Ryan Cheng

Supplementary Data3 from RRAS and RRAS2 Mutations Are Recurrent Oncogenic Drivers in Lung Cancer and Are Sensitive to the Pan-RAS Inhibitor RMC-6236

All supplemental figures and captions

Clinical information of patients harboring RRAS/RRAS mutations

Purpose:RRAS and RRAS2 encode a subfamily of RAS-like small GTPases that share considerable structural and functional similarities with KRAS, HRAS, and NRAS. Whether homologous RRAS/RRAS2 mutations are oncogenic and actionable drivers in lung cancer remains underexplored.Experimental Design:An institutional cohort of 8,488 non–small cell lung carcinomas (NSCLC) sequenced by comprehensive targeted DNA sequencing (MSK-IMPACT) between 2016 and 2024 was evaluated for RRAS/RRAS2 mutations. RRASQ87L or RRAS2Q72L were modeled in murine IL3-dependent Ba/F3 cells and immortalized human bronchiolar epithelial cells (HBEC). The oncogenic potential, signaling characteristics, and sensitivity to MAPK pathway inhibitors, including the novel pan-RAS inhibitor RMC-6236, were evaluated in vitro and in vivo.Results:RRASQ87L or RRAS2Q72L, homologous to KRAS-codon Q61 substitutions, were found in ∼0.45% of NSCLCs (38/8,488), with all but two lacking other MAPK pathway oncogenic drivers. RRASQ87L and RRAS2Q72L mutations transformed Ba/F3 cells and HBECs and robustly activated MAPK and PI3K–mTOR pathway signaling. RMC-6236 suppressed proliferation of RRASQ87L- and RRAS2Q72L-mutant cell lines, reduced ERK phosphorylation, induced apoptosis, and impeded cell-cycle progression. In vivo, RMC-6236 significantly inhibited growth of RRASQ87L/RRAS2Q72L-mutant HBEC-derived xenografts.Conclusions:RRASQ87L and RRAS2Q72L are recurrent, oncogenic, and potentially actionable drivers in NSCLC. Our study supports the inclusion of RRAS/RRAS2 into routine molecular diagnostic panels for precision oncology and provides preclinical rationale for investigating the potential therapeutic utility of pan-RAS inhibitors for patients with RRASQ87L/RRAS2Q72L-mutant lung cancers.

Purpose: RRAS and RRAS2 encode a subfamily of RAS-like small GTPases that share considerable structural and functional similarities with KRAS, HRAS, and NRAS. Whether homologous RRAS/RRAS2 mutations are oncogenic and actionable drivers in lung cancer remains underexplored. Experimental Design: An institutional cohort of 8,488 non–small cell lung carcinomas (NSCLC) sequenced by comprehensive targeted DNA sequencing (MSK-IMPACT) between 2016 and 2024 was evaluated for RRAS/RRAS2 mutations. RRASQ87L or RRAS2Q72L were modeled in murine IL3-dependent Ba/F3 cells and immortalized human bronchiolar epithelial cells (HBEC). The oncogenic potential, signaling characteristics, and sensitivity to MAPK pathway inhibitors, including the novel pan-RAS inhibitor RMC-6236, were evaluated in vitro and in vivo. Results: RRAS Q87L or RRAS2Q72L, homologous to KRAS-codon Q61 substitutions, were found in ∼0.45% of NSCLCs (38/8,488), with all but two lacking other MAPK pathway oncogenic drivers. RRASQ87L and RRAS2Q72L mutations transformed Ba/F3 cells and HBECs and robustly activated MAPK and PI3K–mTOR pathway signaling. RMC-6236 suppressed proliferation of RRASQ87L- and RRAS2Q72L-mutant cell lines, reduced ERK phosphorylation, induced apoptosis, and impeded cell-cycle progression. In vivo, RMC-6236 significantly inhibited growth of RRASQ87L/RRAS2Q72L-mutant HBEC-derived xenografts. Conclusions: RRASQ87L and RRAS2Q72L are recurrent, oncogenic, and potentially actionable drivers in NSCLC. Our study supports the inclusion of RRAS/RRAS2 into routine molecular diagnostic panels for precision oncology and provides preclinical rationale for investigating the potential therapeutic utility of pan-RAS inhibitors for patients with RRASQ87L/RRAS2Q72L-mutant lung cancers.

Table S1. Complete clinical, pathologic, and molecular annotations of oncogenic MET fusions Table S2. Summary of reports describing the use of MET-TKI to treat MET fusion-positive malignancies Table S3. Summary of antibodies used for Western blot and immunohistochemistry Table S4. List of genes targeted by DNA and RNA sequencing of 56 tumors with MET fusions Table S5. List of primers and plasmid systems used for transfection experiments Table S6. List of mutations detected in MET fusion-positive tumors Table S7. List of copy number alterations detected in MET fusion-positive tumors Table S8. Additional intergenic structural variants, other than MET, detected in MET fusion-positive tumors

Supplementary Figure S1. Features of intergenic MET structural variants (SV) classified as SV of unknown significance Supplementary Figure S2. Clinicopathologic features and mutation signature analysis Supplementary Figure S3. Analysis of MET structural variants and fusions in the GENIE cohort v15.1 Supplementary Figure S4. Experimental modeling of ST7::MET revealed retained ligand dependent activity Supplementary Figure S5. Single-cell expression of detected MET fusion partner genes in normal cells, highlighting resident cells of primary sites of MET-fusion-driven tumors Supplementary Figure S6. Clinical vignette: cholangiocarcinoma with MET, HGF, and EGFR co-amplification and nonamplified ST7::MET detected by RNA only (DNA−/RNA+) Supplementary Figure S7. Extended Main Figure 5 Supplementary Figure S8. Summary of MET fusion-positive tumors treated with MET-TKI reported in the literature Supplementary Figure S9. Transfection of CDH1::MET induced TKI resistance in MET exon 14 skipping mutation-positive Hs746T cells Supplementary Figure S10. Confidence models of ZKSCAN1::MET and SYNGR2::MET homodimers depicted in Figures S7A and 6A, respectively