Novel therapeutic intervention of early-stage T1D

AbstractType 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.