Aberrant AKT Activation Drives Ganglioneuroma and Can Be Therapeutically Targeted by mTOR Inhibitors. Aberrant AKT Activation Drives Ganglioneuroma and Can Be Therapeutically Targeted by mTOR Inhibitors

Peripheral sympathetic nervous system tumors are the most common extra-cranial pediatric tumors in children and include neuroblastoma, ganglioneuroma and intermixed ganglioneuroblastoma. Ganglioneuroma can be induced by activating mutations of the RET proto-oncogene or activated Ras in murine models, but the etiology and molecular pathogenesis of this disease is unknown for the majority of human ganglioneuromas. Surgery is the only effective therapy for ganglioneuroma which can be challenging due to tumor location and compression of surrounding structures. Thus, there is great potential benefit to the definition of presurgical therapies that can reduce the size and extent of these tumors, and therefore limit morbidity. We found high levels of phosphorylated AKT in most of human ganglioneuromas, but only in a small portion of human poorly differentiated neuroblastomas (p<0.0001, Fisher’s exact test). As a result, we created zebrafish transgenic for constitutively activated myr-Akt2 in the sympathetic nervous system. These zebrafish were found to develop benign ganglioneuroma without progression to neuroblastoma. Zebrafish tumors displayed high expression of phosphorylated Akt and the downstream Akt targets, phosphorylated mTOR, S6 and EIF4EBP1. Histopathological and comparative genomic analyses revealed that zebrafish ganglioneuroma highly resembles human ganglioneuroma. Inhibition of the downstream AKT target, mTOR, using clinically available inhibitors effectively reduced tumor burden in zebrafish embryos transplanted with primary ganglioneuroma. Our results implicate activated and phosphorylated AKT as a tumorigenic driver in ganglioneuroma, and propose inhibition of the AKT-target kinase mTOR as an ideal candidate to treat patients with ganglioneuroma. Overall design: RNA-Seq for zebrafish tumor cells from MYCN and myrAKT transgenic fish

外周交感神经系统肿瘤（Peripheral sympathetic nervous system tumors）是儿童最常见的颅外儿科肿瘤，涵盖神经母细胞瘤（neuroblastoma）、神经节细胞瘤（ganglioneuroma）以及混合性神经节神经母细胞瘤（intermixed ganglioneuroblastoma）。在小鼠模型（murine models）中，神经节细胞瘤可由RET原癌基因（RET proto-oncogene）或活化Ras（activated Ras）的激活突变诱导，但绝大多数人类神经节细胞瘤的病因与分子发病机制仍未明确。
手术是治疗神经节细胞瘤的唯一有效手段，但受肿瘤位置及对周围结构的压迫影响，手术治疗往往颇具挑战性。因此，开发可缩小肿瘤体积与累及范围、进而减少手术并发症的术前治疗方案，具备极大的潜在临床价值。
本研究团队在多数人类神经节细胞瘤中检测到高水平的磷酸化AKT（phosphorylated AKT），但仅在少数低分化人类神经母细胞瘤中检出该蛋白（p<0.0001，Fisher确切概率法（Fisher’s exact test））。
据此，我们构建了在交感神经系统中组成型激活myr-Akt2的转基因斑马鱼（zebrafish）。研究发现，该转基因斑马鱼可自发形成良性神经节细胞瘤，且不会进展为神经母细胞瘤。斑马鱼肿瘤组织中高表达磷酸化Akt及其下游靶标：磷酸化mTOR（phosphorylated mTOR）、S6及EIF4EBP1。
组织病理学（histopathological）与比较基因组分析（comparative genomic analyses）结果显示，斑马鱼神经节细胞瘤与人类神经节细胞瘤具有高度相似的特征。使用临床可用的mTOR抑制剂靶向抑制AKT下游靶标mTOR，可有效降低移植了原代神经节细胞瘤的斑马鱼胚胎的肿瘤负荷（tumor burden）。
本研究证实，活化的磷酸化AKT是神经节细胞瘤的致瘤驱动因子，并提出靶向抑制AKT下游激酶mTOR可作为治疗神经节细胞瘤患者的理想候选方案。
整体实验设计（Overall design）：针对MYCN与myrAKT转基因斑马鱼的肿瘤细胞开展RNA测序（RNA-Seq）