Pre-clinical Evaluation of TYK2 Inhibitors as a Therapy for Type 1 Diabetes

Farooq Syed (Indiana University)

Farooq Syed, Olivia Ballew, Chi0Chun Lee, Jyoti Rana, Angela Castela, Stephane Demine, Maria Ines Alvelos, Kara Orr, Garrick Chang, Staci A. Weaver, Jacqueline Del Carmen Aquino, Kentaro Yamada, Jing Liu, Donalyn Scheuner, Decio L. Eizirik, Carmella Evans-Molina

T1D results from a complex interaction that occurs between the invading immune cells, which release a variety of chemokines and cytokines, and immunogenic signals produced by injured or dying β cells. Multiple lines of evidence suggest a prominent role for type 1 interferons, particularly interferon-α (IFNα), in T1D pathogenesis. A type I IFN-inducible transcriptional signature is present in the blood of children with a high genetic risk of T1D, even prior to the development of T1D-associated autoantibodies, and interferon-stimulated gene signatures are present in biopsies of pancreatic islets from individuals with recent-onset T1D. IFNα has been shown to induce endoplasmic reticulum stress, chemokine production, and HLA class I overexpression in human β-cells – three histological hallmarks of islets in human T1D. The effects of IFNα are mediated via its receptor (IFNAR1) and the protein tyrosine kinases JAK1 and TYK2 that phosphorylate and activate the signal transducers and activators of transcription (STAT1 and STAT2) proteins. JAK inhibitors have shown promise in mouse and in vitro models of T1D and are being tested in a clinical trial in adolescents and adults with recent-onset T1D. Notably, polymorphisms that decrease TYK2 activity are protective against T1D, and TYK2 inhibitors (TYKi) are being tested in other autoimmune conditions such as psoriasis. However, whether strategies to inhibit TYK2 have similar efficacy in models of T1D has not been tested.

We tested two small molecule inhibitors of TYK2 (BMS-986165 and BMS-986202). Both compounds target the pseudokinase Janus homology 2 regulatory domains (JH2) of TYK2, leading to inhibition of receptor-mediated TYK2 activation. Isolated human islets from cadaveric organ donors and EndoC-βH1 cells were pre-incubated with TYKis for 2hrs followed by exposure to IFNα alone or in combination with IL-1β or TNFα for 24hrs or 48hrs. Expression of HLA class I, CXCL10, and MX1 were evaluated by RT-qPCR; STAT phosphorylation was assessed by immunoblot. Apoptosis was evaluated by Hoechst/Propidium Iodide staining. To determine the in vivo efficacy of TYK2 inhibition, we utilized two mouse models: 1) the RIP-LCMV-GP model, which is a transgenic and virally-induced model of T1D, and 2) the NOD mouse model of spontaneous T1D development. RIP-LCMV mice were pre-treated with BMS-986202 for 2 days prior to the LCMV injection (0.5×105 PFU), and NOD mice were treated with BMS-986202 from 6-12 weeks of age. In both models, vehicle and drug-treated mice were monitored for diabetes incidence. Immunostaining for insulitis and β cell mass and single-molecule RNA Fish (smRNA FISH) was performed in pancreatic sections from treated mice. Flow cytometry was used to profile immune cell populations in the blood, pancreatic lymph node (PLN), and spleen.

Summary of Results
Results from the in vitro studies showed that BMS-986165 and BMS-986202 pre-treatment prevent IFNα-induced upregulation of CXCL10, MX1, and HLA-ABC in EndoC- βH1 and human islets at 24hrs and 48hrs (p<0.05). TYK2 inhibition also decreased STAT1/2 phosphorylation and prevented cytokine (i.e. IFNa + IL1-β) -induced apoptosis in dispersed human islets. BMS-986202 reduced diabetes incidence by 80% in RIP-LCMV mice (n=18 vehicle/18 TYK2i; p<0.001). Flow cytometry analysis of blood, spleen, and PLN 3 days after LCMV injection revealed a significant decrease in the percentage of CD11b+F4/80+ macrophages (M1) and CD11b+CD49+ NK cells in the PLN and blood and increased circulating CD11b-CD49+ tolerogenic-NK cells in TYK2i treated RIP-LCMV mice. At day 7 and 14 post LCMV induction, PDI+CD8+T-cells in the blood, spleen, and PLN and PDI+FOXP3+ Treg cells were increased in the spleen of BMS-986202-treated RIP-LCMV mice. Treatment with BMS-986202 resulted in a 44% decrease in diabetes incidence in NOD mice (n=32 vehicle/ 34 TYK2i; p=0.0075) and significantly reduced insulitis (p<0.05). smFISH analysis revealed decreased β cell expression of STAT1 and MX1 expression in TYK2i-treated RIP-LCMV and NOD mice compared to vehicle-treated mice.

Treatment with TYK2 inhibitors protected human islets in vitro, reduced diabetes incidence in two mouse models of T1D, and was associated with early changes in innate immune and late changes in adaptive immune signatures, most notably leading to increased T regulatory cells and increased PDI expressing CD8+ T cells, a phenotype which has been linked T cell exhaustion, Taken together, these data highlight the potential role for TYK2 inhibitors as a novel disease-modifying therapy in T1D, with beneficial effects in both the immune and β cell compartments.