JAK Inhibition as a Potential Treatment Target in Systemic Lupus Erythematosus

Janus kinase (JAK)/signal transducers and activators of transcription (STATs) are a group of molecules responsible for signal transduction of multiple cytokines and growth factors in different cell types, involved in the maintenance of immune tolerance. Thus, the dysregulation of this pathway plays a crucial role in the pathogenesis of multiple autoimmune, inflammatory, and allergic diseases and is an attractive treatment target. JAK inhibitors (JAKinibs) have been approved in the treatment of multiple autoimmune diseases including rheumatoid arthritis (RA), psoriatic arthritis (PsA) and ankylosing spondylitis (SPA). In SLE, there is a plethora of ongoing trials evaluating their efficacy, with tofacitinib, baricitinib and deucravacitinib showing promising results, without major safety concerns. In this review, we will discuss the rationale of targeting JAKinibs in SLE and summarize the clinical data of efficacy and safety of JAKinibs in SLE patients.


INTRODUCTION
SLE is a chronic autoimmune disease affecting multiple organs with a variable clinical course and prognosis. 1ts pathogenesis is elusive and multifactorial implicating both the innate and adaptive immunity 2 and is characterised by the aberrant production of several cytokines and autoantibodies.The treatment goal is the attainment of remission or Lupus Low Disease Activity State (LLDAS) which is associated with better outcomes including damage accrual, reduction in the number of flares, glucocorticoids' withdrawal, and better quality of life. 3ombination therapy of immunomodulatory agents as well as glucocorticoids may be necessary in achieving the treatment target.To date, unlike other RMDs where a plethora of targeted therapies is available, there are only two biological therapies -belimumab and anifrolumab -that have been approved for the treatment of SLE.The last decade, JAKinibs blocking simultaneously one or more Janus kinases, are a valuable treatment option in the therapeutic armamentarium for patients with RMDs, including those with difficult-to-treat 4 or refractory disease. 5erein, we overview the role of the JAK/STAT pathway in SLE with a focus on ongoing clinical trials of JAKinibs in SLE.STAT5A, STAT5B, STAT6) which are responsible for the signal transduction of over fifty cytokines (including interleukin and interferon members) and growth factors in various cell types regulating gene expression and cell activation, proliferation, and differentiation, playing a crucial role in hematopoiesis, regulation of the immune system, metabolism, and growth. 6JAK1, JAK2, and TYK2 are expressed in almost all tissues whereas JAK3 is only expressed in the bone marrow, lymphatic system, endothelial cells, and vascular smooth muscle cells. 7The activation of the JAK/STAT pathway is complicated and regulated on multiple levels.The critical step is the activation of JAK upon binding of a ligand to its (type I or II) receptor. 8After JAK-mediated phosphorylation at specific receptor tyrosine residues, STATs are recruited to the receptors and become phosphorylated by JAKs. 9STATs are subsequently translocated to the nucleus where they bind to members of the gamma-activated site (GAS) family of enhancers, inducing the transcription of target genes. 9Aberrant JAK/STAT pathway signaling is prevented by the production of inhibitory proteins acting as checkpoints including the suppressor of cytokine signaling (SOCS) family, or the autoinhibitory mechanism of JH2-JH1 domain interaction. 10The breakdown of JAK regulation leads potentially to disease with aberrant activation being linked to tumorigenesis and autoimmunity [11][12][13] and loss-offunction mutations being associated with severe combined immune deficiency. 14

JAK/STAT PATHWAY AND SLE
The pathogenesis of SLE is complex and characterized by the presence of autoreactive T cells and production of autoantibodies by B cells, with the implication of numerous circulating pro-inflammatory cytokines including interleukin (IL)-2, IL-6, IL-10, IL-12/IL-23, IL-19, IL-20, IL-22, and interferon (IFN)-α, IFN-γ. 15,16IFNa is a key cytokine in SLE pathogenesis, and interferon-inducible gene expression is associated with disease activity and lupus nephritis 17,18 whereas the persistence of high levels of ultrasensible IFN-a is related with a higher risk of relapse in patients with quiescent SLE . 19he JAK/STAT pathway is implicated in SLE pathogenesis on multiple levels.First, IFN type I and type II (as well as multiple other proinflammatory cytokines) bind to JAK receptors in order to activate intracellular signals and exert their functions. 20Second, there is growing evidence that the JAK/STAT pathway may regulate IFN-regulatory factor (IRF)-related genes. 21A study of the relation between type I-II IFN trigger activation of the JAK/STAT pathway in 22 SLE patients indicated that the overexpression of STAT1 by B-cells correlated with lupus activity.More specifically, STAT1 appeared to be activated following exposure to IFNa in SLE plasmablasts.Therefore, the use of JAK inhibitors to prevent STAT1 expression and B cell induction in SLE patients would impede two major pathogenic pathways. 22rthermore, several polymorphisms in the STATs have been related to SLE susceptibility according to genome-wide association studies. 23,24Importantly, STAT4 is one of the most dominant risk alleles in SLE 25 and is related with renal disease and anti-ds DNA positivity. 26n the other hand, defects in the deactivation of the JAK/STAT signaling have also been linked to SLE.In the PBMCs from 34 SLE patients the SOCS1 mRNA expression and protein level was reduced compared to 34 healthy controls, 27 whereas SOCS1 mRNA expression was inversely correlated with SLE disease activity. 27own-regulation of SOCS1 has also been linked to lupus nephritis in SLE patients and murine models and seems to ultimately contribute to renal inflammation and fibrosis 28,29 but also with numerous other manifestations including skin and central nervous system. 30Interestingly, treatment with corticosteroids to human PBMCs leads to upregulation of SOCS1 message in a dose-and time-dependent manner. 31

JAK INHIBITION IN SLE: FROM MURINE MODELS TO HUMAN STUDIES Murine lupus: blocking JAKs and STATs
There are multiple experimental studies evaluating the role of the JAK/STAT pathway as a potential treatment target, focusing mostly on cutaneous lupus, lupus nephritis and serological activity.In a study in MRL/lpr mice, treatment with a JAK2-STAT1 inhibitor, led to an improvement in nephritis, sialadenitis and anti-ds DNA levels. 32In another murine study, JAK2 selective inhibition with CEP-33779, led to extended survival, reduced splenomegaly and lymphomegaly, and decreased several cytokines including IL-12, IL-17A, IFN-α, IL-1β, and TNF-α in MRL/lpr or BWF1 mice with established SLE or lupus nephritis. 33mportantly, treatment with CEP-33779 had a significant disease-modifying effect and was able to mitigate several immune parameters associated with SLE disease progression, including the prevention and treatment of mice with lupus nephritis. 33Tofacitinib, a JAK1 and JAK3 inhibitor used in the treatment of multiple autoimmune diseases, has also been evaluated in 3 murine models.5][36] Ruxolitinib, a JAK1 and JAK2 inhibitor approved in the treatment of myelofibrosis, attenuated the development of severe skin lesions in the MRL/lpr mice, reduced lesion severity scores by week 4 and led to a significant reduction in epidermal hyperplasia and inflammatory cell infiltration. 37TAT3 is another attractive treatment target as its activation is highly correlated with different types of glomerulonephritis and is implicated in glomerular and tubulointerstitial cell proliferation, interstitial fibrosis, and the level of renal injury. 38Simultaneous blocking of JAK1 and STAT3 TITLE by CDDO-Me (C-28 methyl ester of 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid) showed promising results in murine models with established LN and prevented the development of LN in two murine models. 39On the other hand, mice treated with compound S31-201 (Stattic), a selective STAT3 inhibitor, had a lesser degree of tubule injury, inflammation, and interstitial fibrosis, 40 whereas Edwards et al. showed that treatment with Stattic delayed onset of proteinuria by 3 weeks compared with controls and lowered levels of anti-dsDNA antibodies and inflammatory cytokines. 41YK2-deficient human T cells exhibit impaired responses to IL-12, IL-23, IFN-α, and IL-10 42 whereas Tyk2 dysfunction has been related to the prevention of immune-mediated inflammatory diseases including psoriasis, psoriatic arthritis and vitiligo 43,44 without immunodeficiency. 45In SLE, TYK2 is implicated in the increased autophagy activity by B cells 46 and NPSLE. 47inical trials and real-world data Tofacitinib After the encouraging results of tofacitinib in SLE murine models, its effectiveness has been evaluated in a phase I double-blinded trial including 30 SLE patients. 48Patients were stratified based on the presence of the STAT4 risk allele, which may correlate with increased production of type I IFNs in PBMCs and thus with a more severe phenotype and increased risk of cardiovascular disease in SLE patients.In this trial, tofacitinib was found to be not only safe but also improve cardiometabolic and immunologic parameters including high-density lipoprotein cholesterol levels, decreasing type I IFN signature and circulating neutrophil extracellular traps (NETs), especially in the subgroup of patients with the STAT4 risk allele. 48In clinical practice, tofacitinib led to rapid remission seven out of ten patients with active skin and/or musculoskeletal disease. 49Interestingly, tofacitinib led to significant clinical improvement in 3 patients with recalcitrant cutaneous SLE. 50ricitinib Baricitinib, an oral selective inhibitor of JAK1 and JAK2, proved to be safe and effective at the dose of 4 mg in the resolution of arthritis or rash at week 24 in combination with standard of care in a phase 2 RCT of 314 SLE patients. 51In this trial, baricitinib reduced anti-dsDNA titres, and other pro-inflammatory cytokines. 52,53In SLE-BRAVE 1, a phase 3 RCT, 760 patients were randomly assigned to receive baricitinib 4mg, 2mg, or placebo. 54he primary endpoint, SLE Responder Index 4 (SRI-4) at week 52, was met in the baricitinib 4mg group. 54These results were not replicated in SLE-BRAVE 2, another phase 3 RCT. 55In this trial, treatment with baricitinib did not meet neither the primary efficacy endpoint of SRI-4 at week 42, nor the major secondary endpoints including glucocorticoid tapering and time to first flare. 55Analysis of the above-mentioned studies indicates that while neither the 2mg nor the 4 mg baricitinib dose correlated with a significant increase in the odds of achieving an LLDAS response, the 4 mg dose was associated with a significant increase in the odds of an SRI-4 response, compared to placebo. 56lcitinib Solcitinib, a selective JAK 1 inhibitor, was evaluated in a phase II study in patients with active, extra-renal SLE. 57n this RCT, patients received solcitinib or placebo twice daily for 12 weeks.Due to absence of significant effect on one of the primary endpoints, mean IFN transcriptional biomarker expression, the study was prematurely terminated after the recruitment of 50 patients. 57Of note, six patients had elevated liver enzymes of whom one confirmed and one suspected case of drug reaction with eosinophilia and systemic symptoms. 57lgotinib Filgotinib, another JAK1 inhibitor has been assessed in a phase 2 trial of patients with cutaneous lupus erythematosus (CLE). 58In this trial, the primary endpoint of change from baseline in Cutaneous Lupus Erythematosus Disease Area and Severity Index Activity (CLASI-A) score at week 12 was not met.Of note, 1 serious adverse event was reported in the filgotinib group.However, filgotinib showed promising results in a small study of nine patients with class V LN. 59 In this study, only four patients in the filgotinib group completed week 16; with a median reduction of 50.7% in 24-hour urine protein. 59ucracitinib Deucracitinib is the first FDA-approved oral TYK2 inhibitor for the treatment of moderate to severe plaque psoriasis. 60In SLE, there are promising results from a phase 2 study including 363 patients with active SLE. 61In this study, patients receiving 3 mg of deucracitinib twice daily met the primary endpoint, SRI-4 at week 32, 61 while deucracitinib treated patients had higher frequencies of attainment of LLDAS, BICLA response and CLASI-50 and improvement in arthritis at week 48. 61pical treatment: ruxolitinib Ruxolitinib is effective in the treatment of several immunologic skin disorders, including alopecia areata. 62The efficacy of ruxolitinib cream will be assessed in a phase 2 trial of patients with active discoid lupus erythematosus. 63adacitinib Upadacitinib is a selective JAK1 inhibitor used in the treatment of autoimmune disorders including RA, atopic dermatitis, psoriatic arthritis, and ulcerative colitis. 64 elsubrutinib/upadacitinib combination therapy, upadacitinib monotherapy or placebo both therapeutic regimens significantly improved disease activity and risk of relapse. 65Both primary and key secondary endpoints were achieved whereas anti-double stranded DNA antibodies also significantly decreased in response to treatment.
No safety concerns were raised, as no malignancies or VTEs were reported, and the 3 non-fatal CV events were not attributed to the treatment. 65Another case study of a 26-year-old female CLE patient receiving upadacitinib, demonstrated significant improvement of lesions using the Revised Cutaneous Lupus Erythematosus Disease Area and Severity Index. 66

SAFETY
Important concerns on the safety of JAKis have been raised after the post hoc analysis of the ORAL surveillance, a randomised, open-label, noninferiority, post-authorisation, safety end-point trial. 67ORAL surveillance showed that RA patients with cardiovascular risk factors treated with tofacitinib had a higher likelihood of developing a cardiovascular event, including myocardial infarction, stroke, and death due to cardiovascular disease, compared to those treated with anti-TNF. 67Treatment with tofacitinib was also related to higher incidence of adjudicated opportunistic infections (including herpes zoster and tuberculosis), and adjudicated nonmelanoma skin cancer. 67It is speculated that the greater inhibition of JAK2, achieved with higher doses of tofacitinib and baricitinib, might be the underlying mechanism responsible for the increased risk of a thromboembolic event. 67owever, a systematic review and meta-analysis of 29 RCTs did not show augmented risk of CVEs and all-cause mortality in patients with immune-mediated inflammatory diseases on tofacitinib treatment in a short-term perspective. 68Data from a 'real-world evidence (RWE) cohort' including 12 852 RA, tofacitinib-treated patients did not find statistically significant evidence for an increased risk of cardiovascular outcomes. 69

CONCLUDING REMARKS
Despite recent advances in the treatment of rheumatic diseases there are substantial unmet needs in the management of SLE patients, mainly due to the heterogenicity and the complexity of the disease and problems in study design including too strict endpoints.JAKis, are nowadays used in the treatment of multiple autoimmune diseases, proving their efficacy even in patients with refractory disease 5,49 51,55ding the risk of serious infection, both Wallace et al. and Petri et al. reported serious adverse events and major infections in patients receiving either 2mg or 4 mg of baricitinib.No deaths attributed to baricitinib use were reported in the aforementioned studies.51,55

Table 1 .
due to their potential of simultaneous inhibition of multiple cytokines.In SLE, tofacitinib, baricitinib, and deucracitinib have shown encouraging results with an acceptable safety profile.Larger trials and real-world data are necessary in order to evaluate the efficacy and safety of JAKis especially in patients with severe manifestations including LN and NPSLE.JAK inhibitor clinical trials of efficacy and safety.

Table 1 .
JAK inhibitor clinical trials of efficacy and safety.