BRAF Mutation in Malignant Astrocytoma Origin, Evolution, and Response to Therapy

描述(申请人提供)：许多儿童脑肿瘤，包括恶性星形细胞瘤(MA)，被认为起源于神经干细胞(NSCs)，由于生长促进基因改变的发生和积累，可能会产生包括肿瘤启动细胞(TICs)在内的各种细胞亚群。抽搐被认为增加了对传统治疗MA的抵抗力，因此是MA复发的重要因素。而神经干细胞经历不对称细胞分裂(ACD)进行自我更新和分化，比例为1：1，而TIC增殖和自我更新，无法产生完全分化的细胞，提示ACD有缺陷。在儿童MA中发现了一种突变的活化形式的BRAF，BRAFVE，以及伴随的编码p16的CDKN2A的纯合子缺失。众所周知，BRAFVE在促进增殖的同时抑制正常的细胞分化。目前尚不清楚BRAFVE诱导的细胞转化是否通过增加对称细胞分裂(即ACD减少)来增加自我更新细胞的产量。为了弥合我们对BRAFVE诱导的转化及其与ACD的关系的理解上的差距，我们将检测BRAFVE在p16缺陷的神经干细胞以及相应的p16缺陷的小鼠和人类来源的星形胶质细胞中的作用。此外，我们还将调查BRAFVE-p16缺陷肿瘤与它们对BRAFVE靶向治疗的适应性之间的关系，我们的主要重点是肿瘤细胞亚群和治疗后ACD的变化。将在以下具体目标范围内开展相关研究。目的1.利用基因工程小鼠模型，研究BRAFVE在神经干细胞和成熟星形胶质细胞中表达对ACD、增殖、分化和存活的影响及其与MA肿瘤发生的关系。目的2.为了补充目标1中的GEMMS研究，我们将通过慢病毒shRNA敲除和BRAFVE基因转移分别抑制p16和BRAFVE在人神经干细胞和正常人星形胶质细胞中的表达。修饰的NCS和NHAS以及表达BRAFVE的MA细胞在体外和体内的特征与AIM中的小鼠模型肿瘤相同。目的3.分别在体外和体内研究BRAFVE肿瘤细胞和肿瘤组织的分子变化、TIC组成和ACD与BRAFVE靶向治疗的反应相关。这项研究将包括比较肿瘤处于治疗反应期以及当它们对治疗产生抵抗力时的效果，并将利用人类肿瘤异种移植和小鼠同种异体移植模型。我们的项目将：1)产生关于BRAFVE诱导MA的细胞起源的新信息；2)提供关于肿瘤转化导致脑肿瘤发展的分子机制的见解；3)提高我们对导致治疗抵抗和肿瘤复发的脑瘤细胞亚群的了解；4)通过这样做，4)最终将改善MA患者的治疗结果。

DESCRIPTION (provided by applicant): Many pediatric brain tumors, including malignant astrocytomas (MA), are thought to originate from neural stem cells (NSCs), which, due to the occurrence and accumulation of growth-promoting gene alterations, may give rise to various cell subpopulations, including tumor-initiating cells (TICs). TICs are considered to have increased resistance to conventional therapy for MA, and consequently are important contributors to MA recurrence. Whereas NSCs undergo asymmetric cell divisions (ACD) to self-renew and differentiate at a one-to-one ratio, TICs proliferate and self-renew, and fail to generate fully differentiated cells, suggestive of defective ACD. A mutant, activated form of BRAF, BRAFVE, and concomitant homozygous deletion of CDKN2A, encoding p16, have been found in a significant fraction of pediatric MA. BRAFVE is known to promote proliferation while suppressing normal cellular differentiation. Whether increased production of self-renewing cells through increasing symmetric cell divisions (i.e., decreased ACD) is manifested in association with BRAFVE induced cell transformation is currently unknown. To bridge the gap in our understanding of BRAFVE-induced transformation, and its relationship with ACD, we will examine effects of BRAFVE in p16 deficient NSCs as well as in corresponding p16 deficient astrocytes of mouse and human origin. In addition, we will investigate relationships between BRAFVE-p16 deficient tumors and their adaptation to BRAFVE targeted therapy, with our primary focus directed to tumor cell subpopulation and ACD changes resulting from treatment. Related research will be performed in the context of the following specific aims. Aim 1. Using genetically engineered mouse models (GEMMs), we will determine effects of BRAFVE expression on ACD, proliferation, differentiation, and survival, and association with MA tumorigenesis in NSCs and mature astrocytes. Aim 2. To complement the GEMMs studies in aim 1, we will suppress p16 expression and force BRAFVE expression in human NSCs and normal human astrocytes, using lentiviral shRNA knockdown and BRAFVE gene transfer, respectively. Modified NCS and NHAs as well as MA cells with BRAFVE expression will be characterized, both in vitro and in vivo, for the same characteristics as for the mouse model tumors in aim 1. Aim 3. Investigate BRAFVE tumor cells and tumor tissues, in vitro and in vivo, respectively, for molecular changes, TIC composition, and ACD in association with response to BRAFVE targeted therapy. This research will include comparison of effects when tumors are in a responsive phase to therapy, as well as when they have acquired resistance to therapy, and will utilize both human tumor xenograft and mouse allograft models. Our project will: 1) generate new information regarding the cellular origin of BRAFVE induced MA; 2) provide insight about the molecular mechanisms of neoplastic transformation resulting in brain tumor development; 3) increase our understanding of brain tumor cell subpopulations that are responsible for therapy resistance and tumor recurrence, and in so doing, 4) will ultimately lead to improved treatment outcomes for MA patients.