Asby Daniel J, Killick-Cole Clare L, Boulter Lisa J, Singleton William Gb, Asby Claire A, Wyatt Marcella J, Barua Neil U, Bienemann Alison S, Gill Steven S
Functional Neurosurgery Research Group, Translational Health Sciences, Bristol Medical School, University of Bristol, Bristol, UK,
Department of Neurosurgery, North Bristol NHS Trust, Southmead Hospital, Bristol, UK,
Cancer Manag Res. 2018 Sep 12;10:3483-3500. doi: 10.2147/CMAR.S167095. eCollection 2018.
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 G 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.
弥漫性脑桥内在型胶质瘤（DIPG）是一种致命的儿童脑肿瘤，对传统化疗具有抗性。帕博西尼是一种有望用于治疗DIPG的新型药物，它通过选择性抑制细胞周期蛋白依赖性激酶（CDK）4和CDK6来限制快速分裂的癌细胞的增殖。然而，将帕博西尼作为DIPG的单一疗法是不可行的，因为CDK4/6抑制剂耐药性很常见，而且帕博西尼不易穿过血脑屏障（BBB）或在中枢神经系统中持续存在。为了抑制DIPG细胞的生长，我们旨在将帕博西尼与雷帕霉素类似物替西罗莫司联合使用，已知替西罗莫司可改善对CDK4/6抑制剂的耐药性并抑制BBB外排。
我们在三种源自患者的DIPG细胞系中测试了帕博西尼和替西罗莫司。分别评估了CDK4/6和雷帕霉素哺乳动物靶点（mTOR）信号通路中关键蛋白的表达谱，以确定对DIPG的可行性。此外，我们研究了对细胞活力的影响并检测了体内药物毒性。
免疫印迹分析显示，帕博西尼和替西罗莫司分别通过对视网膜母细胞瘤（RB）和mTOR蛋白磷酸化的经典扰动来抑制CDK4/6和mTOR信号传导；然而，我们观察到帕博西尼对mTOR有非经典的下调作用。我们证明，帕博西尼和替西罗莫司在所有三种DIPG细胞系中均抑制细胞增殖，联合使用时具有协同作用，可进一步限制细胞生长。流式细胞术分析显示，两种药物均导致G期细胞周期停滞，克隆形成试验表明对细胞增殖有不可逆的影响。帕博西尼在正常大鼠海马原代培养物中或注入大鼠脑内时均未引起神经毒性。
这些数据说明了CDK4/6和mTOR抑制剂在DIPG细胞中的体外抗增殖作用。将帕博西尼直接注入脑内，与替西罗莫司的全身给药联合使用，代表了一种有前景的新方法，有望开发出急需的DIPG治疗方法。
