Mechanisms and therapeutic implications of temozolomide resistance in glioblastoma

项目概要 该奖学金提案描述了一项为期三年的研究和培训计划，旨在为马修博士做好准备 麦考德是西北大学神经病理学临床研究员，职业是医师兼科学家。博士。 麦考德的长期目标是成为诊断神经病理学家和独立资助的大脑专家 肿瘤研究科学家。该研究计划的重点是更好地了解替莫唑胺 (TMZ) 耐药性 TMZ 驱动的胶质母细胞瘤 (GBM) 超突变。替莫唑胺 (TMZ) 化疗是标准疗法 护理 GBM，并暂时延长生存期。然而，肿瘤普遍会复发并产生 TMZ 耐药性， 并且几乎都是致命的。 DNA 错配修复 (MMR) 酶（最常见的是 Msh6）的缺陷 与复发性 GBM 的 TMZ 耐药性有关。 TMZ 治疗后复发性 GBM 的一个子集发展得极高 肿瘤突变负荷 (TMB)，也称为“超突变”表型，也与 MMR 相关 缺陷，如 Msh6。然而，Msh6 受损与反应过度突变之间存在明确的因果关系 TMZ 尚未经过实验证明。某些类型的高度突变癌症发生在其他地方 身体已被证明对免疫检查点抑制（ICI）有反应，但在超突变 GBM 的临床试验中， ICI 的反应参差不齐，原因尚不完全清楚。先前发表的数据，来自 Dr. McCord 等人认为 Msh6 损伤和超突变可能是异质的，仅发生在 通过全局基因组检测，GBM 亚克隆似乎发生了超突变。这可以帮助解释 迄今为止，ICI 在试验中的效果不一致。该提案的中心假设是 MMR 缺陷促进 存在替莫唑胺和 ICI 反应性的超突变，但这些亚克隆变异 缺陷会导致不同的 ICI 疗效。检验该假设的具体目的如下： (1) 证明 通过 MSH6 基因敲除研究 MSH6 在 TMZ 驱动的超突变和 TMZ 耐药中的致病作用 神经胶质瘤细胞实验； (2) 证明 Msh6 损伤的瘤内异质性和 通过单细胞全基因组测序，在 TMZ 治疗后患者来源的神经胶质瘤和 TMZ 耐药性中进行超突变 患者来源的异种移植物（PDX）； (3) 评估超突变与非超突变的体内敏感性 通过创建每个细胞比例不同的原位颅内 GBM，将肿瘤亚克隆到 TMZ 和 ICI 型，然后用 TMZ 和 ICI 治疗。麦考德博士的培训计划是根据拟议的研究量身定制的， 还提供有关生物信息学、免疫学和神经胶质瘤模型的重点指导、研讨会和课程 解决与新生医师科学家相关的具体问题。该奖学金将为 McCord 博士正处于职业生涯的关键时刻，这将使他最终能够竞争 K 级和 R 级 资金，还将促进对 GBM 为何对 TMZ 和 ICI 治疗产生耐药性的理解。

PROJECT SUMMARY This fellowship proposal describes a three-year research and training plan designed to prepare Dr. Matthew McCord, a clinical fellow in neuropathology at Northwestern University, for a career as a physician-scientist. Dr. McCord's long term goal is to become an expert diagnostic neuropathologist and independently-funded brain tumor research scientist. The research plan is focused on better understanding temozolomide (TMZ) resistance and TMZ-driven hypermutation in glioblastoma (GBM). Chemotherapy with temozolomide (TMZ) is standard-of- care for GBM, and temporarily extends survival. However, tumors universally recur and develop TMZ resistance, and are almost uniformly fatal. Defects in DNA mismatch repair (MMR) enzymes, most commonly Msh6, have been linked to TMZ resistance in recurrent GBM. A subset of post-TMZ recurrent GBMs develop extremely high tumor mutation burden (TMB), also known as a “hypermutated” phenotype, which has also been linked to MMR defects, like Msh6. However, a clear causal relationship between impaired Msh6 and hypermutation in response to TMZ has not yet been experimentally proven. Certain types of hypermutated cancer arising elsewhere in the body have proven responsive to immune checkpoint inhibition (ICI), but in clinical trials of hypermutated GBMs, ICI responsiveness has been uneven, for reasons that are not entirely clear. Previously published data, from Dr. McCord and others, suggest that Msh6 impairment and hypermutation may be heterogeneous, occurring only in subclones of GBM that, via global genomic assays, appear to be hypermutated. This could help explain the inconsistent effects of ICI in trials thus far. The central hypothesis of the proposal is that MMR defects facilitate hypermutation in the presence of temozolomide and ICI responsiveness, but that sub-clonal variation in these defects contributes to variable ICI efficacy. Specific Aims to test this hypothesis are as follows: (1) prove a causative role for MSH6 in TMZ-driven hypermutation and TMZ resistance, through MSH6 gene knockout experiments in glioma cells; (2) demonstrate the intratumoral heterogeneity of Msh6 impairment and hypermutation, via single cell whole genome sequencing, in post-TMZ patient-derived gliomas and TMZ-resistant patient-derived xenografts (PDX); (3) evaluate the in vivo sensitivity of hypermutated versus non-hypermutated tumor subclones to TMZ and ICI by creating orthotopic intracranial GBMs with varying proportions of each cell type, then treating with both TMZ and ICI. The training plan for Dr. McCord is tailored to the proposed research, with focused mentoring, workshops, and coursework on bioinformatics, immunology, and glioma models, as well as addressing specific issues relevant for nascent physician-scientists. This fellowship will provide support for Dr. McCord at a critical juncture in his career, will enable him to eventually compete for both K- and R-level funding, and will also advance understanding of why GBMs develop resistance to both TMZ and ICI therapy.