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: 10413181
• 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/10413181
• 关键词: Acute; Adjuvant; Adult; Anatomy; Animals; Ataxia; BRAF gene; Biochemical; Blindness; Brain; C-terminal; CRISPR/Cas technology; Cells; Central Nervous System Neoplasms; 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; Lead; Location; Lung Adenocarcinoma; Malignant Neoplasms; Malignant neoplasm of brain; Methods; Modeling; Molecular; Morbidity - disease rate; Mus; N-terminal; NTRK1 gene; Neonatal; Normal Cell; Oncogenes; Oncogenic; Oncoproteins; Operative Surgical Procedures; Pathology; Patients; Pharmacology; Phosphotransferases; Pilocytic Astrocytoma; Pre-Clinical Model; Precocious Puberty; Prognosis; Proteins; Protocols documentation; RNA; Radiation; Recurrence; Research Personnel; Resistance; Series; Signal Transduction; Site; Symptoms; System; Techniques; Testing; Therapeutic; Toxic effect; Visual Acuity; base; chemotherapy; design; dimer; dosage; efficacy testing; endonuclease; experimental study; fetal; genome editing; human cancer mouse model; implantation; in vivo; inhibitor; insight; interest; metaplastic cell transformation; 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).

项目摘要 毛细胞型星形细胞瘤（PA）和弥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 非浸润性肿瘤肿块，但它们的解剖位置可能会产生严重的后果， 范围从压迫性头痛、颅神经缺陷、共济失调、视力丧失、糖尿病和早熟 青春期手术是这些患者的治疗选择，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化疗一起施用。 可能会使年轻患者虚弱的毒性。事实上，虽然大多数患者都有良好的 就手术切除后的长期生存而言，约50%的患者患有 由于复发或治疗相关的副作用导致的发病率，使PA成为一种需要更好治疗的疾病。 治疗选择DLGT虽然不像PA那么常见，但由于以下原因，在临床上更具挑战性： 弥漫性柔脑膜浸润，妨碍手术干预，导致死亡率较高 接近80% 到目前为止，在近70%的PA和DLTG病例中观察到的最常见的遗传事件是 7号染色体上的反复串联重复。由于这种重排，N-末端 KIAA1549的一部分融合到BRAF的C-末端部分，其包括激酶结构域。 BRAF N末端调节结构域的缺失反过来会导致组成性二聚化和下游 以独立于RAS的方式进行信令。为了生成一个精确的人类癌症小鼠模型， 复杂的染色体重排，我们的实验室最近开发了一种新的基于CRISPR的 在体外，更重要的是，在体内诱导特异性染色体重排的方法。的 CRISPR-Cas9系统理想地适用于体内基因组编辑，因为它仅需要共表达CRISPR-Cas9。 Cas9和适当设计的RNA分子（sgRNA），以将细菌内切核酸酶Cas9引导至细胞。 所需的切割部位。我们开发的方法是基于同时表达Cas9和2 设计为在所需断点处切割的sgRNA。作为概念验证，我们已经证明了 通过生成EML 4-ALK融合驱动肺的新型小鼠模型，证实了该策略的可行性 腺癌和BCAN-NTRK1融合驱动的脑癌。在这些成功的鼓舞下，我们建议 使用体内染色体工程来模拟小鼠脑中的KIAA1549：BRAF重排（Aim 1）。我们已经获得了大量的初步数据，证明了这种方法的可行性。 在成体神经干细胞中离体产生KIAA1549：BRAF融合体在原位移植后产生肿瘤 具有人DLTG特征注射剂。努力忠实地概括PA病理学也将是 在早期发育阶段使用新的体内CRISPR-Cas9建模进行探索。我们将使用我们的模型， 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一种 新的BRAF抑制剂（Aim 3）。