Kinome Profiling to Predict Sensitivity to MAPK Inhibition in Melanoma and to Provide New Insights into Intrinsic and Acquired Mechanism of Resistance Short Title: Sensitivity Prediction to MAPK Inhibitors in Melanoma
Kinome Profiling to Predict Sensitivity to MAPK Inhibition in Melanoma and to Provide New Insights into Intrinsic and Acquired Mechanism of Resistance Short Title: Sensitivity Prediction to MAPK Inhibitors in Melanoma
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DOI：
10.3390/cancers12020512
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发表时间：
2020-02-01
期刊：
影响因子：
5.2
通讯作者：
Ghanem, Ghanem E.
中科院分区：
文献类型：
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作者：
Krayem, Mohamad;Aftimos, Philippe;Ghanem, Ghanem E.
BRAF（快速加速纤维肉瘤）和MEK（丝裂原活化蛋白激酶激酶）抑制剂联合抑制丝裂原活化蛋白激酶（MAPK）已成为BRAF V600突变转移性黑色素瘤一线治疗的标准。然而，大约一半的患者出现原发性耐药，而其余患者在长期治疗下出现继发性耐药。因此，需要预测敏感性和/或耐药性的生物标志物，以进一步细化可能受益于MAPK抑制剂的患者群体。在这项研究中，我们探索了一种自上而下的方法，使用多重激酶测定，首先，发现了一种预测黑色素瘤中MAPK抑制剂敏感性、内在和获得性耐药的kinome特征，其次，利用细胞系了解耐药机制。在体外BRAFi存在和不存在的情况下，PamChip (R)肽芯片分析了患者（4名对BRAFi单药治疗有反应和3名无反应）的给药前组织（4名对BRAFi有反应和3名无反应）的磷酸酪氨酸激酶（PTK）和丝氨酸-苏氨酸激酶（STK）活性。此外，还对4个BRAFi敏感亲本系及其获得性抗性后代和2个内在抗性品系进行了分子研究。在体外BRAFi和/或MEKi存在和不存在的情况下，测定细胞裂解液中PTK和STK的活性。在组织裂解物中，达非尼对STK和PTK活性的体外抑制在应答者中比在无应答者中更强。这种差异在细胞系比较敏感和抗性中得到证实。有趣的是，耐药的共同特征是受体酪氨酸激酶、原癌基因酪氨酸-蛋白激酶Src （Src）家族激酶和蛋白激酶B （PKB、AKT）信号传导活性增加。后一种结果经Western blot证实。在组织和细胞系中，dabrafenib单独抑制STK和PTK活性，而dabrafenib与trametinib合用对STK活性具有拮抗作用，对PTK活性具有协同作用，对酪氨酸激酶总活性的抑制作用更强。完全;这些数据表明，肿瘤和细胞系对MAPK抑制剂的耐药性可以使用多重激酶测定来预测，并且与特定酪氨酸激酶活性的增加和患者组织中整体AKT信号的增加有关。因此，这种预测性的激酶组特征将有助于识别对MAPK双抑制的先天性耐药患者，从而提出其他治疗方法。
Mitogen-activated protein kinase (MAPK) inhibition with the combination of BRAF (Rapidly Accelerated Fibrosarcoma) and MEK (Mitogen-activated protein kinase kinase) inhibitors has become the standard of first-line therapy of metastatic melanoma harbouring BRAF V600 mutations. However, about half of the patients present with primary resistance while the remaining develop secondary resistance under prolonged treatment. Thus, there is a need for predictive biomarkers for sensitivity and/or resistance to further refine the patient population likely to benefit from MAPK inhibitors. In this study, we explored a top-down approach using a multiplex kinase assay, first, to discover a kinome signature predicting sensitivity, intrinsic and acquired resistance to MAPK inhibitors in melanoma, and second, to understand the mechanism of resistance using cell lines. Pre-dose tissues from patients (four responders and three non-responders to BRAFi monotherapy) were profiled for phosphotyrosine kinase (PTK) and serine-threonine kinase (STK) activities on a PamChip (R) peptide microarray in the presence and absence of ex vivo BRAFi. In addition, molecular studies were conducted on four sensitive parental lines, their offspring with acquired resistance to BRAFi and two lines with intrinsic resistance. PTK and STK activities in cell lysates were measured in the presence and absence of ex vivo BRAFi and/or MEKi. In tissue lysates, concentration-dependent ex vivo inhibition of STK and PTK activities with dabrafenib was stronger in responders than in non-responders. This difference was confirmed in cell lines comparing sensitive and resistant ones. Interestingly, common features of resistance were increased activity of receptor tyrosine kinases, Proto-oncogene tyrosine-protein kinase Src (Src) family kinases and protein kinase B (PKB, AKT) signalling. These latter results were confirmed by Western blots. While dabrafenib alone showed an inhibition of STK and PTK activities in both tissues and cell lines, the combination of dabrafenib and trametinib showed an antagonism on the STK activities and a synergism on PTK activities, resulting in stronger inhibitions of overall tyrosine kinase activities. Altogether; these data reveal that resistance of tumours and cell lines to MAPK inhibitors can be predicted using a multiplex kinase assay and is associated with an increase in specific tyrosine kinase activities and globally to AKT signalling in the patient's tissue. Thus, such a predictive kinome signature would help to identify patients with innate resistance to MAPK double inhibition in order to propose other therapies.