Cutaneous JAK Expression in Vitiligo.

BACKGROUND: The Janus kinase-signal transducer and activator of transcription signaling pathway has been suggested as a promising therapeutic target in vitiligo. However, limited data is available on the cutaneous expression of JAK in vitiligo. AIM: This study is designed to analyze the cutaneous expression patterns of JAK1, 2, and 3 in vitiligo and investigate their relation to the disease clinical parameters. METHODS: This case-control study recruited 24 patients having active vitiligo and 20 age, sex, and skin type-matched healthy volunteers. Skin biopsies were obtained from patients (lesional, perilesional and nonlesional) and controls for assessment of JAK1, 2, and 3 expression using RT-PCR. RESULTS: JAK1 and JAK3 were overexpressed in patients' skin compared to control skin and showed a stepwise pattern of upregulation from control to nonlesional, perilesional and lesional skin. However, JAK3 showed much stronger expression. In contrast JAK2 expression showed no significant difference in any of lesional, perilesional or nonlesional skin compared to control skin. JAK1 and JAK3 expression levels showed no correlation with neither the disease activity nor severity. CONCLUSION: JAK1 and more prominently JAK3 are upregulated in vitiliginous skin and possibly contribute to the pathogenesis of the disease. Accordingly, selective JAK3/1 inhibition may provide a favorable therapeutic opportunity for vitiligo patients.This study is registered on the ClinicalTrials.gov Identifier: NCT03185312.

Aminocyanopyridines as anti-lung cancer agents by inhibiting the STAT3 pathway.

Lung cancer is a leading cause of cancer-related death worldwide. Cyanopyridines and aminocyanopyridines with carbon-nitrogen bonds have been proved to exert significant anticancer, antibacterial, and anti-inflammatory effects. In this study, we showed that aminocyanopyridine 3o and 3k displaying potent antitumor activity via inhibiting the signal transducer and activator of transcription 3 (STAT3) pathway. They blocked the constitutive STAT3 phosphorylation in a dose- and time-dependent manner and regulated the transcription of STAT3 target genes encoding apoptosis factors. Most importantly, 3o also inhibited interleukin-6-induced STAT3 activation and nuclear localization. Furthermore, 3o significantly inhibited the tumor growth of H460-derived xenografts. Taken together, these findings suggest that 3o and 3k are promising therapeutic drug candidates for lung cancer by inhibiting persistent STAT3 signaling.

SOCS3 overexpression enhances ADM resistance in bladder cancer T24 cells.

OBJECTIVE: JAK-STAT3 signaling pathway widely participates in cell proliferation and apoptosis. Suppressor of cytokine signaling 3 (SOCS3) is a negative regulator of JAK-STAT3. SOCS3 downregulation is associated with drug resistance in breast cancer and leukemia. However, its role in bladder cancer drug resistance is still unclear. This study established ADM resistant bladder cancer cell model to investigate the role of SOCS3-JAK/STAT3 signaling pathway ADM resistance. MATERIALS AND METHODS: ADM drug resistant cell line T24/ADM was established. SOCS3, p-JAK2, p-JAK3, and Bcl-2 expressions in T24/ADM, T24, and HBEC cells were compared. Cell proliferation and apoptosis were evaluated by flow cytometry. T24/ADM cells were divided into five groups, including control, pSicoR-blank, pSicoR-SOCS3, FLLL32, and pSicoR-SOCS3 + FLLL32 groups. Cell proliferation was determined by EdU staining. RESULTS: SOCS3 was reduced, while p-JAK2, p-STAT3, and Bcl-2 expressions upregulated in T24 cells compared with HBEC cells. T24/ADM cells exhibited lower SOCS3, higher p-JAK2, p-STAT3, and Bcl-2 levels than T24 cells. Cell apoptosis was higher, whereas cell proliferation was weaker in T24 cells compared with T24/ADM cells. SOCS3 overexpression and/or FLLL32 treatment significantly downregulated p-JAK2, p-STAT3, and Bcl-2 expressions, attenuated cell proliferation, and elevated sensitivity to ADM induced cell apoptosis. CONCLUSIONS: SOCS3 reduction was associated with bladder cancer sensitivity to ADM. SOCS3 overexpression decreased JAK-STAT3 signaling pathway activity, declined Bcl-2 expression, inhibited cell proliferation, elevated cell apoptosis, and enhanced ADM sensitivity in T24 cells.

Recent progress and perspective in JAK inhibitors for rheumatoid arthritis: from bench to bedside.

Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by synovial inflammation and joint destruction. However, the combined use of synthetic disease-modifying anti-rheumatic drug (DMARD) such as methotrexate and a biological DMARD targeting tumour necrosis factor (TNF) has revolutionized treatment of RA. Clinical remission is a realistic target to treat and the maintenance of remission has produced significant improvements in structural and function outcomes. However, biological DMARDs are limited to intravenous or subcutaneous uses and orally available small but strong products have been developed. The multiple cytokines and cell surface molecules bind to receptors, resulting in the activation of various signalling, including phosphorylation of kinase proteins. Among multiple kinases, Janus kinase (JAK) plays pivotal roles in the pathological processes of RA. Tofacitinib, a small product targeting JAK, inhibits phosphorylation of JAK1 and JAK3, subsequent Stat1 and expression of Stat1-inducible genes, which contribute to efficient propagation of its anti-inflammatory effects for the treatment of RA. The primary targets of tofacitinib are dendritic cells, CD4(+) T cells such as Th1 and Th17 and activated B cells which leads to multi-cytokine targeting. Six global phase 3 studies revealed that oral administration of 5 or 10 mg tofacitinib was significantly effective than placebo with or without methotrexate in active RA patients with methotrexate-naïve, inadequately responsive to methotrexate or TNF-inhibitors. Therapeutic efficacy of tofacitinib was observed in a short term after administration and was as strong as adalimumab, a TNF-inhibitor. The most commonly observed adverse events were related to infection, hematologic, hepatic and renal disorders and association of tofacitinib with carcinogenicity and infections remains debated. Further investigation on post-marketing survey would help us understand the positioning of this drug.

Sorafenib treatment of FLT3-ITD(+) acute myeloid leukemia: favorable initial outcome and mechanisms of subsequent nonresponsiveness associated with the emergence of a D835 mutation.

Internal tandem duplication (ITD) of the fms-related tyrosine kinase-3 (FLT3) gene occurs in 30% of acute myeloid leukemias (AMLs) and confers a poor prognosis. Thirteen relapsed or chemo-refractory FLT3-ITD(+) AML patients were treated with sorafenib (200-400 mg twice daily). Twelve patients showed clearance or near clearance of bone marrow myeloblasts after 27 (range 21-84) days with evidence of differentiation of leukemia cells. The sorafenib response was lost in most patients after 72 (range 54-287) days but the FLT3 and downstream effectors remained suppressed. Gene expression profiling showed that leukemia cells that have become sorafenib resistant expressed several genes including ALDH1A1, JAK3, and MMP15, whose functions were unknown in AML. Nonobese diabetic/severe combined immunodeficiency mice transplanted with leukemia cells from patients before and during sorafenib resistance recapitulated the clinical results. Both ITD and tyrosine kinase domain mutations at D835 were identified in leukemia initiating cells (LICs) from samples before sorafenib treatment. LICs bearing the D835 mutant have expanded during sorafenib treatment and dominated during the subsequent clinical resistance. These results suggest that sorafenib have selected more aggressive sorafenib-resistant subclones carrying both FLT3-ITD and D835 mutations, and might provide important leads to further improvement of treatment outcome with FLT3 inhibitors.

Janus kinase 3 is expressed in erythrocytes, phosphorylated upon energy depletion and involved in the regulation of suicidal erythrocyte death.

Janus kinase 3, a tyrosine kinase expressed in haematopoetic tissues, plays a decisive role in T-lymphocyte survival. JAK3 deficiency leads to (Severe) Combined Immunodeficiency (SCID) resulting from enhanced lymphocyte apoptosis. JAK3 is activated by phosphorylation. Nothing is known about expression of JAK3 in erythrocytes, which may undergo apoptosis-like cell death (eryptosis) characterized by cell membrane scrambling with phosphatidylserine exposure and cell shrinkage. Triggers of eryptosis include energy depletion. The present study utilized immunohistochemistry and confocal microscopy to test for JAK3 expression and phosphorylation, and FACS analysis to determine phosphatidylserine exposure (annexin binding) and cell volume (forward scatter). As a result, JAK3 was expressed in erythrocytes and phosphorylated following 24h and 48h glucose depletion. Forward scatter was slightly but significantly smaller in erythrocytes from JAK3-deficient mice (jak3(-/-)) than in erythrocytes from wild type mice (jak3(+/+)). Annexin V binding was similarly low in both genotypes. The JAK3 inhibitors WHI-P131/JANEX-1 (4-(4'-Hydroxyphenyl)amino-6,7-dimethoxyquinazoline, 156µM) and WHI-P154 (4-[(3'-Bromo-4'-hydroxyphenyl)amino]-6,7-dimethoxyquinazoline, 11.2µM) did not significantly modify annexin V binding or forward scatter. Glucose depletion increased annexin V binding, an effect significantly blunted in jak3(-/-) erythrocytes and in the presence of the JAK3 inhibitors. The observations disclose a completely novel role of Janus kinase 3, i.e. the triggering of cell membrane scrambling in energy depleted erythrocytes.

JAK3 pathway is constitutively active in B-lineage acute lymphoblastic leukemia.

In this article, we report that primary leukemic B-cell precursors from B-lineage acute lymphoblastic leukemia (ALL) patients overexpress multiple JAK3-activating cytokines as well as their receptors. We also show that amplified expression of JAK3 pathway genes in B-lineage ALL is associated with steroid resistance and relapse. Our findings further demonstrate that several different diagnostic classes of B-lineage lymphoid malignancies exhibit upregulated expression of JAK3 pathway genes, which are associated with an overexpression of genes for JAK3-stimulatory cytokines with concomitant deficiency of JAK3-inhibitory signaling molecules. Thus, despite the rare occurrence of activating JAK3 mutations, JAK3 appears to be constitutively active and represents a viable molecular target in the treatment of a broad range of B-lineage lymphoid malignancies, including B-lineage ALL.

Expression, purification, and characterization of TYK-2 kinase domain, a member of the Janus kinase family.

The Janus kinase family consists of four members: JAK-1, -2, -3 and TYK-2. While JAK-2 and JAK-3 have been well characterized biochemically, there is little data on TYK-2. Recent work suggests that TYK-2 may play a critical role in the development of a number of inflammatory processes. We have carried out a series of biochemical studies to better understand TYK-2 enzymology and its inhibition profile, in particular how the TYK-2 phosphorylated forms differ from each other and from the other JAK family members. We have expressed and purified milligram quantities of the TYK-2 kinase domain (KD) to high purity and developed a method to separate the non-, mono- (pY(1054)) and di-phosphorylated forms of the enzyme. Kinetic studies (k(cat(app))/K(m(app))) indicated that phosphorylation of the TYK-2-KD (pY(1054)) increased the catalytic efficiency 4.4-fold compared to its non-phosphorylated form, while further phosphorylation to generate the di-phosphorylated enzyme imparted no further increase in activity. These results are in contrast to those obtained with the JAK-2-KD and JAK-3-KD, where little or no increase in activity occurred upon mono-phosphorylation, while di-phosphorylation resulted in a 5.1-fold increase in activity for the JAK-2-KD. Moreover, ATP-competitive inhibitors demonstrated 10-30-fold shifts in potency (K(i(app))) as a result of the TYK-2-KD phosphorylation state, while the shifts for JAK-3-KD were only 2-3-fold and showed little or no change for JAK-2-KD. Thus, the phosphorlyation state imparted differential effects on both activity and inhibition within the JAK family of kinases.

Gene expression profiles of infant acute lymphoblastic leukaemia and its prognostically distinct subsets.

This study compared the gene expression profiles of primary leukaemic cells from infants versus children with acute lymphoblastic leukaemia (ALL). Our analyses provided unprecedented evidence that remarkably different pathognomonic transcriptomes dominate the biology of infant versus paediatric high risk ALL. The genetic signature of infant ALL is characterized by concomitant overexpression of mitogenic and anti-apoptotic genes, some of which have been associated with early relapse in ALL. Our study demonstrated that primary leukaemia cells from infant ALL patients expressed significantly higher levels of genes for cytokines that mediate their biological effects through stimulation of the JAK-STAT signal transduction pathway including interleukin 1a, interleukin 1b, interleukin 2, and interleukin 7. We further showed that the JAK/STAT signalling pathway is constitutively active in CD10(-) infant ALL cells and treatment with a JAK3 inhibitor or a pan-JAK kinase inhibitor effectively triggered their apoptosis. These findings identified JAK3 as an attractive molecular target for disrupting the constitutively deregulated anti-apoptotic STAT3 and STAT5 signalling pathways in infant ALL cells.

IL-21 contributes to JAK3/STAT3 activation and promotes cell growth in ALK-positive anaplastic large cell lymphoma.

Interleukin (IL)-21 has been reported to both stimulate cell growth and promote survival in benign lymphoid cells and several types of hematopoietic neoplasms. It induces JAK3/STAT3 signaling, a biologically important cellular pathway activated in most cases of anaplastic lymphoma kinase (ALK)-expressing anaplastic large cell lymphoma (ALK(+)ALCL). Therefore, we hypothesize that IL-21 may contribute to JAK3/STAT3 activation and cell growth in ALK(+)ALCL. By reverse transcription-PCR, we found consistent expression of IL-21 receptor (IL-21R) in all ALK(+)ALCL cell lines and frozen tumors examined. IL-21 was also consistently expressed in ALK(+)ALCL tumors, although its mRNA was detectable in only one of three cell lines tested. By immunohistochemistry, we examined 10 paraffin-embedded ALK(+)ALCL tumors; all cases were positive for both IL-21 and IL-21R in these neoplastic cells. IL-21 signaling is biologically significant in ALK(+)ALCL since the addition of recombinant IL-21 enhanced the activation of JAK3/STAT3 and significantly increased cell growth in ALK(+)ALCL cell lines. However, small interfering RNA down-regulation of IL-21R significantly decreased both STAT3 activation and cell growth. IL-21R expression is not linked to nucleophosmin-ALK since forced expression of nucleophosmin-ALK and small interfering RNA down-regulation of nucleophosmin-ALK did not significantly change the expression of either IL-21R or IL-21. Our findings thus support the enhancement of JAK3/STAT3 activation and cell growth in ALK(+)ALCL via IL-21 signaling. These results further support the concept that constitutive activation of STAT3 in these tumors is multifactorial.

Downregulation of Janus kinase 3 expression by small interfering RNA in rat composite tissue allotransplantation.

RNA interference (RNAi) has recently emerged as an efficient method to silence gene expression in mammalian cells by transfection of small interfering RNAs (siRNAs). The Janus kinase 3 (JAK3) is also pertinent to the development of a new immuno-suppressant. This study aimed to inhibit JAK3 expression using RNAi to determine allograft tolerance. To silence JAK3 expression, one dsRNA was tested to incorporate the JAK3 mRNA sequence. The expression vector containing the pre-mRNA expression cassette was transfected into rat basophilic leukemia cell line, RBL-2H3, for RNAi analysis (in vitro). The alloskin and composite tissue allograft were then transplanted to recipients using RNAi protocol to determine the allograft tolerance (in vivo). The results showed effective in vitro and in vivo downregulation of JAK3 expression by RNAi. Moreover, the histology of alloskin graft and composite tissue allograft (in vivo) under the siRNA showed more prominently diminished inflammatory infiltration than the control group. This is the first time in the literature that the suppressive effect of JAK3 silenced by siRNA has been tested both in vitro and in vivo, and shows that siRNA is capable of specific and functional silencing in allograft rejection.

Constitutive JAK3 activation induces lymphoproliferative syndromes in murine bone marrow transplantation models.

The tyrosine kinase JAK3 plays a well-established role during normal lymphocyte development and is constitutively phosphorylated in several lymphoid malignancies. However, its contribution to lymphomagenesis remains elusive. In this study, we used the newly identified activating JAK3A572V mutation to elucidate the effect of constitutive JAK3 signaling on murine lymphopoiesis. In a bone marrow transplantation model, JAK3A572V induces an aggressive, fatal, and transplantable lymphoproliferative disorder characterized by the expansion of CD8(+)TCRalphabeta(+)CD44(+)CD122(+)Ly-6C(+) T cells that closely resemble an effector/memory T-cell subtype. Compared with wild-type counterparts, these cells show increased proliferative capacities in response to polyclonal stimulation, enhanced survival rates with elevated expression of Bcl-2, and increased production of interferon-gamma (IFNgamma) and tumor necrosis factor-alpha (TNFalpha), correlating with enhanced cytotoxic abilities against allogeneic target cells. Of interest, the JAK3A572V disease is epidermotropic and produces intraepidermal microabscesses. Taken together, these clinical features are reminiscent of those observed in an uncommon but aggressive subset of CD8(+) human cutaneous T-cell lymphomas (CTCLs). However, we also observed a CD4(+) CTCL-like phenotype when cells are transplanted in an MHC-I-deficient background. These data demonstrate that constitutive JAK3 activation disrupts T-cell homeostasis and induces lymphoproliferative diseases in mice.

High yield expression of non-phosphorylated protein tyrosine kinases in insect cells.

The key role of kinases in signal transduction and cell growth regulation has been a long standing interest among academics and the pharmaceutical industry. Recombinant enzymes have been used to understand the mechanism of action as well as to screen for chemical inhibitors. The baculo-insect system has been the primary method used to obtain soluble and active kinases, usually producing a mixture of the kinase in various phosphorylation states in different conformations. To obtain a homogenous preparation of non-phosphorylated kinases is critical for biochemical, biophysical and kinetic studies aimed at understanding the mechanism of kinase activation. Taking advantage of the eukaryotic expression property of insect cells, we were able to obtain high yield expression of non-phosphorylated protein tyrosine kinases BTK, JAK3 and Eph2A through coexpression with the tyrosine phosphatase YopH, which suggests that this method can be applied to protein tyrosine kinases in general. We have demonstrated that the fully non-phosphorylated BTK obtained with this method is suitable for various biochemical and kinetic studies.

Kaempferol inhibits IL-4-induced STAT6 activation by specifically targeting JAK3.

IL-4 is involved in several human diseases including allergies, autoimmunity, and cancer. Its effects are mainly mediated through the transcription factor STAT6. Therefore, investigation of compounds that regulate STAT6 activation is of great interest for these diseases. Natural polyphenols are compounds reported to have therapeutic properties in diseases involving IL-4 and STAT6. The aim of this study was to investigate the effect of these compounds in the activation of this transcription factor. We found that in hemopoietic cells from human and mouse origin, some flavonoids were able to inhibit the activation of STAT6 by IL-4. To identify molecular mechanisms, we focused on kaempferol, the compound that showed the greatest inhibitory effect with the lowest cell toxicity. Treatment of cells with kaempferol did not affect activation of Src kinase by IL-4 but did prevent the phosphorylation of JAK1 and JAK3. Further enzymatic analysis demonstrated that kaempferol blocked the in vitro phosphorylation activity of JAK3 without affecting JAK1, suggesting that it specifically targeted JAK3 activity. Accordingly, kaempferol had no effect on STAT6 activation in nonhemopoietic cell lines lacking JAK3, supporting its selective inhibition of IL-4 responses through type I receptors expressing JAK3 but not type II lacking this kinase. The inhibitory effect of kaempferol was also observed in IL-2 but not IL-3-mediated responses and correlated with the inhibition of MLC proliferation. These findings reveal the potential use of kaempferol as a tool for selectively controlling cell responses to IL-4 and, in general, JAK3-dependent responses.