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<article class="scholarly-article">
<h2>Introduction</h2>
<p>Melanoma is the most aggressive form of skin cancer, with NRAS mutations occurring in approximately 15–20% of cases [3]. Unlike BRAF-mutant melanoma, which has benefited from targeted therapies, NRAS-mutant melanoma lacks effective targeted options. MEK inhibitors (MEKi) such as trametinib have shown modest clinical activity as monotherapy [16], but responses are limited by the rapid development of resistance [2,9]. Resistance mechanisms include activation of parallel signaling pathways, such as PI3K/AKT [11], and metabolic adaptations [24].</p><p>Metabolic reprogramming is a hallmark of cancer, and emerging evidence indicates that resistant melanoma cells become dependent on glutamine metabolism [22]. Glutaminase (GLS) catalyzes the conversion of glutamine to glutamate, which feeds into the tricarboxylic acid (TCA) cycle to support biosynthesis and energy production [28]. In BRAF-mutant melanoma, resistance to BRAF inhibitors induces glutamine dependency [22], but the role of glutamine metabolism in MEKi resistance in NRAS-mutant melanoma remains unclear.</p><p>In this study, we hypothesized that MEKi-resistant NRAS-mutant melanoma cells undergo metabolic reprogramming characterized by increased glutamine utilization, and that pharmacological inhibition of GLS with CB-839 can overcome resistance. We evaluated the efficacy of CB-839 alone and in combination with trametinib in vitro and in vivo, and investigated the underlying metabolic mechanisms.</p>
<h2>Literature Review</h2>
<p>NRAS-mutant melanoma represents a distinct molecular subtype with poor prognosis. MEK inhibitors have been explored as a therapeutic strategy, but clinical trials have shown limited benefit due to intrinsic and acquired resistance [3,16]. Vu and Aplin demonstrated that targeting TBK1 promotes apoptosis in MEKi-resistant NRAS-mutant melanoma cells [2,19]. Additionally, combination strategies with PI3K/mTOR inhibitors have shown synergy in preclinical models [11].</p><p>Metabolic adaptations are increasingly recognized as drivers of therapy resistance. Baenke et al. reported that resistance to BRAF inhibitors induces glutamine dependency in melanoma cells [22]. Similarly, Soumoy et al. identified metabolic reprogramming in metastatic melanoma with acquired resistance to targeted therapies [24]. Glutaminase inhibitors, such as CB-839, have shown promise in various cancers by disrupting glutamine metabolism [30]. In NRAS-mutant melanoma, the role of glutamine dependency in MEKi resistance has not been fully elucidated.</p>
<h2>Methodology</h2>
<h4>Cell lines and reagents</h4><p>NRAS-mutant melanoma cell lines (SK-MEL-2, WM1366) were obtained from ATCC. MEKi-resistant sublines were generated by continuous exposure to increasing concentrations of trametinib (Selleckchem) over 6 months. CB-839 was purchased from MedChemExpress. Antibodies against GLS, cleaved caspase-3, and β-actin were from Cell Signaling Technology.</p><h4>Cell viability and proliferation assays</h4><p>Cells were seeded in 96-well plates and treated with indicated concentrations of trametinib and/or CB-839 for 72 h. Cell viability was measured using MTT assay. Combination indices (CI) were calculated using CompuSyn software.</p><h4>Metabolomics</h4><p>Intracellular metabolites were extracted using 80% methanol and analyzed by LC-MS/MS. Glutamine and glutamate levels were quantified using targeted metabolomics.</p><h4>In vivo xenograft studies</h4><p>Female NSG mice (n=8 per group) were injected subcutaneously with 5×10^6 WM1366-R cells. When tumors reached ~100 mm^3, mice were randomized to vehicle, trametinib (1 mg/kg daily), CB-839 (200 mg/kg BID), or combination. Tumor volumes were measured every 3 days. All animal procedures were approved by the institutional animal care committee.</p><h4>Statistical analysis</h4><p>Data are presented as mean ± SEM. Comparisons were performed using two-tailed unpaired t-test or one-way ANOVA with Tukey's post-hoc test. P < 0.05 was considered significant.</p>
<h2>Results</h2>
<h4>MEKi-resistant cells exhibit increased glutamine dependency</h4><p>Metabolomic profiling revealed that trametinib-resistant SK-MEL-2-R and WM1366-R cells had significantly higher intracellular glutamine levels and lower glutamate levels compared to parental cells, suggesting increased glutamine consumption (Figure 1). GLS mRNA and protein expression were upregulated 2- to 3-fold in resistant cells (p < 0.01).</p><figure class="article-figure"><figcaption>Figure 1. bar chart comparing glutamine and glutamate levels in parental vs resistant cell lines</figcaption></figure><h4>GLS inhibition reduces viability of resistant cells</h4><p>Treatment with CB-839 (1 µM) for 72 h reduced cell viability by 40–60% in resistant cells, while parental cells were less sensitive (IC50 > 10 µM). CB-839 induced apoptosis as evidenced by increased cleaved caspase-3 (Figure 2).</p><figure class="article-figure"><figcaption>Figure 2. western blot images showing cleaved caspase-3 in resistant cells treated with CB-839</figcaption></figure><h4>Combination of CB-839 and trametinib is synergistic</h4><p>Combination treatment with CB-839 and trametinib resulted in synergistic cytotoxicity with CI values < 0.7 at multiple dose ratios (Table 1).</p><figure class="table-figure"><table><thead><tr><th>Cell line</th><th>Trametinib (nM)</th><th>CB-839 (nM)</th><th>Combination Index</th></tr></thead><tbody><tr><td>SK-MEL-2-R</td><td>10</td><td>500</td><td>0.65 ± 0.08</td></tr><tr><td>SK-MEL-2-R</td><td>20</td><td>1000</td><td>0.52 ± 0.06</td></tr><tr><td>WM1366-R</td><td>10</td><td>500</td><td>0.58 ± 0.07</td></tr><tr><td>WM1366-R</td><td>20</td><td>1000</td><td>0.45 ± 0.05</td></tr></tbody></table><figcaption>Table 1. Combination indices for CB-839 and trametinib in MEKi-resistant NRAS-mutant melanoma cell lines. Values are mean ± SEM from three independent experiments.</figcaption></figure><h4>In vivo efficacy of combination therapy</h4><p>In WM1366-R xenografts, combination treatment significantly suppressed tumor growth compared to vehicle or single agents (p < 0.001). Mean tumor volume on day 21 was 450 mm^3 for combination vs 1200 mm^3 for trametinib alone (Table 2).</p><figure class="table-figure"><table><thead><tr><th>Treatment group</th><th>Mean tumor volume (mm^3) at day 21</th><th>Tumor growth inhibition (%)</th></tr></thead><tbody><tr><td>Vehicle</td><td>1800 ± 200</td><td>–</td></tr><tr><td>Trametinib</td><td>1200 ± 150</td><td>33.3</td></tr><tr><td>CB-839</td><td>1400 ± 180</td><td>22.2</td></tr><tr><td>Combination</td><td>450 ± 80</td><td>75.0</td></tr></tbody></table><figcaption>Table 2. In vivo efficacy of CB-839 and trametinib combination in WM1366-R xenografts. Values are mean ± SEM (n=8 per group).</figcaption></figure><p>Metabolomic analysis of tumor tissues showed that combination treatment reduced glutamate and α-ketoglutarate levels, confirming target engagement (Figure 3).</p><figure class="article-figure"><figcaption>Figure 3. scatter plot showing decreased glutamate and α-ketoglutarate levels in combination group compared to controls</figcaption></figure>
<h2>Discussion</h2>
<p>Our findings demonstrate that MEKi resistance in NRAS-mutant melanoma is associated with metabolic reprogramming toward glutamine dependency, and that GLS inhibition effectively overcomes this resistance. These results align with previous studies showing glutamine addiction in BRAF inhibitor-resistant melanoma [22] and extend the concept to NRAS-mutant context.</p><p>The synergistic effect of CB-839 and trametinib can be attributed to dual blockade of MAPK signaling and glutamine metabolism. MEKi resistance often involves activation of compensatory pathways such as PI3K/AKT [11], which can be fueled by glutamine-derived metabolites. By disrupting glutamine anaplerosis, CB-839 may limit the metabolic flexibility required for resistance.</p><p>Our in vivo data confirm the translational potential of this combination. The significant tumor growth inhibition observed in xenografts supports further preclinical development. However, we acknowledge limitations, including the use of only two cell lines and the lack of assessment of immune microenvironment effects. Future studies should explore the impact on anti-tumor immunity, as glutamine metabolism also regulates T cell function [28].</p><p>Clinically, CB-839 is already under investigation in other cancers [30]. Our study provides a rationale for testing CB-839 in combination with MEK inhibitors in NRAS-mutant melanoma patients, particularly those who have progressed on MEKi monotherapy.</p>
<h2>Conclusion</h2>
<p>In conclusion, this study identifies glutamine dependency as a key metabolic vulnerability in MEKi-resistant NRAS-mutant melanoma. GLS inhibition with CB-839 synergizes with trametinib to suppress tumor growth both in vitro and in vivo. These findings support the clinical evaluation of this combination strategy to overcome resistance and improve outcomes for patients with NRAS-mutant melanoma.</p>
<h2>References</h2>
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