The RAS-regulated RAF–MEK1/2–ERK1/2 signalling pathway is activated in cancer due to mutations in RAS proteins (especially KRAS), BRAF, CRAF, MEK1 and MEK2. Whilst inhibitors of KRASG12C (lung adenocarcinoma) and BRAF and MEK1/2 (melanoma and colorectal cancer) are clinically approved, acquired resistance remains a problem. Consequently, the search for new inhibitors (especially of RAS proteins), new inhibitor modalities and regulators of this pathway, which may be new drug targets, continues and increasingly involves cell-based screens with small molecules or genetic screens such as RNAi, CRISPR or protein interference. Here we describe cell lines that exhibit doxycycline-dependent expression KRASG12V or BRAFV600E and harbour a stably integrated EGR1:EmGFP reporter gene that can be detected by flow cytometry, high-content microscopy or immunoblotting. KRASG12V or BRAFV600E-driven EmGFP expression is inhibited by MEK1/2 or ERK1/2 inhibitors (MEKi and ERKi). BRAFi inhibit BRAFV600E-driven EmGFP expression but enhance the response to KRASG12V, recapitulating paradoxical activation of wild type RAF proteins. In addition to small molecules, expression of iDab6, encoding a RAS-specific antibody fragment inhibited KRASG12V- but not BRAFV600E-driven EmGFP expression. Finally, substitution of EmGFP for a bacterial nitroreductase gene allowed KRASG12V or BRAFV600E to drive cell death in the presence of a pro-drug, which may allow selection of pathway inhibitors that promote survival. These cell lines should prove useful for cell-based screens to identify new regulators of KRAS- or BRAF-dependent ERK1/2 signalling (drug target discovery) as well as screening or triaging ‘hits’ from drug discovery screens.

中文翻译：

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用于筛选 KRAS-RAF-MEK1/2-ERK1/2 通路抑制剂或调节剂的报告细胞系


由于 RAS 蛋白（尤其是 KRAS）、BRAF、CRAF、MEK1 和 MEK2 的突变，RAS 调节的 RAF-MEK1/2-ERK1/2 信号通路在癌症中被激活。虽然 KRASG12C（肺腺癌）和 BRAF 和 MEK1/2（黑色素瘤和结直肠癌）抑制剂已获得临床批准，但获得性耐药仍然是一个问题。因此，对该途径的新抑制剂（尤其是 RAS 蛋白）、新抑制剂模式和调节剂（可能是新药物靶标）的探索仍在继续，并且越来越多地涉及基于细胞的小分子筛选或遗传筛选，例如 RNAi、CRISPR 或蛋白质干扰。在这里，我们描述了表现出多西环素依赖性表达 KRASG12V 或 BRAFV600E 并具有稳定整合的 EGR1:EmGFP 报告基因的细胞系，可以通过流式细胞术、高内涵显微镜或免疫印迹检测到该基因。 KRASG12V 或 BRAFV600E 驱动的 EmGFP 表达被 MEK1/2 或 ERK1/2 抑制剂（MEKi 和 ERKi）抑制。 BRAFi 抑制 BRAFV600E 驱动的 EmGFP 表达，但增强对 KRASG12V 的反应，概括了野生型 RAF 蛋白的矛盾激活。除了小分子外，编码 RAS 特异性抗体片段的 iDab6 的表达抑制 KRASG12V 驱动的 EmGFP 表达，但不抑制 BRAFV600E 驱动的 EmGFP 表达。最后，用 EmGFP 替代细菌硝基还原酶基因，使得 KRASG12V 或 BRAFV600E 在前药存在的情况下驱动细胞死亡，这可能允许选择促进存活的途径抑制剂。这些细胞系应该可用于基于细胞的筛选，以识别 KRAS 或 BRAF 依赖性 ERK1/2 信号传导（药物靶点发现）的新调节因子，以及筛选或分类药物发现筛选中的“命中”。