BACKGROUND Pediatric low-grade glioma (pLGG) is the most common childhood brain tumor. Many patients with unresectable or recurrent/refractory tumors have significant lifelong disability. The majority of pLGG have mutations increasing the activity of the Ras/mitogen-activated protein kinase (MAPK) pathway. Activation of mammalian target of rapamycin (mTOR) is also a hallmark of pLGG. We therefore hypothesized that the dual target of rapamycin complexes 1 and 2 (TORC1/2) kinase inhibitor TAK228 would synergize with the mitogen-activated extracellular signal-regulated kinase (MEK) inhibitor trametinib in pLGG. METHODS We tested TAK228 and trametinib in patient-derived pLGG cell lines harboring drivers of pLGG including BRAFV600E and neurofibromatosis type 1 loss. We measured cell proliferation, pathway inhibition, cell death, and senescence. Synergy was analyzed via MTS assay using the Chou-Talalay method. In vivo, we tested for overall survival and pathway inhibition and performed immunohistochemistry for proliferation and vascularization. We performed a scratch assay and measured angiogenesis protein activation in human umbilical vein endothelial cells (HUVECs). RESULTS TAK228 synergized with trametinib in pLGG at clinically relevant doses in all tested cell lines, suppressing proliferation, inducing apoptosis, and causing senescence in a cell line-dependent manner. Combination treatment increased median survival by 70% and reduced tumor volume compared with monotreatment and control cohorts. Vascularization of tumors decreased as measured by CD31 and CD34. Combination treatment blocked activation of focal adhesion kinase (FAK) and sarcoma proto-oncogene non-receptor tyrosine kinase (SRC) in HUVEC cells and reduced HUVEC migration compared with each drug alone. CONCLUSIONS The combination of TAK228 and trametinib synergized to suppress the growth of pLGG. These agents synergized to reduce tumor vascularity and endothelial cell growth and migration by blocking activation of FAK and SRC.

中文翻译：

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mTORC1 / 2激酶和MEK抑制剂的协同活性抑制了小儿低度神经胶质瘤的致瘤性和血管性。

背景技术小儿低度神经胶质瘤（pLGG）是最常见的儿童期脑肿瘤。许多患有无法切除或复发/难治性肿瘤的患者终生患有严重的残疾。大多数pLGG的突变增加了Ras /丝裂原激活的蛋白激酶（MAPK）途径的活性。雷帕霉素（mTOR）哺乳动物靶标的激活也是pLGG的标志。因此，我们假设雷帕霉素复合物1和2（TORC1 / 2）激酶抑制剂TAK228的双重目标将与pLGG中的丝裂原激活的细胞外信号调节激酶（MEK）抑制剂曲美替尼协同作用。方法我们在携带pLGG驱动程序的患者源性pLGG细胞系中测试了TAK228和曲美替尼，这些细胞包括BRAFV600E和1型神经纤维瘤病丢失。我们测量了细胞增殖，途径抑制，细胞死亡和衰老。使用Chou-Talalay方法通过MTS分析对协同作用进行了分析。在体内，我们测试了总生存期和途径抑制，并进行了免疫组织化学的增殖和血管形成。我们进行了刮擦试验，并测量了人脐静脉内皮细胞（HUVEC）中的血管生成蛋白活化。结果在所有测试的细胞系中，TAK228与曲美替尼均以临床相关剂量与pLGG中的曲美替尼协同作用，以细胞系依赖性方式抑制增殖，诱导细胞凋亡并引起衰老。与单一治疗和对照组相比，联合治疗可使中位生存期增加70％，肿瘤体积减小。如通过CD31和CD34所测量，肿瘤的血管化减少。与单独使用每种药物相比，联合治疗可阻断HUVEC细胞中粘着斑激酶（FAK）和肉瘤原癌基因非受体酪氨酸激酶（SRC）的活化，并减少HUVEC迁移。结论TAK228和曲美替尼的组合可协同抑制pLGG的生长。这些药物通过阻断FAK和SRC的活化而协同作用，以减少肿瘤的血管生长以及内皮细胞的生长和迁移。