S5: Elevated cytoplasmic ROS induces apoptosis after the treatment with neocuproine

Table ST2: Summary of the clinical data corresponding to the primary cell cultures used in this publication

Table ST5: Table of the 15 most informative proteins upregulated in MEK inhibitor-resistant cell culture (ranked by log2 fold-change).

Table ST6: Differential expressed lipids and their FDRs in sensitive vs resistant cells (negative values are upregulated in sensitive cells).

S3: Correlation between MEKi responsiveness and sensitivity to nNeocuproine

Table ST3: Differential expressed RNA transcripts between MEKi-sensitive and -resistant melanoma cell cultures.

Table ST1: Summary of growth inhibitory IC50 values (including the corresponding 95% confident intervals (C.I.95%) conducted for neocuproine or MEKi (binimetinib) and primary human cell lines. The table includes the confirmed oncogenic mutation as well as the ratio of neocuproine normalized to the MEKi response.

Metastatic melanoma, the most aggressive form of skin cancer, accounts for the majority of skin cancer-related deaths. While targeted kinase inhibitors have improved outcomes for patients with BRAF-mutated melanomas, their efficacy is often short-lived, and effective treatments for other mutations, such as NRAS, remain scarce. To address this clinical need, we investigated the combination of the novel panRAF inhibitor, brimarafenib, and the MEK inhibitor, mirdametinib, both of which target the MAPK pathway downstream of NRAS. This study demonstrates the efficacy of this combination in NRAS-mutated melanoma and is currently also investigated in a phase I/IIa clinical study. In vitro, the brimarafenib and mirdametinib combination exhibited synergistic effects, significantly inhibiting the growth of patient-derived NRAS-mutated melanoma cell lines. A colony formation assay showed that this combination prevented the emergence of drug-resistant clones, suggesting a strong potential to reduce disease relapse. Transcriptional and proteomic analyses revealed that the observed growth inhibition was due to modulation of MAPK signaling and induction of apoptosis. In vivo studies further validated these findings, showing that the combination treatment inhibited tumor growth and significantly prolonged survival in mouse models bearing patient-derived NRAS-mutated melanoma tumors. Given the tolerability of this combination in vivo, our results suggest that brimarafenib and mirdametinib represent a promising therapeutic strategy for patients with NRAS-mutated melanomas and potentially other RAS-mutated solid tumors.

S2: HPLC analysis of STO13881 and nNeocuproine showed overlapping profiles and confirmed STO13881 to be neocuproine (purchased from Sigma-Aldrich, Switzerland). High-performance liquid chromatography (HPLC) analysis was performed using the Zuürich functional genomic center.

S7: Pathway enrichment (metabolomics)

S9: Treatment of binimetinib-sensitive melanoma cell lines with PPP inhibitor

S11: Immunohistological analysis of orthologic slice cultures.

Table ST7: Clinical data of the 62 melanoma patient which tumor material was used for pharmacoscopy

Table ST4: Table of the 15 most informative proteins upregulated in MEK inhibitor-sensitive cell culture (ranked by log2 fold-change).

S16: scRNA-sequencing analysis of xenograft tumors M130227

S12: Analysis of CyTof data from fresh melanoma tissue

Tumor Profiler Consortium author list

S4: Neocuproine is a copper-dependent ROS inducer