Differential Inhibition of <i>Ex-Vivo</i> Tumor Kinase Activity by Vemurafenib in <i>BRAF</i>(V600E) and <i>BRAF</i> Wild-Type Metastatic Malignant Melanoma

Background Treatment of metastatic malignant melanoma patients harboring BRAF(V600E) has improved drastically after the discovery of the BRAF inhibitor, vemurafenib. However, drug resistance is a recurring problem, and prognoses are still very bad for patients harboring BRAF wild-type. Better markers for targeted therapy are therefore urgently needed. Methodology In this study, we assessed the individual kinase activity profiles in 26 tumor samples obtained from patients with metastatic malignant melanoma using peptide arrays with 144 kinase substrates. In addition, we studied the overall ex-vivo inhibitory effects of vemurafenib and sunitinib on kinase activity status. Results Overall kinase activity was significantly higher in lysates from melanoma tumors compared to normal skin tissue. Furthermore, ex-vivo incubation with both vemurafenib and sunitinib caused significant decrease in phosphorylation of kinase substrates, i.e kinase activity. While basal phosphorylation profiles were similar in BRAF wild-type and BRAF(V600E) tumors, analysis with ex-vivo vemurafenib treatment identified a subset of 40 kinase substrates showing stronger inhibition in BRAF(V600E) tumor lysates, distinguishing the BRAF wild-type and BRAF(V600E) tumors. Interestingly, a few BRAF wild-type tumors showed inhibition profiles similar to BRAF(V600E) tumors. The kinase inhibitory effect of vemurafenib was subsequently analyzed in cell lines harboring different BRAF mutational status with various vemurafenib sensitivity in-vitro. Conclusions Our findings suggest that multiplex kinase substrate array analysis give valuable information about overall tumor kinase activity. Furthermore, intra-assay exposure to kinase inhibiting drugs may provide a useful tool to study mechanisms of resistance, as well as to identify predictive markers.背景 BRAF抑制剂维莫非尼（vemurafenib）问世后，携带BRAF(V600E)突变的转移性恶性黑色素瘤患者的治疗效果得到显著改善。然而，耐药问题反复出现，且携带BRAF野生型的患者预后仍然极差。因此，临床上亟需更为精准的靶向治疗生物标志物。 研究方法 本研究采用包含144种激酶底物的肽芯片，对26例转移性恶性黑色素瘤患者的肿瘤样本进行单个激酶活性谱分析。此外，本研究还考察了维莫非尼与舒尼替尼（sunitinib）在离体条件下对激酶活性整体状态的抑制效应。 结果 相较于正常皮肤组织，黑色素瘤肿瘤裂解物中的整体激酶活性显著升高。进一步研究发现，经维莫非尼与舒尼替尼离体孵育后，激酶底物的磷酸化水平（即激酶活性）出现显著下调。尽管BRAF野生型与BRAF(V600E)突变型黑色素瘤的基础磷酸化谱较为相似，但经离体维莫非尼处理后的分析结果显示，BRAF(V600E)突变型肿瘤裂解物中存在40种激酶底物的受抑制程度更强，可借此区分BRAF野生型与BRAF(V600E)突变型肿瘤。值得注意的是，少数BRAF野生型肿瘤的药物抑制谱与BRAF(V600E)突变型肿瘤相似。随后，本研究在体外培养的、携带不同BRAF突变状态且对维莫非尼敏感性各异的细胞系中，进一步分析了维莫非尼的激酶抑制效应。 结论 本研究结果表明，多重激酶底物芯片分析可有效提供肿瘤整体激酶活性的相关信息。此外，在检测体系中加入激酶抑制剂药物的实验策略，可为耐药机制研究以及预测性生物标志物的筛选提供有效的研究工具。