Characterizing TP53 and PPM1D mutations as resistance drivers to radiation therapy in Diffuse Intrinsic Pontine Gliomas

摘要 诊断为弥漫性内在脑桥胶质瘤（DIPG）的儿童面临100%的死亡率。的 目前的护理标准是放射治疗。尽管达到了最初的反应，肿瘤很快表现出 抵抗并再次开始生长。我们在一项全国性试验DIPG-BAT中处于领先地位， 评价了儿童DIPG的常规活检。我们试图利用通过组织获得的优势， DIPG-BAT试验旨在了解DIPG的遗传基础及其对治疗反应的影响。我们 还寻求鉴定足以防止获得辐射抗性的治疗组合。 在目标1中，我们建议分析迄今为止最大的DIPG全基因组集。我们将联合收割机测序 在DIPG-BAT试验中收集的数据来自新诊断患者，除了之前的数据外， 已发表的基因组在目标2中，我们将评估PPM 1D突变在产生辐射抗性中的作用。 疗法我们对BAT活检的初步分析显示，超过50%的DIPG基因组含有突变， 在PPM 1D或TP 53中，这些突变是相互排斥的。PPM 1D的特点是 TP 53的负调节剂，辐射敏感性的关键促进剂。在目标3中，我们将使用两个假设- 基于和公正的方法，以确定与辐射的治疗组合，提高疗效。我们 将利用遗传和药理学方法抑制PPM 1D和MDM 2，并结合放射治疗 在具有TP 53突变的患者来源的DIPG系中。我们还将进行全基因组CRISPR-cas9筛选， 以鉴定其抑制选择性地增加DIPG相关细胞系中的辐射反应的基因。这些 实验将解决至少三个关于DIPG中对放射治疗的抗性的中心问题： 驱动基因组改变的表征和赋予辐射抗性的那些改变的鉴定 治疗，确定p53信号传导的改变如何赋予对辐射的抗性，以及评估 PPM 1D是一个新的治疗靶点。

Abstract Children diagnosed with Diffuse Intrinsic Pontine Gliomas (DIPGs) are faced with a mortality rate of 100%. The current standard of care is radiation therapy. Despite achieving initial responses, tumors quickly exhibit resistance and start to grow again. We have taken leading positions in a national trial, DIPG-BATs, that has evaluated routine biopsy of DIPGs in children. We seek to take advantage of the tissue obtained through the DIPG-BATs trial to understand the genetic underpinnings of DIPG and their impact on therapeutic response. We also seek to identify therapeutic combinations that are sufficient to prevent the acquisition of radiation resistance. In Aim 1, we propose to analyze the largest set of DIPG whole genomes to date. We will combine sequencing data collected on the DIPG-BATs trial with those from newly diagnosed patients, in addition to previously published genomes. In Aim 2, we will evaluate the role of PPM1D mutations in generating resistance to radiation therapy. Our initial analysis of BAT biopsies has revealed that over 50% of DIPG genomes contain mutations in either PPM1D or TP53, and that these mutations are mutually exclusive. PPM1D has been characterized as a negative regulator of TP53, a critical facilitator of radiation sensitivity. In Aim 3, we will use both hypothesis- based and unbiased approaches to identify therapeutic combinations with radiation that increase efficacy. We will utilize genetic and pharmacological methods of inhibiting PPM1D and MDM2 in combination with radiation in patient-derived DIPG lines with TP53 mutations. We will also perform a genome-wide CRISPR-cas9 screen to identify genes whose suppression selectively increase radiation response in DIPG-relevant cell lines. These experiments will address at least three central questions regarding resistance to radiation therapy in DIPG: characterization of driver genomic alterations and identification of those that confer resistance to radiation therapy, determination of how alterations in p53 signaling confer resistance to radiation, and evaluation of PPM1D as a novel therapeutic target.