Targeting IDH mutations to improve seizure control in glioma patients

项目摘要 弥漫浸润性胶质瘤是成人最常见的原发性脑肿瘤。大多数神经胶质瘤患者 在他们的疾病过程中至少有一次癫痫发作，超过30%的人患有反复癫痫发作，称为 肿瘤相关癫痫（TAE）。目前TAE的一线治疗是左乙拉西坦（LEV）（又名：Keppra®）， 但超过50%的患者无法控制癫痫发作这样的患者需要更强大的二线 抗癫痫药物往往有较大的副作用。TAE在世界卫生组织中更常见 (WHO)II-III级胶质瘤比IV级胶质母细胞瘤多，但原因尚不清楚。绝大多数 的II-III级胶质瘤含有异柠檬酸脱氢酶1和2（统称为“IDHmut”）的突变，其导致 大量D-2-羟基戊二酸（D2 HG）的产生和释放。D2 HG承担了大量的 结构上与谷氨酸相似，谷氨酸是一种与N-甲基-D-天冬氨酸受体结合的兴奋性神经递质 （NMDAR）对神经元的作用。我们的数据表明，D2 HG增加体外神经元膜去极化， 神经元网络活动，并且这可以被NMDA受体（NMDAR）拮抗剂完全阻断。 我们还发现，与IDHwt胶质瘤相比，IDHmut胶质瘤增加了移植小鼠的癫痫发作活性， 这通过用IDHmut酶抑制剂处理而大大降低。最后，我们发现IDHmut胶质瘤是 与IDHwt神经胶质瘤相比更容易引起癫痫发作。这是第一个直接的证据， IDHmut和癫痫发作之间的联系;因此，我们的假设是，D2 HG有助于增加 IDHmut胶质瘤患者癫痫发作的发生率，以及新的靶向治疗策略可以 减少这些患者的癫痫发作。在目标1中，我们将探索D2 HG触发 神经元去极化和增加的神经元网络活性。我们的两个主要假设是：（i）D2 HG直接 刺激NMDA受体;（ii）D2 HG抑制谷氨酸再摄取转运蛋白，通常阻止 突触间隙中谷氨酸的病理性积累。我们将使用膜片钳和多电极阵列 研究D2 HG对体外培养的小鼠皮层神经元电活动的影响，以及对小鼠海马神经元电活动的影响。 脑切片在目标2中，我们将探讨IDHmut胶质瘤对周围非肿瘤组织的影响， 体内，重点关注癫痫特征性变化，包括神经元丢失，NMDAR下调， 氧化应激、炎症、海马损伤和改变的小鼠行为。结果将在 患者来源的IDHwt和IDHmut胶质瘤。在目标3中，我们将比较两个下一个- 第二代IDHmut抑制剂AG-120和AG-881，以及美金刚胺，一种已经被 用来治疗老年痴呆症这些药物中的每一种都将作为单药治疗和与 利未记这些目标的成功完成将确立IDHmut的D2 HG产物作为致癫痫剂， IDHmut如何改变神经胶质瘤周围的非肿瘤性神经组织， 临床试验，以确定IDHmut抑制剂和美金刚对这些患者癫痫发作的疗效。

PROJECT SUMMARY Diffusely infiltrative glioma is the most common primary brain tumor in adults. Most glioma patients experience at least one seizure during the course of their disease, and over 30% suffer from repeated seizures, known as tumor-associated epilepsy (TAE). Current front-line treatment for TAE is levetiracetam (LEV) (a.k.a. Keppra®), but this fails to control seizures in over 50% of patients. Such patients then require more powerful second-line antiepileptic drugs that often have greater side effects. TAE is more common in World Health Organization (WHO) grade II-III gliomas than in grade IV glioblastomas, but the reason for this is not clear. The vast majority of grade II-III gliomas contain mutations in isocitrate dehydrogenases 1 and 2 (collectively “IDHmut”), which lead to the production and release of large amounts of D-2-hydroxyglutarate (D2HG). D2HG bears a great deal of structural similarity to glutamate, an excitatory neurotransmitter that binds to N-methyl-D-aspartate receptor (NMDAR) on neurons. Our data show that D2HG increases in vitro neuronal membrane depolarization and neuronal network activity, and that this can be completely blocked by an NMDA receptor (NMDAR) antagonist. We also found that IDHmut glioma increases seizure activity in engrafted mice compared to IDHwt glioma, and that this is greatly reduced by treatment with IDHmut enzyme inhibitor. Finally, we found that IDHmut gliomas are much more likely to cause seizures compared to IDHwt gliomas. This is the first direct evidence of a mechanistic link between IDHmut and seizures; therefore, our hypothesis is that D2HG contributes to an increased incidence of seizures in patients with IDHmut gliomas, and that new targeted therapeutic strategies can decrease seizures in these patients. In Aim 1, we will explore the mechanisms by which D2HG triggers neuronal depolarization and increased neuronal network activity. Our two main hypotheses are: (i) D2HG directly stimulates NMDA receptors; (ii) D2HG inhibits glutamate reuptake transporters that normally prevent the pathologic accumulation of glutamate in the synaptic cleft. We will use patch clamping and multi-electrode arrays to study the effects of D2HG on the electrical activity of cultured mouse cortical neurons, as well as on mouse brain slices. In Aim 2, we will explore the effects of IDHmut glioma on the surrounding nonneoplastic tissue in vivo, focusing on changes that are characteristic of epilepsy, including neuronal loss, NMDAR downregulation, oxidative stress, inflammation, hippocampal damage, and altered mouse behavior. Results will be validated in patient-derived IDHwt and IDHmut gliomas. In Aim 3, we will compare the anti-seizure effects of two next- generation IDHmut inhibitors, AG-120 and AG-881, as well as memantine, an NMDAR antagonist that is already used to treat Alzheimer’s Disease. Each of these drugs will be tested as monotherapy and in combination with LEV. Successful completion of these Aims will establish the D2HG product of IDHmut as an epileptogenic agent, will shed more light on how IDHmut alters the nonneoplastic neural tissues surrounding glioma, and will foster clinical trials to determine the efficacy of IDHmut inhibitors, and memantine, against seizures in these patients.