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).

项目摘要 毛囊星形细胞瘤（PA）和散布的散布瘦脑膜神经元肿瘤（DLGTS）为两个 小儿患者常见的脑癌类型。 PA是低级神经胶质瘤，通常以 非浸润肿瘤肿块，但它们的解剖位置可能会带来深远的后果，并带有符号 从压力头，颅神经缺陷，共济失调，视力丧失，糖尿病和早熟 青春期。手术是这些患者的选择治疗，尽管根本切除并不总是可能的，但 在这些情况下，经常用急性和长期进行调整的放射和/或化学疗法 年轻患者可能会使人衰弱的毒性。实际上，尽管大多数患者都有良好 手术切除后的长期生存预后，约有50％的患者患有 由于复发或与治疗相关的副作用引起的发病率，使PA成为具有更好需求的疾病 治疗选择。 DLGT虽然不及PAS，但由于 弥漫性瘦素浸润，排除手术干预，导致死亡率较高 接近80％。 到目前为止，最常见的遗传事件 - 在近70％的PA和DLTG病例中观察到A是A 染色体7上的复发串联重复。由于该重排，N末端 KIAA1549的一部分与包括激酶结构域在内的BRAF的C端部分融合在一起。 BRAF的N末端调节域的丢失反过来导致构成二聚化和下游 以不依赖Ras的方式传导。生成由人类癌症的精确小鼠模型 复杂的染色体重排，我们的实验室最近开发了一种基于CRISPR的新型 在体外，更重要的是在体外诱导特定的染色体重排的方法。 CRISPR-CAS9系统非常适合体内基因组编辑，因为它仅需要共表达 CAS9和适当设计的RNA分子（SGRNA）引导细菌核酸内切酶Cas9至 所需的切割站点。我们开发的方法基于Cas9和2的简单表达 SGRNA旨在在所需的断点上清除。作为概念的证明，我们已经证明了 通过生成EML4-Alk融合驱动肺的新型小鼠模型，使该策略的可行性 腺癌和BCAN-NTRK1融合驱动的脑癌。在这些成功的鼓励下，我们提出了 使用体内染色体工程来建模KIAA1549：小鼠大脑中的BRAF重排（AIM 1）。我们已经获得了大量的初步数据，这些数据证明了这种方法的可行性。 KIAA1549的离体生成：成年神经干细胞中的BRAF融合会在原位上产生肿瘤 注射具有人类DLTG的特征。忠实地概括PA病理学的努力也将是 在早期发育阶段使用新颖的体内CRISPR-CAS9建模进行了探索。我们将使用我们的模型 小儿神经胶质瘤研究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A的治疗潜力 新型BRAF抑制剂（AIM 3）。