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

描述（由申请人提供）：许多儿科脑肿瘤，包括恶性星形细胞瘤（MA），被认为起源于神经干细胞（NSC），由于促生长基因改变的发生和积累，可能产生各种细胞亚群，包括肿瘤起始细胞（TIC）。TIC被认为对MA的常规治疗具有增加的抗性，因此是MA复发的重要促成因素。而NSC经历不对称细胞分裂（ACD）以一比一的比例自我更新和分化，TIC增殖和自我更新，并且不能产生完全分化的细胞，这表明ACD缺陷。在相当一部分儿童MA中发现了BRAF、BRAFVE的突变、激活形式以及编码p16的CDKN 2A的伴随纯合缺失。已知BRAFVE促进增殖，同时抑制正常细胞分化。是否通过增加对称细胞分裂（即，ACD降低）与BRAFVE诱导的细胞转化相关的表现目前尚不清楚。为了弥合我们对BRAFVE诱导的转化及其与ACD关系的理解中的差距，我们将研究BRAFVE在p16缺陷型NSC以及小鼠和人来源的相应p16缺陷型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。将在以下具体目标的背景下进行相关研究。目标1.使用基因工程小鼠模型（GEMM），我们将确定BRAFVE表达对ACD，增殖，分化和存活的影响，以及与神经干细胞和成熟星形胶质细胞中MA肿瘤发生的相关性。目标2.为了补充目标1中的GEMM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NSC和正常人星形胶质细胞中的p16表达并迫使BRAFVE表达。修饰的NCS和NHA以及具有BRAFVE表达的MA细胞将在体外和体内表征与目的1中的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中与BRAFVE靶向治疗反应相关的分子变化、TIC组成和ACD。这项研究将包括比较肿瘤处于对治疗的反应阶段以及对治疗产生耐药性时的效果，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）提供关于导致脑肿瘤发展的肿瘤转化的分子机制的见解; 3）增加我们对负责治疗抗性和肿瘤复发的脑肿瘤细胞亚群的理解，并且这样做，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.