There is a clear need to identify targetable drivers of resistance and potential biomarkers for salvage therapy for patients with melanoma refractory to the combination of BRAF and MEK inhibition. In this study, we performed whole-exome sequencing on BRAF -V600E–mutant melanoma patient tumors refractory to the combination of BRAF/MEK inhibition and identified acquired oncogenic mutations in NRAS and loss of the tumor suppressor gene CDKN2A . We hypothesized the acquired resistance mechanisms to BRAF/MEK inhibition were reactivation of the MAPK pathway and activation of the cell-cycle pathway, which can both be targeted pharmacologically with the combination of a MEK inhibitor (trametinib) and a CDK4/6 inhibitor (palbociclib). In vivo , we found that combination of CDK4/6 and MEK inhibition significantly decreased tumor growth in two BRAF/MEK inhibitor–resistant patient-derived xenograft models. In vitro , we observed that the combination of CDK4/6 and MEK inhibition resulted in synergy and significantly reduced cellular growth, promoted cell-cycle arrest, and effectively inhibited downstream signaling of MAPK and cell-cycle pathways in BRAF inhibitor–resistant cell lines. Knockdown of CDKN2A in BRAF inhibitor–resistant cells increased sensitivity to CDK4/6 inhibition alone and in combination with MEK inhibition. A key implication of our study is that the combination of CDK4/6 and MEK inhibitors overcomes acquired resistance to BRAF/MEK inhibitors, and loss of CDKN2A may represent a biomarker of response to the combination. Inhibition of the cell-cycle and MAPK pathway represents a promising strategy for patients with metastatic melanoma who are refractory to BRAF/MEK inhibitor therapy.

中文翻译：

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靶向 CDK4/6 代表获得性 BRAF/MEK 抑制剂耐药黑色素瘤的治疗漏洞

对于 BRAF 和 MEK 抑制剂联合治疗无效的黑色素瘤患者，显然需要确定可靶向的耐药驱动因素和潜在生物标志物，以用于挽救治疗。在这项研究中，我们对 BRAF -V600E 突变的黑色素瘤患者肿瘤进行了全外显子组测序，这些肿瘤对 BRAF/MEK 抑制的联合治疗无效，并确定了 NRAS 中的获得性致癌突变和肿瘤抑制基因 CDKN2A 的缺失。我们假设对 BRAF/MEK 抑制的获得性耐药机制是 MAPK 通路的重新激活和细胞周期通路的激活，这两者都可以通过 MEK 抑制剂（trametinib）和 CDK4/6 抑制剂（palbociclib）的组合进行药理学靶向). 在体内，我们发现 CDK4/6 和 MEK 抑制的组合显着降低了两种 BRAF/MEK 抑制剂耐药患者来源的异种移植模型中的肿瘤生长。在体外，我们观察到 CDK4/6 和 MEK 抑制的组合产生协同作用并显着降低细胞生长，促进细胞周期停滞，并有效抑制 BRAF 抑制剂耐药细胞系中 MAPK 和细胞周期通路的下游信号传导。BRAF 抑制剂耐药细胞中 CDKN2A 的敲低增加了对 CDK4/6 单独抑制和与 MEK 抑制联合抑制的敏感性。我们研究的一个关键意义是 CDK4/6 和 MEK 抑制剂的组合克服了对 BRAF/MEK 抑制剂的获得性抗性，CDKN2A 的丢失可能代表对组合反应的生物标志物。