Modeling BRAF-fusion driven pediatric brain tumors in the mouse

模拟 BRAF 融合驱动的小鼠小儿脑肿瘤

• 批准号: 10672917
• 负责人: ROBERT I BENEZRA
• 金额: $ 61.21万
• 依托单位: SLOAN-KETTERING INST CAN RESEARCH
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-06-01 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10672917
• 关键词: Acute; Adjuvant; Adult; Anatomy; Animals; Ataxia; BRAF gene; Biochemical; Blindness; Brain; C-terminal; CRISPR/Cas technology; Cells; Central Nervous System Neoplasms; Chemotherapy and/or radiation; Child; Childhood Brain Neoplasm; Childhood Glioma; Chimeric Proteins; Chromosomal Rearrangement; Chromosome 7; Chromosomes; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Cranial Nerves; Data; Defect; Development; Diabetes Mellitus; Diffuse; Dimerization; Disease; Disease model; Drug Design; Engineering; Event; Excision; Frequencies; Gene Duplication; Gene Fusion; Generations; Genes; Genetic; Genetic Screening; Glioma; Goals; Grant; Headache; Human; Human Characteristics; In Vitro; Infiltration; Injections; Laboratories; Leptomeninges; Location; Lung Adenocarcinoma; Malignant Neoplasms; Malignant neoplasm of brain; Methods; Modeling; Molecular; Morbidity - disease rate; Mus; N-terminal; NTRK1 gene; Neonatal; Neurological observations; Normal Cell; Oncogenes; Oncogenic; Oncoproteins; Operative Surgical Procedures; Pathology; Patients; Pharmacology; Phosphotransferases; Pilocytic Astrocytoma; Pre-Clinical Model; Precocious Puberty; Prognosis; Proteins; Protocols documentation; RNA; Recurrence; Research Personnel; Resistance; Series; Signal Transduction; Site; Symptoms; System; Techniques; Testing; Therapeutic; Toxic effect; Tumor Promotion; Visual Acuity; design; dimer; dosage; efficacy testing; endonuclease; experimental study; fetal; genome editing; human cancer mouse model; implantation; in vivo; inhibitor; insight; interest; metaplastic cell transformation; monomer; mortality; mouse model; mutant; nerve stem cell; new therapeutic target; novel; novel therapeutic intervention; pediatric patients; preclinical study; pressure; rapid growth; side effect; small molecule; small molecule inhibitor; somatic cell gene editing; success; targeted treatment; therapeutic evaluation; treatment choice; tumor; tumor initiation; tumor progression; tumorigenesis

PROJECT SUMMARY Pilocytic astrocytomas (PAs) and disseminated diffuse leptomeningeal glioneuronal tumors (DLGTs) are two types of brain cancer common among pediatric patients. PAs are low grade gliomas, generally presenting as non-infiltrating tumor masses, but their anatomical location can have profound consequences, with symptoms ranging from pressure headaches, cranial nerve defects, ataxia, loss of visual acuity, diabetes, and precocious puberty. Surgery is the treatment of choice for these patients, although radical resection is not always possible, In these cases, adjuvant radiation and/or chemotherapy is often administered with acute and long-term toxicities that can be debilitating in young patients. In fact, although the majority of patients have a good prognosis in terms of long-term survival following surgical resection, approximately 50% of patients suffer from morbidity due to recurrence or therapy-related side effects, making PA a disease with an unmet need for better therapeutic options. DLGTs, although less common than PAs, are even more challenging clinically due to diffuse leptomenigial infiltration which precludes surgical intervention, leading to higher mortality rates approaching 80%. By far the most common genetic event—observed in nearly 70% of cases of both PA and DLTG—is a recurrent tandem duplication on chromosome 7. As a consequence of this rearrangement, the N-terminal portion of KIAA1549 becomes fused to the C-terminal portion of BRAF, which includes the kinase domain. Loss of BRAF’s N-terminal regulatory domain in turn, results in constitutive dimerization and downstream signaling in a RAS-independent manner. To generate an accurate mouse model of human cancer driven by complex chromosomal rearrangements, our laboratory has recently developed a novel CRISPR-based approach to induce specific chromosomal rearrangements in vitro and, more importantly, in vivo . The CRISPR-Cas9 system is ideally suited for in vivo genome editing because it only requires co-expression of Cas9 and an appropriately designed RNA molecule (sgRNA) to guide the bacterial endonuclease Cas9 to the desired cut site. The method we have developed is based on the simultaneous expression of Cas9 and 2 sgRNAs designed to cleave at the desired breakpoints. As a proof of concept, we have demonstrated the feasibility of this strategy by generating novel mouse models of EML4-ALK fusion driven lung adenocarcinomas and BCAN-NTRK1 fusion-driven brain cancer. Encouraged by these successes, we propose to use in vivo chromosomal engineering to model the KIAA1549:BRAF rearrangement in the mouse brain (Aim 1). We have already obtained a large body of preliminary data that demonstrate the feasibility of this approach. Ex vivo generation of the KIAA1549:BRAF fusion in adult neural stem cells produces tumors upon orthotopic injection that have characteristics of human DLTG. Efforts to faithfully recapitulate PA pathology will also be explored using novel in vivo CRISPR-Cas9 modeling at early developmental stages. We will use our models of pediatric glioma to investigate the molecular mechanisms through which KIAA159-BRAF promotes tumor initiation and progression (Aim 2). Finally, we will use this model to directly test the therapeutic potential of a novel BRAF inhibitor (Aim 3).

项目概要 毛细胞星形细胞瘤 (PA) 和播散性弥漫性软脑膜胶质神经元肿瘤 (DLGT) 是两种 儿科患者中常见的脑癌类型。 PA 是低级别胶质瘤，通常表现为 非浸润性肿瘤块，但其解剖位置可能会产生深远的影响，并伴有症状 包括压力性头痛、脑神经缺陷、共济失调、视力丧失、糖尿病和性早熟 青春期。手术是这些患者的首选治疗方法，尽管根治性切除并不总是可能的， 在这些情况下，辅助放疗和/或化疗通常与急性和长期的治疗一起进行。 可能使年轻患者衰弱的毒性。事实上，尽管大多数患者的病情良好 就手术切除后的长期生存而言，约 50% 的患者患有以下疾病： 由于复发或治疗相关副作用而导致的发病率，使得 PA 成为一种对更好的治疗需求未得到满足的疾病 治疗选择。 DLGT 虽然不如 PA 常见，但由于以下原因在临床上更具挑战性 弥漫性软脑膜浸润妨碍手术干预，导致死亡率更高 接近80%。 迄今为止，最常见的遗传事件（在近 70% 的 PA 和 DLTG 病例中观察到）是 7 号染色体上的反复串联重复。由于这种重排，N 端 KIAA1549 的部分与 BRAF 的 C 端部分融合，其中包括激酶结构域。 BRAF N 端调控域的丢失进而导致组成型二聚化和下游 以独立于 RAS 的方式发送信号。生成精确的人类癌症小鼠模型 为了解决复杂的染色体重排问题，我们的实验室最近开发了一种基于 CRISPR 的新型 在体外，更重要的是在体内诱导特定染色体重排的方法。这 CRISPR-Cas9系统非常适合体内基因组编辑，因为它只需要共表达 Cas9 和适当设计的 RNA 分子 (sgRNA) 可引导细菌核酸内切酶 Cas9 到达 所需的切割位点。我们开发的方法是基于Cas9和2的同时表达 sgRNA 设计用于在所需断点处进行切割。作为概念证明，我们展示了 通过生成 EML4-ALK 融合驱动肺的新型小鼠模型来验证该策略的可行性 腺癌和 BCAN-NTRK1 融合驱动的脑癌。受到这些成功的鼓舞，我们建议 使用体内染色体工程模拟小鼠大脑中的 KIAA1549:BRAF 重排（Aim 1）。我们已经获得了大量初步数据，证明了这种方法的可行性。 成体神经干细胞中 KIAA1549:BRAF 融合体的离体生成在原位产生肿瘤 具有人DLTG特征的注射液。忠实再现 PA 病理学的努力也将是 在早期发育阶段探索使用新型体内 CRISPR-Cas9 模型。我们将使用我们的模型 儿童胶质瘤研究 KIAA159-BRAF 促进肿瘤的分子机制 启动和进展（目标 2）。最后，我们将使用该模型直接测试药物的治疗潜力 新型 BRAF 抑制剂（目标 3）。