(PQD3) Mechanisms of prolonged initial disease-free survival in glioblastoma

描述(由申请人提供)：诊断为多形性胶质母细胞瘤(GBM)的患者存活时间延长是极其罕见的，大约2%的患者存活超过5年。在存活时间较长的患者中，约有三分之一的基底膜由异柠檬酸脱氢酶突变控制，但其余长期存活的患者在积极手术、放射治疗(RT)和替莫唑胺(TMZ)治疗后，肿瘤的中位初始无进展生存期(PFS)通常不到6个月。这一应用的重点是确定导致这些罕见的非IDH突变肿瘤患者初始PFS延长的分子机制。为了研究GBM的治疗效果，Mayo脑瘤孢子已经开发了一组43个来自新诊断的原发GBM患者的原发肿瘤异种移植。这些模型中的三个(GBM5、75和84)来自于长时间的PFS患者，每个模型都对细胞毒治疗表现出独特的超敏反应。这些结果支持这样的假设，即至少在一些患者中，延长的PFS是由于对初始治疗的独特敏感性所致。 而不仅仅是来自于普遍懒惰的肿瘤生物学。在这项应用中，患者肿瘤和异种移植模型将使用下一代测序(NGS)和功能蛋白质组学工具进行分析，然后异种移植模型将被操纵，以强有力地确定对RT和TMZ治疗的超敏机制。在目标1中，收集的15个长期存活的患者样本将通过完整外显子序列(WES)和mRNAseq进行分析，配对的患者/异种移植样本将接受更详细的全基因组序列和表观基因组图谱的分子图谱分析。通过对这些数据的综合分析，并与TCGA中短期幸存者的WES/mRNAseq数据进行比较，将产生与长期疾病控制有关的机械性假设，并将在随后的AIMS中进行测试。目的2将在GBM5、75和84异种移植模型中专门测试极端治疗敏感性的机制。整合的NGS和功能蛋白质组学分析将确定超敏反应的可能机制，然后将利用基因表达的操纵来确定特定途径对治疗反应的重要性。这项应用的最终目标是将对与长期疾病控制相关的分子机制的理解转化为新的治疗策略，从而显著提高典型GBM肿瘤的治疗效果。因此，在目标3中，我们将利用从目标1和2获得的见解来设计定制的shRNA文库，然后使用体内shRNA筛选和后续验证研究来确定可以显著提高辐射和/或替莫唑胺疗效的途径靶点。

DESCRIPTION (provided by applicant): Prolonged survival for patients diagnosed with glioblastoma multiforme (GBM) is exceedingly rare with approximately 2% of patients surviving beyond 5 years. Approximately a third of patients with prolonged survival have a more indolent sub-type of GBM governed by mutation of isocitrate dehydrogenase, but the remainder of long survivors has tumors that are typically associated with less than a 6 month median initial progression-free survival (PFS) following aggressive surgery, radiation (RT) and temozolomide (TMZ) therapy. The focus of this application is to define the molecular mechanisms that result in extended initial PFS in these rare patients with non-IDH mutant tumors. To study the spectrum of treatment efficacy in GBM, the Mayo Brain Tumor SPORE has developed a panel of 43 primary tumor xenografts derived from patients with newly diagnosed primary GBM. Three of these models (GBM5, 75 and 84) were derived from patients with long initial PFS, and each demonstrates unique hypersensitivity to cytotoxic therapy. These results support the hypothesis that prolonged PFS, in at least some patients, results from a unique sensitivity to initial therapy and not just from a generally indolent tumor biology. In this application, patient tumors and xenograft models will be analyzed with next generation sequencing (NGS) and functional proteomic tools, and then the xenograft models will be manipulated to robustly define mechanisms of hypersensitivity to RT and TMZ therapies. In Aim 1 a collection of 15 long-surviving patient samples will be analyzed by whole exome seq (WES) and mRNAseq, and paired patient/xenograft samples will be subjected to a more detailed molecular landscape analysis with whole genome seq and epigenomic profiling. Through an integrated analysis of these data and comparison to WES/mRNAseq data from short survivors in TCGA, mechanistic hypotheses linked to prolonged disease control will be generated that will be tested in subsequent aims. Aim 2 specifically will test mechanisms of extreme therapy sensitivity in the GBM5, 75 and 84 xenograft models. Integrated NGS and functional proteomic analyses will define putative mechanisms of hypersensitivity, and manipulation of gene expression then will be used to define the importance of specific pathways on therapy response. The ultimate goal of this application is to translate an understanding of molecular mechanisms associated with prolonged disease control into novel therapeutic strategies that could significantly enhance the efficacy of therapy for typical GBM tumors. Thus, in Aim 3 we will use the insights gained from Aims 1 and 2 to design a custom shRNA library, and then use an in vivo shRNA screen and subsequent validation studies to define pathway targets that can increase significantly the efficacy of radiation and/or temozolomide.