Targeted therapy against TERT oncogene-rearranged neuroblastoma

项目总结/摘要 背景转录超级增强子导致的TERT癌基因重排导致大量的TERT 过度表达和神经母细胞瘤的高危患者。端粒酶抑制剂几乎不显示 在临床试验中，对儿童脑癌患者的抗癌作用和危及生命的副作用。 转录激酶CDK 7和CDK 9在超增强子相关癌基因中起关键作用 CKIα诱导p53蛋白降解。 CDK 7/CDK 9/CKIα共抑制剂A51抑制超级增强子相关癌基因表达， 激活p53蛋白表达，导致小鼠白血病消退，对正常组织无毒性。 初步数据。我们已经发现，CDK 7/CDK 9/CKIα与shRNA共敲低或用CDK 9/CKIα治疗， CDK 7/CDK 9/CKIα共抑制剂A51显著降低TERT基因表达和MDM 2蛋白 表达，激活p53蛋白表达，并诱导大量的细胞凋亡，在TERT癌基因- 重排的神经母细胞瘤，而不是正常细胞。一例RNA测序基因表达谱分析 来自493例神经母细胞瘤患者的预后数据集显示，CDK 7基因在神经母细胞瘤患者中的高水平表达， 人肿瘤组织与高水平的TERT基因表达呈正相关， 人神经母细胞瘤组织中CDK 7和CKIα的表达与患者预后不良相关。 具体目标。(1)目的：研究CDK 7和CDK 9在诱导TERT基因转录中的作用， 启动、延伸和过度表达及CKIα诱导p53蛋白降解的研究 CDK 7、CDK 9和CKIα在神经母细胞瘤细胞中的作用 体外神经母细胞瘤细胞增殖和存活以及体内肿瘤进展;（3）确定抗癌药物 CDK 7/CDK 9/CKIα共抑制剂A51在体外和体内对TERT重排的神经母细胞瘤的疗效。 结果和意义。我们希望证明CDK 7和CDK 9协同诱导TERT 基因转录起始、暂停释放、延伸和过表达，CKIα诱导p53蛋白 CDK 7、CDK 9和CKIα协同诱导TERT重排神经母细胞瘤细胞 体外增殖和存活以及体内肿瘤进展;以及CDK 7/CDK 9/CKIα共抑制剂A51 有效阻断TERT基因转录起始、暂停释放、延伸和表达，激活p53 蛋白表达，诱导体外神经母细胞瘤细胞凋亡，并阻断肿瘤进展， 用TERT重排的神经母细胞瘤细胞系或患者来源的细胞系异种移植的小鼠中的肿瘤消退 神经母细胞瘤组织。由于A51目前正在白血病患者中进行临床试验，该项目的完成将 为A51治疗TERT基因重排患者的临床试验提供重要依据 神经母细胞瘤，针对其没有靶向治疗现在可用于或曾经在临床试验中测试。

PROJECT SUMMARY/ABSTRACT Background. TERT oncogene rearrangement with transcriptional super-enhancers leads to substantial TERT over-expression and neuroblastoma in high-risk neuroblastoma patients. Telomerase inhibitors show little anticancer effects and cause life-threatening side effects in childhood brain cancer patients in clinical trials. The transcriptional kinases CDK7 and CDK9 play critical roles in super-enhancer-associated oncogene transcriptional initiation, pause release and elongation respectively, and CKIα induce p53 protein degradation. The CDK7/CDK9/CKIα co-inhibitor A51 suppresses super-enhancer-associated oncogene expression and activates p53 protein expression, resulting in leukemia regression in mice with no toxicity to normal tissues. Preliminary Data. We have found that CDK7/CDK9/CKIα co-knockdown with shRNAs or treatment with the CDK7/CDK9/CKIα co-inhibitor A51 considerably reduced TERT gene expression and MDM2 protein expression, activated p53 protein expression, and induced substantial apoptosis in TERT oncogene- rearranged neuroblastoma, but not normal cells. Analysis of a RNA sequencing gene expression-patient prognosis dataset from 493 neuroblastoma patients, showed that high levels of CDK7 gene expression in human tumor tissues positively correlated with high levels of TERT gene expression, and that high levels of CDK7 and CKIα expression in human neuroblastoma tissues correlated with poor patient prognosis. Specific Aims. (1) To identify the critical roles of CDK7 and CDK9 in inducing TERT gene transcriptional initiation, elongation and over-expression and CKIα in inducing p53 protein degradation in TERT-rearranged neuroblastoma cells; (2) To identify the critical roles of CDK7, CDK9 and CKIα in TERT-rearranged neuroblastoma cell proliferation and survival in vitro and tumor progression in vivo; (3) To define the anticancer efficacy of the CDK7/CDK9/CKIα co-inhibitor A51 against TERT-rearranged neuroblastoma in vitro and in vivo. Outcomes and Significance. We hope to demonstrate that CDK7 and CDK9 co-operatively induce TERT gene transcriptional initiation, pause release, elongation and over-expression, and CKIα induces p53 protein degradation; that CDK7, CDK9 and CKIα co-operatively induce TERT-rearranged neuroblastoma cell proliferation and survival in vitro and tumor progression in vivo; and that the CDK7/CDK9/CKIα co-inhibitor A51 efficiently blocks TERT gene transcriptional initiation, pause release, elongation and expression, activates p53 protein expression, induces neuroblastoma cell apoptosis in vitro, and blocks tumor progression and causes tumor regression in mice xenografted with TERT-rearranged neuroblastoma cell lines or patient-derived neuroblastoma tissues. As A51 is currently in clinical trials in leukemia patients, completion of this project will provide the vital evidence for clinical trials of A51 therapy in patients with TERT gene-rearranged neuroblastoma, against which no targeted therapy is now available for, or has ever be tested in, clinical trials.