Sorting Out the Stats from the Jaks

If you browse the medical literature over the past year, you’re likely to come across one or more references to the 20th anniversary of the discovery of the Jak-Stat signal pathway.Stark GR, Darnell JE Jr. The Jak-Stat pathway at twenty. Immunity 2012;36:4:503-14.,O’Shea JJ, Holland SM, Staudt LM. JAKs and STATs in Immunity, Immunodefi ciency, and Cancer. N Engl J Med 2013;368:161-70. Who could forget the year Jak-Stat signaling was first identified? Perhaps you also recall 1992 was the year nitrous oxide won the Molecule of the Year award from Science magazine.Koshland DE. The molecule of the year. Science 1992;258:1861. No? That same year, H. Ross Perot ran a spirited independent presidential campaign and Jack Nicholson proclaimed “You can’t handle the truth!” in “A Few Good Men.” The point is this: 1992 was quite a while ago. Since then, we’ve learned just how crucial the Jaks, the Stats and all their various associates are in the function and malfunction of hundreds of cellular signaling pathways. In the eye, a host of pathological processes, from allergy, dry eye and glaucoma to uveitis and macular edema can be related to some disruption in a Jak-Stat pathway.Krishnan S, Lakshmanan S, Iyer GK, UmaMaheswari K, Krishnakumar S. The role of signaling pathways in the expansion of corneal epithelial cells in serum-free B27 supplemented medium. Mol Vis 2010;16:1169-77.,Malvitte L, Montange T, Vejux A, et al. Measurement of infl ammatory cytokines by multicytokine assay in tears of patients with glaucoma topically treated with chronic drugs. Br J Ophthalmol. 2007;91:1:29-32.,Koyasu S. Natural helper cells and TH2-type innate immunity. Cornea 2012;31:Suppl 1:S20-4 So perhaps it is an anniversary worthy of note.

Early on in studies of Jak-Stat function, it was clear that this signaling pathway had great promise as a target for therapeutic intervention. This month, we’ll catch up on the many roles Jak-Stat signaling plays in ocular physiology, describe some examples of conditions in which pathology includes dysregulation of those pathways and discuss the possibilities for new drugs to target Jak-Stat pathways in the eye.

Jak-Stat and Cytokines

The Jak-Stat pathway refers to two families of functionally related proteins, the Janus kinases, or Jaks, and the signal transducers and activators of transcription, or Stats. Together, these two convey the signals of extracellular cytokines, directing the on and off switches of genes and gene products responsible for the cells’ response to cytokine activation. The Jaks are named after Janus, the two-faced Roman god who is associated with doorways, transition and change. Janus, the protein kinase, monitors the extracellular atmosphere and guides cellular signals from the cell surface to the nucleus.

Cytokines are a diverse, multiplelineage family of autocoids that serve as regulatory signals for numerous cell types. Growth hormone, erythropoietin and prolactin are cytokines, as are the 23 or so interleukins, the interferons, the colony stimulating factors, the bone morphogenic factors—and the list goes on. Studies of interferon signaling led to the identifi cation of the Jak-Stat pathway that has since been shown to be a critical part of all cytokine signaling.Velazquez L, Fellous M, Stark GR, Pellegrini S. A protein tyrosine kinase in the interferon alpha/beta signaling pathway. Cell 1992;70:313-322. (See the diagram, opposite page.)

Jak-Stat signalingIn Jak-Stat signaling, Interleukin-2 initiates cellular responses by binding to the cell’s surface receptor, activating intracellular Jak1 and Jak3 kinases. Phosphorylation of kinase and receptor promotes association of Stat5B proteins, which are phosphorlyated, form a dimer and then translocate to the cell nucleus. There, they activate an IL-2 specifi c program of gene transcription and expression.

All cytokines signal by activating a specifi c variant of the Jak-Stat pathway. The general structure of a cytokine receptor is a trans-membrane, dimeric protein assembled from a pool of cytokine receptor monomers, each with an associated, intracellular Jak kinase.,O’Shea JJ, Plenge R. JAK and STAT Signaling Molecules in Immunoregulation and Immune-Mediated Disease. Immunity 2012;36:542-550. Cytokine binding initiates a cascade of phosphorylations: The associated Jak undergoes auto-phosphorylation and then phosphorylates intracellular sites on the cytokine receptor. These become binding sites for Stat proteins. The bound Stat is also phosphorylated, and this leads to formation of Stat dimers, which can translocate to the cell nucleus, associate with specifi c target DNA sequences, and turn on the expression of these gene products. While different cytokines may share a receptor, a Jak or a Stat, each assembles a unique combination of components in constructing its signaling pathway.

Another part of the diversity exhibited by Jak-Stat signaling comes from combinations of the four different Jaks (Jak1, Jak2, Jak3, Tyk2) plus seven different Stats (Stats1-4, 5A, 5B and 6). Both Jak1 and Jak2 are essential for survival (based on gene knockout studies in mice), while loss of Jak3 leads to failed immune system development and severe combined immunodeficiency, or SCID. The fourth Jak, Tyk2, is also essential for immune system development and homeostasis and is prominently associated with interferon signaling. Each of the seven Stats is also associated with specific physiological functions. Stat 1 is a key mediator of interferon signaling, and Stat 1 defi cits are associated with infectious diseases. Stat 5A and 5B are important for growth and development, and mediate actions of GH, prolactin, Epo and IL-2.

Stat4 and Stat6 are primary signaling molecules for many interleukins, and polymorphisms of each are associated with a host of immunological disorders including rheumatoid arthritis, lupus, allergy and asthma.

Sample of small-molecule jak inhibitors

Two of the better-known pathways employing Jak-Stat transduction are those of growth hormone and erythropoietin. Beyond its effects on muscle and bone, GH stimulates synthesis of insulin-like growth factor 1 via a Jak2/ Stat5B pathway in the liver. It’s this IGF-1 that mediates the majority of anabolic effects ascribed to GH. The blood-forming cells of the marrow respond to Epo (and several other cytokines) via a Jak2/Stat5A pathway to stimulate proliferation and differentiation of red cells, platelets and other myeloid cells. These examples highlight the importance of Jak-Stat signaling, and also provide clues to possible adverse effects of drugs designed to disrupt Jak-Stat pathways.

Jak-Stats in the Eye

The central function of Jak-Stat signaling in the eye involves infl ammatory responses. In the retina, infl ammation associated with glaucoma, macular edema or macular degeneration has been linked to TNF-α, a cytokine that signals, in part, by activating Jak-Stat pathways.Tezel G, Li LY, Patil RV, Wax MB. TNF-alpha and TNF-alpha receptor-1 in the retina of normal and glaucomatous eyes. Invest Ophthalmol Vis Sci 2001;42:8:1787-94. A recent study showed that human retinal epithelial cells produced VEGF in response to TNF-α, interferon-γ or IL-1β, demonstrating a link between Jak-Stat signaling and the neovascular response that is central to age-related macular degeneration and diabetic retinopathy. Moreover, TNF-α can perpetuate a cycle of Th1, pro-infl ammatory signaling by stimulating other cytokines such as IL-2 and IL-17.

Inflammation in the front of the eye is central to late-phase allergy, chronic allergy, dry eye and uveitis.Barabino S, Chen Y, Chauhan S, Dana R. Ocular surface immunity: Homeostatic mechanisms and their disruption in dry eye disease. Prog Retin Eye Res 2012;31:3:271-85. These disorders involve infi ltration of ocular tissues by immune cells such as macrophages, neutrophils and eosinophils. Inflammation creates an environment with increased secretion of chemokines, metalloproteinases and cytokines that can promote surface damage and a disruption of epithelial barriers. For the ocular surface, this can also mean the loss of protective functions provided by goblet cells and a healthy tear fi lm. Virtually all of these events involve activation of Jak-Stat signaling pathways, and, in particular, require participation by Jak3. Mild allergic infl ammation can be mitigated by reductions in allergen exposure, antihistamines and topical steroids, but therapeutic choices for dry-eye sufferers and other types of ocular infl ammation are limited.

Break the Inflammation Cycle

Among the anti-infl ammatory drugs available, systemic or topical glucocorticoids are the most effective at reducing inflammation, but they have adverse effects that limit them. Despite this, it’s interesting to note that GC inhibition of Jak-Stat signaling is known to be a key to their therapeutic effi cacy.Bianchi M, Meng C, Ivashkiv LB. Inhibition of IL-2-induced Jak- STAT signaling by glucocorticoids. PNAS 2000;97:9573. This is primarily due to the GC inhibition of IL-2, Jak3-Stat5 mediated infl ammatory pathway, so it’s reasonable to presume that other drugs that can interfere with this or other Jak-Stats might also be effective anti-infl ammatories.

In 20 years of research and clinical development, a number of biologicals and small-molecule inhibitors of Jak- Stat signaling have been developed and tested.Tayal V, Kalra BS. Cytokines and anti-cytokines as therapeutics — An update. Eur. J Pharm 2008;579: 1–12.,Kontzias A, Kotlyar A, Laurence A, et al. Jakinibs. A new class of kinase inhibitors in cancer and autoimmune disease. Cur Opin Pharm 2012;12:464-470. Many of these drugs block activation by cytokines or their receptors, so the inhibition of Jak-Stat signaling is indirect. A major focus has been in the areas of cancer treatment and autoimmune disease. Monoclonal antibodybased therapies targeting cytokines such as TNF (adalimumab, etanercept, and infl iximab), IL-1 (canakinumab) or cytokine receptors (IL-2 basiliximab, IL-6 tocilizumab) have all been shown effective in autoimmune diseases, for prevention of transplant rejection, and for some cancers. Recent small-scale, prospective studies testing intravitreal injection of agents such as anti-TNF (adalimumab)Androudi S, Tsironi E, Kalogeropoulos C, et al. Intravitreal adalimumab for refractory uveitis-related macular edema. Ophthalmology 2010 Aug;117:8:1612-6. or anti-IL-6 (tocilizumab)Muselier A, Bielefeld P, Bidot S, Vinit J, Besancenot JF, Bron A. Effi cacy of tocilizumab in two patients with anti-TNF-alpha refractory uveitis. Ocul Immunol Infl amm. 2011;19:5:382-3. for refractory uveitis have shown mixed results, but several larger prospective trials are under way. Based on the success of anti-VEGF treatments for neovascular AMD, the prospects for success with biologicals for retinal inflammation seem high, but remain to be seen.

Use of biologicals for anterior segment conditions is diffi cult because of drug delivery issues. Here, the focus of Jak-Stat inhibition is on small-molecule inhibitors. A key issue for this drug development task is kinase specifi city: A Jak-specifi c anti-infl ammatory agent should preferentially target Jak3 and Jak1. To date, two drugs that act as Jak inhibitors have gained Food and Drug Administration approval. Ruxolitinib (Jakafi ; Incyte, Novartis) is a Jak1/Jak2 inhibitor used to treat myelofibrosis and polycythemia vera, and Tofacitinib (Xeljanz; Pfi zer), a nonspecifi c Jak antagonist, was approved for rheumatoid arthritis in 2012.h t t p : / / w w w.fda.gov/NewsEvents/Newsroom/ PressAnnouncements/ucm327152.htm. Accessed Feb 26, 2013. Both are systemic medications in an oral formulation, and both come with the potential for adverse effects including infection, anemia and neutropenia. Other agents in the pipeline generally focus on RA or on specific cancers, but all must deal with a similar spectrum of adverse effects of oral delivery.

Compounds such as these that have already met the FDA standard for systemic disease are well-positioned to be screened for topical use. Several recent clinical trials have tested topical formulations of Jak inhibitors for either skin conditionsPunwani N, Scherle P, Flores R, et al. Preliminary clinical activity of a topical JAK1/2 inhibitor in the treatment of psoriasis. J Am Acad Dermatol 2012;67:4:658-64. or for treatment of ocular surface disease.Liew SH, Nichols KK, Klamerus KJ, et al. Tofacitinib, a Janus Kinase Inhibitor for Dry Eye Disease. Ophthalmology 2012;119:1328-1335. Topical tofacitinib underwent a Phase I/II trial for treatment of dry-eye disease that included both a dose-ranging, safety component and a prospective effi cacy trial. Tofacitinib was superior to cyclosporine emulsion (the positive control) in terms of both frequency of adverse events and reports of ocular discomfort. While the drug did not reach its primary endpoint, it’s possible this refl ects issues of study design more than drug effi cacy. For example, neither tofacitinib nor cyclosporine was signifi – cantly better than vehicle in reducing corneal staining. Large placebo effects are often seen in dry-eye studies, and such confounding results can be mitigated by alterations in study design. (For a broader discussion of placebo effects in dry-eye trials see Therapeutic Topics, May 2011.)

A second Jak inhibitor in development for dry eye is R932348, also known as R348 (Rigel Pharma), a compound with preferential action against Jak3 that has demonstrated effi cacy in a number of preclinical studies.Deuse T, Velotta JB, Hoyt G, et al. Novel immunosuppression: R348, a JAK3- and Syk-inhibitor attenuates acute cardiac allograft rejection. Transplantation 2008;85:6:885-92.,Chang BY, Zhao F, He X, et al. JAK3 inhibition signifi cantly attenuates psoriasiform skin infl ammation in CD18 mutant PL/J mice. J Immunol 2009;183:3:2183-92.,Velotta JB, Deuse T, Haddad M, et al. A novel JAK3 inhibitor, R348, attenuates chronic airway allograft rejection. Transplantation 2009;87:5:653-9. This compound has a secondary activity as it also inhibits Syk kinase, another key intracellular target in immune signaling. The simultaneous inhibition of Jak and Syk may provide for reduced dosing, greater effi cacy or both when R932348 is compared to other Jak inhibitors.Kyttaris VC. Kinase inhibitors: a new class of anti-rheumatic drugs. Drug Des Devel Ther 2012;6:245-50. The combination is particularly appealing for topical formulation trials based upon the known impact of these targets in ocular surface inflammation. Currently in the midst of a Phase I safety/ dose ranging study (NCT01733992), it’s likely that R932348 will be an early candidate in the topical Jak/Syk inhibitor drug development race.

With a dozen or more Jak inhibitors in early- to mid-stage development, it seems likely that one or more of these will find a suitable formulation for topical delivery, and should become a lynchpin of therapy for ocular surface inflammation in the future. Perhaps 20 years from now, we’ll be celebrating the anniversary of the fi rst Jak-targeted topical drugs, the family of therapeutics that changed that way we treat ocular infl ammation.

Dr. Abelson is a clinical professor of ophthalmology at Harvard Medical School and senior clinical scientist at the Schepens Eye Research Institute. Dr. McLaughlin is a medical writer at Ora Inc.