Combined use of CDK4/6 and mTOR inhibitors induce synergistic growth arrest of diffuse intrinsic pontine glioma cells via mutual downregulation of mTORC1 activity.
Combined use of CDK4/6 and mTOR inhibitors induce synergistic growth arrest of diffuse intrinsic pontine glioma cells via mutual downregulation of mTORC1 activity.
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DOI：
10.2147/cmar.s167095
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发表时间：
2018
影响因子：
3.3
通讯作者：
Gill SS
中科院分区：
文献类型：
--
作者：
Asby DJ;Killick-Cole CL;Boulter LJ;Singleton WG;Asby CA;Wyatt MJ;Barua NU;Bienemann AS;Gill SS
弥漫性内在脑桥胶质瘤（DIPG）是一种致命的小儿脑肿瘤，对常规化疗有抵抗力。Palbociclib是一种公认的新型DIPG治疗药物，通过选择性抑制细胞周期蛋白依赖性激酶（CDK）4和CDK 6来限制快速分裂癌细胞的增殖。然而，将palbociclib作为DIPG的单药治疗是不可行的，因为CDK 4/6抑制剂耐药是常见的，palbociclib不易穿过血脑屏障（BBB）或在中枢神经系统中持续存在。为了抑制DIPG细胞的生长，我们的目的是将palbociclib与雷帕霉素类似物temsirolimus联合使用，已知temsirolimus可改善对CDK 4/6抑制剂的耐药性并抑制BBB外排。我们在三种患者源性DIPG细胞系中测试了palbociclib和temsirolimus。分别评估CDK 4/6和哺乳动物雷帕霉素靶蛋白（mTOR）信号通路中关键蛋白的表达谱，以确定针对DIPG的可行性。此外，我们研究了对细胞活力的影响，并检查了体内药物毒性。免疫印迹分析显示，palbociclib和temsirolimus分别通过视网膜母细胞瘤（RB）和mTOR蛋白磷酸化的典型扰动抑制CDK 4/6和mTOR信号传导;然而，我们观察到palbociclib对mTOR的非典型下调。我们证明了Palbociclib和替西罗莫司在所有三种DIPG细胞系中均抑制细胞增殖，联合作用协同作用，进一步限制细胞生长。流式细胞仪分析显示，这两种药物引起G1期细胞周期阻滞，克隆形成试验显示对细胞增殖的不可逆影响。Palbociclib在正常大鼠海马原代培养物中或注入大鼠大脑时不会引起神经毒性。这些数据说明了CDK 4/6和mTOR抑制剂在DIPG细胞中的体外抗增殖作用。将palbociclib直接输注到大脑中，结合全身给药坦罗莫司，代表了一种有前途的新方法，可开发急需的DIPG治疗方法。
Diffuse intrinsic pontine glioma (DIPG) is a lethal type of pediatric brain tumor that is resistant to conventional chemotherapies. Palbociclib is a putative novel DIPG treatment that restricts the proliferation of rapidly dividing cancer cells via selective inhibition of cyclin-dependent kinase (CDK) 4 and CDK6. However, implementing palbociclib as a monotherapy for DIPG is unfeasible, as CDK4/6 inhibitor resistance is commonplace and palbociclib does not readily cross the blood–brain barrier (BBB) or persist in the central nervous system. To inhibit the growth of DIPG cells, we aimed to use palbociclib in combination with the rapamycin analog temsirolimus, which is known to ameliorate resistance to CDK4/6 inhibitors and inhibit BBB efflux. We tested palbociclib and temsirolimus in three patient-derived DIPG cell lines. The expression profiles of key proteins in the CDK4/6 and mammalian target of rapamycin (mTOR) signaling pathways were assessed, respectively, to determine feasibility against DIPG. Moreover, we investigated effects on cell viability and examined in vivo drug toxicity. Immunoblot analyses revealed palbociclib and temsirolimus inhibited CDK4/6 and mTOR signaling through canonical perturbation of phosphorylation of the retinoblastoma (RB) and mTOR proteins, respectively; however, we observed noncanonical downregulation of mTOR by palbociclib. We demonstrated that palbociclib and temsirolimus inhibited cell proliferation in all three DIPG cell lines, acting synergistically in combination to further restrict cell growth. Flow cytometric analyses revealed both drugs caused G1 cell cycle arrest, and clonogenic assays showed irreversible effects on cell proliferation. Palbociclib did not elicit neurotoxicity in primary cultures of normal rat hippocampi or when infused into rat brains. These data illustrate the in vitro antiproliferative effects of CDK4/6 and mTOR inhibitors in DIPG cells. Direct infusion of palbociclib into the brain, in combination with systemic delivery of temsirolimus, represents a promising new approach to developing a much-needed treatment for DIPG.