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

描述（由申请人提供）：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，可能产生各种细胞亚群，包括肿瘤启动细胞（tic）。tic被认为增加了对MA常规治疗的耐药性，因此是MA复发的重要因素。NSCs通过非对称细胞分裂（ACD）以一对一的比例进行自我更新和分化，而tic增殖和自我更新，不能产生完全分化的细胞，提示ACD缺陷。BRAF、BRAFVE和编码p16的CDKN2A的纯合子缺失在很大一部分儿童MA中被发现。已知BRAFVE可促进细胞增殖，同时抑制正常细胞分化。是否通过增加对称细胞分裂增加自我更新细胞的产生（即ACD降低）与BRAFVE诱导的细胞转化有关目前尚不清楚。为了弥合我们对BRAFVE诱导的转化及其与ACD关系的理解差距，我们将研究BRAFVE在p16缺陷的NSCs以及相应的p16缺陷的小鼠和人类星形胶质细胞中的作用。此外，我们将研究BRAFVE-p16缺陷肿瘤与其对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引起的ACD变化。相关研究将在以下具体目标的背景下进行。目的1。使用基因工程小鼠模型（GEMMs），我们将确定BRAFVE表达对NSCs和成熟星形胶质细胞中ACD、增殖、分化和存活的影响，以及与MA肿瘤发生的关系。目标2。为了补充aim 1中的GEMMs研究，我们将分别使用慢病毒shRNA敲低和BRAFVE基因转移来抑制人类NSCs和正常人类星形胶质细胞中p16的表达和强迫BRAFVE的表达。修饰后的NCS和NHAs以及表达BRAFVE的MA细胞在体外和体内均具有与目的1小鼠模型肿瘤相同的特征。目标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靶向治疗反应的关系。这项研究将包括肿瘤处于治疗反应期和对治疗产生耐药性时的效果比较，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)深入了解肿瘤转化导致脑肿瘤发展的分子机制；3)增加我们对导致治疗抵抗和肿瘤复发的脑肿瘤细胞亚群的了解，这样做，4)最终将改善MA患者的治疗效果。

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.