IDO1 and Immunotolerance in Glioblastoma

项目总结 这份R01续订提案旨在延长PI过去的生产力，包括35种出版物，因为 2016年，包括3篇临床癌症研究资深作者手稿和2个额外的工作机构 这些研究正在《神经肿瘤学和自然》杂志上进行综述。PI续展申请的科学前提是 延续NIH/NINDS F32(博士后NRSA)、NIH/NINDS K99/R00(途径 独立)和NIH/NINDS R01支持，共同专注于吲哚胺的调查 2，3-双加氧酶1(IDO；IDO1)在胶质母细胞瘤中的表达在所有患者切除的GBM中，IDO表达的比例为90%。 并能有效抑制抗GBM免疫反应。GBM浸润性T细胞促进IDO的表达 细胞，因此是抵抗基于免疫的治疗的关键机制，因为靶向 旨在增强T细胞对基底膜的渗透的方法，反过来将导致IDO的代偿性增加 表达和抑制抗GBM免疫反应。重要的是，肿瘤内的高IDO 表达与GBM患者存活率显著降低和免疫抑制增加相关 调节性T细胞(CD4+CD25+FoxP3+)。由于IDO通常被描述为一种限速酶， 将必需的氨基酸色氨酸(Trp)代谢成下游代谢物犬尿氨酸(Kyn)， 色氨酸的耗尽和/或Kyn的积累一直是IDO抑制 抗GBM免疫反应。然而，我们的工作挑战了这一假说，这在一定程度上是通过 用(I)空表达载体、(Ii)载体重组的IDO缺陷型小鼠GBM细胞的建立 表达野生型IDO，(Iii)或表达非酶活性IDO的载体。当被植入脑内时 同基因小鼠脑内，GBM细胞IDO表达增加，Tregs增加，存活率降低，不依赖于 色氨酸代谢。我们的集体结果表明，GBM细胞IDO代表了一种复杂的 免疫抵抗力推动了针对嵌合体的IDO蛋白分解(IDO-PROTACs)的发展 -与IDO蛋白结合并招募E3泛素连接酶以促进蛋白酶体降解的化合物。 IDO-PROTAC使实验能够解决IDO免疫抑制效应，这些作用不依赖于酶。 因此，我们建议的目的是：(I)研究GBM细胞IDO酶的作用机制 独立免疫抑制和(Ii)设计、创建和优化介导IDO的新型IDO-PROTAC 人肾小球系膜细胞中蛋白质的降解。确定IDO如何抑制联合免疫反应 随着强大的IDO-PROTAC的产生，将解决关于GBM免疫生物学的知识空白，同时还 改善与免疫疗法和IDO-PROTAC联合治疗的GBM患者的治疗结果。

PROJECT SUMMARY This R01 renewal proposal intends to extend the PI's past productivity that includes 35 publications since the year, 2016, including 3 senior-authored manuscripts in Clinical Cancer Research and 2 additional bodies of work that are under review in Neuro-Oncology and Nature. The scientific premise of the PI's renewal application is an extension of continuous NIH/NINDS F32 (Postdoctoral NRSA), NIH/NINDS K99/R00 (Pathway To Independence) and NIH/NINDS R01 support that have collectively focused on the investigation of indoleamine 2,3 dioxygenase 1 (IDO; IDO1) in glioblastoma (GBM). IDO is expressed in >90% of all patient-resected GBM and potently suppresses the anti-GBM immune response. IDO expression is increased by GBM-infiltrating T cells and therefore represents a key mechanism of resistance to immune-based therapies, since targeted approaches that aim to enhance T cell infiltration into GBM, will in-turn, result in a compensatory increase of IDO expression and suppression of the anti-GBM immune response. Importantly, the high intratumoral IDO expression is associated with significantly decreased GBM patient survival and increased immunosuppressive regulatory T cells (CD4+CD25+FoxP3+). Since IDO is canonically characterized as a rate-limiting enzyme that metabolizes the essential amino acid, tryptophan (Trp), into the downstream metabolite, kynurenine (Kyn), the depletion of Trp and/or accumulation of Kyn has been the presumed mechanism of how IDO suppresses the anti-GBM immune response. However, our work has challenged this hypothesis, supported in-part through the creation of IDO-deficient murine GBM cells reconstituted with either (i) empty expression vector, (ii) a vector expressing wild-type IDO, (iii) or a vector expressing enzyme-inactive IDO. When intracranially-engrafted into the syngeneic mouse brain, GBM cell IDO expression increases Tregs and decreases survival independent of tryptophan metabolism. Our collective results indicate that GBM cell IDO represents a complex mechanism of immune resistance that has motivated our development of IDO-proteolysis targeting chimeras (IDO-PROTACs) - compounds that bind to IDO protein and recruit an E3 ubiquitin ligase to facilitate proteasome degradation. IDO-PROTACs enable experiments to address IDO immunosuppressive effects that are enzyme independent. The aims of our proposal are therefore to (i) investigate the mechanism responsible for GBM cell IDO enzyme independent immunosuppression and (ii) design, create and optimize novel IDO-PROTACs that mediate IDO protein degradation in human GBM cells. Determination of how IDO suppresses the immune response combined with the generation of potent IDO-PROTACs will address knowledge gaps about GBM immunobiology while also improving treatment outcomes of GBM patients that are co-treated with immunotherapy and IDO-PROTACs.