Novel therapeutic intervention of early-stage T1D

摘要 1型糖尿病(T1D)是一种慢性自身免疫性疾病，分泌胰岛素的β细胞被 渗入胰岛的免疫细胞(即，胰岛炎症)。与其阻止或逆转T1D，大多数 治疗的重点是通过胰岛素替代疗法缓解症状。同时，T1D是一个异质的 这种疾病对定义发病机制并最终发展为有效的疾病构成了重大挑战 治疗学。使用我们的基于染色质活性的化学蛋白质组学(CHAC)来剖析T1D异质性 发现了一种新的T1D免疫发病的翻译调控机制，其中G9a，a 组蛋白甲基转移酶非典型地激活一组T1D驱动蛋白的翻译。此外， 我们已经推导出一种药物作用机制，在非肥胖糖尿病(NOD)小鼠中，G9a抑制T1D 模型通过特异性抑制T1D相关蛋白的翻译减轻β细胞自身免疫 致病效应T细胞(T细胞)。因此，我们(TransChroMix和UNC)将开发新一代 以机制为基础的、特效的T1D疗法。流行病学证据表明，G9a是结构性的 在T1D患者的淋巴细胞中活性，暗示G9a在T1D致病中的相互作用途径。使用字符 以生物素化的G9a抑制剂为探针，我们捕获并鉴定了与 高浸润性胰岛NOD小鼠和外周血单核细胞与G9a的相互作用 (PBMCs)。因此，我们发现G9a抑制或抑制Ezh2，一种Chac-鉴定的G9a- 在NOD小鼠中，相互作用特异性地降低了驱动β细胞自身免疫的胰腺浸润性T细胞。此外， 对抑制剂处理的T1D小鼠的定量蛋白质组学分析表明，G9a或Ezh2抑制下调了T1D小鼠的基因表达 调节TEF致病力的蛋白质，特别是与葡萄糖相关的蛋白质在体内的表达 糖尿病中的代谢、胰腺疾病途径和T细胞增殖。重要的是，受影响的抑制物 代表临床T1D病理的T1D蛋白质组从鼠到人是保守的，表明抑制 G9a介导的基因特异性翻译在临床上可用于T1D的有效治疗。 由于蛋白质直接介导促进致病性的事件，我们将检验靶向G9a- 自身反应性TEF中的中介翻译机制为预防和/或逆转提供了一种有效的策略 T1D进展。在第一阶段，代表疾病发展不同阶段的NOD小鼠将接受 抑制剂，我们将(1)进行研究，以验证G9a或Ezh2抑制剂的体内Tef特异性，并 测定抑制剂的毒性，(2)确定G9a或/和Ezh2抑制的特异性和长期疗效 NOD小鼠在T1D的早期阶段，以及(3)对于人类临床验证，我们将通过蛋白质组学来确定 探讨T1D患者外周血单核细胞体外培养的抑制作用。我们的机械发现 G9a和Ezh2调节驱动T细胞介导的b细胞自身免疫的蛋白的翻译提供了一个新的 治疗T1D的途径。

Abstract Type 1 diabetes (T1D) is a chronic autoimmune disease in which insulin-secreting β-cells are destroyed by immune cells that infiltrate the pancreatic islets (i.e., insulitis). Instead of preventing or reversing T1D, most treatments focus on alleviating symptoms with insulin-replacement therapy. Meanwhile, T1D is a heterogeneous disease that poses significant challenges to define mechanisms of pathogenesis and ultimately develop effective therapeutics. Using our chromatin-activity-based chemoproteomics (ChaC) to dissect T1D heterogeneity we have discovered a novel translational regulatory mechanism of T1D immunopathogenesis wherein G9a, a histone methyltransferase, noncanonically activates the translation of a battery of T1D-driving proteins. Further, we have deduced a mechanism of drug action wherein G9a inhibition, in nonobese diabetic (NOD) mice, a T1D model, mitigated β cell autoimmunity by specifcially suppressing the translation of T1D-related proteins in pathogenic effector T cells (Teff). Thus, we (TransChromix and UNC) will develop a new generation of mechanism-based, Teff-specific T1D therapeutics. Epidemiologic evidence showed that G9a is constitutively active in lymphocytes from T1D patients, implicating G9a-interacting pathways in T1D pathogenicity. Using ChaC with a biotinylated G9a inhibitor as a probe we captured and identified the same translation regulators that interact with G9a in both the NOD mice with highly infiltrated islets and in peripheral blood mononuclear cells (PBMCs) of T1D patients. Accordingly, we found that G9a inhibition or inhibition of Ezh2, a ChaC-identified G9a- interactor, in NOD mice, specifically reduced pancreas-infiltrating Teff that drive β cell autoimmunity. Further, quantitative proteomic analysis of inhibitor treated T1D mice revealed that G9a or Ezh2 inhibition downregulates in vivo expression of proteins regulating Teff pathogenicity, particularly those proteins related to glucose metabolism in diabetes, pancreatic disease pathway, and T cell proliferation. Importantly, the inhibitor-affected T1D proteome that represents clinical T1D pathology is mouse-to-human conserved, indicating that suppressing G9a-mediated, gene-specific translation in Teff cells is clinically practical for effective therapeutics of T1D. Because proteins directly mediate events promoting pathogenicity, we will test the hypothesis that targeting G9a- mediated translational mechanisms in autoreactive Teff provides an effective strategy to prevent and/or reverse T1D progression. In Phase I, NOD mice representing varying stages of disease progression will be treated with inhibitors, and we will (1) conduct studies to validate the in vivo Teff specificity of G9a or Ezh2 inhibitors, and to measure inhibitor toxicity, (2) determine the specificity and long-term efficacy of G9a or/and Ezh2 inhibition on NOD mice at early stages of T1D, and (3) for the human clinical validation we will determine by proteomic approaches the inhibitor effects on ex vivo cultures of the PBMCs from T1D patients. Our mechanistic discovery that G9a and Ezh2 regulate translation of proteins driving Teff-mediated b cell autoimmunity provides a new approach to treat T1D.