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

鉴定 H3.3 G34R/V 高级神经胶质瘤中的转录核心调节电路和其他关键转录因子依赖性

• 批准号: 10462271
• 负责人: Jordan Trent Roach
• 金额: $ 4.28万
• 依托单位: UNIVERSITY OF TENNESSEE HEALTH SCI CTR
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-01 至 2027-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10462271
• 关键词: ATAC-seq; Address; Adolescent and Young Adult; Age; Anatomy; Apoptotic; Biological Assay; Biology; Brain; Brain Neoplasms; CRISPR/Cas technology; Cancer Etiology; Cell Line; Cell Proliferation; Cell Survival; Cells; Cerebral cortex; ChIP-seq; Chemotherapy and/or radiation; Child; Childhood; Childhood Brain Neoplasm; Childhood Glioma; Chromatin; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Collaborations; Collection; Complex; DNA Binding; DNA Binding Domain; DNA Methylation; Data; Dependence; Development; Developmental Gene; Disease; Enhancers; Epigenetic Process; Etiology; Excision; Exhibits; Frequencies; Gene Expression Profile; Gene Expression Regulation; Genes; Genetic Transcription; Genomics; Glioma; Histone H3; Individual; Institutes; Interneurons; Investigation; Label; Laboratories; Malignant Childhood Neoplasm; Malignant Neoplasms; Maps; Mediating; Mentors; Modeling; Molecular; Molecular Profiling; Morbidity - disease rate; Mutate; Mutation; Neuraxis; Neurons; Oncogenic; Operative Surgical Procedures; Patients; Pattern; Physiological; Primary Neoplasm; Quantitative Reverse Transcriptase PCR; RNA Interference; Radiation therapy; Recurrence; Regulator Genes; Research; Role; Small Interfering RNA; TdT-Mediated dUTP Nick End Labeling Assay; Techniques; Therapeutic; Training; Western Blotting; Xenograft procedure; base; cancer cell; curative treatments; experience; genome-wide; genomic platform; glioma cell line; human stem cells; improved; knock-down; loss of function; member; mortality; mutant; neoplastic cell; neuro-oncology; neurosurgery; novel; novel therapeutic intervention; novel therapeutics; programs; spatiotemporal; standard of care; stem cell model; stem cells; transcription factor; transcription regulatory network; transcriptome sequencing; tumor; tumorigenesis

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.

项目概要 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 对这些致命的儿童脑肿瘤的调查，积极的手术切除结合传统的 化疗和放射治疗仍然是标准治疗方法。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，仍低于 20%。这 HGG 患者缺乏有效的治疗方案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上用于知情的治疗方法。鉴定 我的导师 Suzanne J. Baker 博士和其他人对儿科 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 HGG 病因学中的关键作用。复发性 Gly34Arg/Val 超过 15% 的大脑皮层 HGG 发生组蛋白 H3.3 (H3.3 G34R/V) 取代 青少年和年轻人。皮质 HGG 子集中这些突变的频率强调了 表观遗传失调的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 发育中的大脑中独特的时空背景下的肿瘤发生仍有待确定。这 该提案将贝克实验室在 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，他们已经识别并功能验证了转录 儿科癌症模型中的核心调节电路（CRC）和其他关键的肿瘤依赖性。经过 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领域，我打算破译复杂的转录失调模式 H3.3 G34R/V HGG 解决目前治疗这种棘手的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 纠正了 HGG 转录失调实验询问的 H3.3 背景。目标1 利用基于染色质的测定和 RNA 干扰来识别转录因子 (TF)，其中包括 转录 CRC 对于维持 H3.3 G34R/V HGG 中的致癌细胞状态至关重要。目标 2 雇用 CRISPR/Cas9 负选择筛选，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 中的其他关键 TF 基因依赖性。关键 TF 基因依赖性的鉴定 H3.3 G34R/V HGG 将增强我们对癌细胞对转录调控依赖性的理解 计划并阐明潜在的可行生物学，以开发新的治疗策略。