mTOR as a Central Regulator of iMCD Pathogenesis and Novel Therapeutic Target

项目概要/摘要： 人类疱疹病毒（HHV）-8阴性，特发性多中心Castleman病（iMCD）是一种致命的血液病， 涉及多克隆淋巴细胞增生和多器官系统功能障碍的疾病。iMCD诊断为 美国每年约有600- 1000人; 35%在5年内死亡。细胞毒性化疗是 66%的siltuximab（难治性iMCD）IL-6阻断难治性患者的唯一选择;复发是 共同没有新药正在开发中。病因，！病理细胞类型和失调信号 路径未知。提高对疾病机制的理解对于确定新的治疗方法是必要的。 我们的初步数据显示，血清血管内皮生长因子（VEGF）的上调，激活 复发期间难治性iMCD患者的CD 8 + T细胞和不受控制的PI 3 K/Akt/mTOR信号传导。中位血清 16例iMCD患者的VEGF水平高于正常上限3倍。蛋白质组学定量 难治性iMCD指数病例（IC）的315项血清分析物发现，VEGF是上调最多的细胞因子 在耀斑。在IC中观察到循环活化的HLA-DR+ CD 8 + T细胞的分数显著增加， 与对照组相比，另一例难治性iMCD病例。磷酸-S6，mTOR活性的读数， 与6个反应性和狼疮性淋巴结相比，IC和2/2个其他iMCD淋巴结增加。此外，长期 在来自IC的T细胞受体（TCR）刺激的CD 8 + T细胞中观察到Akt的磷酸化，而另一个来自IC的T细胞受体刺激的CD 8 + T细胞中观察到Akt的磷酸化。 难治性iMCD病例。重要的是，向IC施用mTOR抑制剂西罗莫司导致完全的免疫抑制。 缓解持续时间比之前的平均缓解持续时间长5倍。另一名患者在临床上 反应持续两个月。我们还鉴定了IC的CABIN 1复合杂合错义突变， 基因，T细胞活化的负调节因子，作为iMCD中T细胞失调的潜在机制基础。 我们假设活化的CD 8 + T细胞中不受控制的PI 3 K/Akt/mTOR信号传导对于 iMCD发病机制中，对TCR介导的T细胞活化的超敏反应是机制基础，西罗莫司 通过抑制mTOR、T细胞活化和VEGF中断iMCD。在目标1中，我们将测试是否存在 在其他难治性iMCD患者中，VEGF、T细胞活化和PI 3 K/Akt/mTOR信号传导上调。在Aim中 2，我们将严格评估难治性iMCD T细胞如何在体外响应TCR刺激。然后，我们将测试 IC的CABIN 1突变是否会导致可能易患TCR超敏反应的功能丧失， 在更多病例中寻找CABIN 1突变。目的3概述了西罗莫司概念研究的机理证明 在一些实施方案中，将PI 3 K/Akt/mTOR施用至难治性iMCD患者以研究体内PI 3 K/Akt/mTOR信号传导并记录功效。 这些研究将通过阐明一种新的失调信号来促进我们对iMCD的理解 途径、细胞类型、细胞因子和基因组改变，并可能导致一种新的治疗模式， iMCD和相关炎症性疾病。使用临床和发现数据集来揭示 现有的药物对于确定95%的罕见疾病的治疗方法至关重要，这些疾病没有FDA批准的治疗方法。

Project Summary/Abstract: Human herpesvirus(HHV)-8-negative, idiopathic multicentric Castleman disease (iMCD) is a deadly hematologic illness involving polyclonal lymphoproliferation and multiple organ system dysfunction. iMCD is diagnosed in approximately 600-1,000 individuals annually in the USA; 35% die within 5 years. Cytotoxic chemotherapies are the only options for the 66% of patients refractory to IL-6 blockade with siltuximab (refractory-iMCD); relapse is common. No new drugs are in development. The etiology,!pathological cell types, and dysregulated signaling pathways are unknown. Improved understanding of disease mechanisms is necessary to identify new treatments. Our preliminary data reveal upregulation of serum vascular endothelial growth factor (VEGF), activated CD8+ T cells, and uncontrolled PI3K/Akt/mTOR signaling in refractory-iMCD patients during flares. Median serum VEGF levels were three-fold above the upper limits of normal in 16 iMCD patients. Proteomic quantification of 315 serum analytes in a refractory-iMCD index case (IC) found that VEGF was the most up-regulated cytokine in flare. A significantly increased fraction of circulating activated HLA-DR+ CD8+ T cells was observed in IC and another refractory-iMCD case compared to controls. Phospho-S6, a read-out of mTOR activity, was dramatically increased in IC and 2/2 other iMCD lymph nodes compared to six reactive and lupus nodes. Moreover, prolonged phosphorylation of Akt was observed in T cell receptor (TCR)-stimulated CD8+ T cells from IC and another refractory-iMCD case. Importantly, administration of the mTOR inhibitor, sirolimus, to IC led to a complete remission lasting five-fold longer than the previous average remission duration. Another patient has had a clinical response lasting two months. We also identified compound heterozygous missense mutations in IC’s CABIN1 gene, a negative regulator of T cell activation, as a potential mechanistic basis for T cell dysregulation in iMCD. We hypothesize that uncontrolled PI3K/Akt/mTOR signaling in activated CD8+ T cells is critical to iMCD pathogenesis, hypersensitivity to TCR-mediated T cell activation is the mechanistic basis, and sirolimus interrupts iMCD by inhibiting mTOR, T cell activation, and VEGF. In Aim 1, we will test whether there is upregulated VEGF, T cell activation, and PI3K/Akt/mTOR signaling in additional refractory-iMCD patients. In Aim 2, we will rigorously evaluate how refractory-iMCD T cells respond to TCR stimulation in vitro. Then, we will test whether the IC’s CABIN1 mutations result in loss-of-function that could predispose to TCR hypersensitivity and search for CABIN1 mutations in more cases. Aim 3 outlines a mechanistic proof of concept study of sirolimus administration to refractory-iMCD patients to investigate PI3K/Akt/mTOR signaling in vivo and document efficacy. The proposed studies will advance our understanding of iMCD by elucidating a novel dysregulated signaling pathway, cell type, cytokine, and genomic alteration in iMCD and may lead to a new treatment paradigm for iMCD and related inflammatory conditions. Using clinical and discovery datasets to uncover a novel use for an existing drug is essential to identify therapies for the 95% of rare diseases with no FDA-approved treatments.