Differential resistance mechanisms to monofunctional vs bifunctional alkylating agents in glioma

项目摘要 对烷化剂的抗性，包括单官能团替莫唑胺(TMZ)和双官能团CCNU， 仍然是改善胶质瘤患者预后的主要障碍。两者之间的一个重要区别 这些疗法是单官能化烷基化形成几种类型的烷基化核苷酸，而双官能化核苷酸 烷化剂形成剧毒的链间交联剂(ICL)。我们假设CCNU和TMZ杀死胶质瘤 细胞通过不同的机制，在某些情况下导致不同的抗性和 对结果的独立影响，可用于治疗效益。对两者都有抵触 药物可由甲基鸟嘌呤甲基转移酶(MGMT)蛋白的表达产生。TMZ电阻 也可由错配修复缺陷(MMR-d)引起。对CCNU的耐药性尚未被系统地 具有特征性，但涉及几个DNA损伤修复(DDR)途径的成员。我们将延长之前的 通过使用各种具有代表性的模型和最先进的技术系统地探索DDR途径 基因组和功能基因组技术，以检验以下假设： 目的1：通过MMR-d产生的TMZ耐药预测对CCNU的敏感性。我们最近的研究表明，MMR-d 不会引起CCNU耐药性。使用同基因和患者衍生模型，我们将确定哪个TMZ 耐药机制也会导致对CCNU的耐药性，并评估在 CCNU治疗。然后我们将使用竞争分析和条形码分析来检验这一假设 在某些情况下，TMZ和CCNU的联合治疗可能比单一治疗更有疗效。 目的2：修复蛋白参与CCNU诱导的ICL，决定对CCNU的敏感性。我们将测试 已知有助于ICL修复的基因转录激活对CCNU耐药性的影响。到时候我们会的 确定这些蛋白质的化学抑制剂是否可以提高CCNU的疗效。最后，我们将表演完整的 CCNU治疗前后脑胶质瘤的基因组特征：突变和SV图谱 CCNU抗性以及CCNU处理对基因组不稳定性的影响。这项建议将使 浅谈临床上可能出现的单官能团烷基化药物和双官能团烷基化药物的耐药性差异 可用于改善神经胶质瘤患者的预后。 布罗德研究所和达纳·法伯癌症研究所的这项培训将为西蒙娜·达林博士提供 教职员工，包括系统和基因组技术、基因组数据集的计算分析、 算法开发和DDR生物学。她还将在该项目的各个方面指导学生。

Project Summary Resistance to alkylating agents, including monofunctional temozolomide (TMZ) and bifunctional CCNU, remains a major obstacle to improving outcomes for patients with glioma. An important difference between these therapies is that monofunctional alkylators form several types of alkylated nucleotides, while bifunctional alkylators form highly toxic interstrand crosslinks (ICLs). We hypothesize that CCNU and TMZ kill glioma cells through different mechanisms, leading in some instances to different patterns of resistance and independent effects on outcome that may be leveraged for therapeutic benefit. Resistance to both agents can result from expression of the methyl guanine methyl transferase (MGMT) protein. TMZ resistance can also result from mismatch repair deficiency (MMR-d). Resistance to CCNU has not been systematically characterized but involves members of several DNA damage repair (DDR) pathways. We will extend previous work by systematically probing DDR pathways using varied and representative models and state-of-the-art genomic and functional genomic technologies to test the following hypotheses: Aim 1: TMZ resistance via MMR-d predicts sensitivity to CCNU. Our recent work suggests that MMR-d does not cause CCNU resistance. Using isogenic and patient derived models, we will determine which TMZ resistance mechanisms also result in resistance to CCNU and assess changes in mutational signatures after CCNU treatment. We will then use competition assays and barcoding assays to test the hypothesis that the combination of TMZ and CCNU may provide therapeutic benefit over monotherapy in some contexts. Aim 2: Repair proteins that engage CCNU-induced ICLs determine sensitivity to CCNU. We will test the effect of transcriptional activation of genes known to contribute to ICL repair on CCNU resistance. We will then determine if chemical inhibitors of those proteins can improve CCNU efficacy. Finally, we will perform complete genomic characterization of gliomas pre- and post- CCNU treatment to characterize mutational and SV profiles of CCNU resistance as well as the effect of CCNU treatment on genomic instability. This proposal will shed light on differences in resistance to mono- and bi-functional alkylating agents that may be clinically exploitable to improve outcomes for patients with glioma. This training at the Broad Institute and Dana Farber Cancer Institute will provide Dr. Simona Dalin skills of a faculty member, including systematic and genomic techniques, computational analysis of genomic datasets, algorithm development, and DDR biology. She will also mentor students in aspects of the project.