Pilocytic astrocytomas as well as other pediatric low-grade gliomas (pLGG) exhibit genetic events leading to aberrant activation of the MAPK pathway. The most common alterations are KIAA1549:BRAF fusions and BRAFV600E and NF1 mutations. Novel drugs targeting the MAPK pathway (MAPKi) are prime candidates for the treatment of these single-pathway diseases. We aimed to develop an assay suitable for preclinical testing of MAPKi in pLGGs with the goal to identify novel MAPK pathway–suppressing synergistic drug combinations. A reporter plasmid (pDIPZ) with a MAPK-responsive ELK-1–binding element driving the expression of destabilized firefly luciferase was generated and packaged using a lentiviral vector system. Pediatric glioma cell lines with a BRAF fusion (DKFZ-BT66) and a BRAFV600E mutation (BT-40) background, respectively, were stably transfected. Modulation of the MAPK pathway activity by MAPKi was measured using the luciferase reporter and validated by detection of phosphorylated protein levels. A screening of a MAPKi library was performed, and synergy of selected combinations was calculated. Screening of a MAPKi library revealed MEK inhibitors as the class inhibiting the pathway with the lowest IC50s, followed by ERK and next-generation RAF inhibitors. Combination treatments with different MAPKi classes showed synergistic effects in BRAF fusion as well as BRAFV600E mutation backgrounds. Here, we report a novel reporter assay for medium- to high-throughput preclinical drug testing in pLGG cell lines. The assay confirmed MEK, ERK, and next-generation RAF inhibitors as potential treatment approaches for KIAA1549:BRAF and BRAFV600E-mutated pLGGs. In addition, the assay revealed that combination treatments synergistically suppressed MAPK pathway activity.

中文翻译：

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基于细胞的 MAPK 报告基因检测揭示了 MAPK 抑制剂组合在 BRAF 驱动的儿童低级别胶质瘤细胞中协同抑制 MAPK 通路活性

毛细胞星形细胞瘤以及其他小儿低级别胶质瘤 (pLGG) 表现出导致 MAPK 通路异常激活的遗传事件。最常见的改变是 KIAA1549:BRAF 融合以及 BRAFV600E 和 NF1 突变。针对 MAPK 通路 (MAPKi) 的新型药物是治疗这些单通路疾病的主要候选药物。我们旨在开发一种适用于 pLGG 中 MAPKi 临床前测试的检测方法，目的是确定新的 MAPK 通路抑制协同药物组合。使用慢病毒载体系统生成并包装具有 MAPK 响应性 ELK-1 结合元件的报告质粒 (pDIPZ)，该元件驱动不稳定的萤火虫荧光素酶的表达。分别具有 BRAF 融合 (DKFZ-BT66) 和 BRAFV600E 突变 (BT-40) 背景的小儿神经胶质瘤细胞系被稳定转染。使用荧光素酶报告基因测量 MAPKi 对 MAPK 通路活性的调节，并通过检测磷酸化蛋白质水平进行验证。进行了 MAPKi 文库的筛选，并计算了所选组合的协同作用。MAPKi 文库的筛选显示 MEK 抑制剂是抑制 IC50 最低的途径的类别，其次是 ERK 和下一代 RAF 抑制剂。具有不同 MAPKi 类别的联合治疗在 BRAF 融合以及 BRAFV600E 突变背景中显示出协同作用。在这里，我们报告了一种用于 pLGG 细胞系中高通量临床前药物测试的新型报告基因检测。该测定证实 MEK、ERK 和下一代 RAF 抑制剂是 KIAA1549:BRAF 和 BRAFV600E 突变 pLGG 的潜在治疗方法。此外，