Deucravacitinib, a tyrosine kinase 2 pseudokinase inhibitor, protects human EndoC-βH1 β-cells against proinflammatory insults

Introduction Type 1 diabetes is characterized by pancreatic islet inflammation and autoimmune-driven pancreatic β-cell destruction. Interferon-α (IFNα) is a key player in early human type 1 diabetes pathogenesis. IFNα activates the tyrosine kinase 2 (TYK2)-signal transducer and activator of transcription (STAT) pathway, leading to inflammation, HLA class I overexpression, endoplasmic reticulum (ER) stress, and β-cell apoptosis (in synergy with IL-1β). As TYK2 inhibition has raised as a potential therapeutic target for the prevention or treatment of type 1 diabetes, we investigated whether the selective TYK2 inhibitor deucravacitinib could protect β-cells from the effects of IFNα and other proinflammatory cytokines (i.e., IFNγ and IL-1β). Methods All experiments were performed in the human EndoC-βH1 β-cell line. HLA class I expression, inflammation, and ER stress were evaluated by real-time PCR, immunoblotting, and/or immunofluorescence. Apoptosis was assessed by the DNA-binding dyes Hoechst 33342 and propidium iodide or caspase 3/7 activity. The promoter activity was assessed by luciferase assay. Results Deucravacitinib prevented IFNα effects, such as STAT1 and STAT2 activation and MHC class I hyperexpression, in a dose-dependent manner without affecting β-cell survival and function. A comparison between deucravacitinib and two Janus kinase inhibitors, ruxolitinib and baricitinib, showed that deucravacitinib blocked IFNα- but not IFNγ-induced signaling pathway. Deucravacitinib protected β-cells from the effects of two different combinations of cytokines: IFNα + IL-1β and IFNγ + IL-1β. Moreover, this TYK2 inhibitor could partially reduce apoptosis and inflammation in cells pre-treated with IFNα + IL-1β or IFNγ + IL-1β. Discussion Our findings suggest that, by protecting β-cells against the deleterious effects of proinflammatory cytokines without affecting β-cell function and survival, deucravacitinib could be repurposed for the prevention or treatment of early type 1 diabetes.


Introduction
Type 1 diabetes is characterized by pancreatic islet inflammation and specific destruction of pancreatic b-cells by an autoimmune assault, which develops in the context of an inadequate "dialogue" between b-cells and the invading immune cells (1,2).
A growing body of evidence places type I interferons (IFNs) as key players in the early stages of human type 1 diabetes pathogenesis (3).IFNa was found in islets from type 1 diabetes patients (4)(5)(6), and laser-captured islets from living donors with recent-onset type 1 diabetes showed increased expression of IFNstimulated genes (ISGs) (7).In genetically susceptible children, an IFN signature was temporarily amplified preceding the development of autoantibodies and throughout the progress of type 1 diabetes (8,9).Recently, three type I IFN response markers, namely human MX Dynamin Like GTPase 1 (MX1), double-stranded RNA sensor protein kinase R, and HLA class I, were found to be expressed in a significantly higher percentage of insulin-containing islets from autoantibody-positive and/or recentonset type 1 diabetes donors (10).In human b-cells, IFNa induced inflammation, endoplasmic reticulum (ER) stress as well as a longlasting overexpression of HLA class I via activation of the tyrosine kinase 2 (TYK2)-signal transducer and activator of transcription (STAT) pathway.Moreover, IFNa induced apoptosis in the presence of .
Targeting the type I IFN signaling pathway has been proposed as a potential adjuvant therapy to treat at-risk individuals or patients still in the very early stages of the disease (3,15).Among some of the strategies that have been suggested, inhibitors of Janus kinase (JAK) proteins (JAK1-3 and TYK2) show great promise.Treatment with AZD1480 (a JAK1/JAK2 inhibitor) and ABT 317 (a JAK1-selective inhibitor) protected non-obese diabetic mice against autoimmune diabetes and reversed diabetes in newly diagnosed non-obese diabetic mice (16,17).In human b-cells, clinically used JAK inhibitors, namely ruxolitinib, cerdulatinib, and baricitinib, prevented MHC class I overexpression, ER stress, chemokine production, and apoptosis (13,14).
Lately, attention has focused on TYK2, a candidate gene for type 1 diabetes whose genetic variants that decrease TYK2 activity are associated with protection against the disease (18-20).TYK2 is crucial for cell development and IFNa-mediated responses in human b-cells (11,21,22).Partial TYK2 knockdown protected human b-cells against apoptosis and inflammation induced by polyinosinic-polycitidilic acid, a mimic of double-stranded RNA produced during viral infection (21).In mature stem cell-islets, TYK2 knockout or pharmacologic inhibition decreased T-cellmediated cytotoxicity by preventing IFNa-induced antigen processing and presentation, including MHC class I expression (22).As these findings place TYK2 as a critical regulator of the type I IFN signaling pathway in b-cells, selective TYK2 inhibition has emerged as a drug target to treat type 1 diabetes.Recently, two novel small molecule inhibitors binding to the TYK2 pseudokinase domain protected human b-cells against the deleterious effects of IFNa without compromising b-cell function and susceptibility to potentially diabetogenic viruses (23).
Deucravacitinib, a small molecule that selectively targets the TYK2 pseudokinase domain, has shown great therapeutic potential for immune-mediated diseases, such as lupus nephritis and systemic lupus erythematosus (24,25).In fact, deucravacitinib has been recently approved for treatment of plaque psoriasis (26).However, no preclinical studies have deeply explored the possible use of deucravacitinib in the context of type 1 diabetes.Notably, Chandra et al. recently used deucravacitinib to validate their CRISPR-Cas9generated TYK2 knockout in human induced pluripotent stem cells, but did not provide further characterisation of its effects on b-cells (22).
In this study, we report the effects of deucravacitinib on the human insulin-producing EndoC-bH1 cells, including its ability to prevent IFNa-triggered signaling pathway and damaging effects on b-cells.

Cell treatments
Proinflammatory cytokine concentrations were selected according to previously established experiments in human b-cells (11, 28): recombinant human IFNa (PeproTech Inc., Rocky Hill, NJ) at 1000 U/mL; recombinant human IFNg (PeproTech Inc., Rocky Hill, NJ) at 1000 U/mL; and recombinant human IL-1b (R&D Systems, Abingdon, UK) at 50 U/mL.Ruxolitinib, baricitinib, or deucravacitinib (Selleckchem, Planegg, Germany) were prepared in DMSO (used as vehicle) and cells were treated as indicated in the figures.Ruxolitinib and baricitinib concentrations were selected based on previous doseresponse experiments (unpublished data).For treatments involving cytokines, 2% FBS was added to the culture medium.

Cell viability assessment
The percentage of apoptosis was measured by fluorescence microscopy upon staining with the DNA-binding dyes Hoechst 33342 and propidium iodide (Sigma-Aldrich, Saint Louis, MO, USA) as described (29).At least 600 cells were counted for each experimental condition.Viability was assessed by two independent researchers, one of whom was unaware of sample identity, with >90% agreement between results.

C-X-C motif chemokine ligand 10 measurements
The release of C-X-C motif chemokine ligand 10 (CXCL10) to the culture medium was detected using Human ProcartaPlex immunoassays (Invitrogen, Vienna, Austria) following the manufacturer's recommendations.Reactions were read with a MagPix system (Luminex, Austin, TX, USA).

Luciferase reporter assays
Cells were transfected using Lipofectamine 2000 (Invitrogen) with pRL-CMV encoding Renilla luciferase (Promega) and luciferase reporter constructs for either gamma-interferon activation site (GAS) (Panomics, Fremont, CA, USA) or IFNstimulated regulatory element (ISRE) (kindly provided by Dr Izortze Santin, University of the Basque Country, Spain).After recovery, cells were treated with either IFNa for 2 h or IFNg for 24 h (30).Luciferase activity was measured in a POLASTAR plate reader (BMG Labtech) using the Dual-Luciferase Reporter Assay System (Promega) and corrected for the luciferase activity of the internal control plasmid, i.e., pRL-CMV.

Real-time PCR
Poly(A) + mRNA was extracted using Dynabeads mRNA DIRECT kit (Invitrogen) and cDNA synthesis was performed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems).Real-time PCR was performed on the CFX96 Real Time System (Bio-Rad) as described (31) and the housekeeping gene b-actin was used to correct expression values.Of note, b-actin expression was not altered by the experimental conditions used herein.All primers used here are listed in Supplementary Table 1.
All antibodies used here are listed in Supplementary Table 2.
All the original, uncropped images representing immunoblots and microscopic photos are provided in the Supplementary Material.

Glucose-stimulated insulin secretion
After preincubation in modified Krebs-Ringer for 1 h, cells were sequentially stimulated with low (0 mmol/L) and high glucose (20 mmol/L) for 1 h (each stimulation) as previously described (34).Insulin secreted and insulin content from lysed cells were measured using a human insulin ELISA kit (Mercodia, Uppsala, Sweden) following the manufacturer's instructions.The amount of secreted insulin as % of total insulin was calculated as previously described (35) and data were normalized to insulin secretion at 20 mmol/L glucose in vehicle-treated cells without IFNa (considered as 100%).See Supplementary Material for further details.

Statistical analyses
The GraphPad Prism 7.0 software (GraphPad Software, La Jolla, CA, USA) was used for statistical analyses.Data are shown as mean ± SEM of independent experiments (i.e.considering EndoC-bH1 cells from different passages as n = 1).The statistical significance of differences between groups was evaluated using one-way ANOVA followed by Dunnett's test or two-way ANOVA followed by Sidak's test or Dunnett's test, as appropriate.Differences were considered statistically significant when p ≤ 0.05.

Deucravacitinib prevented IFNa effects without affecting b-cell survival and function
IFNa-mediated TYK2 activation leads to STAT1 and STAT2 phosphorylation, which will eventually upregulate several ISGs, including HLA-ABC, CXCL10, and MX1 (Supplementary Figure 1A).Pre-treatment with deucravacitinib inhibited IFNa-induced STAT1 and STAT2 phosphorylation in a dose-dependent manner, where deucravacitinib showed greater potency against IFNa-stimulated STAT1 phosphorylation (Figures 1A, B).We then selected two doses, 10 and 1000 nmol/L, for the follow-up experiments.Next, we examined how deucravacitinib affects the kinetics of IFNa-induced STAT activation.IFNa increased P-STAT1 and P-STAT2 levels, with a maximum effect at 1-4 h post-treatment and a return to baseline by 24 h (Figures 1C, D; Supplementary Figure 1B).Although STAT1 and STAT2 protein levels were already upregulated by 8 h, STAT2 expression reached peak level at 16 h, while STAT1 expression was still increasing by 24 h (Supplementary Figures 1C, D).Exposure to 1000 nmol/L deucravacitinib abrogated the IFNa-stimulated STAT1 and STAT2 phosphorylation and protein expression, whereas 10 nmol/L deucravacitinib had only a minor effect (Figures 1C, D and Supplementary Figures 1B-D).Furthermore, IFNa-induced MHC class I protein overexpression was blocked by 1000 nmol/L deucravacitinib (Figures 1E, F).Finally, deucravacitinib did not affect b-cell viability nor changed glucose-stimulated insulin secretion and insulin content in the absence or presence of IFNa (Supplementary Figures 1E-G).

Deucravacitinib blocked IFNa-induced upregulation of ISGs, but not ER stress markers
Assessement of the expression of some ISGs and ER stress markers showed that all three inhibitors prevented IFNa-induced upregulation of HLA-ABC, CXCL10, and MX1 in a dose-dependent manner (Figures 2F-K).Although ruxolitinib and baricitinib inhibited the mRNA expression of the ER stress markers C/EBP homologous protein (CHOP) and spliced isoform of XBP1 X-box binding protein 1 (XBP1s), only 10 nmol/L deucravacitinib reduced CHOP expression (Figures 2I, J).None of these inhibitors changed the expression of activating transcription factor 3 (ATF3) (Figure

The harmful effects of cytokines were partially inhibited by deucravacitinib
So far, we investigated whether pre-treatment with deucravacitinib prevents the effects of different cytokines in b-cells.Here, we assessed if deucravacitinib could abrogate these damaging effects.EndoC-bH1 cells were pre-treated with either IFNa + IL-1b or IFNg + IL-1b for 24 h.Afterwards, 1000 nmol/L deucravacitinib was added for an additional 24 h still in the presence of cytokines (Figure 4A).Deucravacitinib partially decreased IFNa + IL-1b-induced apoptosis (60% decrease) (Figure 4B).IFNa + IL-1b-stimulated HLA-ABC mRNA expression remained unchanged in deucravacitinib-treated cells (Figure 4D), which agrees with previous data showing an IFNatriggered long-lasting expression of HLA-ABC (13).STAT1 protein levels, CXCL10 secretion, and CHOP mRNA expression were reduced by 26-42% (Figures 4C, F, H; Supplementary Figure 3E), while the expression of MX1 and CXCL10 was completely inhibited by deucravacitinib ( Figures 4E, G).

Discussion
Targeting the JAK-STAT pathway has emerged as a promising therapeutic approach for type 1 diabetes prevention/early treatment (3,15).Although this strategy has been approved for treatment of some autoimmune diseases, including rheumatoid arthritis and psoriatic arthritis (37), there are no JAK inhibitors approved for type 1 diabetes.Nonetheless, recent preclinical data suggest that these inhibitors could be repurposed for this disease (13,14,16,17,22,23,38) and a clinical trial investigating whether baricitinib prevents the progressive, immune-mediated destruction of b-cells in type 1 diabetes patients is ongoing (39).
In the current study, we tested whether the TYK2 inhibitor deucravacitinib could protect human b-cells against the deleterious effects of IFNa and other cytokines.We focused on this TYK2 inhibitor for two reasons: first, due to TYK2 importance for type 1 diabetes pathogenesis.For instance, TYK2 regulates IFNa-mediated pro-apoptotic and proinflammatory pathways in b-cells (21,22).Second, exploring a drug recently approved by the U.S. Food and Drug Administration to treat another autoimmune disease, namely plaque psoriasis (26), increases its repositioning potential for type 1 diabetes and facilitates the bench-to-bedside transition.
Deucravacitinib is a small-molecule ligand that binds to and stabilizes the TYK2 pseudokinase domain, leading to highly potent and selective allosteric TYK2 inhibition (24,40).Inhibition of IFNainduced STAT phosphorylation by deucravacitinib has been shown in several cell types, such as CD3 + T cells, CD19 + B cells, and CD14 + monocytes (24).Here we showed that deucravacitinib also prevents IFNa-stimulated STAT1 and STAT2 phosphorylation in human EndoC-bH1 cell line.Furthermore, in agreement with previous findings (24), deucravacitinib also showed higher potency against TYK2-mediated phosphorylation of STAT1 compared with STAT2 phosphorylation in our experimental model.Notably, at the concentrations used in our study, deucravacitinib did not affect bcell function and viability, which is a desired feature for a drug with therapeutic potential.
Compared with ruxolitinib and baricitinib, two clinically available JAK1/JAK2 inhibitors, deucravacitinib was more potent against IFNa-stimulated STAT phosphorylation, ISRE activity, and mRNA expression of HLA-ABC, MX1, and CXCL10.However, unlike ruxolitinib and baricitinib, deucravacitinib did not affect the IFNa-mediated upregulation of the ER stress markers CHOP and XBP1s.Our results partially agree with a previous publication reporting that two TYK2 inhibitors failed to prevent IFNa-induced CHOP expression in EndoC-bH1 cells (23).Prior studies have shown that other JAK/TYK2 inhibitors could prevent the detrimental effects of IFNa + IL-1b, such as apoptosis and inflammation (14, 23).Therefore, we investigated whether deucravacitinib could protect b-cells against the harmful effects of two different combinations of cytokines: IFNa + IL-1b (early insulitis) and IFNg + IL-1b (late insulitis).In both scenarios, pre-treatment with deucravacitinib protected against cytokine-induced apoptosis and CXCL10 mRNA expression.Additionally, in cells treated with IFNa + IL-1b, pre-treatment with deucravacitinib blocked the overexpression of MHC class I at the cell surface and CXCL10 secretion to the medium.Interestingly, while the IFNa + IL-1b-induced upregulation of HLA-ABC, MX1, and CHOP was inhibited by the pre-treatment with deucravacitinib, this inhibitor did not change the expression of HLA-ABC stimulated by IFNg + IL-1b.Moreover, MX1 and CHOP mRNA expression was only partially reduced by the pre-treatment with deucravacitinib in IFNg + IL-1b-treated cells.Importantly, the addition of deucravacitinib when cytokine exposure was already ongoing could reduce the deleterious effects of these cytokines.Although it seems clear that deucravacitinib confers protection against IFNa + IL-1b by directly inhibiting the TYK2-mediated pathway, it remains to be answered how deucravacitinib protects against IFNg + IL-1b-induced effects.Indeed, our present data suggest that deucravacitinib does not interfere with the IFNg-mediated signaling pathway.One possibility might be the following: in b-cells, either IFNg alone or in combination with IL-1b induce the expression of members of the interferon regulatory factor (IRF) family, such as IRF3 and IRF7 (41,42).As IRF3 and IRF7 are potent activators of IFNa and IFNb gene expression (43,44), it is conceivable that IFNg + IL-1binduced IRF3 and IRF7 could lead to type I IFN expression and secretion.Then, secreted IFNa and/or IFNb could stimulate the type I IFN receptor-TYK2 pathway in an autocrine fashion.In this context, deucravacitinib could inhibit this positive-feedback loop stimulated by IFNg + IL-1b-induced IRF3 and IRF7 expression.
Based on our findings, it will be interesting to test whether novel small molecule TYK2 pseudokinase ligands (45) could also protect bcells from IFNa deleterious effects.Nevertheless, we must bear in mind that completely inhibiting TYK2 may be counterproductive, as it might lead to susceptibility to microorganisms (e.g., mycobacteria and virus) and immunodeficiency (46).Thus, regardless of the TYK2 inhibitor chosen, we should focus on doses that induce a partial inhibition, as seen in individuals with a protective single nucleotide polymorphism in the TYK2 gene (18), as it could offer maximal efficacy with reduced risk of developing secondary infections.Moreover, our data suggest that partial TYK2 inhibition obtained with low doses of deucravacitinib was enough to prevent most IFNainduced harmful effects in b-cells, such as upregulation of the proapoptotic CHOP, MHC class I overexpression, and apoptosis (in the presence of IL-1b).One potential limitation of our study is its purely in vitro nature, which may limit our conclusions regarding the use of deucravacitinib to treat a disease as complex as type 1 diabetes.Conversely, our findings, along with others (22,23), provide further preclinical evidence that TYK2 inhibitors could be considered a strategy for an early therapy for type 1 diabetes.The next logical step would be to investigate whether our in vitro findings could be translated to animal models of type 1 diabetes (e.g., NOD and RIP-B7.1 mice).
In conclusion, we provided evidence that deucravacitinib protects b-cells against the deleterious effects of proinflammatory cytokines, such as IFNa, IFNg and IL-1b, without affecting b-cell function and survival.Our present findings add to the existing evidence that TYK2 inhibition may be an efficient treatment strategy for type 1 diabetes.Moreover, these preclinical findings suggest that deucravacitinib could be repurposed to presymptomatic 1 diabetes subjects (i.e., positive for 2-3 autoantibodies but still normoglycemic) or be introduced in the early stages of type 1 diabetes onset.