Anti-proliferative effects of continued mitogen activated protein kinase pathway inhibition following acquired resistance to BRAF and/or MEK inhibition in melanoma: Implications for treatment beyond disease progression. Homo sapiens

Inhibitors of the MAPKs, BRAF and MEK, induce tumor regression in the majority of patients with BRAF-mutant metastatic melanoma. The clinical benefit of MAPK inhibitors is restricted by the development of acquired resistance with half of those who benefit having progressed by 6-7 months and long-term responders uncommon. There remains no agreed treatment strategy on disease progression in these patients. Without published evidence, fears of accelerated disease progression on inhibitor withdrawal have led to the continuation of drugs beyond formal disease progression. We now demonstrate that treatment with MAPK inhibitors beyond disease progression can provide significant clinical benefit, and the withdrawal of these inhibitors led to a marked increase in the rate of disease progression in two patients. We also show that MAPK inhibitors retain partial activity in acquired resistant melanoma by examining drug-resistant clones generated to dabrafenib, trametinib or the combination of these drugs. All resistant sublines displayed a markedly slower rate of proliferation when exposed to MAPK inhibitors, and this coincided with a reduction in MAPK signalling, decrease in BrdU incorporation and S-phase inhibition. This cytostatic effect was also associated diminished levels of cyclin D1 and p-pRb.. Two short-term melanoma cultures generated from resistant tumour biopsies also responded to MAPK inhibition with comparable inhibitory changes in proliferation and MAPK signalling. These data provide a rationale for the continuation of BRAF and MEK inhibitors after disease progression and support the development of clinical trials to examine this strategy. Overall design: Total RNA obtained from melanooma cell lines treated for 24h with dabrafenib, trametinib or combination of dabrafenib and trametinib

丝裂原活化蛋白激酶（Mitogen-Activated Protein Kinases, MAPKs）、BRAF与MEK抑制剂，可使绝大多数BRAF突变型转移性黑色素瘤患者实现肿瘤消退。MAPK抑制剂的临床获益会因获得性耐药的产生而受限：半数获益患者会在6~7个月内出现疾病进展，长期应答者极为罕见。目前针对这类患者疾病进展后的治疗策略尚未达成共识。由于缺乏已发表的相关证据，学界对停用抑制剂后疾病进展加速的担忧，使得部分患者在正式确认疾病进展后仍持续服用此类药物。本研究证实，在疾病进展后继续使用MAPK抑制剂仍可带来显著临床获益；而在2例患者中，停用此类药物后疾病进展速率显著加快。通过针对达拉非尼（dabrafenib）、曲美替尼（trametinib）或二者联合用药诱导产生的耐药克隆进行分析，本研究还证实，MAPK抑制剂对获得性耐药黑色素瘤仍具有部分活性。所有耐药亚细胞系在暴露于MAPK抑制剂后，增殖速率均显著减慢；这一现象与MAPK信号通路活性降低、溴脱氧尿苷（Bromodeoxyuridine, BrdU）掺入量减少以及S期阻滞相吻合。这种细胞生长抑制效应还与细胞周期蛋白D1（cyclin D1）以及磷酸化视网膜母细胞瘤蛋白（phosphorylated retinoblastoma protein, p-pRb）水平降低相关。2株从耐药肿瘤活检组织中建立的短期黑色素瘤培养细胞系，同样对MAPK抑制产生应答，其增殖与MAPK信号通路的抑制变化特征与前述耐药亚细胞系一致。上述数据为疾病进展后继续使用BRAF与MEK抑制剂提供了理论依据，并支持开展临床试验以验证该治疗策略。实验整体设计：从经达拉非尼、曲美替尼或二者联合用药处理24小时的黑色素瘤细胞系中提取总RNA。