S19A)) () were a kind gift from Erik Sahai and Tohru Takaki (The Francis Crick Institute). HEK293T cells were transfected with MLC2-lentivectors and packaging plasmids using standard procedures, and after 48 hr supernatants were collected and spun down to remove debris. A375 cells were transduced with lentiviral supernatants for 8 hr, and 48 hr later cells were selected with 1 μg/ml puromycin for 5 days, then cells were used for subsequent experiments.

Immunofluorescence and Confocal Imaging Cells were fixed with 4% formaldehyde, permeabilised with 0.2% Triton X-100 for 5 min, blocked with 5% BSA-PBS for 1 hr at room temperature, and incubated with anti-p-MLC2 (p-MLC2S19, 1:200 in 5% BSA-PBS) overnight at 4°C. Alexa-488 anti-rabbit secondary antibody (Life Technologies) was used at 1:500 for 1 hr at room temperature. F-actin was detected with Phalloidin (1 hr RT) and nuclei were stained with Hoechst 33258 (Life Technologies). Imaging was carried out on a Zeiss LSM 510 Meta confocal microscope (Carl Zeiss) with C-Apochromat × 40/1.2 NA (water) or a Plan Apochromat × 63/1.4 NA (oil) objective lenses and Zen software (Carl Zeiss). Line scan analysis was performed in ImageJ.

Immunoblotting Cells were lysed in Laemmli buffer and snap frozen. Lysates were then boiled, sonicated for 15 s and spun down. Cell lysates were fractionated using sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gels in non-reducing conditions, and transferred subsequently to PVDF filters. Membranes were blocked in 5% BSA in 0.1% Tween 20-TBS. Primary antibodies were incubated overnight at 4°C. For detection, ECL or Prime ECL detection systems coupled to HRP-conjugated secondary antibodies (GE Healthcare) with X-ray films and an Amersham Imager 600 were used. Bands were quantified using ImageJ. Levels of phospho-proteins were calculated after correction to total levels of the relevant protein.

TGF-β1 ELISA Cells were seeded on T6-well plates (150,000 cells/well), next day cells were washed 3 times and then grown in serum-free media with or without ROCKi GSK269962A (5 μM). Forty-eight hr later supernatants were collected, spun down and assayed fresh or frozen at -80°C. TGF-β1 levels were detected by ELISA using Total TGF-β1 Legend Max™ ELISA Kit with Pre-coated plate (#436707, Biolegend) on neat samples diluted 1/5 following the manufacturer’s instructions.

Phospho-proteomics Preparation of tandem mass tagged (TMT)-multivariate phosphoproteomic samples. Cells treated with MEKi (200 nM GSK1120212 trametinib or 200 nM PD184352) or vehicle (DMSO) for 24 hr were lysed in 6 M urea, sonicated, centrifuged to clear cell debris and protein concentration was determined by BCA (Pierce 23225). 100 μg of each condition was individually digested by FASP (PMID: 19377485 ) using 1:100 Lys-C (Wako 125-05061), 1:100 Trypsin (Worthington), and amine-TMT-10 plex labeled (Pierce 90111) on membrane (iFASP) (PMID: 23692318 ). TMT channel assignment: 126 = Control (Bio. Rep. 1); 127N = Control (Bio. Rep. 2), 127C = Control (Bio. Rep. 3); 128N = Control (Bio. Rep. 4); 128C = MEKi A (Bio. Rep. 1); 129N = MEKi A (Bio. Rep. 2); 129C = MEKi A (Bio. Rep. 3); 130N = MEKi B (Bio. Rep. 1); 130C = MEKi B (Bio. Rep. 2); 131 = MEKi B (Bio. Rep. 3) (A= GSK1120212, B= PD184352). Peptides were then eluted, pooled, lyophilized and subjected to automated phosphopeptide enrichment (APE) (PMID: 25233145 ). Phosphopeptides were desalted using OLIGO R3 resin (Life Technologies 1-1339-03) and lyophilised prior to LC-MS/MS analysis (see below). Data-dependent acquisition LC-MS/MS. Phosphopeptide samples were resuspended in 0.1% formic acid and analyzed on a Q-Exactive Plus mass spectrometer (Thermo Scientific) coupled to a Dionex Ultimate 3000 RSLCnano System (Thermo Scientific). Reversed-phase chromatographic separation was performed on a C18 PepMap 300 Å trap cartridge (0.3 mm i.d. x 5 mm, 5 μm bead size; loaded in a bi-directional manner), a 75 μm i.d. x 50 cm column (5 μm bead size) using a 120 min linear gradient of 0-50% solvent B (MeCN 100% + 0.1% formic acid (FA)) against solvent A (H2O 100% + 0.1% FA) with a flow rate of 300 nL/min. The mass spectrometer was operated in the data-dependent mode to automatically switch between dual Orbitrap MS and MS/MS acquisition. Survey full scan MS spectra (from m/z 400-2000) were acquired in the Orbitrap with a resolution of 70,000 at m/z 400 and FT target value of 1 x 106 ions. The 20 most abundant ions were selected for fragmentation using higher-energy collisional dissociation (HCD) and dynamically excluded for 30 s. Fragmented ions were scanned in the Orbitrap at a resolution 35,000 at m/z 400. The isolation window was reduced to 1.2 m/z (to reduce ion co-isolation) and a MS/MS fixed first mass of 120 m/z was used (to ensure consistent TMT reporter ion coverage). For accurate mass measurement, the lock mass option was enabled using the polydimethylcyclosiloxane ion (m/z 445.120025) as an internal calibrant. For peptide identification, raw data files produced in Xcalibur 2.1 (Thermo Scientific) were processed in Proteome Discoverer 1.4 (Thermo Scientific) and searched against Human Unitprot database using Mascot (v2.2). Searches were performed with a precursor mass tolerance set to 10 ppm, fragment mass tolerance set to 0.05 Da and a maximum number of missed cleavages set to 2. Static modifications were limited to carbamidomethylation of cysteine, and variable modifications used were oxidation of methionine, deamidation of asparagine/glutamine, and phosphorylation of serine, threonine and tyrosine residues. Peptides were further filtered using a mascot significance threshold <0.05, a peptide ion Score >20 and a FDR <0.01 (evaluated by Percolator (PMID: 17952086 )). Phospho-site localization probabilities were calculated with phosphoRS 3.1 (>75%) (PMID: 22073976 ). For relative phosphopeptide quantification, MEKi/vehicle ratios were calculated by Proteome Discoverer 1.4. See Data and Code Availability section below for further details. Phosphoproteomic data analysis. Phosphopeptides from Proteome Discoverer 1.4 were normalised against total protein levels (from SILAC in-gel digest experiments), and protein-level phospho-site locations (phosphoRS 3.1 score >75%, maximum 4-PTM/peptide) were manually annotated using PhosphoSitePlus. Precursor ion spectra, extracted ion chromatograms, and product ion spectra were manually inspected for each regulated phosphopeptide. Empirical parent kinases were manually identified by referenced Uniprot annotation and putative parent kinases were manually assigned using ScanSite (PMID: 12824383 ) 3 (top 1 percentile of all sites, lowest score). Phospho-sites that did not meet these conditions were not annotated. Regulated phospho-peptides in Table S1 were those which were significant across both MEKi (GSK1120212 and PD184352) compared to vehicle-treated cells.

Phospho-Peptide Enrichment Analysis Girotti et al., 2013 Girotti M.R.

Pedersen M.

Sanchez-Laorden B.

Viros A.

Turajlic S.

Niculescu-Duvaz D.

Zambon A.

Sinclair J.

Hayes A.

Gore M.

et al. Inhibiting EGF receptor or SRC family kinase signaling overcomes BRAF inhibitor resistance in melanoma. Titz et al., 2016 Titz B.

Lomova A.

Le A.

Hugo W.

Kong X.

Ten Hoeve J.

Friedman M.

Shi H.

Moriceau G.

Song C.

et al. JUN dependency in distinct early and late BRAF inhibition adaptation states of melanoma. Pathway enrichment analyzes of the list of phospho-peptides increased in MEKi-treated A375 compared to vehicle-treated A375 cells (this study, see Phospho-proteomics section; Table S1 ); A375/PLX/R compared to A375 cells (data from ()) and M229- and M238-vemurafenib-resistant vs parental cells from () were performed using MetaCore from GeneGo Inc. ( https://portal.genego.com/ ).

Quantitative Real Time One-Step PCR RNA was isolated using TriZol (Life technologies). For experiments comparing expression in parental vs BRAFi-resistant cells (A375- and Colo829-derivatives), resistant cells were cultured with 1 μM PLX4720 and sensitive cells with equivalent volume of DMSO for 24 hr. QuantiTect Primer Assays (Qiagen) and Brilliant II SYBR Green QRT-PCR 1-step system (Agilent Technologies) with 100 ng RNA were used following the manufacturer’s instructions. GAPDH was used as loading control. The following QuantiTect Primers were used (Qiagen): GAPDH (QT00079247), LIMK1 (QT00008680), LIMK2 (QT00084357), MKL1 (QT00067921), MKL2 (QT00010115), MYH9 (QT00073101), MYL9 (QT00072268), MYL12A (QT01665741), MYL12B (QT00075264), ROCK1 (QT00034972), ROCK2 (QT00011165). Primer sequences are not provided by Qiagen, as stated in their website: ‘Sequences of the QuantiTect Primer Assays are not provided. Approximate location of primers within a specific gene can be viewed on the Product Detail pages retrieved via our GeneGlobe data base.’

Gene Enrichment Analyzes Gene sets for cross-resistance processes (EMT, metastasis, angiogenesis, hypoxia, wound healing, TGF-β, STAT3, NF-κB, YAP) were downloaded and analyzed using Gene Set Enrichment Analysis (GSEA) software ( http://www.broadinstitute.org/gsea/index.jsp ) with the settings: permutations-1,000, permutation type-gene set, metric for ranking genes-t-test. Significantly enriched gene sets in resistant vs baseline samples were considered according to p value <0.05 and FDR <0.25 in at least 2 of the 5 comparisons performed. To calculate the gene-signature score in each sample, we used single-sample Gene Set Enrichment Analysis (ssGSEA) Projection Software from GenePattern platform ( https://www.broadinstitute.org/cancer/software/genepattern ). Cantelli et al., 2015 Cantelli G.

Orgaz J.L.

Rodriguez-Hernandez I.

Karagiannis P.

Maiques O.

Matias-Guiu X.

Nestle F.O.

Marti R.M.

Karagiannis S.N.

Sanz-Moreno V. TGF-b-induced transcription sustains amoeboid melanoma migration and dissemination. Sanz-Moreno et al., 2011 Sanz-Moreno V.

Gaggioli C.

Yeo M.

Albrengues J.

Wallberg F.

Viros A.

Hooper S.

Mitter R.

Feral C.C.

Cook M.

et al. ROCK and JAK1 signaling cooperate to control actomyosin contractility in tumor cells and stroma. For the transcriptional signature of melanoma cells with high myosin II activity, genes upregulated in high myosin II activity compared to low myosin II activity melanoma cells (cells treated with ROCKi and blebbistatin) () were selected using a fold change ≥ 1.5 and a p value <0.01. GSEA analysis was performed as described above. Enrichment plot (green line) show upregulation of gene signature in indicated samples (resistant, non-responders or on-treatment). Nominal p values are shown along plot, false discovery rate (FDR) in figure legend. 10 ) p value. For analysis of ROS-related gene signatures, all available ROS/oxidative stress gene sets were downloaded from GSEA Broad Institute ( http://www.broadinstitute.org/gsea/index.jsp ). Graph shows (-Log) p value. Mjelle et al., 2015 Mjelle R.

Hegre S.A.

Aas P.A.

Slupphaug G.

Drablos F.

Saetrom P.

Krokan H.E. Cell cycle regulation of human DNA repair and chromatin remodeling genes. Peng et al., 2014 Peng G.

Chun-Jen Lin C.

Mo W.

Dai H.

Park Y.Y.

Kim S.M.

Peng Y.

Mo Q.

Siwko S.

Hu R.

et al. Genome-wide transcriptome profiling of homologous recombination DNA repair. For analysis of expression of DNA repair genes, we compiled a DNA repair gene signature from the list in () and the homologous recombination defect signature (). Network enrichment analysis of genes commonly downregulated (<0.65-fold) in at least 4 of 7 cell lines from Group 1 ( Figure 2 B) was performed using Ingenuity Pathway Analysis (Qiagen).

Tumor Xenografts A375/PLX/R cells (1 x 106) were injected subcutaneously into the right flank of 5-week-old female nude CD-1 mice (Charles River). Patient #2 cells (4 x 106) or Patient #35 cells (6 x 106) were injected into 5-8-week old NOD/SCID/ IL-2Rγ-/- (NSG, Charles River) mice (male and female). Tumors were allowed to establish, sizes (average 60-100 mm3) were matched and then mice were randomly allocated to groups of 7-8 animals. Treatment was by orogastric gavage with 45 mg/kg PLX4720, 10-25 mg/kg GSK269962A or both drugs together. GSK269962A was used at 25 mg/kg for A375/PLX/R and 10 mg/kg for Patient #2, #35. Drugs were dissolved in 5% DMSO or in 6% DMSO+50% PEG300+ 9% Tween 80. All the drugs were administered daily, 7 days a week. Tumor size was determined by caliper measurements of tumor length, width and depth and tumor volume was calculated as volume = 0.5236 x length x width x depth (mm).

Immunotherapy Experiments 5555 cells (100,000, 250,000 or 1 million) were subcutaneously injected into the right flank of 5-7-week-old female C57BL/6J mice. After 7-14 days, mice with tumors (50-80 mm3) were randomly allocated into groups of 6-7 animals and treated daily with ROCKi GSK269962A (10 mg/kg, oral gavage) or vehicle and every 3 days with anti-PD-1 monoclonal antibody (InVivoPlus clone RMP1-14, BioXCell #BE0146) (10 mg/kg, intraperitoneally (i.p.)) or rat IgG2a isotype control (clone 2A3 BioXCell # BE0089). Vehicle for ROCKi was 5% DMSO or 5% DMSO, 10% Tween 80, 6.5% ethanol. Tumor volume was determined as above. Anti-PD-1-non-responder (NR) lines were established in culture by digesting tumors with a mixture of Liberases (TH and TM, 75 μg/ml each, Roche Diagnostics) and 1 μg/ml DNase I (Sigma) in HBSS for 1 hr at 37°C with shaking, and then passed through 100 μM strainers. For experiments using 5555-anti-PD-1/NR cells, 1 million cells were injected subcutaneously into 7-week old C57BL/6J mice. Next day, all mice were given 1 dose of anti-PD-1 (10 mg/kg) i.p., and then again 3 days later. At day 7, mice were randomized into 4 treatment groups (ROCKi, anti-PD-1, combo or control) as above.

Survival in the Lung Assay Patient #2 cells were pre-treated for 24 hr with 5 μM PLX4720, 5 μM GSK269962A or both (control had DMSO), then cells were labelled with 10 μM CMFDA-Green in OptiMem (Life Technologies) for 10 min, trypsinized and equal numbers were injected into the tail vein of NSG mice in 100 μl PBS along with drugs (same concentrations as pre-treatment). At the time of injection, mice (male and female) were 6-10 weeks old and weighed around 20-22 g; mice were age and sex-matched between the groups. Mice were sacrificed 30 min (to confirm that equal numbers arrived at the lung) and 24 hr after tail vein injection. The lungs were extracted, washed twice with PBS, fixed (4% formaldehyde for 16 hr at 4°C) and examined for fluorescently-labelled cells under a Zeiss LSM 510 Meta confocal microscope (Carl Zeiss) with a 20X objective. Lung retention is represented as fluorescence area (CMFDA-Green from melanoma cells) per field, and approximately 20 fields per mouse lung were analyzed. Each experiment had 4-5 mice/condition, and experiments were replicated twice and data pooled together. Quantification of survival in the lung 24 hr after injection is shown as mean fluorescence area/field.

Immunohistochemistry Tumors and spleens were formalin-fixed and paraffin-embedded using standard protocols. For cell pellets, transfected cells were harvested 48 hr after transfection using a cell scraper, spun down, fixed with 4% formalin for 30 min and washed with PBS. Cell pellet was resuspended in 2% agarose and then embedded in paraffin. Four μm thick sections were incubated at 60°C for 20 min and then subjected to antigen retrieval using Access Super Tris pH 9 buffer (A.Menarini Diagnostics) at 110°C for 6 min in a Decloaking Chamber NxGen (Biocare Medical). Samples were blocked with Dual Endogenous Enzyme-Blocking Reagent (Dako) for 10 min and then were incubated with primary antibodies for 40 min at RT, washed and then incubated with biotinylated secondary antibodies (rabbit, mouse or rat; 1:200; Vector-Labs) for 30 min at RT. Signal was then amplified using VECTASTAIN ABC HRP kit (PK-4000) for 20 min at RT and the reaction was developed using VIP substrate (SK-4600, Vector-Labs) for 10 min at RT. Stainings were counterstained with Hematoxylin. Positive and negative controls were included in each experiment, including staining of melanoma markers HMB45/Melan-A or S100. For ECM staining, samples were fixed in Bouin’s solution (HT10132, Sigma) for 1 hr at 60°C, then stained with Weigert’s iron hematoxylin solution (HT1079, Sigma) for 5 min at RT and with Trichrome Stain (Masson) Kit (HT15-1KT, Sigma) following the manufacturer’s instructions.