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

恶性星形细胞瘤的起源、演变和治疗反应中的 BRAF 突变

• 批准号: 8402661
• 负责人: Charles David James
• 金额: $ 35.22万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-01 至 2017-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8402661
• 关键词: Address; Affect; Allografting; Anaplastic astrocytoma; Apoptosis; Apoptotic; Astrocytes; Astrocytoma; BRAF gene; Basic Science; Brain Neoplasms; CDKN2A gene; Cell division; Cells; Characteristics; Childhood; Childhood Brain Neoplasm; Clinical Research; Complement; Cytotoxic Chemotherapy; Development; Elements; Etiology; Evolution; Gene Mutation; Gene Transfer; Genetic; Genetically Engineered Mouse; Growth; Heterogeneity; Human; Implant; In Vitro; Investigation; Lead; Malignant - descriptor; Malignant Neoplasms; Modeling; Modification; Molecular; Molecular Target; Mus; Mutation; Neoplastic Cell Transformation; Outcome; Parents; Patients; Phase; Process; Production; Proliferating; Radiosurgery; Recurrence; Regulation; Research; Resistance; Rodent; Testing; Treatment outcome; Tumor Tissue; Tumorigenicity; base; brain cell; cell transformation; chemotherapy; conventional therapy; daughter cell; effective therapy; improved; in vivo; insight; interest; metaplastic cell transformation; mouse model; mutant; neoplastic cell; nerve stem cell; novel; novel therapeutics; outcome forecast; pre-clinical research; response; self renewing cell; self-renewal; small hairpin RNA; therapy resistant; tumor; tumor xenograft; tumorigenesis; tumorigenic

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. PUBLIC HEALTH RELEVANCE: Despite the investigation of numerous novel therapeutics and treatment approaches, the outcome for pediatric malignant astrocytoma (MA) patients has seen little improvement over several decades of basic, preclinical, and clinical research. Our research is expected to 1) inform regarding the cellular origin of MA; 2) provide new insight about the molecular mechanisms of neoplastic transformation, especially those involving asymmetric cell division (ACD), that promote brain tumor development; 3) increase our understanding of brain tumor cell subpopulations responsible for therapy resistance and tumor recurrence, and as such our studies will 4) ultimately facilitate the identification of molecular targets for improved MA treatment.

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（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抵抗力，因此是 MA 复发的重要因素。 NSC 经历不对称细胞分裂 (ACD)，以一对一的比例进行自我更新和分化，而 TIC 会增殖和自我更新，但无法生成完全分化的细胞，这表明 ACD 有缺陷。在很大一部分儿科 MA 中发现了 BRAF、BRAFVE 的突变激活形式，以及编码 p16 的 CDKN2A 的伴随纯合缺失。已知 BRAFVE 可以促进增殖，同时抑制正常细胞分化。目前尚不清楚通过增加对称细胞分裂（即减少 ACD）来增加自我更新细胞的产生是否与 BRAFVE 诱导的细胞转化有关。为了弥合我们对 BRAFVE 诱导的转化及其与 ACD 关系的理解上的差距，我们将检查 BRAFVE 在 p16 缺陷型 NSC 以及相应的 p16 缺陷型小鼠和人类来源的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，主要关注肿瘤细胞亚群和治疗引起的 ACD 变化。相关研究将围绕以下具体目标进行。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 NSC 和成熟星形胶质细胞中 MA 肿瘤发生的关系。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 基因转移来抑制人类 NSC 和正常人星形胶质细胞中的 p16 表达并强制 BRAFVE 表达。将在体外和体内表征具有 BRAFVE 表达的修饰 NCS 和 NHA 以及 MA 细胞，其特征与目标 1 中的小鼠模型肿瘤相同。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获得治疗耐药性时的效果，并将利用人类肿瘤异种移植模型和小鼠同种异体移植模型。我们的项目将：1）生成有关 BRAFVE 诱导的 MA 细胞起源的新信息； 2）深入了解导致脑肿瘤发展的肿瘤转化的分子机制； 3) 增加我们对导致治疗耐药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 患者的治疗结果。 公共健康相关性：尽管对许多新疗法和治疗方法进行了研究，但在数十年的基础、临床前和临床研究中，儿科恶性星形细胞瘤 (MA) 患者的结局几乎没有改善。我们的研究预计将 1) 揭示 MA 的细胞起源； 2）提供关于肿瘤转化的分子机制的新见解，特别是涉及不对称细胞分裂（ACD）的分子机制，促进脑肿瘤的发展； 3) 增加我们对导致治疗耐药和肿瘤复发的脑肿瘤细胞亚群的了解，因此我们的研究将 4) 最终促进分子靶标的识别，以改善 MA 治疗。