Mechanisms and therapeutic implications of temozolomide resistance in glioblastoma

项目总结 这份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 麦考德是西北大学神经病理学的临床研究员，他的职业生涯是内科科学家。Dr。 麦考德的长期目标是成为一名专家诊断神经病理学家和独立资助的大脑 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)的耐药性 和TMZ驱动的胶质母细胞瘤(GBM)的过度突变。替莫唑胺(TMZ)的化疗是- 治疗基底膜，暂时延长存活期。然而，肿瘤普遍复发并产生对TMZ的耐药性， 几乎都是致命的。DNA错配修复(MMR)酶的缺陷，最常见的是MSH6，有 在复发的GBM中与TMZ耐药有关。TMZ后复发的GBM的一个子集发展得非常高 肿瘤突变负荷(TMB)，也被称为“高突变”表型，它也与MMR有关 缺陷，如MSH6。然而，MSH6受损和应答中的高突变之间存在明确的因果关系 对TMZ的影响尚未得到实验证明。某些类型的高度突变的癌症出现在 人体已被证明对免疫检查点抑制(ICI)有反应，但在高突变的GBM的临床试验中， ICI的反应参差不齐，原因尚不完全清楚。之前发布的数据来自Dr。 McCord和其他人提出，MSH6损伤和超突变可能是异质性的，只发生在 通过全球基因组分析，似乎发生了超突变的GBM亚克隆。这可能有助于解释 到目前为止，ICI在试验中的影响是不一致的。该提议的中心假设是，MMR缺陷促进了 替莫唑胺存在的高度突变和ICI反应性，但这些亚克隆变异 缺陷导致不同的ICI疗效。检验这一假设的具体目的如下：(1)证明一个 MSH6基因敲除在TMZ诱导的高度突变和TMZ抗性中的作用 在胶质瘤细胞中的实验；(2)证实了MSH6损伤和肿瘤内的异质性。 通过单细胞全基因组测序，在TMZ后患者来源的胶质瘤和TMZ耐药的胶质瘤中发生超突变 患者来源的异种移植(PDX)；(3)评价高突变与非高突变的体内敏感性 肿瘤亚克隆到TMZ和ICI通过创建具有不同比例的每个细胞的原位颅内基底膜 类型，然后同时使用TMZ和ICI进行治疗。麦考德博士的培训计划是为拟议的研究量身定做的， 重点是生物信息学、免疫学和胶质瘤模型方面的指导、研讨会和课程工作 作为解决与新生的内科科学家相关的具体问题。这项奖学金将为 麦考德博士在他职业生涯的关键时刻，将使他最终能够同时参加K级和R级的竞争 资助，也将促进理解为什么GBM对TMZ和ICI疗法都产生耐药性。

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.