(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）的患者生存期延长非常罕见，约2%的患者生存期超过5年。大约三分之一的生存期延长的患者具有由异柠檬酸脱氢酶突变控制的更惰性的GBM亚型，但其余的长期存活者具有通常与侵袭性手术、放射（RT）和替莫唑胺（TMZ）治疗后的中位初始无进展生存期（PFS）小于6个月相关的肿瘤。本申请的重点是确定导致这些罕见的非IDH突变型肿瘤患者的初始PFS延长的分子机制。为了研究GBM的治疗效果谱，马约脑肿瘤孢子已经开发了一组43个原发性肿瘤异种移植物，这些移植物来自新诊断的原发性GBM患者。其中3个模型（GBM 5、75和84）来自初始PFS较长的患者，每个模型均表现出对细胞毒性治疗的独特超敏反应。这些结果支持这样的假设，即至少在某些患者中，PFS延长是由于对初始治疗的独特敏感性所致 而不仅仅是从一个通常惰性的肿瘤生物学。在本申请中，将使用下一代测序（NGS）和功能性蛋白质组学工具分析患者肿瘤和异种移植模型，然后操作异种移植模型，以稳健地确定对RT和TMZ治疗的超敏反应机制。在目标1中，将通过全外显子组测序（WES）和mRNAseq分析15个长期存活的患者样品的集合，并且配对的患者/异种移植物样品将通过全基因组测序和表观基因组分析进行更详细的分子景观分析。通过对这些数据的综合分析，并与TCGA中短期存活者的WES/mRNAseq数据进行比较，将产生与长期疾病控制相关的机制假设，并将在后续目标中进行测试。目标2将专门测试GBM 5、75和84异种移植模型中极端治疗敏感性的机制。综合NGS和功能蛋白质组学分析将定义超敏反应的推定机制，然后将使用基因表达的操纵来定义特定途径对治疗反应的重要性。本申请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，这些策略可以显着提高典型GBM肿瘤的治疗效果。因此，在目标3中，我们将使用从目标1和2中获得的见解来设计定制的shRNA文库，然后使用体内shRNA筛选和随后的验证研究来定义可以显著增加放射和/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.