Introduction: Patients with NRAS-mutant metastatic melanoma often have an aggressive disease requiring a fast-acting, effective therapy. The MEK inhibitor binimetinib shows an overall response rate of 15% in patients with NRAS-mutant melanoma, providing a backbone for combination strategies. Our previous studies demonstrated that in NRAS-mutant melanoma, the antitumor activity of the MEK inhibitor binimetinib was significantly potentiated by the BRAFV600E/K inhibitor encorafenib through the induction of ER stress, leading to melanoma cell death by apoptotic mechanisms. Encorafenib combined with binimetinib was well tolerated in a phase III trial showing potent antitumor activity in BRAF-mutant melanoma, making a rapid evaluation in NRAS-mutant melanoma imminently feasible. These data provide a mechanistic rationale for the evaluation of binimetinib combined with encorafenib in preclinical and clinical studies on NRAS-mutant metastatic melanoma. Methods: The combination of BRAFi plus MEKi was tested in a monolayer culture of patient-derived cell lines and in corresponding patient-derived tissue slice cultures of NRAS-mutant melanoma. To investigate the treatment in vivo, NSG (NOD. Cg-PrkdcscidIl2rgtm1Wjl/SzJ) mice were subcutaneously injected with three different BRAF wild-type melanoma models harboring oncogenic NRAS mutations and treated orally with encorafenib (6 mg/kg body weight, daily) with or without binimetinib (8 mg/kg body weight, twice daily). In parallel, an individual healing attempt was carried out by treating one patient with an NRAS-mutated tumor. Results: Encorafenib was able to enhance the inhibitory effect on cell growth of binimetinib only in the cell line SKMel147 in vitro. It failed to enhance the apoptotic effect found in two other NRAS-mutated cell lines. Encorafenib led to a hyperactivation of ERK which could be reduced with the combinational treatment. In two of the three patient-derived tissue slice culture models of NRAS-mutant melanomas, a slight tendency of a combinatorial effect was seen which was not significant. Encorafenib showed a slight induction of the ER stress genesATF4,CHOP, andNUPR1. The combinational treatment was able to enhance this effect, but not significantly. In the mouse model, the combination therapy of encorafenib with binimetinib resulted in reduced tumor growth compared to the control and encorafenib groups; however, the best effect in terms of tumor growth inhibition was measured in the binimetinib therapy group. The therapy showed no effect in an individual healing attempt for a patient suffering from metastatic, therapy-refractory NRAS-mutated melanoma. Conclusion: In in vitro and ex vivo settings, the combination therapy was observed to elicit a response; however, it did not amplify the efficacy observed with binimetinib alone, whereas in a patient, the combinational treatment remained ineffective. The preclinical in vivo data showed no increased combinatorial effect. However, the in vivo effect of binimetinib as monotherapy was unexpectedly high in the tested regimen. Nevertheless, binimetinib proved to be advantageous in the treatment of melanoma in vivo and led to high rates of apoptosis in vitro; hence, it still seems to be a good base for combination with other substances in the treatment of patients with NRAS-mutant melanoma.
引言：NRAS突变型转移性黑色素瘤患者常表现为侵袭性疾病，需要快速起效的有效治疗方案。MEK抑制剂比美替尼在NRAS突变型黑色素瘤患者中显示出15%的总体缓解率，为联合治疗策略提供了基础。我们先前的研究表明，在NRAS突变型黑色素瘤中，BRAFV600E/K抑制剂恩考芬尼通过诱导内质网应激，显著增强了MEK抑制剂比美替尼的抗肿瘤活性，从而通过凋亡机制导致黑色素瘤细胞死亡。恩考芬尼联合比美替尼在III期试验中耐受性良好，并在BRAF突变型黑色素瘤中显示出强大的抗肿瘤活性，这使得在NRAS突变型黑色素瘤中进行快速评估成为可能。这些数据为在NRAS突变型转移性黑色素瘤的临床前和临床研究中评估比美替尼联合恩考芬尼提供了机制依据。 方法：在NRAS突变型黑色素瘤的患者来源细胞系单层培养物及相应的患者来源组织切片培养物中测试了BRAF抑制剂联合MEK抑制剂的疗效。为研究体内治疗效果，将三种不同的携带致癌性NRAS突变的BRAF野生型黑色素瘤模型皮下注射至NSG（NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ）小鼠体内，并口服恩考芬尼（6毫克/千克体重，每日一次）联合或不联合比美替尼（8毫克/千克体重，每日两次）进行治疗。同时，对一名NRAS突变肿瘤患者进行了个体化治疗尝试。 结果：体外实验中，恩考芬尼仅在SKMel147细胞系中增强了比美替尼对细胞生长的抑制作用，而在另外两种NRAS突变细胞系中未能增强其促凋亡效应。恩考芬尼导致ERK过度激活，联合治疗可降低此效应。在三种NRAS突变型黑色素瘤患者来源组织切片培养模型中，有两种观察到微弱的联合治疗趋势，但未达显著水平。恩考芬尼轻微诱导了内质网应激基因ATF4、CHOP和NUPR1的表达，联合治疗虽能增强此效应，但同样不显著。在小鼠模型中，与对照组和恩考芬尼单药组相比，恩考芬尼联合比美替尼治疗组肿瘤生长减缓；然而，在抑制肿瘤生长方面，比美替尼单药组显示出最佳效果。在一例转移性、难治性NRAS突变型黑色素瘤患者的个体化治疗尝试中，该联合方案未显现疗效。 结论：在体外和离体实验中观察到联合治疗能引发一定反应，但并未放大比美替尼单药的疗效；而在临床患者中，联合治疗仍无效。临床前体内数据显示联合治疗未增强疗效。然而，在测试方案中比美替尼单药的体内疗效意外显著。尽管如此，比美替尼在黑色素瘤体内治疗中仍显示出优势，并在体外诱导高凋亡率；因此，它似乎仍是NRAS突变型黑色素瘤患者治疗中与其他药物联合的良好基础。
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