CDK9-mediated processive transcription in H3K27M+ diffuse intrinsic pontine glioma

建议书摘要 弥漫性桥脑胶质瘤(DIPGs)是儿童侵袭性脑干肿瘤，目前尚无治疗方法。 可用。DIPGs通常是由组蛋白3基因(H3K27M)的反复突变驱动的。这一替代 对调节招募的组蛋白翻译后修饰(PTM)造成广泛的失调 以及转录机器的启动。过程转录，或RNA POL II的主动机制 跨染色质转录，既依赖于并主动传播染色质状态，如动态 可及性和转录相关的PTM(TPTM)。转录动力学的紊乱 已证实致病在癌症发展中的作用，抑制转录机制是一种有效的 在这些模型中进行治疗。我们最近发现，H3K27M突变激活了 转录延伸，包括CDK9。我们已经证明了对CDK9依赖的抑制 转录延长是治疗DIPG的有效方法，但过程转录在DIPG中的作用 致癌转化尚不清楚。这一提议的总体假设是H3K27M突变 促进依赖CDK9的新生转录，这反过来又有助于建立 致癌染色质状态以及对标准护理放射治疗的适应性反应。使用 CRISPR编辑的模型系统、患者来源的培养以及患者来源的异种移植和 同基因工程小鼠模型，我们将通过1)定义过程中的作用来检验这一假说 转录在H3K27M介导的肿瘤发生中的作用，2)CDK9抑制对过程的影响 转录，以及3)转录诱导在电离辐射反应中的作用。 提案的成功完成将使我们能够全面绘制H3K27M突变的影响图 在新生的转录图景上。这些数据将使我们能够定义一个新的转录框架 了解DIPG的染色质介导的肿瘤发生，并将演示如何利用这一点 转录依赖性可以被用来改善患者从放射治疗中获得的益处。 拟议的职业发展计划利用这些研究提供以下方面的高级培训 严格的假设驱动的研究，转录调控的分子研究，以及代表性的Pre 临床癌症模型。导师团队反映了拥有这两项技能的国家公认的资深科学家 专注于这些研究领域的专业知识，并对我的职业发展做出坚定的承诺。培训 计划概述了我将如何通过授课课程作业、专注的研讨会、 国家会议和导师指导。总的来说，这个培训平台将促进我向 作为基础-翻译研究人员的独立性-长期目标是应用新的染色质-和 基于转录的策略，以改善DIPG患者的预后。

PROPOSAL SUMMARY Diffuse intrinsic pontine gliomas (DIPGs) are aggressive brainstem tumors in children with no curative therapies available. DIPGs are canonically driven by recurrent mutations in the histone 3 gene (H3K27M). This substitution imparts broad dysregulation of the histone post-translational modifications (PTMs) that regulate the recruitment and initiation of transcriptional machinery. Processive transcription, or the mechanics of RNA Pol II as it actively transcribes across chromatin, is both dependent on and actively propagates chromatin states such as dynamic accessibility and transcription-associated PTMs (tPTMs). Disorders of transcription dynamics have demonstrated pathogenic roles in cancer development, and inhibition of transcription machinery is an effective therapy in these models. We have recently shown that the H3K27M mutation activates regulators of transcriptional elongation, including CDK9. We have demonstrated that inhibition of CDK9-dependent transcriptional elongation is an effective therapy in DIPG, but the contribution of processive transcription to DIPG oncogenic transformation is unknown. The overall hypothesis of this proposal is that the H3K27M mutation promotes CDK9-dependent nascent transcription, which in turn contributes to both the establishment of an oncogenic chromatin state as well as the adaptive response to standard-of-care radiation therapy. Using a combination of CRISPR-edited model systems, patient derived cultures, and both patient-derived xenograft and syngeneic engineered mouse models, we will test this hypothesis by 1) defining the role of processive transcription in H3K27M-mediated oncogenesis, 2) determining the impact of CDK9 inhibition on processive transcription, and 3) characterizing the role of transcriptional induction in response to ionizing radiation. Successful completion of the proposal will allow us to comprehensively map the impact of the H3K27M mutation on the nascent transcriptional landscape. This data will enable us to define a novel transcriptional framework for understanding DIPG’s chromatin-mediated oncogenesis, and it will demonstrate how exploiting this transcriptional dependence may be leveraged to improve the patient benefit derived from radiation therapy. The proposed career development plan leverages these studies to provide advanced training in the conduct of rigorous hypothesis-driven research, the molecular study of transcriptional regulation, and representative pre- clinical cancer modeling. The mentorship team reflects nationally-recognized senior scientists who possess both focused expertise in these areas of study as well as a strong commitment to my career development. The training plan outlines how I will refine my expertise through a combination of didactic course work, focused workshops, national meetings, and mentorship guidance. Collectively, this training platform will facilitate my transition to independence as a basic-translational researcher with a long-term goal of applying novel chromatin- and transcription-based strategies to improve patient outcomes in DIPG.