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The efficacy of ritlecitinib, an oral JAK3/TEC family kinase inhibitor, on active and stable lesions was evaluated in patients with active non-segmental vitiligo in a phase 2b trial (NCT03715829). Patients were randomized to placebo or daily ritlecitinib 50 mg (with or without 4-week 100-mg or 200-mg loading dose), 30 mg, or 10 mg for 24 weeks. Active lesions showed greater baseline expression of inflammatory/immune markers IFNG and CCL5, levels of CD103, and T-cell infiltrates than stable lesions. Patients with more active than stable vitiligo lesions showed higher baseline serum levels of CXCL9 and PD-L1, while patients with more stable than active lesions showed higher baseline serum levels of HO-1. At Week 24, ritlecitinib 50 mg significantly stabilized mean percent change from baseline in depigmentation extent in both active lesions and stable lesions vs. placebo-response, with stable lesions showing greater repigmentation. After 24 weeks of treatment, ritlecitinib 50 mg increased expression of melanocyte markers in stable lesions, while Th1/Th2-related and co-stimulatory molecules decreased significantly in both stable and active lesions. Serum from patients with more active than stable lesions showed decreased levels of ICOS and NK cell activation markers. These data, confirmed at transcription/protein levels, indicate that stable lesion repigmentation occurs early with ritlecitinib, while active lesions require stabilization of inflammation first. ClinicalTrials.gov: NCT03715829.

Objective To explore the relationship between the expression of Toll like receptor 4 (TLR4) and Janus protein tyrosine kinase 3 (JAK3) genes and the imbalance of helper T cell 17 (Th17)/regulatory T cells (Treg) in peripheral blood mononuclear cells (PBMCs) of patients with ankylosing spondylitis (AS). Methods A total of 101 AS patients were selected as the AS group. According to the AS Disease Activity Index (BASDAI) score, patients were divided into the AS stable phase (ASS) group (＜4 points, 42 cases) and the AS active phase (ASA) group (≥ 4 points, 59 cases). Additionally, 50 healthy volunteers who underwent physical examinations at our hospital during the same period were selected as the control group (health group). Real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) was used to determine the mRNA expression of TLR4 and JAK3 in PBMCs, and the proportion of serum Th17 and Treg was measured and Th17/Treg levels were calculated. Results The levels of TLR4 mRNA and JAK3 mRNA in PBMCs of peripheral blood were higher in the AS group than those in the health group (P＜0.05). The levels of Th17, proportion and Th17/Treg in peripheral blood were higher in the AS group than those of the health group (P＜0.05). Higher levels of TLR4 mRNA, JAK3 mRNA, Th17, and Th17/Treg were risk factors for AS, while higher level of Treg was protective factors for AS. TLR4 mRNA and JAK3 mRNA in AS patients were positively correlated with Th17 ratio and Th17/Treg ratio, and negatively correlated with Treg ratio (P＜0.05). Conclusion TLR4 and JAK3 genes are highly expressed and Th17/Treg is unbalanced in PBMC of AS patients. The high expression of TLR4 and JAK3 genes is closely related to Th17/Treg imbalance in AS patients. [ABSTRACT FROM AUTHOR]

BackgroundDi(2-ethylhexyl) phthalate (DEHP) is the highest consumed and the most widely used phthalic acid ester, their effects on lipid metabolism have attracted the attention of many scholars. However, the associated mechanism is still unclear.ObjectiveTo observe the effect of DEHP on lipid metabolism in rats, probe its possible mechanism, and provide a research basis for the effect of DEHP on human lipid metabolism.MethodsForty healthy male SD rats were randomly divided into 4 groups: solvent control (0 mg·kg−1 DEHP), low DEHP (187 mg·kg−1), medium DEHP (375 mg·kg−1), and high DEHP (750 mg·kg−1) groups. DEHP was administered by oral gavage for 6 d per week, consecutively 8 weeks. The rats were weighed once a week during the exposure period. At 24 h after the last exposure, the rats were anesthetized with 20% urethane and sacrificed by apical puncture. Rat livers were harvested and weighed before hematoxylin-eosin (HE) histopathological observation. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the mRNA levels of lipid metabolism-related genes Janus kinase 3 (JAK3), signal transducer and activator of transcription 5b (STAT5b), and peroxisome proliferator-activated receptor γ (PPARγ) in liver, and Western blot was used to detect the expression levels of lipid metabolism-related proteins JAK3, STAT5b, and PPARγ in liver.ResultsCompared with the control group, there was no significant difference in the body weight gain of the rats in each group (P>0.05). The liver organ coefficients of the DEHP exposure groups were higher than that of the control group (P

Background: T-cell-mediated acute kidney transplant rejection (TCMR) is a post-transplantation complication characterized by the infiltration of immune cells into the tissue and sudden dysfunction of the allograft. To develop effective diagnostic and therapeutic methods, it is crucial to understand the molecular mechanisms involved in the initiation and progression of TCMR. Janus kinase (JAK) enzymes, which are involved in proinflammatory cytokine signal transduction, may play a role in TCMR. Given the recent development of JAK inhibitors, the role of JAK molecules should be explored to determine whether they could be potential targets for the treatment of TCMR. Objectives: The study aimed to compare the relative expression of JAK-1, JAK-2, and JAK-3 genes between patients diagnosed with TCMR and stable recipients. Methods: The expression of JAK-1, JAK-2, and JAK-3 genes was evaluated in peripheral blood monocytes of 30 patients with TCMR and 30 stable patients using the RT-PCR method. Results: Janus kinase-1 gene expression in TCMR patients was significantly higher compared to stable patients (P-value = 0.02). However, the relative expression of JAK-2 and JAK-3 genes was comparable between the two groups. Additionally, there was a negative correlation between JAK-1 and JAK-3 gene expression and the estimated glomerular filtration rate in the studied population (P-value = 0.01 and 0.029, respectively). Conclusions: Janus kinase-1 gene expression increased significantly during TCMR of the kidney transplant. Therefore, specific inhibition of this molecule by JAK inhibitor drugs might be considered a therapeutic option for treating TCMR. [ABSTRACT FROM AUTHOR]

Biallelic null or hypomorphic variants in JAK3 cause SCID and less frequently Omenn syndrome. We investigated homozygous hypomorphic JAK3 mutations in two patients, and expression and function of a novel JAK3R431P variant in Omenn syndrome. Immunophenotyping of PBMC from the patient with the novel JAK3R431P variant was undertaken, by flow cytometry and Phosflow after stimulation with IL-2, IL-7, and IL-15. JAK3 expression was investigated by Western blotting. We report two patients with homozygous hypomorphic JAK3 variants and clinical features of Omenn syndrome. One patient had a previously described JAK3R775H variant, and the second had a novel JAK3R431P variant. One patient with a novel JAK3R431P variant had normal expression of JAK3 in immortalised EBV-LCL cells but reduced phosphorylation of STAT5 after stimulation with IL-2, IL-7, and IL-15 consistent with impaired kinase activity. These results suggest the JAK3R431P variant to be hypomorphic. Both patients are alive and well after allogeneic haematopoietic stem cell transplantation. They have full donor chimerism, restitution of thymopoiesis and development of appropriate antibody responses following vaccination. We expand the phenotype of hypomorphic JAK3 deficiency and demonstrate the importance of functional testing of novel variants in disease-causing genes. [ABSTRACT FROM AUTHOR]

Abstract The disruptor of telomeric silencing 1-like (DOT1L), a specific histone methyltransferase that catalyzed methylation of histone H3 on lysine 79, was associated with the pathogenesis of many diseases, but its role in peritoneal fibrosis remained unexplored. Here, we examined the role of DOT1L in the expression and activation of protein tyrosine kinases and development of peritoneal fibrosis. We found that a significant rise of DOT1L expression in the fibrotic peritoneum tissues from long-term PD patients and mice. Inhibition of DOT1L significantly attenuated the profibrotic phenotypic differentiation of mesothelial cells and macrophages, and alleviated peritoneal fibrosis. Mechanistically, RNA sequencing and proteomic analysis indicated that DOT1L was mainly involved in the processes of protein tyrosine kinase binding and extracellular matrix structural constituent in the peritoneum. Chromatin immunoprecipitation (ChIP) showed that intranuclear DOT1L guided H3K79me2 to upregulate EGFR in mesothelial cells and JAK3 in macrophages. Immunoprecipitation and immunofluorescence showed that extranuclear DOT1L could interact with EGFR and JAK3, and maintain the activated signaling pathways. In summary, DOT1L promoted the expression and activation of tyrosine kinases (EGFR in mesothelial cells and JAK3 in macrophages), promoting cells differentiate into profibrotic phenotype and thus peritoneal fibrosis. We provide the novel mechanism of dialysis-related peritoneal fibrosis (PF) and the new targets for clinical drug development. DOT1L inhibitor had the PF therapeutic potential.

The disruptor of telomeric silencing 1-like (DOT1L), a specific histone methyltransferase that catalyzed methylation of histone H3 on lysine 79, was associated with the pathogenesis of many diseases, but its role in peritoneal fibrosis remained unexplored. Here, we examined the role of DOT1L in the expression and activation of protein tyrosine kinases and development of peritoneal fibrosis. We found that a significant rise of DOT1L expression in the fibrotic peritoneum tissues from long-term PD patients and mice. Inhibition of DOT1L significantly attenuated the profibrotic phenotypic differentiation of mesothelial cells and macrophages, and alleviated peritoneal fibrosis. Mechanistically, RNA sequencing and proteomic analysis indicated that DOT1L was mainly involved in the processes of protein tyrosine kinase binding and extracellular matrix structural constituent in the peritoneum. Chromatin immunoprecipitation (ChIP) showed that intranuclear DOT1L guided H3K79me2 to upregulate EGFR in mesothelial cells and JAK3 in macrophages. Immunoprecipitation and immunofluorescence showed that extranuclear DOT1L could interact with EGFR and JAK3, and maintain the activated signaling pathways. In summary, DOT1L promoted the expression and activation of tyrosine kinases (EGFR in mesothelial cells and JAK3 in macrophages), promoting cells differentiate into profibrotic phenotype and thus peritoneal fibrosis. We provide the novel mechanism of dialysis-related peritoneal fibrosis (PF) and the new targets for clinical drug development. DOT1L inhibitor had the PF therapeutic potential. [ABSTRACT FROM AUTHOR]

Background/Objectives: This subgroup analysis of the ALLEGRO phase 2b/3 trial (NCT03732807) evaluated the efficacy and safety of ritlecitinib, an oral, selective dual JAK3/TEC family kinase inhibitor, for the treatment of alopecia areata (AA) in patients aged 12–17 years. Methods: In ALLEGRO‐2b/3, patients aged ≥12 years with AA and ≥50% scalp hair loss received once‐daily ritlecitinib 50 or 30 mg (±4‐week 200‐mg loading dose) or 10 mg or placebo for 24 weeks. In a subsequent 24‐week extension period, ritlecitinib groups continued their doses, and patients initially assigned to placebo switched to 200/50 or 50 mg daily. Clinician‐ and patient‐reported hair regrowth outcomes and safety were assessed. Results: In total, 105 adolescents were randomized. At Week 24, 17%–28% of adolescents achieved a Severity of Alopecia Tool (SALT) score ≤20 (≤20% scalp without hair) in the ritlecitinib 30 mg and higher treatment groups versus 0% for placebo. At Week 48, 25%–50% of patients had a SALT score ≤20 across ritlecitinib treatment groups (30 mg and higher). Adolescents reporting that their AA "moderately" or "greatly" improved were 45%–61% in the ritlecitinib groups (30 mg and higher) (vs. 10%–22% for placebo) at Week 24 and 44%–80% at Week 48. The most common adverse events in adolescents were headache, acne, and nasopharyngitis. No deaths, major adverse cardiovascular events, malignancies, pulmonary embolisms, opportunistic infections, or herpes zoster infections were reported. Conclusion: Ritlecitinib treatment demonstrated clinician‐reported efficacy, patient‐reported improvement, and an acceptable safety profile through Week 48 in adolescents with AA with ≥50% scalp hair loss. [ABSTRACT FROM AUTHOR]

The antigen-independent, strong proliferative responses of naive CD8+ T cells have been well demonstrated in a particular strain of mice lacking IL-2 receptors. This type of proliferation is mainly driven by common gamma-chain (γc) cytokines, such as IL-2, IL-7, and IL-15, present at abnormally high levels in these mice. Similarly, in the present study, we showed that mice lacking Janus kinase 3 (Jak3), a tyrosine kinase crucial for γc cytokine signaling, could induce strong proliferation of adoptively transferred naive CD8+ T cells. This proliferation was also independent of antigenic stimulation, but heavily dependent on IL-2, as evidenced by the failure of proliferation of adoptively transferred IL-2 receptor alpha- and beta-chain-deficient naive CD8+ T cells. Consistent with this, Jak3-/- mice showed elevated serum levels of IL-2 compared to wild-type mice, and interestingly, IL-2 production was due to high levels of accumulation of activated CD4+ T cells in Jak3-/- mice along with defective CD4+ T regulatory cells. Collectively, these findings reveal previously unidentified unique immune contexts of Jak3-/- mice that cause robust IL-2-driven T cell expansion and have a clinical implication for designing a treatment strategy for human patients with loss-of-function genetic mutations of Jak3.

Kinases are involved in numerous critical cell signaling processes, and dysregulation in kinase signaling is implicated in many types of human cancers. In this study, we applied a parallel-reaction monitoring (PRM)-based targeted proteomic method to assess kinome reprogramming during melanoma metastasis in three pairs of matched primary/metastatic human melanoma cell lines. Around 300 kinases were detected in each pair of cell lines, and the results showed that Janus kinase 3 (JAK3) was with reduced expression in the metastatic lines of all three pairs of melanoma cells. Interrogation of The Cancer Genome Atlas (TCGA) data showed that reduced expression of JAK3 is correlated with poorer prognosis in melanoma patients. Additionally, metastatic human melanoma cells/tissues exhibited diminished levels of JAK3 mRNA relative to primary melanoma cells/tissues. Moreover, JAK3 suppresses the migration and invasion of cultured melanoma cells by modulating the activities of matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9). In summary, our targeted kinome profiling method provided by far the most comprehensive dataset for kinome reprogramming associated with melanoma progression, which builds a solid foundation for examining the functions of other kinases in melanoma metastasis. Moreover, our results reveal a role of JAK3 as a potential suppressor for melanoma metastasis.

Introduction: Janus kinase 3 (JAK3) is a well-established oncogene in clear cell renal cell carcinoma (ccRCC). The methylation status of oncogene promoters has emerged as biomarkers for cancer diagnosis and prognosis. Objective: This study aims to investigate the biological and clinical significance of JAK3 promoter methylation in ccRCC. Methods: We analyzed the relationship of JAK3 promoter methylation with its mRNA expression, overall survival, and immune cell infiltration in a cohort obtained from The Cancer Genome Atlas (TCGA), which was further validated by another independent cohort. We further validated correlations of JAK3 promoter methylation with JAK3 expression, overall survival, and immune cell infiltration in an independent ccRCC cohort (Sun Yat-sen University Cancer Center (SYSUCC) cohort) by methods of immunohistochemistry (IHC) and pyrosequencing. Results: We found JAK3 promoter was significantly hypomethylated in tumor tissues compared to normal adjacent tissues in ccRCC, and JAK3 promoter hypomethylation was strongly correlated with high JAK3 mRNA expression in all three ccRCC cohorts we examined. JAK3 promoter hypomethylation predicted advanced clinicopathological characteristics and shorter overall survival (TCGA cohort and SYSUCC cohort). Furthermore, we found that JAK3 promoter methylation was significantly associated with immune cell infiltration and expression of immune checkpoint molecules (TCGA cohort and CPTAC cohort). Finally, our SYSUCC cohort validated that JAK3 promoter methylation was correlated with CD4+ and CD8+ T cell infiltration in ccRCC tumor tissues. Conclusion: Our data demonstrated that the crucial role of JAK3 promoter methylation in its expression regulation and tumor microenvironment. JAK3 promoter methylation and expression are associated with clinicopathological characteristics, overall survival, and immune cell infiltration in ccRCC. We propose a rationale for further validation of JAK3 promoter methylation as a molecular biomarker for predicting responses to immune checkpoint inhibitors in ccRCC.

[Display omitted] • JAK3 promoter methylation was correlated with JAK3 expression in ccRCC. • JAK3 promoter methylation was associated with clinicopathological characteristics in ccRCC. • JAK3 promoter methylation was associated with overall survival in ccRCC. • JAK3 promoter methylation was correlated with immune cell infiltration in ccRCC. • JAK3 promoter methylation was correlated with the expression of immune checkpoint molecules in ccRCC. • JAK3 promoter methylation was a potential biomarker for predicting responses to immune checkpoint inhibitors in ccRCC. Janus kinase 3 (JAK3) is a well-established oncogene in clear cell renal cell carcinoma (ccRCC). The methylation status of oncogene promoters has emerged as biomarkers for cancer diagnosis and prognosis. This study aims to investigate the biological and clinical significance of JAK3 promoter methylation in ccRCC. We analyzed the relationship of JAK3 promoter methylation with its mRNA expression, overall survival, and immune cell infiltration in a cohort obtained from The Cancer Genome Atlas (TCGA), which was further validated by another independent cohort. We further validated correlations of JAK3 promoter methylation with JAK3 expression, overall survival, and immune cell infiltration in an independent ccRCC cohort (Sun Yat-sen University Cancer Center (SYSUCC) cohort) by methods of immunohistochemistry (IHC) and pyrosequencing. We found JAK3 promoter was significantly hypomethylated in tumor tissues compared to normal adjacent tissues in ccRCC, and JAK3 promoter hypomethylation was strongly correlated with high JAK3 mRNA expression in all three ccRCC cohorts we examined. JAK3 promoter hypomethylation predicted advanced clinicopathological characteristics and shorter overall survival (TCGA cohort and SYSUCC cohort). Furthermore, we found that JAK3 promoter methylation was significantly associated with immune cell infiltration and expression of immune checkpoint molecules (TCGA cohort and CPTAC cohort). Finally, our SYSUCC cohort validated that JAK3 promoter methylation was correlated with CD4+ and CD8+ T cell infiltration in ccRCC tumor tissues. Our data demonstrated that the crucial role of JAK3 promoter methylation in its expression regulation and tumor microenvironment. JAK3 promoter methylation and expression are associated with clinicopathological characteristics, overall survival, and immune cell infiltration in ccRCC. We propose a rationale for further validation of JAK3 promoter methylation as a molecular biomarker for predicting responses to immune checkpoint inhibitors in ccRCC. [ABSTRACT FROM AUTHOR]

Objectives: To explore the effectiveness of tofacitinib for immunoglobulin G4-related disease (IgG4-RD) and idiopathic retroperitoneal fibrosis (IRF), and investigate the expression of JAKs in the lesion of these diseases., Methods: Clinical data of patients with IgG4-RD or IRF who were administered with tofacitinib monotherapy were collected. IgG4-RD responder index (IgG4-RD RI) was assessed. The expression of JAK1, JAK2, JAK3, and TYK2 were analysed with immunohistochemistry staining in three salivary glands specimens of IgG4-RD and one retroperitoneal tissue of IRF., Results: Two patients with IRF and two patients with IgG4-RD used tofacitinib monotherapy. Two patients with IRF achieved complete remission with diminished retroperitoneal mass and decreased CRP, as IgG4-RD RI decreased from 6 to 1 in both of them. One with IgG4-RD achieved complete remission with alleviated enlargement of pancreas and IgG4 level decreased from 13.7 g/L to 2.4 g/L, as IgG4-RD RI decreased from 12 to 1. One with IgG4-RD achieved partial response with IgG4 level decreased from 77.1g/L to 25.8g/L as IgG4-RD RI from 18 to 6. JAK1, JAK2, JAK3, and TYK2 expression were detected in biopsy tissues. The staining intensity of the JAK family on the lesion from one IRF patient was similar to those from IgG4-RD patients., Conclusions: Tofacitinib is a potentially effective treatment for IgG4-RD and IRF and it is reasonable to conduct clinical trial to validate its efficacy. The JAKs were expressed in the inflammatory lesions of IgG4-RD and IRF and they may share a common pathogenesis pathway that is independent of IgG4 production.

JAK3 genetic mutations effects The Janus kinase-signal transducerand activator of transcription(JAK-STAT) pathway was supposedto be involved in the frequently alteredmechanism in HCC. Keywords: Hepatocellular carcinoma; Next generation sequencing (NGS); Janus kinase 3 EN Hepatocellular carcinoma Next generation sequencing (NGS) Janus kinase 3 Liver diseases are a significantcause of mortality and morbiditythroughout the world, causing twomillion deaths per year worldwide(3.5% of all death), with 50% relatedto cirrhosis complications and 50%associated with Hepatocellular Carcinoma(HCC) and viral hepatitis infections(Asrani et al., 2019). JAK3 mutations detected inthe current study might play a minimalrole in HCV-associated hepatocarcinogenesis.A study conducted by(Hin Tang et al., 2020) showed thatmany intracellular signaling pathwayscontribute to hepatocarcinogenesis,including the JAK/STAT pathway,which has normal roles in regulatingcell proliferation, survival, and differentiation. [Extracted from the article]

Lung cancer is the leading cause of cancer deaths worldwide, with the majority of mortality resulting from metastatic spread. However, the molecular mechanism by which cancer cells acquire the ability to disseminate from primary tumors, seed distant organs, and grow into tissue-destructive metastases remains incompletely understood. We combined tumor barcoding in a mouse model of human lung adenocarcinoma with unbiased genomic approaches to identify a transcriptional program that confers metastatic ability and predicts patient survival. Small-scale in vivo screening identified several genes, including Cd109, that encode novel pro-metastatic factors. We uncovered signaling mediated by Janus kinases (Jaks) and the transcription factor Stat3 as a critical, pharmacologically targetable effector of CD109-driven lung cancer metastasis. In summary, by coupling the systematic genomic analysis of purified cancer cells in distinct malignant states from mouse models with extensive human validation, we uncovered several key regulators of metastatic ability, including an actionable pro-metastatic CD109-Jak-Stat3 axis.

To drive lymphocyte proliferation and differentiation, common γ-chain (γc) cytokine receptors require hours to days of sustained stimulation. JAK1 and JAK3 kinases are found together in all γc-receptor complexes, but how their respective catalytic activities contribute to signaling over time is not known. Here we dissect the temporal requirements for JAK3 kinase activity with a selective covalent inhibitor (JAK3i). By monitoring phosphorylation of the transcription factor STAT5 over 20 h in CD4(+) T cells stimulated with interleukin 2 (IL-2), we document a second wave of signaling that is much more sensitive to JAK3i than the first wave. Selective inhibition of this second wave is sufficient to block cyclin expression and entry to S phase. An inhibitor-resistant JAK3 mutant (C905S) rescued all effects of JAK3i in isolated T cells and in mice. Our chemical genetic toolkit elucidates a biphasic requirement for JAK3 kinase activity in IL-2-driven T cell proliferation and will find broad utility in studies of γc-receptor signaling.

Introduction and hypothesis: As a notable chronic disorder, the incidence of interstitial cystitis (IC) has been documented to have increased among the female population with activity in microRNA-495 (miR-495) implicated in this disease. The current study was aimed at elucidating the effects associated with miR-495 on the inflammatory response and bladder fibrosis in rats with ulcerative IC via the JAK–STAT pathway by targeting JAK3. Methods: Ulcerative IC rat models were established. The targeting relationship between JAK3 and miR-495 was evaluated using luciferase reporter assay. After gain- or loss-of-function assays, mast-cell infiltration was assessed using toluidine blue staining, bladder fibrosis using Masson staining, and NO content using nitrate reductase method. JAK3 protein expression was detected by immunohistochemistry, JAK3, STAT1, STAT3, TGFβ-1, Col-I, Col-III, JAK1, JAK2, p-STAT1, and p-STAT3 expression by RT-qPCR and Western blot analysis, and serum IL-6, IL-8, IL-10, IL-17, and TNF-α levels in rats by ELISA. Results: Following transfection of overexpressed miR-495 or siRNA-JAK3, a diminished degree of mast-cell infiltration, number of mast cells, bladder fibrosis, NO content, JAK3-positive expression, mRNA expression of JAK3, STAT1, STAT3, TGFβ-1, Col-I, Col-III, protein expression of JAK1, JAK2, JAK3, p-STAT1, p-STAT3, and expression of IL-6, IL-8, IL-10, IL-17, and TNF-α were identified. Conclusions: Collectively, our key findings provide evidence supporting the notion that the overexpression of miR-495 ameliorates inflammatory response and bladder fibrosis in ulcerative IC rat models via inactivation of the JAK–STAT signaling pathway by inhibiting JAK3. [ABSTRACT FROM AUTHOR]

The antigen-independent, strong proliferative responses of naive CD8+ T cells have been well demonstrated in a particular strain of mice lacking IL-2 receptors. This type of proliferation is mainly driven by common gamma-chain (γc) cytokines, such as IL-2, IL-7, and IL-15, present at abnormally high levels in these mice. Similarly, in the present study, we showed that mice lacking Janus kinase 3 (Jak3), a tyrosine kinase crucial for γc cytokine signaling, could induce strong proliferation of adoptively transferred naive CD8+ T cells. This proliferation was also independent of antigenic stimulation, but heavily dependent on IL-2, as evidenced by the failure of proliferation of adoptively transferred IL-2 receptor alpha- and beta-chain-deficient naive CD8+ T cells. Consistent with this, Jak3–/– mice showed elevated serum levels of IL-2 compared to wild-type mice, and interestingly, IL-2 production was due to high levels of accumulation of activated CD4+ T cells in Jak3–/– mice along with defective CD4+ T regulatory cells. Collectively, these findings reveal previously unidentified unique immune contexts of Jak3–/– mice that cause robust IL-2-driven T cell expansion and have a clinical implication for designing a treatment strategy for human patients with loss-of-function genetic mutations of Jak3.

The antigen-independent, strong proliferative responses of naive CD8+ T cells have been well demonstrated in a particular strain of mice lacking IL-2 receptors. This type of proliferation is mainly driven by common gamma-chain (γc) cytokines, such as IL-2, IL-7, and IL-15, present at abnormally high levels in these mice. Similarly, in the present study, we showed that mice lacking Janus kinase 3 (Jak3), a tyrosine kinase crucial for γc cytokine signaling, could induce strong proliferation of adoptively transferred naive CD8+ T cells. This proliferation was also independent of antigenic stimulation, but heavily dependent on IL-2, as evidenced by the failure of proliferation of adoptively transferred IL-2 receptor alpha- and beta-chain-deficient naive CD8+ T cells. Consistent with this, Jak3 –/– mice showed elevated serum levels of IL-2 compared to wild-type mice, and interestingly, IL-2 production was due to high levels of accumulation of activated CD4+ T cells in Jak3 –/– mice along with defective CD4+ T regulatory cells. Collectively, these findings reveal previously unidentified unique immune contexts of Jak3 –/– mice that cause robust IL-2-driven T cell expansion and have a clinical implication for designing a treatment strategy for human patients with loss-of-function genetic mutations of Jak3. [ABSTRACT FROM AUTHOR]

JAK-STAT signalling pathway inhibitors have emerged as promising therapeutic agents for the treatment of hair loss. Among different JAK isoforms, JAK3 has become an ideal target for drug discovery because it only regulates a narrow spectrum of γc cytokines. Here, we report the discovery of MJ04, a novel and highly selective 3-pyrimidinylazaindole based JAK3 inhibitor, as a potential hair growth promoter with an IC 50 of 2.03 nM. During in vivo efficacy assays, topical application of MJ04 on DHT-challenged AGA and athymic nude mice resulted in early onset of hair regrowth. Furthermore, MJ04 significantly promoted the growth of human hair follicles under ex-vivo conditions. MJ04 exhibited a reasonably good pharmacokinetic profile and demonstrated a favourable safety profile under in vivo and in vitro conditions. Taken together, we report MJ04 as a highly potent and selective JAK3 inhibitor that exhibits overall properties suitable for topical drug development and advancement to human clinical trials., (© 2024. The Author(s).)

Background: The ALLEGRO phase 2a and 2b/3 studies demonstrated that ritlecitinib, an oral JAK3/TEC family kinase inhibitor, is efficacious at doses of ≥ 30 mg in patients aged ≥ 12 years with alopecia areata (AA)., Objective: The objective of this study was to evaluate the safety of ritlecitinib in an integrated analysis of four studies in AA., Methods: Two cohorts were analyzed: a placebo-controlled and an all-exposure cohort. Proportions and study size-adjusted incidence rates (IRs) of adverse events (AEs) of interest and laboratory abnormalities are reported., Results: In the placebo-controlled cohort (n = 881; median exposure: 169 days), the proportion of ritlecitinib-treated patients with AEs was 70.2-75.4% across doses versus 69.5% in the placebo group; serious AEs occurred in 0-3.2% versus 1.9% for the placebo. A total of 19 patients permanently discontinued due to AEs (5 while receiving the placebo). In the all-exposure cohort (n = 1294), median ritlecitinib exposure was 624 days [2091.7 total patient-years (PY)]. AEs were reported in 1094 patients (84.5%) and serious AEs in 57 (4.4%); 78 (6.0%) permanently discontinued due to AEs. The most common AEs were headache (17.7%; 11.9/100 PY), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) positive test (15.5%; 9.8/100 PY), and nasopharyngitis (12.4%; 8.2/100 PY). There were two deaths (breast cancer and acute respiratory failure/cardiorespiratory arrest). Proportions (IRs) were < 0.1% (0.05/100 PY) for opportunistic infections, 1.5% (0.9/100 PY) for herpes zoster, 0.5% (0.3/100 PY) for malignancies (excluding nonmelanoma skin cancer), and 0.2% (0.1/100 PY) for major adverse cardiovascular events., Conclusions: Ritlecitinib is well tolerated with an acceptable safety profile up to 24 months in patients aged ≥ 12 years with AA (video abstract and graphical plain language summary available)., Trial Registries: ClinicalTrials.gov: NCT02974868 (date of registration: 11/29/2016), NCT04517864 (08/18/2020), NCT03732807 (11/07/2018), and NCT04006457 (07/05/2019)., (© 2024. The Author(s).)

Cortistatin A is a natural product isolated from the marine sponge Corticium simplex has been considered as a therapeutic agent for autoimmune disorders, HIV infection as well as cancer. Janus kinases (JAKs) and signal transducer and activator of transcription (STAT) transcription factors play a promising role in either causing or participating in the tumorigenesis. Following our earlier studies on JAK-STAT pathway genes varied expression among the various types of breast cancer samples, JAK3 protein plays a crucial role in cancer pathway, thus we implement a feasible systems pharmacology method to explore JAK3 function. Our systematic Insilco approaches involve systems-based assessment of JAK3 role in cytokine signaling, ADMET a scale for pharmacokinetic and pharmacodynamics assessment, pharmacophore mapping of JAK3 and ligand Cortistatin A. Sequence level annotation of functional domains revealed similarity between the JAK family members namely JAK1, JAK2, JAK 3 and TYK2. Thus intermolecular interaction, molecular dynamics and simulation assessment of JAK3 with Cortistatin docked complex pattern applicable other members that shared the domain similarity. Thus, our present study proposes the system level of understanding about Cortistatin A agonist activity on JAK3 and suggesting the compound as a potent antiproliferative agent for cancer treatment. [ABSTRACT FROM AUTHOR]

In the present study, the mechanism by which carboxyl terminal activating region 3 (CTAR3) of latent membrane protein 1 (LMP1), encoded by the Epstein-Barr virus, regulated cell proliferation and protein expression was investigated in the nasopharyngeal epithelial cell line NP69. The deletion mutant LMP1 (LMP1Δ232-351; amino acid residues including 232–351 codons in CTAR3 deleted) was generated by polymerase chain reaction. An NP69-LMP1Δ232-351 cell line was established by retroviral infection. Finally, cell proliferation and protein expression of NP69 cells expressing LMP1Δ232-351 were examined using a cell growth curve and western blot analysis. The results demonstrated: i) The proliferation of NP69-LMP1Δ232-351 cells was significantly decreased compared with cells expressing wild type LMP1 (LMP1WT; n=3; P<0.05); ii) 17 proteins exhibited differential protein expression (>2-fold change) in NP69-LMP1Δ232-351 cells compared with NP69-LMP1WT cells; and iii) LMP1WT was involved in activating the Janus kinase 3 (JAK3) promoter and regulating the expression of JAK3 protein, while LMP1Δ232-351 was almost defective in ability to activate the JAK promoter. These results suggested that LMP1-CTAR3 may be an important functional domain for regulating cell proliferation and protein expression in nasopharyngeal epithelial cells. [ABSTRACT FROM AUTHOR]

Background: Vitiligo is an autoimmune depigmenting disorder with no effective and safe treatments. Its pathogenesis is not fully elucidated., Objective: This substudy of a randomized, double-blind, placebo-controlled phase 2b trial (NCT03715829) evaluated effects of ritlecitinib, an oral JAK3/TEC family kinase inhibitor, on skin and blood biomarkers in participants with nonsegmental vitiligo (NSV)., Methods: Sixty-five adults with NSV participated in the substudy and received daily treatment for 24 weeks with placebo (n = 14) or ritlecitinib with or without a 4-week loading dose: 200 (loading dose)/50 mg (n = 13), 100/50 mg (n = 12), 50 mg (n = 11), 30 mg (n = 8), or 10 mg (n = 6). Skin (lesional and nonlesional) biopsy samples were obtained at baseline and at 4 and 24 weeks. Changes from baseline to weeks 4 and 24 in skin and blood molecular and cellular biomarkers were evaluated by RNA sequencing, quantitative real-time PCR, proteomic analysis, and flow cytometry., Results: Ritlecitinib-treated groups showed downregulation of immune biomarkers and upregulation of melanocyte-related markers at weeks 4 and 24 compared to baseline and/or placebo. Significant reductions were seen in CD3 + /CD8 + T-cell infiltrates, with significant increases in melanocyte markers (tyrosinase; Melan-A) in NSV lesions in the 50 mg ritlecitinib groups (both P < .05). There was significant, dose-dependent downregulation in T-cell activation, NK, cytotoxic, and regulatory markers in lesional skin (IL-2, IL2-RA, IL-15, CCR7, CD5, CRTAM, NCR1, XCL1, KIR3DL1, FASLG, KLRD; P < .05). T H 1 and T H 2 markers were also downregulated in lesional skin and blood in a dose-dependent manner (P < .05). Changes in immune biomarkers correlated with clinical response., Conclusions: Ritlecitinib significantly downregulated proinflammatory biomarkers and increased melanocyte products in skin and blood of participants with NSV, suggesting its potential in treatment. Ritlecitinib-mediated changes positively correlated with clinical response., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Peripheral T-cell lymphoma (PTCL) is a clinically heterogeneous group that represents 10%-15% of all lymphomas. Despite improved genetic and molecular understanding, treatment outcomes for PTCL have not shown significant improvement. Although Janus kinase-2 (JAK2) plays an important role in myeloproliferative neoplasms, the critical role of JAK isoforms in mediating prosurvival signaling in PTCL cells is not well defined. Immunohistochemical analysis of PTCL tumors (n = 96) revealed high levels of constitutively active JAK3 (pJAK3) that significantly (p < 0.04) correlated with the activation state of its canonical substrate STAT3. Furthermore, constitutive activation of JAK3 and STAT3 positively correlated, at least in part, with an oncogenic tyrosine phosphatase PTPN11. Pharmacological inhibition of JAK3 but not JAK1/JAK2 significantly (p < 0.001) decreased PTCL proliferation, survival and STAT3 activation. A sharp contrast was observed in the pJAK3 positivity between ALK+ (85.7%) versus ALK-negative (10.0%) in human PTCL tumors and PTCL cell lines. Moreover, JAK3 and ALK reciprocally interacted in PTCL cells, forming a complex to possibly regulate STAT3 signaling. Finally, combined inhibition of JAK3 (by WHI-P154) and ALK (by crizotinib or alectinib) significantly (p < 0.01) decreased the survival of PTCL cells as compared to either agent alone by inhibiting STAT3 downstream signaling. Collectively, our findings establish that JAK3 is a therapeutic target for a subset of PTCL, and provide rationale for the clinical evaluation of JAK3 inhibitors combined with ALK-targeted therapy in PTCL., (© 2023 John Wiley & Sons Ltd.)

Ritlecitinib is a small molecule in clinical development that covalently and irreversibly inhibits Janus kinase 3 (JAK3) and the TEC family of kinases (BTK, BMX, ITK, TXK, and TEC). This phase 1, open-label, parallel-group study assessed target occupancy and functional effects of ritlecitinib on JAK3 and TEC family kinases in healthy participants aged 18-60 years who received 50 or 200 mg single doses of ritlecitinib on day 1. Blood samples to assess ritlecitinib pharmacokinetics, target occupancy, and pharmacodynamics were collected over 48 hours. Target occupancy was assessed using mass spectroscopy. Functional inhibition of JAK3-dependent signaling was measured by the inhibition of the phosphorylation of its downstream target signal transducer and activator of transcription 5 (pSTAT5), following activation by interleukin 15 (IL-15). The functional inhibition of Bruton's tyrosine kinase (BTK)-dependent signaling was measured by the reduction in the upregulation of cluster of differentiation 69 (CD69), an early marker of B-cell activation, following treatment with anti-immunoglobulin D. Eight participants received one 50 mg ritlecitinib dose and 8 participants received one 200 mg dose. Ritlecitinib plasma exposure increased in an approximately dose-proportional manner from 50 to 200 mg. The maximal median JAK3 target occupancy was 72% for 50 mg and 64% for 200 mg. Ritlecitinib 50 mg had >94% maximal target occupancy of all TEC kinases, except BMX (87%), and 200 mg had >97% for all TEC kinases. For BTK and TEC, ritlecitinib maintained high target occupancy throughout a period of 48 hours. Ritlecitinib reduced pSTAT5 levels following IL-15- and BTK-dependent signaling in a dose-dependent manner. These target occupancy and functional assays demonstrate the dual inhibition of the JAK3- and BTK-dependent pathways by ritlecitinib. Further studies are needed to understand the contribution to clinical effects of inhibiting these pathways., (© 2023 Pfizer Inc. The Journal of Clinical Pharmacology published by Wiley Periodicals LLC on behalf of American College of Clinical Pharmacology.)

Steroids are the standard treatment for allergic airway inflammation in asthma, but steroid-refractory asthma poses a challenge. Group 2 innate lymphoid cells (ILC2s), such as T helper 2 (T H 2) cells, produce key asthma-related type 2 cytokines. Recent insights from mouse and human studies indicate a potential connection between ILC2s and steroid-resistant asthma. Here, we highlight that lung ILC2s, rather than T H 2 cells, can develop steroid resistance, allowing them to persist and maintain their disease-driving activity even during steroid treatment. The emergence of multipotent IL-5 + IL-13 + IL-17A + ILC2s is associated with steroid-resistant ILC2s. The Janus kinase 3 (JAK3)/signal transducer and activator of transcription (STAT) 3, 5, and 6 pathways contribute to the acquisition of steroid-resistant ILC2s. The JAK3 inhibitor reduces ILC2 survival, proliferation, and cytokine production in vitro and ameliorates ILC2-driven Alternaria -induced asthma. Furthermore, combining a JAK3 inhibitor with steroids results in the inhibition of steroid-resistant asthma. These findings suggest a potential therapeutic approach for addressing this challenging condition in chronic asthma.

This work aimed to find new inhibitors of the CYP3A4 and JAK3 enzymes, which are significant players in autoimmune diseases such as rheumatoid arthritis. Advanced computer-aided drug design techniques, such as pharmacophore and 3D-QSAR modeling, were used. Two strong 3D-QSAR models were created, and their predictive power was validated by the strong correlation (R 2 values > 80%) between the predicted and experimental activity. With an ROC value of 0.9, a pharmacophore model grounded in the DHRRR hypothesis likewise demonstrated strong predictive ability. Eight possible inhibitors were found, and six new inhibitors were designed in silico using these computational models. The pharmacokinetic and safety characteristics of these candidates were thoroughly assessed. The possible interactions between the inhibitors and the target enzymes were made clear via molecular docking. Furthermore, MM/GBSA computations and molecular dynamics simulations offered insightful information about the stability of the binding between inhibitors and CYP3A4 or JAK3. Through the integration of various computational approaches, this study successfully identified potential inhibitor candidates for additional investigation and efficiently screened compounds. The findings contribute to our knowledge of enzyme-inhibitor interactions and may help us create more effective treatments for autoimmune conditions like rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronically systemic autoimmune disorder, which is related with various cellular signal pathways. Both BTK (Bruton's Tyrosine Kinase) and JAK3 (Janus Kinase 3) play important roles in the pathogenesis of rheumatoid arthritis. Herein, we reported the discovery of dual BTK/JAK3 inhibitors through bioisosterism and computer-aided drug design based on the structure of BTK inhibitor ibrutinib. We reported the discovery of dual BTK/JAK3 inhibitors which are based on the structure of BTK inhibitor ibrutinib via the method of bioisosterism and computer-aided drug design) Most of the target compounds exhibited moderate to strong inhibitory activities against BTK and JAK3. Among them, compound XL-12 stood out as the most promising candidate targeting BTK and JAK3 with potent inhibitory activities (IC 50  = 2.0 nM and IC 50  = 14.0 nM respectively). In the in vivo studies, compound XL-12 (40 mg/kg) exhibited more potent antiarthritic activity than ibrutinib (10 mg/kg) in adjuvant arthritis (AA) rat model. Furthermore, compound XL-12 (LD 50  > 1600 mg/kg) exerted improved safety compared with ibrutinib (LD 50  = 750 mg/kg). These results indicated that compound XL-12, the dual BTK/JAK3 inhibitor, might be a potent drug candidate for the treatment of RA., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Ltd.)

Natural killer (NK) cell is an essential cytotoxic lymphocyte in our innate immunity. Activation of NK cells is of paramount importance in defending against pathogens, suppressing autoantibody production and regulating other immune cells. Common gamma chain ( γ c) cytokines, including IL-2, IL-15, and IL-21, are defined as essential regulators for NK cell homeostasis and development. However, it is inconclusive whether γ c cytokine-driven NK cell activation plays a protective or pathogenic role in the development of autoimmunity. In this study, we investigate and correlate the differential effects of γ c cytokines in NK cell expansion and activation. IL-2 and IL-15 are mainly responsible for NK cell activation, while IL-21 preferentially stimulates NK cell proliferation. Blockade of Janus tyrosine kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway by either JAK inhibitors or antibodies targeting γ c receptor subunits reverses the γ c cytokine-induced NK cell activation, leading to suppression of its autoimmunity-like phenotype in vitro . These results underline the mechanisms of how γ c cytokines trigger autoimmune phenotype in NK cells as a potential target to autoimmune diseases., Competing Interests: WCW, CS, TKT, and CWH are employed by SinoMab BioScience Limited. SOL is the chief executive officer of SinoMab BioScience Limited., (Copyright © 2023 Wai Chung Wu et al.)

LITFULO TM (ritlecitinib) capsules were recently approved for the treatment of severe alopecia areata in adolescents and adults, aged ≥12 years. Ritlecitinib is the active ingredient and a dual inhibitor of Janus kinase 3 and the tyrosine kinase expressed in hepatocellular carcinoma kinase family. It prevents immune attack on the hair follicles that leads to hair loss. In a phase 2b-3 dose-dependant study, five doses of oral ritlecitinib and placebo administered once daily (QD) were investigated. Ritlecitinib demonstrated efficacy in achieving the primary outcome, Severity of Alopecia Tool (SALT) score of ≤20, at week 24 (31% [38/124] 200-mg ritlecitinib QD for 4 weeks, then 50 mg QD for 20 weeks; 22% [27/121] 200-mg ritlecitinib QD for 4 weeks, then 30 mg QD for 20 weeks; 23% [29/124] 50-mg ritlecitinib QD; 14% [17/119] 30-mg ritlecitinib QD; 2% [1/59] 10-mg ritlecitinib QD; and 2% [2/130] placebo). Mild to moderate common adverse effects were observed, which included headache, nasopharyngitis, and upper respiratory tract infection. The recommended regimen of ritlecitinib capsules is 50 mg QD with without food and swallowed whole.

Background: Inflammatory autoimmune arthritis like that present in rheumatoid arthritis (RA) is treated by medications with many side effects. This study was a trial to benefit from Toxoplasma immune-modulatory effects on its host to treat arthritis in rat model resembling joints affection of RA. To avoid hazards of infection, Toxoplasma lysate antigen (TLA) was given instead of the whole infection, in addition to giving its encapsulated niosomes form, assuming that it would enhance the effect of TLA alone, to compare effects of both on disease activity with that of prednisolone., Methods: Swiss albino rats were divided into 6 groups: normal control group and the remaining 5 groups were injected by CFA adjuvant to induce arthritis; one of those groups was the untreated model. Each of the other groups received one of the following (TLA, TLA-encapsulated niosomes, prednisolone or niosomes) for comparison of their results. Inflammatory markers measured at the end of the experiment were: interleukin 17 (IL-17), IL-10 and CRP by ELISA technique; histopathological assessment of the biopsied hind paw joints was done and also, Janus kinase 3 (JAK3) expression was assessed by immunohistochemistry., Results: TLA and TLA-encapsulated niosomes both mitigated the signs of clinical and histopathological arthritis and were having anti-inflammatory effects (decreased CRP, IL-17 and JAK3 expressions, while increased IL-10 levels) with better effects in TLA-encapsulated niosomes-treated RA group, both groups' results were comparable to prednisolone. Niosomes also gave some anti-inflammatory effects but were mild in comparison to TLA and TLA-encapsulated niosomes., Conclusion: Vaccination with both TLA and TLA-encapsulated niosomes for the first time in adjuvant-induced arthritis ameliorated the disease through diversion of immune system and JAK3 downregulation. Both vaccinations should be further tested to evaluate the possibility of their introduction for disease treatment and in other autoimmune diseases., (© 2023. The Author(s).)

The nonreceptor tyrosine TEC kinases are key regulators of the immune system and play a crucial role in the pathogenesis of diverse hematological malignancies. In contrast to the substantial efforts in inhibitor development for Bruton’s tyrosine kinase (BTK), specific inhibitors of the other TEC kinases, including the bone marrow tyrosine kinase on chromosome X (BMX), remain sparse. Here we present a novel class of dual BMX/BTK inhibitors, which were designed from irreversible inhibitors of Janus kinase (JAK) 3 targeting a cysteine located within the solvent-exposed front region of the ATP binding pocket. Structure-guided design exploiting the differences in the gatekeeper residues enabled the achievement of high selectivity over JAK3 and certain other kinases harboring a sterically demanding residue at this position. The most active compounds inhibited BMX and BTK with apparent IC50 values in the single digit nanomolar range or below showing moderate selectivity within the TEC family and potent cellular target engagement. These compounds represent an important first step towards selective chemical probes for the protein kinase BMX.

The products of the Philadelphia chromosome translocation, P210 and P190(BCR/ABL), are cytoplasmic protein tyrosine kinases that share the ability to transform hematopoietic cytokine-dependent cell lines to cytokine independence but differ in the spectrum of leukemia induced in vivo. We have analyzed the Janus kinase (JAK) and signal transducer and activator of transcription (STAT) pathways in hematopoietic cells transformed by Bcr/Abl. STAT5 and, to a lesser extent, STATs 1 and 3 were constitutively activated by tyrosine phosphorylation and induction of DNA binding activity in both P210 and P190(BCR/ABL)-transformed cells, but P190 differed in that it also prominently activated STAT6. There was low level tyrosine phosphorylation of JAKs 1, 2, and 3 in Bcr/Abl-transformed cells, but no detectable complex formation with Bcr/Abl, and activation of STAT5 by P210 was not blocked by two different dominant-negative JAK mutants. These results suggest that P210 and P190(BCR/ABL) directly activate specific STAT family members and may help explain their overlapping yet distinct roles in leukemogenesis.

Background/Aims: Oscillations of cytosolic Ca2+ activity ([Ca2+]i) participate in the orchestration of tumor cell proliferation. [Ca2+]i could be increased by intracellular Ca2+ release followed by store-operated Ca2+-entry (SOCE). [Ca2+]i could be decreased by Ca2+ extrusion via Na+/Ca2+ exchange. Mechanisms accomplishing SOCE include the pore-forming ion channel unit Orai1 and its regulator STIM1, Na+/Ca2+ exchanger isoforms include NCX1. In MCF-7 breast carcinoma cells Orai1 and NCX1 have previously been shown to be modified by pharmacological inhibition of Janus activated kinase JAK2. The present study explored whether SOCE and Na+/Ca2+ exchange are similarly sensitive to pharmacological JAK3 inhibition. Methods: MCF-7 breast carcinoma cells were studied in the absence and presence of the JAK3 inhibitor WHI-P154 (22 µM). [Ca2+]i was estimated from Fura-2-fluorescence, SOCE from increase of [Ca2+]i following Ca2+ re-addition after Ca2+-store depletion with sarcoendoplasmatic Ca2+-ATPase (SERCA) inhibitor thapsigargin (1 µM), and Na+/Ca2+ exchanger activity from increase of [Ca2+]i following extracellular Na+ removal. Transcript levels were quantified with RT-PCR. Results: Addition of ATP (100 µM) was followed by a rapid increase of [Ca2+]i, which was significantly blunted by WHI-P154. Thapsigargin-induced intracellular Ca2+ release was not appreciably influenced by WHI-P154. Subsequent SOCE was, however, significantly blunted by WHI-P154. WHI-P154 further significantly decreased Orai1 transcript levels. The increase of [Ca2+]i following extracellular Na+-removal and the NCX1 transcript levels were similarly decreased by WHI-P154. Conclusions: The JAK3 inhibitor WHI-P154 decreases both, Orai1 and NCX1 transcript levels and thus impairs SOCE and Na+/Ca2+ exchange.

Glioblastoma multiforme (GBM) is the most deadly brain tumor, effective treatment options for which still remain elusive. The current treatment procedure of maximal resection followed by chemotherapy has proved to be grossly insufficient to prevent disease progression and death. Despite best efforts, the maximum survival post-diagnosis is a mere 1.5 years. Therefore, there is a huge unmet clinical need to find effective therapeutic procedures to prevent the pathogenesis and relapse of GBM. Small-molecule inhibitors of signaling pathways are an attractive option to prevent various types of tumors. However, no effective small-molecule inhibitors have been successful against GBM in clinical trials. Various signaling pathways are altered and an array of signaling molecules, transcription factors (TFs), and epigenetic modifying factors have been implicated in the pathogenesis of GBM. JAK-STAT pathway alteration is an important contributor to GBM pathogenesis and relapse. Many small-molecule inhibitors of JAKs, or STAT TFs, especially JAK2 and STAT3, have been assessed for their anti-tumor activity in GBM. However, no definitive success so far has been achieved. Herein, by using two small-molecule inhibitors of JAK3, we show that they are quite effective in inhibiting GBM cell proliferation and neurosphere formation, downregulating their stemness character, and inducing differentiation into neuronal origin cells. The effect of a single treatment with the drugs, both in a serum-containing differentiation medium and in a proliferation medium containing EGF and FGF, was really strong in limiting GBM cell growth, suggesting a potential therapeutic application for these JAK inhibitors in GBM therapy.

The drug development process suffers from low success rates and requires expensive and time-consuming procedures. The traditional one drug-one target paradigm is often inadequate to treat multifactorial diseases. Multitarget drugs may potentially address problems such as adverse reactions to drugs. With the aim to discover a multitarget potential inhibitor for B-cell lymphoma treatment, herein, we developed a general pipeline combining machine learning, the interpretable model SHapley Additive exPlanation (SHAP), and molecular dynamics simulations to predict active compounds and fragments. Bruton's tyrosine kinase (BTK) and Janus kinase 3 (JAK3) are popular synergistic targets for B-cell lymphoma. We used this pipeline approach to identify prospective potential dual inhibitors from a natural product database and screened three candidate inhibitors with acceptable drug absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties. Ultimately, the compound CNP0266747 with specialized binding conformations that exhibited potential binding free energy against BTK and JAK3 was selected as the optimum choice. Furthermore, we also identified key residues and fingerprint features of this dual-target inhibitor of BTK and JAK3.

Graphical abstract The two generated pharmacophore models (PBDD) were coupled with docking simulations (LBDD) to identify new compounds with dual JAK1 and JAK3 inhibitory activity. Highlights • Pharmacophore models of JAK1 and JAK3 were generated followed by rigorous validation. • Virtual screening through the generated molecules was carried out. • Molecular docking and molecular dynamics analysis was incorporated to confirm the interactions with respective enzymes. • ADME filter was done to eliminate the hits with poor pharmacokinetic profile. Abstract Janus kinase 1 and 3 are non-receptor protein tyrosine kinases, involved in the regulation of various cytokines implicated in the pathogenesis of autoimmune and inflammatory disease conditions. Thus, they serve as therapeutic targets for the designing of multi-targeted agents for the treatment of inflammatory-mediated pathological conditions. In the present study, diverse inhibitors of JAK1 and JAK3 were considered for the development of ligand-based pharmacophore models, followed by docking analysis to design putative dual inhibitors. The pharmacophore models were generated in PHASE 3.4, and top five models for each target were selected on the basis of survival minus inactive score. The best model for JAK1 (AAADH.25) and JAK3 (ADDRR.142) were selected corresponding to the highest value of Q2 test. Both models were employed for the screening of a PHASE database, and subsequently, the retrieved hits were filtered employing molecular docking in JAK1 and JAK3 proteins. The stable interactions between retrieved hits and proteins were confirmed using molecular dynamics simulations. Finally, ADME properties of screened dual inhibitors displaying essential interactions with both proteins were calculated. Thus, the new leads obtained in this way may be prioritized for experimental validation as potential novel therapeutic agents in the treatment of various autoimmune and inflammatory disorders related to JAK1 and JAK3. [ABSTRACT FROM AUTHOR]

Janus kinase 3 (JAK3) is associated with the common gamma chain of several interleukin (IL) receptors essential to inflammatory signaling. To study the potential role of JAK3 in stroke-induced neuroinflammation, we subjected mice to permanent middle cerebral artery occlusion and investigated the effects of JAK3 inhibition with decernotinib (VX-509) on infarct size, behavior, and levels of several inflammatory mediators. Results from our double immunofluorescence staining showed JAK3 expression on neurons, endothelial cells, and microglia/macrophages in the ischemic mouse brain (n = 3). We found for the first time that total and phosphorylated/activated JAK3 are dramatically increased after stroke in the ipsilateral hemisphere (**P

Janus kinases (JAKs) are a family of four cytosolic protein kinases with a high degree of structural similarity. Due to its very restricted role in immune regulation, JAK3 was promoted as an excellent target for immunosuppression for more than a decade, but clinical validation of this concept is still elusive. During the last years, speculation arose that kinase activity of JAK1, which cooperates with JAK3 in cytokine receptor signaling, may have a dominant role over the one of JAK3. Until recently, however, this issue could not be appropriately addressed due to a lack of highly isoform-selective tool compounds. With the recent resurgence of covalent drugs, targeting of a specific cysteine that distinguishes JAK3 from other JAK family members became an attractive design option. By applying this strategy, a set of JAK3 inhibitors with excellent selectivity against other JAK isoforms and the kinome was developed during the last three years and used to decipher JAK3-dependent signaling. The data obtained with these tool compounds demonstrates that selective JAK3 inhibition is sufficient to block downstream signaling. Since one of these inhibitors is currently under evaluation in phase II clinical studies against several inflammatory disorders, it will soon become apparent whether selective JAK3 inhibition translates into clinical efficacy. [ABSTRACT FROM AUTHOR]

Janus kinase 3 (JAK3) is associated with the common gamma chain of several interleukin (IL) receptors essential to inflammatory signaling. To study the potential role of JAK3 in stroke-induced neuroinflammation, we subjected mice to permanent middle cerebral artery occlusion and investigated the effects of JAK3 inhibition with decernotinib (VX-509) on infarct size, behavior, and levels of several inflammatory mediators. Results from our double immunofluorescence staining showed JAK3 expression on neurons, endothelial cells, and microglia/macrophages in the ischemic mouse brain (n = 3). We found for the first time that total and phosphorylated/activated JAK3 are dramatically increased after stroke in the ipsilateral hemisphere (**P < 0.01; n = 5-13/group) in addition to increased IL-21 expression after stroke (**P < 0.01; n = 5-7/group). However, inhibition of JAK3 confirmed by reduced phosphorylation of its activation loop at tyrosine residues 980/981 does not reduce infarct volume measured at 48 h after stroke (n = 6-10/group) nor does it alter behavioral outcomes sensitive to neurological deficits or stroke-induced neuroinflammatory response (n = 9-10/group). These results do not support a detrimental role for JAK3 in acute neuroinflammation following permanent focal cerebral ischemia. The functional role of increased JAK3 activation after stroke remains to be further investigated. [ABSTRACT FROM AUTHOR]

Background: Janus kinase (Jak) 3 has recently been shown as a beneficial target for the treatment of chronic inflammatory diseases, such as psoriasis and alopecia areata. The role of Jak3 in tissue repair and remodeling is emerging., Objective: This study aimed to investigate the role of Jak3 signaling in the remodeling of the sebaceous gland (SG) during skin wound repair, and the development of in vitro SGs., Methods: Mouse skin tissue (ICR mouse) was obtained from the recovered skin eight days after a 4 mm biopsy punch wound. To observe the role of Jak3, the selective inhibitors WHI-p131 and PF06651600 was administered. Formation of in vitro SG was examined using primary sebocyte cultures obtained postnatally from 3-day-old mice., Results: The data showed that SGs showed highly positive signals with anti-isolectin B4, which also used for detection of angiogenetic vessels and the basal epidermis. Isolectin B4 could be a good indicator of SGs. The Jak3 inhibitors significantly reduced the area and volume of SG remodeling with reduced expression of p-Jak3. In addition, the area of cultured intact SG in vitro was significantly decreased in a concentration-dependent manner by Jak3 inhibition., Conclusion: These data showed that Jak3 signaling is a potent regulator to develop SGs. Jak3 inhibition did not decrease the number of sebocytes in SGs but decreased the area and volume of SG remodeling. Therefore, Jak3 inhibition may be a potential target for the treatment of SG hyperplasia and associated skin diseases., Competing Interests: The authors have nothing to disclose., (Copyright © The Korean Dermatological Association and The Korean Society for Investigative Dermatology.)

An unmet medical need remains for patients suffering from dry eye disease (DED). A fast-acting, better-tolerated noncorticosteroid anti-inflammatory eye drop could improve patient outcomes and quality of life. Herein, we describe a small-molecule drug discovery effort to identify novel, potent, and water-soluble JAK inhibitors as immunomodulating agents for topical ocular disposition. A focused library of known 3-(4-(2-(arylamino)pyrimidin-4-yl)-1 H -pyrazol-1-yl)propanenitriles was evaluated as a molecular starting point. Structure-activity relationships (SARs) revealed a ligand-efficient (LE) JAK inhibitor series, amenable to aqueous solubility. Subsequent in vitro analysis indicated the potential for off-target toxicity. A KINOME scan selectivity profile of 5 substantiated the likelihood of widespread series affinity across the human kinome. An sp 2 -to-sp 3 drug design strategy was undertaken to attenuate off-target kinase activity while driving JAK-STAT potency and aqueous solubility. Tactics to reduce aromatic character, increase fraction sp 3 (Fsp 3 ), and bolster molecular complexity led to the azetidin-3-amino bridging scaffold in 31 .

Developing selective inhibitors for Janus kinase 1 (JAK1) is a significant focus for improving the efficacy and alleviating the adverse effects in treating immune-inflammatory diseases. Herein, we report the discovery of a series of C-5 pyrazole-modified pyrrolopyrimidine derivatives as JAK1-selective inhibitors. The potential hydrogen bond between the pyrazole group and E966 in JAK1 is the key point that enhances JAK1 selectivity. These compounds exhibit 10- to 20-fold JAK1 selectivity over JAK2 in enzyme assays. Compound 12b also exhibits excellent JAK1 selectivity in Ba/F3-TEL-JAK cellular assays. Metabolism studies and the results of the hair growth model in mice indicate that compound 12b may be a viable lead compound for the development of highly JAK1-selective inhibitors for immune and inflammatory diseases.

Acquired aplastic anemia (AA) is recognized as an immune-mediated disorder resulting from active destruction of hematopoietic cells in bone marrow (BM) by effector T lymphocytes. Bulk genomic landscape analysis and transcriptomic profiling have contributed to a better understanding of the recurrent cytogenetic abnormalities and immunologic cues associated with the onset of hematopoietic destruction. However, the functional mechanistic determinants underlying the complexity of heterogeneous T lymphocyte populations as well as their correlation with clinical outcomes remain to be elucidated. To uncover dysfunctional mechanisms acting within the heterogeneous marrow-infiltrating immune environment and examine their pathogenic interplay with the hematopoietic stem/progenitor pool, we exploited single-cell mass cytometry for BM mononuclear cells of severe AA (SAA) patients pre- and post-immunosuppressive therapy, in contrast to those of healthy donors. Alignment of BM cellular composition with hematopoietic developmental trajectories revealed potential functional roles for non-canonically activated CD4 + naïve T cells in newly-diagnosed pediatric cases of SAA. Furthermore, single-cell transcriptomic profiling highlighted a population of Th17-polarized CD4 + CAMK4 + naïve T cells showing activation of the IL-6/JAK3/STAT3 pathway, while gene signature dissection indicated a predisposition to proinflammatory pathogenesis. Retrospective validation from our SAA cohort of 231 patients revealed high plasma levels of IL-6 as an independent risk factor of delayed hematopoietic response to antithymocyte globulin-based immunosuppressive therapy. Thus, IL-6 warrants further investigation as a putative therapeutic target in SAA., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The Janus kinases (JAKs) are a family of non-receptor cytosolic protein kinases critical for immune signaling. Many covalently bound ligands of JAK3 inhibitors have been reported. To help design selective JAK inhibitors, in this paper, we used five model proteins to study the subtype selectivity of and the mutational effects on inhibitor binding. We also compared the Covalent Dock programs from the Schrodinger software suite and the MOE software suite to determine which method to use for the drug design of covalent inhibitors. Our results showed that the docking affinity from 4Z16 (JAK3 wild-type model), 4E4N (JAK1), 4D1S (JAK2), and 7UYT (TYK2) from the Schrödinger software suite agreed well with the experimentally derived binding free energies with small predicted mean errors. However, the data from the mutant 5TTV model using the Schrödinger software suite yielded relatively large mean errors, whereas the MOE Covalent Dock program gave small mean errors in both the wild-type and mutant models for our model proteins. The docking data revealed that Leu905 of JAK3 and the hydrophobic residue at the same position in different subtypes (Leu959 of JAK1, Leu932 of JAK2, and Val981 of TYK2) is important for ligand binding to the JAK proteins. Arg911 and Asp912 of JAK3, Asp939 of JAK2, and Asp988 of TYK2 can be used for selective binding over JAK1, which contains Lys965 and Glu966 at the respective positions. Asp1021, Asp1039, and Asp1042 can be utilized for JAK1-selective ligand design, whereas Arg901 and Val981 may help guide TYK2-selective molecule design.

Severe combined immunodeficiency ( SCID) is the most severe form of primary immunodeficiency disease, and it is characterized by marked impairment in cellular and humoral immunity. Mutations in several genes cause SCID, one of which is Janus kinase 3 ( JAK3), resulting in autosomal recessive T(-)B(+) NK(-) SCID. Only three patients with JAK3-deficient SCID have been reported in Japan. We herein describe the case of a 6-month-old girl with pneumocystis pneumonia, who was diagnosed with SCID with compound heterozygous JAK3 mutations (c.1568G>A + c.421-10G>A). One of the mutations was previously reported in another Japanese patient. The other mutation was a novel and de novo relatively deep intronic mutation causing aberrant RNA splicing. The patient was successfully treated with bone marrow transplantation from a haploidentical donor. [ABSTRACT FROM AUTHOR]

Background/Aims: Oscillations of cytosolic Ca2+ activity ([Ca2+]i) participate in the orchestration of tumor cell proliferation. [Ca2+]i could be increased by intracellular Ca2+ release followed by store-operated Ca2+-entry (SOCE). [Ca2+]i could be decreased by Ca2+ extrusion via Na+/ Ca2+ exchange. Mechanisms accomplishing SOCE include the pore-forming ion channel unit Orai1 and its regulator STIM1, Na+/Ca2+ exchanger isoforms include NCX1. In MCF-7 breast carcinoma cells Orai1 and NCX1 have previously been shown to be modified by pharmacological inhibition of Janus activated kinase JAK2. The present study explored whether SOCE and Na+/ Ca2+ exchange are similarly sensitive to pharmacological JAK3 inhibition. Methods: MCF- 7 breast carcinoma cells were studied in the absence and presence of the JAK3 inhibitor WHI-P154 (22 μM). [Ca2+]i was estimated from Fura-2-fluorescence, SOCE from increase of [Ca2+]i following Ca2+ re-addition after Ca2+-store depletion with sarcoendoplasmatic Ca2+- ATPase (SERCA) inhibitor thapsigargin (1 μM), and Na+/Ca2+ exchanger activity from increase of [Ca2+]i following extracellular Na+ removal. Transcript levels were quantified with RT-PCR. Results: Addition of ATP (100 μM) was followed by a rapid increase of [Ca2+]i, which was significantly blunted by WHI-P154. Thapsigargin-induced intracellular Ca2+ release was not appreciably influenced by WHI-P154. Subsequent SOCE was, however, significantly blunted by WHI-P154. WHI-P154 further significantly decreased Orai1 transcript levels. The increase of [Ca2+]i following extracellular Na+-removal and the NCX1 transcript levels were similarly decreased by WHI-P154. Conclusions: The JAK3 inhibitor WHI-P154 decreases both, Orai1 and NCX1 transcript levels and thus impairs SOCE and Na+/Ca2+ exchange. [ABSTRACT FROM AUTHOR]

ABSTRACT The small molecule WHI-131 is a potent therapeutic agent with anti-inflammatory, antiallergic, and antileukemic potential. However, the regulatory effects of WHI-131 on osteoblast and osteoclast activity are unclear. We examined the effects of WHI-131 on osteoblast and osteoclast differentiation with respect to bone remodeling. The production of receptor activator of nuclear factor kappa-B ligand (RANKL) by osteoblasts in response to interleukin (IL)-1 or IL-6 stimulation decreased by 56.8% or 50.58%, respectively, in the presence of WHI-131. WHI-131 also abrogated the formation of mature osteoclasts induced by IL-1 or IL-6 stimulation. Moreover, WHI-131 treatment decreased RANKL-induced osteoclast differentiation of bone marrow-derived macrophages, and reduced the resorbing activity of mature osteoclasts. WHI-131 further decreased the mRNA and protein expression levels of c-Fos and nuclear factor of activated T cells, cytoplasmic 1 (NFATc1) by almost twofold, and significantly downregulated the mRNA expression of the following genes: tartrate-resistant acid phosphatase ( TRAP), osteoclast-associated receptor ( OSCAR), DC-STAMP, OC-STAMP, ATP6v0d2, and cathepsin K ( CtsK) compared with the control group. WHI-131 further suppressed the phosphorylation of protein kinase B (Akt) and degradation of inhibitor of kappa B (IκB); Ca2+ oscillation was also affected, and phosphorylation of the C-terminal Src kinase (c-Src)-Bruton agammaglobulinemia tyrosine kinase (Btk)-phospholipase C gamma 2 (PLCγ2) (c-Src-Btk-PLCg2 calcium signaling pathway) was inhibited following WHI-131 treatment. The Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway was activated by WHI-131, accompanied by phosphorylation of STAT3 Ser727 and dephosphorylation of STAT6. In osteoblasts, WHI-131 caused an approximately fourfold increase in alkaline phosphatase activity and Alizarin Red staining intensity. Treatment with WHI-131 increased the mRNA expression levels of genes related to osteoblast differentiation, and induced the phosphorylation of Akt, p38, and Smad1/5/8. Furthermore, 5-week-old ICR mice treated with WHI-131 exhibited antiresorbing effects in a lipopolysaccharide-induced calvaria bone loss model in vivo and increased bone-forming activity in a calvarial bone formation model. Therefore, the results of this study show that WHI-131 plays a dual role by inhibiting osteoclast differentiation and promoting osteoblast differentiation. Thus, WHI-131 could be a useful pharmacological agent to treat osteoporosis by promoting bone growth and inhibiting resorption. © 2015 American Society for Bone and Mineral Research. [ABSTRACT FROM AUTHOR]

Background: The etiology of axial spondyloarthritis (axSpA) is not fully elucidated. Research continues in determining the mechanisms responsible for initiation of the disease process, its maintenance and development. Objectives: The aim of this study was to evaluate the expression of transcription factors STAT (signal transducer and activator of transcription) and NF-κB (nuclear factor kappa B) as well as Janus kinase3 (JAK3) in the peripheral blood leukocytes. We also analyzed the connection between the degree of activation of transcription factors and the disease activity. Material/Methods: The study involved 46 patients with axSpA and 19 healthy individuals who comprised the control group. The expression of NF-κB, STAT1, STAT3, STAT4, STAT5, STAT6, and JAK3 in peripheral blood leukocytes was assessed. To determine the degree of activation of transcription factors STAT-s and NF-κB and JAK3 kinase, the immunocytochemistry method was used. For location of the factors, the primary monoclonal anti-NF-κB, anti-JAK3 and polyclonal anti-STAT-s antibodies were used (Chemicon International, USA, Abcam, Cambridge, UK), and the set of antibodies Novocastain Super ABC Kit (Novocastra, UK). Results: Expression of STAT1, STAT3, STAT4, STAT5, STAT6, NF-κB and JAK3 was statistically higher in the group of patients with axSpA than in the control group. There was a positive correlation with ESR values and expression of STAT4. There was no correlation between STAT, NF-κB, and JAK3 expression and ASDAS, BASDAI, and BASFI. Nine patients were treated with TNF-α inhibitors. The expression of NF-κB and STAT6 was higher in the group treated with TNF-α inhibitors, even though disease activity in these patients was shown to be lower than in those not receiving such treatment (ASDAS = 1.34±0.51 vs. 3.52±0.90, BASDAI = 2.34±1.92 vs. 5.51±2.41). Conclusions: In the group of patients with axSpA compared with the control group, higher expression of the transcription factors STAT and NF-κB as well as JAK3 was observed. Due to its crucial roles in inflammation and autoimmunity, STAT4 may have promise as an effective therapeutic target for axSpA. [ABSTRACT FROM AUTHOR]