Mechanisms and therapeutic implications of temozolomide resistance in glioblastoma

项目摘要 这个奖学金提案描述了一个为期三年的研究和培训计划，旨在培养马修博士 他是西北大学神经病理学的临床研究员。博士 麦考德的长期目标是成为一个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）耐药性 和TMZ驱动的胶质母细胞瘤（GBM）超突变。替莫唑胺（TMZ）化疗是标准的- 照顾GBM，并暂时延长生存期。然而，肿瘤普遍复发并产生TMZ抗性， 几乎都是致命的DNA错配修复（MMR）酶的缺陷，最常见的是Msh 6， 与复发性GBM的TMZ耐药性有关。TMZ后复发性GBM的一个子集发展非常高 肿瘤突变负荷（TMB），也称为“超突变”表型，也与MMR相关 缺陷，如Msh 6。然而，受损的Msh 6和应答中的超突变之间存在明确的因果关系， TMZ还没有被实验证明。某些类型的高度突变的癌症发生在其他地方， 身体已经被证明对免疫检查点抑制（ICI）有反应，但在高度突变的GBM的临床试验中， 由于不完全清楚的原因，伊斯兰合作组织的反应不均衡。此前公布的数据，从博士。 McCord等人的研究表明，Msh 6损伤和超突变可能是异质性的，仅发生在 GBM的亚克隆，通过全局基因组测定，似乎是高度突变的。这可能有助于解释 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缺陷有助于 在替莫唑胺和ICI反应性的存在下，高突变，但这些亚克隆变异， 缺陷导致ICI功效可变。检验这一假设的具体目的如下：（1）证明一个 通过MSH 6基因敲除，MSH 6在TMZ驱动的超突变和TMZ抗性中的致病作用 神经胶质瘤细胞中的实验;（2）证明Msh 6损伤的肿瘤内异质性， 通过单细胞全基因组测序，在TMZ后患者来源的胶质瘤和TMZ耐药胶质瘤中的超突变 患者来源的异种移植物（PDX）;（3）评价超突变与非超突变的异种移植物（PDX）的体内敏感性。 通过创建具有不同比例的每种细胞的原位颅内GBM，将肿瘤亚克隆至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， 同时，还提供生物信息学、免疫学和神经胶质瘤模型方面的重点指导、研讨会和课程 作为解决与新生的医学科学家相关的具体问题。该奖学金将为以下方面提供支持： 博士麦考德在他的职业生涯的关键时刻，将使他最终竞争的K和R级 此外，还将进一步了解为什么GBM对TMZ和ICI治疗产生耐药性。

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.