ALK signaling in CNS development, ischemic brain injury and gliomagenesis

描述（由申请人提供）：本指导研究的目标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、对缺氧损伤的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在小鼠中枢神经系统发育过程中ALK及其配体多营养蛋白（PTN）和Midkine （MK）的高表达水平。PTN-ALK信号轴在成人中枢神经系统缺血性损伤后也具有神经保护作用，并在成人GBMs中被重新激活；然而，在这些正常和患病状态下，ALK的功能和作用机制仍有待确定。为了确定ALK信号如何促进或调节NSC生物学，将利用先前生成的人类和小鼠胎儿NSC细胞系来测试ALK表达的增加或减少如何改变NSC增殖、凋亡或分化。此外，细胞内信号级联和转录网络在存在或不存在ALK配体（多营养因子和midkine）时被激活，将在正常的NSCs中被绘制出来。初步数据表明，ALK在GBM中的表达定义了对ALK抑制剂克唑替尼敏感的独特患者群体；因此，我们设计了一项研究来测试ALK是否是GBM患者的预后生物标志物，并确定新的ALK融合或突变是否参与GBM的发病机制。ALK融合或突变的功能影响将使用科学相关的原代GBM细胞系来确定。达纳-法伯癌症研究所脑肿瘤生物库和活组织库提供了大量临床注释的GBM组织和原代细胞系的独特途径，这些组织和原代细胞系将用于进行拟议的实验。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PTN-ALK信号轴在正常中枢神经系统发育和胶质瘤形成过程中的作用机制。具体目的如下：目的一：表征ALK重排并评估ALK作为GBM预后生物标志物的作用。目的二：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。目的三：通过体内ALK的缺失来检测多营养因子-ALK信号轴的破坏是否会改变神经干细胞的生物学特性或对缺氧脑损伤的反应。

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.