Novel Approaches to Modeling and Treating IDH1 Mutant Glioma

项目摘要 异柠檬酸脱氢酶1（IDH 1）基因的热点突变在低级别胶质瘤中高度复发 （LGG）和继发性胶质母细胞瘤（GBM）并且编码突变IDH 1-R132 H酶。这种酶 显示新形态活性，将2-酮戊二酸（2 OG）转化为癌代谢产物（R）-2-羟基戊二酸 （（R）-2HG）。（R）-2HG在IDH 1突变胶质瘤中积累至毫摩尔水平，并通过以下方式促进胶质瘤的发生： 竞争性调节调节神经胶质细胞转化的2 OG依赖性酶。虽然这些 这些发现深刻地改变了我们对这些疾病的分子发病机理的理解， 这些见解还没有转化为新的、有效的神经胶质瘤治疗策略的发展 患者突变IDH酶的直接抑制剂在早期临床前和临床试验中显示出较差的活性。 IDH突变型胶质瘤的研究相对于它们对IDH突变型白血病显示出的强大功效。这些 这些发现强调了临床上需要替代治疗策略来治疗IDH 1突变型胶质瘤。我 假设新的治疗策略可以通过利用IDH 1- R132 H癌基因。我用IDH 1的同基因突变体和野生型进行了化学合成致死性筛选， 神经胶质瘤细胞系，并发现多种嘧啶合成抑制剂优先杀死IDH 1突变细胞。 我的初步数据表明，（R）-2 HG介导的2 OG依赖性支链氨基酸的抑制作用， 转氨酶BCAT 1和BCAT 2损害氮掺入嘧啶生物合成途径。 因此，在目标1中，我将测试BCAT活性降低是否代表潜在的分子机制 突变IDH 1诱导的嘧啶合成抑制剂超敏反应。我将继续评估IDH 1- R132 H突变是一种预测生物标志物，通过测试嘧啶合成抑制剂的反应， 这些试剂对一组IDH 1突变体和野生型胶质瘤神经球系的细胞毒性。最后我 将测试一种新的嘧啶生物合成酶二氢乳清酸脱氢酶的脑渗透抑制剂 在IDH 1突变胶质瘤的异种移植模型中，单独使用DHODH和与放射疗法组合使用DHODH。另一 IDH 1突变型胶质瘤的新的、有效的治疗策略的发展的主要障碍是 缺乏这种疾病的基因工程小鼠（GEM）模型，可用于评估这种疾病， 战略布局在目标2中，我提出建立第一个基于Crispr/Cas9的胶质瘤GEM模型，该模型由突变体驱动， IDH 1.我已经成功地在小鼠中产生了突变的Idh 1驱动的III级间变性星形细胞瘤，我将 优化我的方法来增加GEM模型中星形细胞瘤的转移率。我还将修改我的策略， 产生IDH 1突变体和野生型GBM的同基因GEM模型，可用于未来的体内研究 Idh 1-R132 H致癌基因的合成致死性。如果成功，这些项目将建立以下理由： 用于IDH 1突变型胶质瘤治疗的嘧啶合成抑制剂的临床试验，并产生 这种疾病，以支持未来的测试新设计的治疗策略。

PROJECT SUMMARY Hot-spot mutations in the isocitrate dehydrogenase 1 (IDH1) gene are highly recurrent in lower grade gliomas (LGGs) and secondary glioblastomas (GBMs) and encode the mutant IDH1-R132H enzyme. This enzyme displays neomorphic activity, converting 2-oxoglutarate (2OG) to the oncometabolite (R)-2-hydroxyglutarate ((R)-2HG). (R)-2HG accumulates to millimolar levels in IDH1 mutant gliomas and promotes gliomagenesis by competitively modulating 2OG-dependent enzymes that regulate glial cell transformation. Although these discoveries have profoundly reshaped our understanding of the molecular pathogenesis of these diseases, these insights have not yet translated to the development of new, effective therapeutic strategies for glioma patients. Direct inhibitors of the mutant IDH enzyme have shown poor activity in early preclinical and clinical studies of IDH mutant glioma relative to the robust efficacy they display against IDH mutant leukemias. These findings highlight the clinical need for alternative therapeutic strategies to treat IDH1 mutant gliomas. I hypothesize that novel treatment strategies can be developed by exploiting synthetic lethality with the IDH1- R132H oncogene. I performed a chemical synthetic lethality screen using isogenic IDH1 mutant and wild-type glioma cell lines and found that multiple pyrimidine synthesis inhibitors preferentially killed IDH1 mutant cells. My preliminary data shows that (R)-2HG-mediated inhibition of the 2OG-dependent branched chain amino acid transaminases BCAT1 and BCAT2 impairs nitrogen incorporation into the pyrimidine biosynthesis pathway. Therefore, in Aim 1 I will test whether reduced BCAT activity represents the molecular mechanism underlying mutant IDH1-induced pyrimidine synthesis inhibitor hypersensitivity. I will go on to assess whether the IDH1- R132H mutation is a predictive biomarker for response to pyrimidine synthesis inhibitors by testing the cytotoxicity of these agents against a panel of IDH1 mutant and wild-type glioma neurosphere lines. Finally, I will test a novel brain-penetrant inhibitor of the pyrimidine biosynthesis enzyme dihydroorotate dehydrogenase (DHODH) alone and in combination with radiotherapy in xenograft models of IDH1 mutant glioma. Another major impediment to the development of new, effective therapeutic strategies for IDH1 mutant glioma is a paucity of genetically-engineered mouse (GEM) models of this disease that can be used to evaluate such strategies. In Aim 2 I propose to establish the first Crispr/Cas9-based GEM models of glioma driven by mutant IDH1. I have successfully produced mutant Idh1-driven Grade III anaplastic astrocytomas in mice and I will optimize my approach to increase astrocytoma penetrance in this GEM model. I will also modify my strategy to generate isogenic GEM models of IDH1 mutant and wild-type GBM that can be used for future in vivo studies of synthetic lethality with the Idh1-R132H oncogene. If successful, these projects will establish rationale for clinical testing of pyrimidine synthesis inhibitors for IDH1 mutant glioma therapy and generate GEM models of this disease to support future testing of newly devised treatment strategies.