JAKpot! New small molecules in autoimmune and inflammatory diseases

Abstract

Cytokines are key mediators of the development and homeostasis of hematopoietic cells, critical for host defense, but also for the development of autoimmune and inflammatory diseases like psoriasis or rheumatoid arthritis (RA). Blocking cytokines activity by interfering with the ligand-receptor association has been successfully employed to treat several immune disorders. A subgroup of cytokines signals through receptors requiring the association with a family of cytoplasmic protein tyrosine kinases known as Janus kinases (Jaks). Jaks have recently gained significant attention as therapeutic targets in inflammation and autoimmunity and several Jak inhibitory small molecules have been developed. The first two Jak inhibitors, tofacitinib and ruxolitinib, have been approved for the treatment of RA and primary myelofibrosis, respectively. Efficacy and safety data suggest that some of these oral Jak inhibitors as well as their topical formulations may soon enter the daily clinical practice for treating patients with psoriasis, lupus erythematosus or other inflammatory skin diseases. While biologics typically target one single cytokine, these new immunomodulators can inhibit signals from multiple cytokines intracellularly and therefore could be useful when other therapies are ineffective. Thus, Jak inhibitors may replace some traditional immunosuppressive agents and help patients not responding to previous therapies.

Introduction

Given the importance that cytokines have in development and homeostasis of the immune system it is not surprising that these soluble factors are critical players in immune mediated disorders including inflammatory autoimmune diseases. For example, the pathogenesis of psoriasis is characterized by the activation of numerous immune cells, which interact with resident tissue cells. In the skin, these cells are primarily composed by keratinocytes and endothelia, in the joints by synoviocytes, fibroblasts and osteoblasts (1, 2). Cellular contact and, more importantly, secreted factors like cytokines, can cause persistent inflammation of skin and joints. The cytokines network in psoriasis’ pathogenesis is well studied (1, 3) and some similarities are found in other pathologies like RA or ulcerative colitis (4, 5). Innate cytokines determining lineage-specification of CD4⁺ T helper (Th) cells, such as interleukin (IL)-1 and IL-6, and cytokines released by T cells and resident tissue cells like tumor necrosis factor (TNF), interferons (IFNs), IL-17 or IL-23 are indispensable for disease manifestation and perpetuation.

Given the role these molecules play in inflammatory pathologies limiting their interaction with their specific receptors has been successfully exploited for therapeutic purposes through the use of biologics. For immunological purposes, biologics such as monoclonal antibodies, recombinant soluble receptors and fusion proteins of receptor moieties with antibodies fragments, block the interaction of a specific cytokine with its receptor. In the past 15 years, biologics have completely revolutionized the clinical approach to the treatment of autoimmunity and inflammatory pathologies.

The basis for this success was our better understanding of cellular and molecular players. In psoriasis, clearance was observed after bone marrow transplantation, in those receiving the immunosuppressant cyclosporine or antibodies targeting CD4 suggesting a prominent role for T cells (6, 7) and, in particular, IFN-γ-producing Th1 cells (8, 9). Based on such analysis of disease-associated or tissue-infiltrating Th cells in patients and in animal models, Th1 cells were thought to drive the inflammatory responses in organ-specific autoimmune diseases and inflammatory pathologies like psoriasis.

Surprisingly, mice lacking IFN-γ, the IFN-γ receptor or its downstream signaling element signal transducer and activator of transcription (STAT)1 exhibit exaggerated autoimmune inflammation (10). Furthermore, the Th1-promoting cytokines IFN-α and IFN-γ both exacerbate psoriasis whereas therapies with recombinant IL-4, IL-10 or IL-11 showed some clinical improvements (9, 11, 12). At the beginning of this millennium, a second CD4⁺ T cell population known as Th17, a T cell subset producing IL-17, IL-22 and TNF was recognized to play a major role. Th17 cells with potential to induce inflammatory pathology typically require signals from IL-23 (13, 14). High numbers of IL-17-expressing Th17 cells and IL-23-expressing dendritic cells (DC) are present in psoriatic skin (Figure 1) (15, 16). The knowledge on the underlying cytokine network in psoriasis allowed the establishment of therapies with biologics targeting TNF (17–19) or the IL-12/IL-23 p40 subunit (20). Targeting IL-17 or its receptor is being evaluated in phase III studies for psoriasis (21–23). In RA, biologics targeting TNF, IL-1 or the IL-6 receptor have been approved (2).

Figure 1. The critical role of cytokines in the pathogenesis of psoriasis: Targeted therapies inhibiting extracellular cytokine actions and the new approach of blocking intracellular cytokine signaling.

Immune sensors like dendritic cells release innate cytokines including IL-12, IL-1, IL-6 or IL-23 that drive the differentiation of T cells towards pathogenic Th1 and Th17 cells. Th1 cells produce IFN-γ, IL-2 and TNF, while Th17 cells express IL-17, IL-22, TNF and, in humans, IFN-γ. The resident tissue cells respond to the inflammatory cells and their secreted cytokines by uncontrolled proliferation. Biologics can neutralize single cytokines to dampen the inflammation. In contrast, Jak inhibitors interfere with cytokine signaling by blocking signal transmission in innate and adaptive immune cells, keratinocytes as well as endothelial cells.

The knowledge on the underlying cytokine network in inflammatory diseases allowed the establishment of therapies with biologics. Conversely, therapeutic targeting of single cytokines has also led to great advancements in the understanding of the distinctive roles cytokines play in autoimmune diseases. Nonetheless, these drugs have limitations. The endovenous administration, which most of these drugs require, is less than ideal. Not all patients show sufficient response and fulfill the expected treatment goals. Furthermore, these proteins harbor immunogenic potential which sometimes results in loss of response during continuous therapy (24). Notably, biologics with long half-life bear some risks during infections and may increase the risk of cancer and major cardiovascular events. Finally, their costs is beyond the means for some patients and health care systems.

Thus, development of alternative therapeutic strategies is desirable particularly for patients not responding to classical immunosuppressive drugs or targeted antibody therapies. Upon receptor binding, cytokines trigger signaling events involving several cytosolic substrates. These substrates, and their enzymatic activities, are ideal targets for the development of small molecules aiming at modulating cytokine-driven cellular responses.

In this viewpoint we discuss the arrival, in the clinical arena, of a new class of intracellular immunomodulators, which block kinases of the Janus family, better known as Jaks, which may re-revolutionize the treatment of autoimmune diseases and inflammatory disorders beyond biologics.

Cytokine signaling - moving from the cell surface to the inside

The diverse functions of cytokines in tissues are initiated by the binding of the cytokines to their binding to specific receptors and subsequent signal transmission. The subgroup of cytokines that uses type I and type II cytokine receptors comprises more than 60 members including hormone-like mediators, interleukins and IFNs. Among these, IL-6, IL-12, IL-19, IL-20, IL-21, IL-22 and IL-23 as well as IFNs are relevant in the pathogenesis of psoriasis, RA and many other inflammatory and autoimmune pathologies (25). Cytokine signaling is also important for the biology of non-immune cells like keratinocytes, fibroblasts, osteoblasts, synoviocytes or endothelial cells.

Upon binding of these cytokines to their specific type I or type II receptor, the receptor undergoes oligomerization. Since the receptor itself lacks intrinsic kinase activity, this oligomerization recruits Jaks which autophosphorlyate and then phosphorylate tyrosine residues within the receptor’s chains. This provides binding sites for STATs which bind and also become phosphorylated by Jaks. Activated STATs then dimerize and translocate into the nucleus where they regulate gene expression (Figure 1) Complexity in signaling in part is due to the fact that one or more members of the Jak family (Jak1, Jak2, Jak3, and Tyk2) and one or more members of STATs may be recruited by any specific receptor. For example, while receptors for hormone-like cytokines (erythropoietin (EPO), granulocyte macrophage colony-stimulating factor (GMCSF) use exclusively Jak2 (26), the type I IFNs use a combination of Jak1 and Tyk2, the IFN-γ receptor uses a combination of Jak1 and Jak2. The p40-sharing cytokines IL-12 and IL-23 activate receptors associating with a combination of Jak2 and Tyk2 (27). The group of γc-chain containing receptors, utilized by IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21, use Jak1 and Jak3 (Table 1) (28–30).

Table 1.

Cytokines, their associated JAK/STAT proteins and their role in Th cell differentiation and function.

Ligand (Cytokine)	Associated Jak kinases	Activated Stat proteins	Important for differentiation of	Effector cytokine secreted by
IL-2	Jak1, Jak3	Stat5, Stat3	Treg, Th2	Th1
IL-4	Jak1, Jak3	Stat6	Th2, Th9	Th2
IL-6	Jak1, Jak2, Tyk2	Stat3, Stat1	Th17
IL-7	Jak1, Jak3	Stat5, Stat3
IL-9	Jak1, Jak3	Stat5, Stat3		Th9
IL-10	Jak1, Tyk2	Stat3, Stat1		Treg, Th2, Th17
IL-12	Jak2, Tyk2	Stat4	Th1
IL-13	Jak1, Jak2, Tyk2	Stat6		Th2
IL-15	Jak1, Jak3	Stat5, Stat3
IL-21	Jak1, Jak3	Stat3, Stat1	Th17	Th17
IL-22	Jak1, Tyk2	Stat3, Stat1		Th17, Th22
IL-23	Jak2, Tyk2	Stat3, Stat4	Th17
IL-27	Jak1, Jak2, Tyk2	Stat1, Stat3	Th1
IFNα/β	Jak1, Tyk2	Stat1, Stat2	Th1
IFN-γ	Jak1, Jak2	Stat1	Th1	Th1
EPO	Jak2	Stat5
GM-CSF	Jak2	Stat3, Stat5

Inhibiting protein tyrosine kinases - a new old concept

Protein kinases like Jaks are enzymes, which transfer phosphate groups from adenosine triphosphate (ATP) or guanosine triphosphate to hydroxyl groups of amino acids of their substrates. Tyrosine kinases phosphorylate tyrosine residues and blocking their enzymatic activity has proved to be a successful strategy. Imatinib, a small, orally available, competitive molecule which blocks the ATP binding activity of the BCR-Abl tyrosine kinase, was developed for the treatment of patients with chronic myeloid leukemia (CML) (31). Imatinib has become a successful therapy in CML, demonstrating that the development of specific kinase inhibitors, which inhibit the activity of the cytokines involved in Th1/Th17-mediated diseases was clearly feasible.

Individuals with mutations within the JAK3 gene suffer from severe combined immunodeficiency, lacking T and NK and having reduced numbers of B cells (32, 33). These patients present with recurrent infections but do not have any other organ abnormalities. Thus, Jak3 was viewed as an excellent target for immunosuppressive therapies and resulted in the development of tofacitinib (CP-690,550). Preclinical trials with tofacitinib showed significant improvement in experimental models of autoimmunity and organ transplantation (34–36)

Currently, tofacitinib is approved in the USA, Japan, Switzerland, Russia, Argentina, Kuwait and the United Arab Emirates for the treatment of patients with RA. The European Medicines Agency (EMA) has yet to approve the marketing of tofacitinib in the European Union for RA. Clinical phase 2 and phase 3 trials showed efficacy in RA with American College of Rheumatology responses in patients failing disease-modifying antirheumatic drugs (DMARDs) or even TNF antagonists. Importantly, clinical responses were similar to those achieved with TNF antagonists (37–42). In psoriasis, oral tofacitinib showed a dose-dependent efficacy in phase 2 trials (43). Furthermore, tofacitinib demonstrated clinical effectiveness in a phase 2 study for ulcerative colitis (44). Adverse events such as leukopenia or lymphopenia were only observed in a subset of patients, arguing against a profound block of T or B cell homoeostasis. Moreover, neutropenia occurred rarely in tofacitinib-treated patients. Notably, anemia was documented in some patients (37–40, 45, 46). This adverse event could not be explained by selective Jak3 inhibition, since the erythropoietin receptor associates with Jak2 (26). In fact, when tofacitinib’s activity was carefully analyzed in vitro and in vivo, the results indicated that, although Jak3 was inhibited at nanomolar potency, other Jak family members especially Jak1, but also Jak2 and to a much lesser degree also Tyk2 were affected (35, 47). Thus, tofacitinib is a potent inhibitor of Jak3 and Jak1 with some activity against Jak2.

Targeting Jak1 or Jak2 was thought to be helpful for interfering with the signaling pathways implicated in the generation of pathogenic Th1 and Th17 cells in autoimmunity and inhibition of Jak2 was the initial intent, which resulted in the development of ruxolitinib (INCB018424). In 2005, independent groups identified the importance of a mutated Jak2 variant in myeloproliferative disorders (MPD) such as primary polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (48–51). These hematologic malignancies are characterized by excess proliferation of one or more myeloid lineages. The most common cause is a point mutation (JAK2V617F) present in 95% of patients with PV and over 50% of patients with ET and PMF.

Jak2 inhibitors like ruxolitinib SB1518, SAR302503 and lestaurtinib can interfere with the autophosphorylation of Jak2 in JAK2V617F-positive cells. In patients with primary myelofibrosis, ruxolitinib demonstrated significant clinical benefits reducing spleen size, myelofibroisis-related symptoms and prolonging overall survival (52–54). As described for tofacitinib, the selectivity of these inhibitors for Jak2 was analyzed in detail. Ruxolitinib has been reported to inhibit both Jak2 and Jak1; nonetheless, the toxicity of ruxolitinib in humans seems to be limited. As expected, Jak2 inhibition frequently resulted in anemia and thrombocytopenia. As a Jak1/Jak2 inhibitor, ruxolitinib became the first Jak inhibitor approved by the Food and Drug Administration and EMA with therapeutic indication for the treatment of intermediate or high-risk myelofibrosis (55). In several preclinical studies, ruxolitinib showed efficacy in rodent models of arthritis and in animal models of skin inflammation (56, 57), and, therefore is also under clinical evaluation for RA and psoriasis.

To be or not to be… selective?

Imatinib was designed and thought to be a selective BCR-Abl inhibitor. In reality it blocks multiple kinases (47) and this off-target activity allowed the use of imatinib in other diseases (58). Similarly, most of the Jak inhibitors developed so far are selective for these kinases, but, as mentioned above, do not discriminate well among the Jak family members (47, 58). The Jak inhibitors approved so far target at least two Jaks with nanomolar potency. Such non-specificity may indeed explain their efficacy in Th1/Th17-mediated autoimmunity and pathologies. Such promiscuity in inhibition leads often to concerns of toxicity, however, although not yet assessed in long-term studies, the toxicity of the Jak inhibitors seems to be limited. The limited toxicity observed so far could be due to the rapid kinetics of action of such compounds. It is likely that Jak inhibitors lower the degree of STAT activation in vivo without causing permanent or total blockade of this pathway. Furthermore, cytokines have in immune cells their major targets and therefore Jak inhibitors could preferentially act on activated immune cells.

The generation of highly selective inhibitors, with no off-target activity against other Jaks, may result in increased efficacy and safety, as some of the anti-inflammatory cytokines could be spared. For instance, a selective Tyk2 inhibitor would target the signals of IL-12 and IL-23 and probably mimic, at least to some extent, the therapeutic effects of an anti-p40 antibody.

Immunological mode of action of Jak inhibitors

Tofacitinib and ruxolitinib both inhibit STAT3 phosphorylation (Table 1) (36, 56). In the case of tofacitinib, it was predicted that this Jak3/Jak1 inhibitor would block IL-17 production. Yet, surprisingly, when naïve Th cells were treated with the Th17-promoting cytokines, a combination of IL-6 and TGF-β, in the presence of tofacitinib, IL-17 production was dramatically enhanced (36). This observation can be explained by the opposing roles that STAT3 and STAT5 have on Th17 differentiation (59). STAT5 inhibition by tofacitinib enhances IL-17 expression. In contrast, in the absence of TGF-β, tofacitinib is a potent inhibitor of Th17 cells (36). Tofacitinib limits the production of IL-17A, IL-17F and IL-22, expression of the IL-23R as well as differentiation of Th1 cells. Other Jak1 inhibitors have similar effects on pathogenic Th1 and Th17 responses. Ruxolitinib can reduce the expression of the Th1 cytokine IFN-γ and the Th17 cytokines IL-17, IL-21 and IL-22 in a rodent model of arthritis (56). In patients with myelofibrosis, ruxolitinib decreased circulating inflammatory cytokines like IL-6 and TNF (52).

However, additional actions on other cell types of the immune system like macrophages, NK and B cells may contribute to the antirheumatic and antipsoriatic activity of Jak inhibitors. Decreased numbers of NK cells were observed in tofacitinib-treated individuals (45). Surprisingly, tofacitinib can even modulate innate immune responses as demonstrated in a model of LPS-induced endotoxemic shock (36).

Lowering both innate and adaptive immune responses seems to be advantageous for the efficacy of oral immunomodulating compounds in psoriasis and arthritis (60, 61). Some of the immunological effects observed when using Jak inhibitors resemble the effects when inhibiting the IL-6 receptor or p40. Yet, only highly selective inhibitors for single Jaks or STAT3-targeting compounds may allow decipher the relevance of each signaling molecule.

Jak inhibitors in dermatology

As mentioned above, oral tofacitinib is already in phase 3 for the treatment of patients with psoriasis. Baricitinib (also known as LY3009104) and the Jak1 inhibitor GSK2586184 are both in phase 2 (Table 2). Interestingly, both orally approved Jak inhibitors tofacitinib and ruxolitinib are also studied as topical formulations in psoriasis (62, 63). The data on efficacy and safety of topical Jak inhibitors formulations are important since these therapeutics could revolutionize the treatment modalities in dermatology. Topical treatment of skin inflammation is mainly done with corticosteroids and their continuous use can result in skin atrophy and teleangiectasia. So far, only a few immunomodulating ointments are available like topical vitamin D derivatives for psoriasis (64) and topical calcineurin inhibitors for atopic dermatitis (65).

Table 2.

Clinical stage of some Jak inhibitors in inflammatory autoimmune diseases and myeloproliferative disorders

Inhibitor	Primary targets	Indications and developmental stage
Tofacitinib (Xeljanz®)	Jak1, Jak3	PSO (phase 3), RA*, UC (phase 2)
Ruxolitinib (Jakafi®)	Jak1, Jak2	PSO (phase 2), RA (phase 2), PMF#, PV and ET (phase 3)
Baricitinib	Jak1, Jak2	PSO (phase 2), RA (phase 3),
R333 and R348	Jak3, Syk	LUPUS (phase 2), DED (phase 1)
SAR302503	Jak2	PMF (phase 2), PV and ET (phase 2)
VX-509	Jak3	RA (phase 2)
GLPG0634	Jak1	RA (phase 2)
GSK2586184	Jak1	PSO (phase 2), LUPUS (phase 2)
Lestaurtinib	Jak2, Flt3	PSO (Phase 2), PMF (phase 2), PV and ET (phase 2)
CYT387	Jak1, Jak2	PMF (phase 2), PV and ET (phase 2)
AZD 1480	Jak1, Jak2	PMF, PV and ET (phase 2)
Pacritinib	Jak2	PMF (phase 2)
AC430	Jak2	RA (phase 1)

RA, rheumatoid arthritis; PSO, psoriasis; PMF, primary myelofibrosis; ET, essential thrombocythemia; PV, polycythemia vera; LUPUS, lupus erythematosus; DED, drye eye disease;

^*approved in US and Japan;

^#approved in US and EU

Interestingly, both compounds also affect keratinocyte biology. In psoriatic skin, IL-22-mediated STAT3 activation in keratinocytes results in acanthosis (66). Topical Jak inhibitors could directly dampen STAT3 activation in keratinocytes and affect the expression of numerous genes implicated in epidermal differentiation (67, 68). The anti-inflammatory and anti-proliferative potential of topical Jak inhibitors, if accompanied with limited side effects, could be helpful in a number of skin diseases like psoriasis, contact dermatitis, atopic dermatitis, lupus erythematosus, lichen planus and alopecia.

Conclusions

Given the success of tofacitinib and ruxolitinib, several other compounds are in various phases of development (Table 2). Inhibitors currently in trials for RA include baricitinib (Jak1/Jak2), VX-509 (Jak3), GLPG0634 (Jak1) and AC430 (Jak2). Safety and efficacy in the setting of myelofibrosis was also recently demonstrated for the presumably more selective Jak2 inhibitor SAR302503 (69). Other Jak inhibitors tested for myeloproliferative disorders include AZD1480 and CYT387.

It has been 20 years since we started to understand signaling downstream of cytokine receptors. Importantly we can now manipulate cytokine activity and limit their actions by inhibiting proximal cytoplasmic molecules like the Jaks. So far, the clinical results are promising and we believe that with newer and more specific inhibitors safety and efficacy will improve. The biologics were a huge clinical success and we may be on the brink of another therapeutic revolution. We may, once again… hit the Jakpot!