Differential resistance mechanisms to monofunctional vs bifunctional alkylating agents in glioma

项目摘要 对烷化剂耐药，包括单功能替莫唑胺（TMZ）和双功能CCNU， 仍然是改善神经胶质瘤患者预后的主要障碍。之间的一个重要区别 这些疗法是单官能烷基化剂形成几种类型的烷基化核苷酸，而双官能烷基化剂形成几种类型的烷基化核苷酸， 烷基化剂形成高毒性链间交联（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耐药模式， 对结果的独立影响，可用于治疗获益。对两者的抵抗 药物可以由甲基鸟嘌呤甲基转移酶（MGMT）蛋白的表达产生。TMZ抗性 错配修复缺陷（MMR-d）也可能导致。对环己亚硝脲的抗药性尚未得到系统的控制。 其特征在于，其涉及几种DNA损伤修复（DDR）途径的成员。我们将延续以往的 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途径来开展工作 基因组和功能基因组技术，以测试以下假设： 目的1：通过MMR-d检测TMZ耐药可预测CCNU敏感性。我们最近的研究表明，MMR-d 不会引起CCNU耐药。使用同基因和患者衍生模型，我们将确定哪种TMZ 耐药机制也导致对CCNU的耐药性，并评估在CCNU治疗后突变特征的变化。 CCNU治疗。然后，我们将使用竞争测定和条形码测定来测试以下假设： 在某些情况下，TMZ和CCNU的组合可以提供优于单一疗法的治疗益处。 目的2：参与CCNU诱导的ICL的修复蛋白决定对CCNU的敏感性。我们将测试 已知有助于ICL修复的基因的转录激活对CCNU抗性的影响。然后我们将 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，我们将完成 CCNU治疗前后神经胶质瘤的基因组表征，以表征突变和SV谱 以及CCNU处理对基因组不稳定性的影响。这项提案将使 对单官能和双官能烷化剂的耐药性差异可能在临床上 可用于改善神经胶质瘤患者的预后。 在布罗德研究所和达纳法伯癌症研究所的培训将为西蒙娜达林博士提供一个 教员，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.