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

项目摘要/摘要： 人类疱疹病毒(HHV)-8阴性的特发性多中心Castleman病(IMCD)是一种致命的血液病 涉及多克隆淋巴增殖和多器官系统功能障碍的疾病。IMCD被诊断为 在美国，每年大约有600-1000人死亡；35%的人在5年内死亡。细胞毒性化疗是 对于66%的患者来说，用西尔图昔单抗(难治性-IMCD)阻断IL-6无效的唯一选择是复发 很普通。目前还没有新药在研发中。病因学、病理细胞类型和信号转导失调 路径是未知的。为了找到新的治疗方法，有必要提高对疾病机制的理解。 我们的初步数据显示，激活的血清血管内皮生长因子(VEGF)上调 难治性IMCD患者发作期CD8+T细胞和PI3K/Akt/mTOR信号失控。中位数血清 在16例IMCD患者中，血管内皮生长因子水平是正常上限的三倍。蛋白质组学定量研究 315例难治性IMCD指数病例(IC)的血清分析发现，血管内皮生长因子是上调最多的细胞因子 在照明弹里。外周血中活化的HLA-DR+CD8+T细胞比例显著增加。 与对照组相比，另一例难治性IMCD病例。磷酸化S6，mTOR活性的读数，戏剧性地 与6个反应性和狼疮性淋巴结相比，IC和2/2其他IMCD淋巴结增加。此外，延长了 在T细胞受体(TCR)刺激的CD8+T细胞中观察到Akt的磷酸化 难治性IMCD病例。重要的是，给IC应用mTOR抑制剂西罗莫司导致了完全的 缓解期比以前的平均缓解期长五倍。另一名患者进行了临床检查 回复持续两个月。我们还在IC的CABIN1中发现了复合杂合性错义突变 基因是T细胞激活的负调节因子，是IMCD中T细胞调节失调的潜在机制基础。 我们推测，激活的CD8+T细胞中不受控制的PI3K/Akt/mTOR信号对 IMCD发病机制中，对TCR介导的T细胞活化超敏是其机制基础，而西罗莫司 通过抑制mTOR、T细胞活化和血管内皮生长因子来阻断IMCD。在目标1中，我们将测试是否有 在其他难治性IMCD患者中，上调了VEGF、T细胞激活和PI3K/Akt/mTOR信号转导。在AIM 2、我们将严格评估难治性IMCD T细胞在体外对TCR刺激的反应。然后，我们将测试 IC的CABIN1突变是否导致功能丧失，从而易患TCR超敏反应和 在更多的病例中寻找CABIN1突变。目标3概述了西罗莫司概念研究的机械证明 给难治性IMCD患者用药，研究体内PI3K/Akt/mTOR信号转导并记录疗效。 拟议的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 IMCD的途径、细胞类型、细胞因子和基因组改变，并可能导致新的治疗方案 IMCD及相关炎症状态。使用临床和发现数据集揭示一种新的 现有的药物对于确定治疗95%没有FDA批准的治疗方法的罕见疾病是必不可少的。

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.