ALK signaling in CNS development, ischemic brain injury and gliomagenesis

描述（由申请人提供）：本指导研究职业发展提案的目的是研究间变性淋巴瘤激酶（ALK）受体在中枢神经系统发育（CNS）、神经干细胞（NSC）生物学、缺氧损伤反应和胶质母细胞瘤（GBM）中的作用。本研究的基本原理是基于ALK及其配体多效营养因子（PTN）和中期因子（MK）在小鼠CNS发育期间的高表达水平。PTN-ALK信号传导轴还在成人CNS缺血性损伤后提供神经保护作用，并在成人GBM中重新激活;然而，仍有机会确定ALK在这些正常和疾病状态下的功能和作用机制。为了确定ALK信号传导如何促进或调节NSC生物学，将使用先前生成的人和鼠胎儿NSC系来检测ALK表达的获得或丧失如何改变NSC增殖、凋亡或分化。此外，在存在或不存在ALK配体（多效生长因子和中期因子）的情况下激活的细胞内信号级联和转录网络将在正常NSC中定位。初步数据表明，GBM中的ALK表达定义了对ALK抑制剂克唑替尼敏感的独特患者人群;因此，设计了一项研究，以检测ALK是否是GBM患者的预后生物标志物，并确定新型ALK融合或突变是否参与GBM的发病机制。将使用科学相关的原代GBM细胞系确定ALK融合或突变的功能效应。达纳-法伯癌症研究所脑肿瘤生物储存库和活组织库提供了对将用于进行所提出的实验的大量临床注释的GBM组织和原代细胞系的独特访问。这些研究涵盖了广泛的学科，包括神经发育、缺血性脑损伤、胶质瘤生物学、下一代测序和临床病理分析，这些研究将共同描述正常CNS发育和胶质瘤形成期间PTN-ALK信号传导轴的作用机制。具体目的是：目的一：表征ALK重排并评估ALK作为GBM的预后生物标志物。 目标二：在原代GBM细胞系和正常神经干细胞中，确定野生型或突变型ALK（p.P399S、p.H1030P或p.K1612N）激活的细胞内信号通路和转录网络。目标三：检测通过体内ALK丢失破坏多效营养因子-ALK信号传导轴是否会改变神经干细胞生物学或对缺氧性脑损伤的反应。

DESCRIPTION (provided by applicant): The objective of this mentored research career development proposal is to investigate the role of the anaplastic lymphoma kinase (ALK) receptor in central nervous system development (CNS), neural stem cell (NSC) biology, response to hypoxic injury and glioblastoma (GBM). The rationale for this study is based on the high expression levels of ALK and its ligands Pleiotrophin (PTN) and Midkine (MK) during murine CNS development. The PTN-ALK signaling axis also provides neuroprotective effects after ischemic injury in the adult CNS and is reactivated in adult GBMs; however, an opportunity remains to define the function and mechanisms of action for ALK during these normal and diseased states. To determine how ALK signaling contributes to or regulates NSC biology, previously generated human and murine fetal NSC lines will be utilized to test how gain- or loss-of ALK expression alters NSC proliferation, apoptosis or differentiation. Additionally the intracellular signaling cascades and transcriptional networks activated in the presence or absence of ALK ligands (pleiotrophin and midkine) will be mapped in normal NSCs. Preliminary data suggests that ALK expression in GBM defines a distinct patient population with sensitivity to the ALK inhibitor crizotinib; therefore, a research study was designed to test whether ALK is a prognostic biomarker for GBM patients and determine if novel ALK fusions or mutations are involved in the pathogenesis of GBM. The functional effects of ALK fusions or mutations will be determined using scientifically relevant primary GBM cell lines. The Dana- Farber Cancer Institute Brain Tumor Biorepository and Living Tissue Banks provide unique access to a large cohort of clinically annotated GBM tissues and primary cell lines that will be used to conduct the proposed experiments. These research studies encompass a wide array of disciplines including neurodevelopment, ischemic brain injury, glioma biology, next generation sequencing, and clinic pathologic analyses, which together will delineate the mechanisms of action for the PTN-ALK signaling axis during normal CNS development and gliomagenesis. The specific aims are: Aim One: To characterize ALK rearrangements and evaluate ALK as a prognostic biomarker for GBM. Aim Two: To determine intracellular signaling pathways and transcriptional networks activated by wild-type or mutant ALK (p.P399S, p.H1030P, or p.K1612N) in primary GBM cell lines and normal neural stem cells. Aim Three: To test whether disruption of the Pleiotrophin-ALK signaling axis through in vivo loss of ALK alters neural stem cell biology or response to hypoxic brain injury.