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，其中分泌胰岛素的β细胞被糖尿病性疾病破坏。 浸润胰岛的免疫细胞（即，胰岛炎）。大多数T1D患者并没有预防或逆转T1D， 治疗集中于用胰岛素替代疗法减轻症状。同时，T1D是一种异质性的 这种疾病对确定发病机制和最终开发有效的 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， 已经发现了T1D免疫发病机制的新的翻译调节机制，其中G9a， 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，本发明还 我们已经推导出药物作用机制，其中在非肥胖糖尿病（NOD）小鼠中，G9a抑制，T1D 模型，通过特异性抑制T1D相关蛋白的翻译来减轻β细胞自身免疫， 致病性效应T细胞（Teff）。因此，我们（transchromix和zh）将开发新一代的 基于机制的Tef特异性T1D疗法。流行病学证据表明G9 a是组成性的 在T1D患者的淋巴细胞中有活性，暗示T1D致病性中的G9a相互作用途径。使用ChaC 用生物素化的G9a抑制剂作为探针，我们捕获并鉴定了相同的翻译调节子， 在具有高度浸润的胰岛的NOD小鼠和外周血单核细胞中与G9a相互作用 T1D患者的外周血单个核细胞（PBMC）。因此，我们发现G9a抑制或Ezh2（ChaC鉴定的G9a- 在NOD小鼠中，相互作用物特异性地减少了驱动β细胞自身免疫的胰腺浸润性Teff。此外，本发明还 对抑制剂处理的T1D小鼠的定量蛋白质组学分析显示，G9a或Ezh2抑制下调 调节Teff致病性的蛋白质，特别是那些与葡萄糖相关的蛋白质的体内表达 糖尿病代谢、胰腺疾病途径和T细胞增殖。重要的是， 代表临床T1D病理学的T1D蛋白质组在小鼠到人之间是保守的，这表明抑制T1D蛋白质组的表达是可能的。 Teff细胞中G9a介导的基因特异性翻译对于T1D的有效治疗是临床实用的。 由于蛋白质直接介导促进致病性的事件，我们将测试靶向G9a- 自身反应性Teff介导的翻译机制提供了一种有效的策略，以防止和/或逆转 T1D进展。在I期，代表疾病进展的不同阶段的NOD小鼠将用以下药物治疗： 我们将（1）进行研究以验证G9a或Ezh2抑制剂的体内Teff特异性，并 测量抑制剂毒性，（2）确定G9a或/和Ezh2抑制对G9a或/和Ezh2抑制的特异性和长期功效。 NOD小鼠在T1D的早期阶段，和（3）对于人类临床验证，我们将通过蛋白质组学确定 接近对来自T1D患者的PBMC的离体培养物的抑制剂作用。我们的机械发现 G9 α和Ezh2调节驱动Tef介导的B细胞自身免疫的蛋白质的翻译，这提供了一种新的 治疗T1D的方法

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.