Journal: bioRxiv

Article Title: Mutation-Resolved Drug Sensitivity Atlas Reveals Broad RAS(ON) Inhibitor Vulnerabilities and a STAT3 Co-Dependency in NRAS-Mutant Melanoma

doi: 10.64898/2026.02.18.706707

Figure Lengend Snippet: Saturation mutagenesis mapping of NRAS variant fitness and RAS(ON) inhibitor sensitivity in MeWo melanoma cells. (A) Schematic overview of the NRAS saturation mutagenesis workflow, competitive growth profiling, and inhibitor-sensitivity screening. (B) Temporal dynamics of NRAS variant abundance relative to synonymous controls during pooled competitive assays. MeWo cells expressing all 95 missense mutants were cultured either as 3D spheroids in low-attachment plates with MEM + 10% FBS (panels i and iii) or injected subcutaneously into nu/J mice (panels ii and iv). For each time point, variant frequencies were averaged across biological replicates (n = 4) and normalized to day-0 values. Colours denote individual variant trajectories. (C) Drug-response profiling of pooled NRAS mutants under treatment with sotorasib, adagrasib, BI-2865, ADT-007, RMC-6236, and RMC-7977. Read counts for each mutant abundance in inhibitor-treated pools (day 6) normalized to DMSO controls. Relative viability (%) is colour-coded according to the scale shown. (D) Structural basis for differential inhibitor binding among NRAS Q61 mutants. Modelled cocrystal structures displaying the van der Waals surfaces of residue 61, RMC-6236, and GMP-PNP. In NRAS WT , Q61 maintains optimal molecular distances with both RMC-6236 and GMP-PNP, without steric interference. In contrast, the Q61P substitution introduces steric clashes between PRO61 and the thiazole moiety of RMC-6236 (HG2–C9), as well as with GMP-PNP (HD3–HO2G, C10–HG2, and CD–H2OG), disrupting key molecular contacts at the inhibitor–RAS interface. (E) ICLL distributions for selected NRAS mutants derived from the saturation mutagenesis screen treated with RMC-6236 or RMC-7977. Significance relative to NRAS WT was evaluated by unpaired t-test from three independent experiments (*p < 0.05, **p < 0.01). (F) Validation of oncogenic fitness in MeWo isogenic clones. (Top) Western blot confirming expression of selected NRAS variants and associated signaling changes. (Bottom) 3D spheroid proliferation over 8 days. Data confirmed with independent experiments.

Article Snippet: MeWo melanoma cell line was obtained from ATCC and maintained in MEM supplemented with 10% FBS and 1% PenStrep.

Techniques: Mutagenesis, Variant Assay, Expressing, Cell Culture, Injection, Binding Assay, Residue, Derivative Assay, Biomarker Discovery, Clone Assay, Western Blot

Journal: bioRxiv

Article Title: Mutation-Resolved Drug Sensitivity Atlas Reveals Broad RAS(ON) Inhibitor Vulnerabilities and a STAT3 Co-Dependency in NRAS-Mutant Melanoma

doi: 10.64898/2026.02.18.706707

Figure Lengend Snippet: STAT3 is required for survival of melanoma cells treated with active RAS(ON) inhibitors. (A) Signaling responses to RAS(ON) inhibition in isogenic MeWo cells expressing WT, Q61R, Q61K, or Q61L NRAS. Bubble plot summarizing Western blot–derived signaling changes after 8-hour treatment with the indicated inhibitors. Band intensities were quantified by densitometry, normalized to vehicle controls (set to 100%), and represented as bubble size. Cells were treated with dose ranges appropriate for each inhibitor class: sotorasib and adagrasib (0.1, 1, 10 μM); ADT-007, BI-2865, RMC-6236, and RMC-7977 (0.01, 0.1, 1, 10 μM). (B) Western blot analysis of phospho-STAT3 (Tyr705) in MeWo isogenic cells treated with 1 μM RMC-6236 or RMC-7977 for 0, 12, or 24 hours. (C) Apoptosis followed by STAT3 knockdown combined with RAS(ON) inhibition. (i) Representative Annexin V–FITC/PI density plot of MeWo NRAS WT , NRAS Q61R , NRAS Q61K , and NRAS Q61L cells treated for 48 hours with 1 μM RMC-6236, 1 μM RMC-7977, or DMSO. Red gate denotes apoptotic cells (Annexin VL). (ii) Quantification of total apoptotic cells. Data are presented as mean ± SD (n = 3). Statistical significance was assessed using Two-way ANOVA with Sidak’s multiple-comparisons test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). (D) Western blot of MYC, p-STAT3, and cleaved PARP following siSTAT3 combined with 24-hour treatment with 1 μM RMC-6236 or RMC-7977. From (A) to (D), data were confirmed with independent experiments. (E) Receptor Tyrosine Kinase activation following RAS(ON) inhibitor treatment. (i) Representative phospho-RTK array from NRAS Q61R MeWo cells treated with 1 μM RMC-6236 or vehicle for 18 hours. (ii) Quantification of significantly upregulated RTKs. Data are mean ± SD. Statistical significance was determined by Two-way ANOVA with Sidak’s multiple-comparisons test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Article Snippet: MeWo melanoma cell line was obtained from ATCC and maintained in MEM supplemented with 10% FBS and 1% PenStrep.

Techniques: Inhibition, Expressing, Western Blot, Derivative Assay, Knockdown, Activation Assay

Journal: bioRxiv

Article Title: Mutation-Resolved Drug Sensitivity Atlas Reveals Broad RAS(ON) Inhibitor Vulnerabilities and a STAT3 Co-Dependency in NRAS-Mutant Melanoma

doi: 10.64898/2026.02.18.706707

Figure Lengend Snippet: RAS(ON) inhibitors and napabucasin combination shows enhanced antitumor activity in NRAS-mutant melanoma. (a) (i) Representative Annexin V/PI flow cytometry density plots and (ii) quantification of apoptotic cells following 48-hour treatment with vehicle, 1 µM of RMC6236, 0.5 µM napabucasin, or combo. Data are displayed as mean ± SD (n=3). Significance was determined by Two-way ANOVA with Sidak’s multiple-comparisons test (***p < 0.001, ****p < 0.0001). Data confirmed with independent experiments. (b) Western blot analysis of cleaved PARP, phospho-STAT3 (Tyr705), and MYC levels in NRAS WT and NRAS Q61R/K/L MeWo cells following 48-hour treatment with vehicle, 1 µM RMC-6236 or RMC-7977, 0.5 µM napabucasin, or the combination. Densitometry was performed to quantify STAT3 suppression by normalizing phospho-STAT3 band intensities to vehicle-treated controls. Data are confirmed with independent experiments. (c) Viability-based synergy matrix and 3D interaction landscape of isogenic MeWo cells expressing NRAS WT, Q61K, Q61L, or Q61R treated with the RAS(ON) inhibitor RMC-6236 and the STAT3 inhibitor napabucasin. Quantification of synergy by the HSA model. The combination demonstrates strong synergy in NRAS-mutant cells (HSA score ≈ 10), compared to moderate synergy in NRAS WT cells (HSA score ≈ 3–4). Data are from a representative experiment among three independent replicates. Drug concentrations are expressed in μM. (d) (I) Tumor weight and (ii) final tumor weights in mice bearing subcutaneous MeWo xenografts expressing NRAS WT or Q61R following treatment with vehicle, RMC-6236 (10 mg/kg daily via oral gavage), napabucasin (10 mg/kg daily via intraperitoneal injection), or the combination for 14 days. Data points and bars represent mean ± SEM (N=6). Statistical significance for tumor growth was analyzed by Two-way ANOVA with Sidak’s multiple-comparisons test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Article Snippet: MeWo melanoma cell line was obtained from ATCC and maintained in MEM supplemented with 10% FBS and 1% PenStrep.

Techniques: Activity Assay, Mutagenesis, Flow Cytometry, Western Blot, Expressing, Injection

Journal: bioRxiv

Article Title: Functional and sensitivity profiling of the KIT Mutation Landscape in Melanoma

doi: 10.64898/2026.02.18.706482

Figure Lengend Snippet: KIT mutations shows differential responses towards common KIT inhibitors in vitro. (A) Western Blot analysis revealed changes in cell growth and proliferation pathways upon expression of KIT mutants in MeWO melanoma cells. (B) Upper panel: Monitoring relative growth of melanoma cell spheroids expressing KIT mutants using high-content microscopy. Lower panel: Representative image depicting spheroid size after 8 days of growth in 3D environment. (C) Western Blot demonstrating the expression of KIT in Ba/F3 cells. (D) Assessment of relative growth of Ba/F3 cells expressing different KIT mutants following withdrawal of IL-3 from the culture medium, measured using cell-titre glo assay. (E) Heatmap shows the relative IC50 of Ba/F3 cell with KIT mutants against KIT inhibitors. (F) Violin plot illustrating the IC50 values of MeWo cells expressing KIT mutants when treated with various KIT inhibitors. Results were obtained from three independent experiments. The horizontal line represents the mean, and statistical analysis was performed using one-way ANOVA.

Article Snippet: The IL-3-dependent murine pro-B cell line Ba/F3 and the human melanoma cell line MeWo (ATCC) were used for functional characterization.

Techniques: In Vitro, Western Blot, Expressing, Microscopy, Glo Assay

Journal: Cell reports

Article Title: MITF, TFEB, and TFE3 drive distinct adaptive gene expression programs and immune infiltration in melanoma

doi: 10.1016/j.celrep.2025.116499

Figure Lengend Snippet: (A) Left, western blot of IGR37 melanoma cell line transfected with control or MITF-specific small interfering RNA (siRNA). Right, RT-qPCR for PTEN mRNA from corresponding IGR37 cells. (B and C) Heatmaps showing relative mRNA expression in CCLE melanoma or Tsoi et al. melanoma cell lines. (B) Right, Pearson correlation of gene expression. (D) TCGA melanomas ranked by MITF expression ( MITF ; black line). Gray bars indicate expression of RRAGD or FNIP2 in each melanoma, with the moving average of each per 20 melanoma window indicated by colored lines. (E) MITF ChIP-seq showing binding to the RRAGD or FNIP2 genes. (F) Western blot of melanoma cell lines. (G and H) Box and whisker plots showing relative expression of MITF , RRAGD , and FNIP2 based on triplicate RNA-seq of IGR37 cells expressing doxycycline-inducible β-catenin (iCTNNB1) (G) or in MeWo cells after short hairpin RNA (shRNA)-mediated depletion of MITF (H). See also .

Article Snippet: MeWo Human melanoma cell line (male) , Obtained from the ATCC , Cornil et al. .

Techniques: Western Blot, Transfection, Control, Small Interfering RNA, Quantitative RT-PCR, Expressing, Gene Expression, ChIP-sequencing, Binding Assay, Whisker Assay, RNA Sequencing, shRNA