Identifying a transcriptional core regulatory circuitry and other critical transcription factor dependencies in H3.3 G34R/V high-grade glioma

项目摘要 儿童高级别胶质瘤（HGG）是一种毁灭性的中枢神经系统恶性肿瘤。尽管经过数十年的 研究这些致命的儿童脑肿瘤，积极的手术切除结合传统的 化疗和放疗仍然是标准治疗。不幸的是，这种治疗方法 未能改善皮质HGG患者的5年生存率，其持续低于20%。的 HGG患者的治愈性治疗选择的缺乏强调了我们迫切需要进一步了解 这些疾病实体在细胞和分子水平的知情治疗方法。识别 我的导师Suzanne J. Baker博士和其他人强烈认为， 支持表观遗传失调在儿童HGG病因学中的关键作用。复发性Gly 34 Arg/瓦尔 组蛋白H3.3（H3.3 G34 R/V）的取代发生在大脑皮质HGG中的15%以上， 青少年和年轻人。这些突变在皮质HGG亚群中的频率强调了 表观遗传学失调的时空基因调控程序的功能意义 发展然而，H3.3 G34 R/V对转录调控程序的影响有助于 在发育中的脑内的不同时空背景下的肿瘤发生仍有待确定。这 该提案将Baker实验室在HGG功能和机制研究方面的专业知识与 一组合作者的开创性专业知识，他们已经确定并功能验证了转录 核心调节电路（CRC）和其他关键的肿瘤依赖性在儿科癌症模型。通过 将尖端的基因组平台和其他实验技术与临床培训经验相结合 在儿科神经肿瘤学和神经外科学中，我打算破译复杂的转录模式失调， 在H3.3 G34 R/V HGG中，以解决目前治疗这种难治性中枢神经系统的局限性 恶性肿瘤贝克实验室建立了一个新的年龄和解剖学匹配的原发性肿瘤集合， 患者来源的原位异种移植物，以及一系列模拟野生型、突变型和CRISPR的细胞系。 校正H3.3背景，用于HGG中转录失调的实验询问。要求1 利用基于染色质的测定和RNA干扰来鉴定转录因子（TF）， 在H3.3 G34 R/V HGG中，转录CRC对于维持致癌细胞状态是必需的。Aim 2采用 CRISPR/Cas9阴性选择筛选以全面靶向患者来源的TF DNA结合结构域 H3.3 G34 R HGG的细胞系，并扩展到包含转录CRC的核心TF之外，以揭示 H3.3 G34 R HGG中的其他关键TF基因依赖性。关键TF基因依赖性的鉴定 H3.3 G34 R/V HGG将增强我们对癌细胞依赖于转录调控的理解 程序和照亮潜在的可操作的生物学为新的治疗策略的发展。

Project Summary Pediatric high-grade gliomas (HGGs) are devastating central nervous system malignancies. Despite decades of investigation into these lethal childhood brain tumors, aggressive surgical resection combined with conventional chemotherapy and radiation therapy remains the standard of care. Unfortunately, this treatment approach has failed to improve the 5-year survival of patients with cortical HGGs, which continues to be less than 20%. The paucity of curative treatment options for patients with HGGs emphasizes our urgent need to further understand these disease entities at the cellular and molecular level for informed therapeutic approaches. The identification of recurrent histone H3 mutations in pediatric HGGs by my mentor Dr. Suzanne J. Baker and others strongly supports a critical role for epigenetic dysregulation in the etiology of childhood HGGs. Recurrent Gly34Arg/Val substitutions in histone H3.3 (H3.3 G34R/V) occur in more than 15% of cerebral cortex HGGs found in adolescents and young adults. The frequency of these mutations in a subset of cortical HGGs underscores the functional significance of epigenetically dysregulated spatiotemporal gene regulatory programs during development. However, the impact of H3.3 G34R/V on transcriptional regulatory programs contributing to tumorigenesis in a distinct spatiotemporal context within the developing brain remains to be determined. This proposal integrates the expertise of the Baker lab for functional and mechanistic studies in HGG with the pioneering expertise of a group of collaborators who have identified and functionally validated transcriptional core regulatory circuitries (CRC) and other critical tumor dependencies in models of pediatric cancer. By combining cutting-edge genomic platforms and other experimental techniques with clinical training experiences in pediatric neuro-oncology and neurosurgery, I intend to decipher complex transcriptional patterns dysregulated in H3.3 G34R/V HGG to address current limitations in treating this intractable central nervous system malignancy. The Baker lab established a novel collection of age and anatomically matched primary tumors, patient-derived orthotopic xenografts, and a range of cell lines modeling wild-type, mutant, and CRISPR- corrected H3.3 backgrounds for experimental interrogation of transcriptional dysregulation in HGG. Aim 1 leverages chromatin-based assays and RNA interference to identify transcription factors (TF) comprising a transcriptional CRC essential to maintaining an oncogenic cellular state in H3.3 G34R/V HGG. Aim 2 employs CRISPR/Cas9 negative selection screens to comprehensively target TF DNA-binding domains in patient-derived cell lines from H3.3 G34R HGG and expands beyond core TFs comprising a transcriptional CRC to uncover other critical TF gene dependencies in H3.3 G34R HGG. The identification of critical TF gene dependencies in H3.3 G34R/V HGG will enhance our understanding of cancer cell dependency on transcriptional regulatory programs and illuminate potentially actionable biology for the development of novel therapeutic strategies.