From the ‡Department of Chemistry and Biochemistry.
§BioFrontiers Institute.
Mol Cell Proteomics. 2018 Apr;17(4):550-564. doi: 10.1074/mcp.RA117.000335. Epub 2017 Dec 18.
The BRAF-MKK1/2-ERK1/2 pathway is constitutively activated in response to oncogenic mutations of BRAF in many cancer types, including melanoma. Although small molecules that inhibit oncogenic BRAF and MAP kinase kinase (MKK)1/2 have been successful in clinical settings, resistance invariably develops. High affinity inhibitors of ERK1/2 have been shown in preclinical studies to bypass the resistance of melanoma and colon cancer cells to BRAF and MKK1/2 inhibitors, and are thus promising additions to current treatment protocols. But still unknown is how molecular responses to ERK1/2 inhibitors compare with inhibitors currently in clinical use. Here, we employ quantitative phosphoproteomics to evaluate changes in phosphorylation in response to the ERK inhibitors, SCH772984 and GDC0994, and compare these to the clinically used MKK1/2 inhibitor, trametinib. Combined with previous studies measuring phosphoproteomic responses to the MKK1/2 inhibitor, selumetinib, and the BRAF inhibitor, vemurafenib, the outcomes reveal key insights into pathway organization, phosphorylation specificity and off-target effects of these inhibitors. The results demonstrate linearity in signaling from BRAF to MKK1/2 and from MKK1/2 to ERK1/2. They identify likely targets of direct phosphorylation by ERK1/2, as well as inhibitor off-targets, including an off-target regulation of the p38α mitogen activated protein kinase (MAPK) pathway by the MKK1/2 inhibitor, trametinib, at concentrations used in the literature but higher than drug concentrations. In addition, several known phosphorylation targets of ERK1/2 are insensitive to MKK or ERK inhibitors, revealing variability in canonical pathway responses between different cell systems. By comparing multiple inhibitors targeted to multiple tiers of protein kinases in the MAPK pathway, we gain insight into regulation and new targets of the oncogenic BRAF driver pathway in cancer cells, and a useful approach for evaluating the specificity of drugs and drug candidates.
BRAF-MKK1/2-ERK1/2 通路在许多癌症类型中，包括黑色素瘤，对 BRAF 的致癌突变会持续激活。虽然抑制致癌 BRAF 和 MAP 激酶激酶（MKK）1/2 的小分子在临床环境中取得了成功，但耐药性不可避免地会出现。临床前研究表明，ERK1/2 的高亲和力抑制剂可绕过黑色素瘤和结肠癌对 BRAF 和 MKK1/2 抑制剂的耐药性，因此有望成为当前治疗方案的补充。但目前仍不清楚 ERK1/2 抑制剂的分子反应与临床使用的抑制剂相比如何。在这里，我们采用定量磷酸化蛋白质组学来评估 ERK 抑制剂 SCH772984 和 GDC0994 对磷酸化反应的影响，并将其与临床上使用的 MKK1/2 抑制剂 trametinib 进行比较。结合以前的研究，测量了 MKK1/2 抑制剂 selumetinib 和 BRAF 抑制剂 vemurafenib 对磷酸化蛋白质组的反应，结果揭示了这些抑制剂的途径组织、磷酸化特异性和非靶点效应的关键见解。结果表明，从 BRAF 到 MKK1/2 和从 MKK1/2 到 ERK1/2 的信号传递具有线性关系。它们确定了 ERK1/2 直接磷酸化的可能靶点，以及抑制剂的非靶点，包括 MKK1/2 抑制剂 trametinib 在文献中使用的浓度下，但高于药物浓度时对 p38α 有丝分裂原激活蛋白激酶（MAPK）途径的非靶点调节。此外，ERK1/2 的几个已知磷酸化靶标对 MKK 或 ERK 抑制剂不敏感，这表明不同细胞系统之间经典途径反应的可变性。通过比较靶向 MAPK 途径中多个蛋白激酶级别的多种抑制剂，我们深入了解了癌症细胞中致癌 BRAF 驱动途径的调节和新靶点，以及评估药物和候选药物特异性的有用方法。
