Abstract NRAS mutations occur in 20-30% of cutaneous melanomas and are associated with poor outcomes. Immune checkpoint inhibitors (ICI) have improved survival rates for advanced and metastatic melanoma, but most patients with NRAS mutant metastatic melanoma will eventually experience disease progression with ICI. In various tumor types, it is established that mutant RAS reprograms the tumor immune microenvironment (TIME) to promote ICI resistance. Therefore, we sought to identify effectors of mutant-NRAS that may be implicated in TIME and ICI resistance in melanoma. To gain a broader view of mutant NRAS-induced transcriptomic changes, we first engineered isogenic non-malignant RPE1 cells to over-express wild-type or mutant (Q61K) NRAS vs. an empty vector control. Second, NRAS Q61K WM3623 melanoma cells were transfected with non-targeting or NRAS siRNA. RNAseq analysis was performed on RPE1 and WM3623 cells. We compared these data with transcriptomic data from (n=11) NRAS Q61 mutant vs. (n=9) NRAS/BRAF WT melanoma cell lines from the DepMap portal. By integrating these three analyses, we identified 61 genes that were common differentially expressed and associated with mutant NRAS. Of these, n=30 genes were specifically upregulated in NRAS-mutant, while n=31 were downregulated. Amongst the upregulated genes, we identified SH2B3, a gene that encodes LNK as a potential immunomodulator of interest. LNK is an adapter protein and a negative regulator of IFN-JAK/STAT signalling, and thus may play a role in NRAS-induced TIME modulation. Using NRAS-targeted siRNA, qPCR and immunoblots, we validated that mutant NRAS is both necessary and sufficient for inducing LNK expression at the mRNA and protein levels in RPE-1 cells and in human melanoma cells (WM3623, WM1960, SkMel2). Next, we evaluated whether the novel pan-RAS inhibitor, RMC-7977 is capable of inhibiting MAPK and AKT signalling and LNK expression in human melanoma cells. We performed 2hr dose response and 72-hour time course treatments with RMC-7977 in a panel of NRAS mutant (WM3623, SkMel2, WM1960), BRAF mutant (SkMel28), and WT (WM3311) cell lines. RMC-7977 caused rapid MAPK inhibition but no significant changes in AKT signalling on the protein level, with near-complete pERK loss by 2 hours in all NRAS-mutant melanoma lines. RMC-7977 reduced LNK protein levels ∼24 hours after treatment, suggesting that LNK responds to sustained NRAS inhibition. To interrogate the functional roles of LNK in NRAS mutant melanoma, we generated CRISPR–Cas9 LNK knockout (KO) in SkMel2 melanoma cells. Preliminary analysis shows that LNK KO cells exhibit substantial differences in baseline STAT1 phosphorylation compared to control LNK expressing cells. Our on-going experiments evaluate the role of LNK in modulating IFN-γ signalling, tumor growth and ICI response in vitro and in vivo. In conclusion, this work identifies LNK as an effector of mutant NRAS in melanoma and highlights its potential role as an important immunomodulator and therapeutic target that could be exploited to optimize precision therapy strategies. Citation Format: Meghan Vrkoc, Sadaf Solati, Isabel Soria-Bretones, Theo Goullet de Rugy, Huijie Wang, Karen Christensen, April A. N. Rose. Identifying and characterizing the role of the adapter protein, LNK in NRAS mutant melanoma abstract. In: Proceedings of the AACR Special Conference in Cancer Research: RAS Oncogenesis and Therapeutics; 2026 Mar 5-8; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Res 2026;86 (5Suppl₁): Abstract nr B050.
Vrkoc et al. (Thu,) studied this question.