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。尽管几十年来 儿童致死性脑瘤的研究--侵袭性手术联合常规 化疗和放射治疗仍然是护理的标准。不幸的是，这种治疗方法已经 未能提高皮质HGGs患者的5年生存率，继续低于20%。这个 HGGS患者治疗选择的匮乏凸显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上获得知情的治疗方法。身份识别 我的导师苏珊娜·J·贝克博士和其他人对儿童HGG中复发性组蛋白H3突变的研究 支持表观遗传失调在儿童HGGs病因学中的关键作用。复发性Gly34Arg/Val 组蛋白H3.3(H3.3 G34R/V)的替换发生在超过15%的大脑皮层HGG中 青少年和青壮年。这些突变在皮质HGG的子集中的频率强调了 表观遗传异常时空基因调控程序的功能意义 发展。然而，H3.3 G34R/V对转录调控程序的影响有助于 在发育中的大脑中，肿瘤发生在不同的时空背景下仍有待确定。这 该方案将Baker实验室在HGG中进行功能和机械研究的专业知识与 一组合作者的开创性专业知识，他们已经确定并在功能上验证了转录 儿童癌症模型中的核心调控电路(CRC)和其他关键的肿瘤依赖性。通过 将尖端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，我打算破译失调的复杂转录模式。 在H3.3 G34R/V HGG中解决目前治疗这种顽固中枢神经系统的局限性 恶毒。贝克实验室建立了一个新的年龄和解剖匹配的原发肿瘤集合， 患者来源的原位异种移植，以及一系列模拟野生型、突变型和CRISPR的细胞系- 更正了HGG转录失调的实验询问的H3.3背景。目标1 利用基于染色质的分析和RNA干扰来鉴定转录因子(Tf)，包括 H3.3 G34R/V HGG中维持致癌细胞状态所必需的转录CRC。AIM 2员工 CRISPR/Cas9阴性选择筛选可全面靶向患者来源的Tf DNA结合域 来自H3.3 G34R 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 G34R HGG中其他关键的TF基因依赖性。关键的转铁蛋白基因依赖关系的鉴定 H3.3 G34R/V HGG将增强我们对癌细胞对转录调控的依赖性的理解 计划和阐明潜在的可操作的生物学，以开发新的治疗策略。

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.