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 细胞被破坏 免疫细胞浸润胰岛（即胰岛炎）。大多数人并没有预防或逆转 T1D，而是 治疗重点是通过胰岛素替代疗法减轻症状。同时，T1D 是一种异质性 疾病给定义发病机制并最终开发有效的方法带来了重大挑战 疗法。我们使用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 T1D 免疫发病机制的一种新的翻译调节机制，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， 我们推断出一种药物作用机制，其中 G9a 抑制作用在非肥胖糖尿病 (NOD) 小鼠中是一种 T1D 模型中，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 UNC）将开发新一代 基于机制的 Teff 特异性 T1D 疗法。流行病学证据表明 G9a 是组成型 在 T1D 患者的淋巴细胞中活跃，表明 G9a 相互作用途径与 T1D 致病性有关。使用 ChaC 使用生物素化的 G9a 抑制剂作为探针，我们捕获并鉴定了与 在胰岛高度浸润的 NOD 小鼠和外周血单核细胞中与 G9a 相互作用 T1D 患者的 (PBMC)。因此，我们发现 G9a 抑制或 Ezh2（ChaC 鉴定的 G9a-）的抑制 在 NOD 小鼠中，相互作用蛋白特异性地减少了驱动 β 细胞自身免疫的胰腺浸润 Teff。此外， 对抑制剂治疗的 T1D 小鼠的定量蛋白质组学分析表明，G9a 或 Ezh2 抑制会下调 调节 Teff 致病性的蛋白质的体内表达，特别是那些与葡萄糖相关的蛋白质 糖尿病、胰腺疾病途径和 T 细胞增殖中的代谢。重要的是，受抑制剂影响 代表临床 T1D 病理学的 T1D 蛋白质组在小鼠与人类之间是保守的，表明抑制 Teff 细胞中 G9a 介导的基因特异性翻译在临床上可用于有效治疗 T1D。 由于蛋白质直接介导促进致病性的事件，我们将测试针对 G9a- 的假设 自身反应性苔麸介导的翻译机制提供了预防和/或逆转的有效策略 T1D 进展。在第一阶段，代表疾病进展不同阶段的 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 Teff 特异性，并 测量抑制剂毒性，(2) 确定 G9a 或/和 Ezh2 抑制的特异性和长期疗效 T1D 早期阶段的 NOD 小鼠，以及 (3) 对于人类临床验证，我们将通过蛋白质组学确定 探讨抑制剂对 T1D 患者 PBMC 离体培养物的影响。我们的机械发现 G9a 和 Ezh2 调节驱动 Teff 介导的 b 细胞自身免疫的蛋白质翻译，这提供了一种新的方法 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.