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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 CD4Our 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.

Fos-related antigen-2 (Fra-2) is a transcription factor belonging to the activator protein 1 (AP-1) family, which is associated with many chronic airway diseases such as asthma. Alternatively activated (M2) macrophages are associated with Fra2 in airway diseases such as pulmonary fibrosis. However, there is no specific study that explores the relationship between M2 macrophages and Fra2 in asthma.We hypothesized that a potential mechanism of allergic asthma could be that Fra2 is highly expressed in M2 macrophages through JAK3-STAT5 and facilitates the production of downstream T-helper 2 (Th2) cytokines, thus promoting the pathogenesis of asthma.Peripheral venous blood and airway tissue samples of patients with asthma and controls were obtained. Moreover, a C57BL/6 mouse model of asthma was established. Fra2 expression was detected using immunohistochemistry and immunofluorescence. Macrophages were obtained by flow sorting, and expression of the JAK3-STAT5-Fra2 signaling pathway was determined using PCR and western blotting. Enzyme-linked immunosorbent assay was used to determine M2 macrophage-associated Th2-type cytokine levels.Fra2 was highly expressed in patients with asthma and asthmatic mice. The JAK3-STAT5 was a signal pathway related to the high expression of Fra2 in M2 macrophages. Moreover, we found that Fra2 could affect the production of Th2 cytokines downstream of M2 macrophages, including interleukin 4 (IL-4) and IL-13.M2 macrophages could promote airway inflammation through JAK3-STAT5-Fra2 to induce allergic asthma. Our study offers a new insight to further understand the pathogenesis of asthma and also provides a new direction for targeted treatment.

Background. The role of ketamine as an adjuvant for morphine in the treatment of cancer pain and immune functions has been confirmed. This study aimed to explore the role of morphine and ketamine on cancer pain and T cells of patients with cervical cancer (CC). Methods. T cells were isolated from peripheral blood mononuclear cells (PBMC) of CC patients by positive selection using anti-CD3 beads. The isolated T cells were assigned into three groups: the control group, the morphine group, and the morphine + ketamine (Mor + Ket) group. The percentages of CD4+ and CD8+ were analyzed by flow cytometry. The levels of interferon (IFN)-γ, interleukin (IL)-2, and IL-17 and the corresponding mRNA expression in vitro were determined using ELISA and qRT-PCR, respectively. Western blotting was used for detection of JAK3/STAT5 pathway-related proteins after naltrexone treatment in vitro. Afterwards, all the patients were further divided into the morphine group and the Mor + Ket group in accordance with the principles of the randomized and double-blind method to assess pain intensity. Results. Our in vivo results showed that drug combinations relieved cancer pain more effectively than morphine intervention. The in vitro results demonstrated that the combination of morphine and ketamine may decrease CD4+ percentage, CD4+/CD8+ ratio, and the levels of IFN-γ, IL-2, and IL-17 via the JAK3/STAT5 pathway. Conclusions. Our finding indicated that morphine-ketamine combination could improve cancer pain and repress immune function via the JAK3/STAT5 pathway in the progression of CC.

Resistance to mitochondrial apoptosis predicts inferior treatment outcomes in patients with diverse tumor types, including T-cell acute lymphoblastic leukemia (T-ALL). However, the genetic basis for variability in this mitochondrial apoptotic phenotype is poorly understood, preventing its rational therapeutic targeting. Using BH3 profiling and exon sequencing analysis of childhood T-ALL clinical specimens, we found that mitochondrial apoptosis resistance was most strongly associated with activating mutations of JAK3. Mutant JAK3 directly repressed apoptosis in leukemia cells, because its inhibition with mechanistically distinct pharmacologic inhibitors resulted in reversal of mitochondrial apoptotic blockade. Inhibition of JAK3 led to loss of MEK, ERK and BCL2 phosphorylation, and BH3 profiling revealed that JAK3-mutant primary T-ALL patient samples were characterized by a dependence on BCL2. Treatment of JAK3-mutant T-ALL cells with the JAK3 inhibitor tofacitinib in combination with a spectrum of conventional chemotherapeutics revealed synergy with glucocorticoids, in vitro and in vivo. These findings thus provide key insights into the molecular genetics of mitochondrial apoptosis resistance in childhood T-ALL, and a compelling rationale for a clinical trial of JAK3 inhibitors in combination with glucocorticoids for patients with JAK3-mutant T-ALL.

Jak3, a member of the Janus kinase family, is essential for the cytokine receptor common gamma chain (γc)-mediated signaling. During activation of Jak3, tyrosine residues are phosphorylated and potentially regulate its kinase activity. We identified a novel tyrosine phosphorylation site within mouse Jak3, Y820, which is conserved in human Jak3, Y824. IL-2-induced tyrosine phosphorylation of Jak3 Y824 in human T cell line HuT78 cells was detected by using a phosphospecific, pY824, antibody. Mutation of mouse Jak3 Y820 to alanine (Y820A) showed increased autophosphorylation of Jak3 and enhanced signal transducer and activator of transcription 5 (STAT5) tyrosine phosphorylation and transcriptional activation. Stably expressed Jak3 Y820A in F7 cells, an IL-2 responsive mouse pro-B cell line Ba/F3, exhibited enhanced IL-2-dependent cell growth. Mechanistic studies demonstrated that interaction between Jak3 and STAT5 increased in Jak3 Y820A compared to wild-type Jak3. These data suggest that Jak3 Y820 plays a role in negative regulation of Jak3-mediated STAT5 signaling cascade upon IL-2-stimulation. We speculate that this occurs through an interaction promoted by the tyrosine phosphorylated Y820 or a conformational change by Y820 mutation with either the STAT directly or with the recruitment of molecules such as phosphatases via a SH2 interaction. Additional studies will focus on these interactions as Jak3 plays a crucial role in disease and health.

Up to now, a total of eight Janus kinase (JAK) inhibitors have been approved for the treatment of autoimmune and myeloproliferative disease. The JAK family belongs to the non-receptor tyrosine kinase family, consisting of JAK1, JAK2, JAK3, and Tyk2. Among these four subtypes, only JAK3 is mainly expressed in hematopoietic tissue cells and is exclusively associated with the cytokines shared in the common gamma-chain receptor subunit. Due to its specific tissue distribution and functional characteristics that distinguish it from the other JAKs family subtypes, JAK3 is a promising target for the treatment of autoimmune disease. This study aimed to provide a comprehensive review of the available patent literature on JAK-family inhibitors published from 2016 to the present. In addition, an overview of the clinical activities of selective JAK3 inhibitors in recent years was provided. To date, no selective JAK3 inhibitors have been approved for use in clinics. Over the last 5 years, an increasing number of studies on JAK3 inhibitors, particularly ritlecitinib by Pfizer, have demonstrated their promising therapeutic potential. In this review, recent studies reported that selective JAK3 inhibitors may offer valid, interesting, and promising therapeutic potential in inflammatory and autoimmune diseases.

Abivertinib, a third-generation tyrosine kinase inhibitor, is originally designed to target epidermal growth factor receptor (EGFR)-activating mutations. Previous studies have shown that abivertinib has promising antitumor activity and a well-tolerated safety profile in patients with non-small-cell lung cancer. However, abivertinib also exhibited high inhibitory activity against Bruton’s tyrosine kinase and Janus kinase 3. Given that these kinases play some roles in the progression of megakaryopoiesis, we speculate that abivertinib can affect megakaryocyte (MK) differentiation and platelet biogenesis. We treated cord blood CD34+ hematopoietic stem cells, Meg-01 cells, and C57BL/6 mice with abivertinib and observed megakaryopoiesis to determine the biological effect of abivertinib on MK differentiation and platelet biogenesis. Our in vitro results showed that abivertinib impaired the CFU-MK formation, proliferation of CD34+ HSC-derived MK progenitor cells, and differentiation and functions of MKs and inhibited Meg-01-derived MK differentiation. These results suggested that megakaryopoiesis was inhibited by abivertinib. We also demonstrated in vivo that abivertinib decreased the number of MKs in bone marrow and platelet counts in mice, which suggested that thrombopoiesis was also inhibited. Thus, these preclinical data collectively suggested that abivertinib could inhibit MK differentiation and platelet biogenesis and might be an agent for thrombocythemia.

Alopecia areata (AA) is an autoimmune disease caused by T cell-mediated destruction of the hair follicle (HF). Therefore, approaches that effectively disrupt pathogenic T cell responses are predicted to have therapeutic benefit for AA treatment. T cells rely on the duality of T cell receptor (TCR) and gamma chain (γc) cytokine signaling for their development, activation, and peripheral homeostasis. Ifidancitinib is a potent and selective next-generation JAK1/3 inhibitor predicted to disrupt γc cytokine signaling. We found that Ifidancitinib robustly induced hair regrowth in AA-affected C3H/HeJ mice when fed with Ifidancitinib in chow diets. Skin taken from Ifidancitinib-treated mice showed significantly decreased AA-associated inflammation. CD44+CD62L- CD8+ T effector/memory cells, which are associated with the pathogenesis of AA, were significantly decreased in the peripheral lymphoid organs in Ifidancitinib-treated mice. We observed high expression of co-inhibitory receptors PD-1 on effector/memory CD8+ T cells, together with decreased IFN-γ production in Ifidancitinib-treated mice. Furthermore, we found that γc cytokines regulated T cell exhaustion. Taken together, our data indicate that selective induction of T cell exhaustion using a JAK inhibitor may offer a mechanistic explanation for the success of this treatment strategy in the reversal of autoimmune diseases such as AA.

JAK3 is principally activated by members of the interleukin-2 receptor family and plays an essential role in lymphoid development, with inactivating JAK3 mutations causing autosomal-recessive severe combined immunodeficiency (SCID). This study aimed to generate an equivalent zebrafish model of SCID and to characterize the model across the life-course. Genome editing of zebrafish jak3 created mutants similar to those observed in human SCID. Homozygous jak3 mutants showed reduced embryonic T lymphopoiesis that continued through the larval stage and into adulthood, with B cell maturation and adult NK cells also reduced and neutrophils impacted. Mutant fish were susceptible to lymphoid leukemia. This model has many of the hallmarks of human SCID resulting from inactivating JAK3 mutations and will be useful for a variety of pre-clinical applications.

Terminal T-cell exhaustion poses a significant barrier to effective anticancer immunotherapy efficacy, with current drugs aimed at reversing exhaustion being limited. Recent investigations into the molecular drivers of T-cell exhaustion have led to the identification of chronic IL2 receptor (IL2R)–STAT5 pathway signaling in mediating T-cell exhaustion. We targeted the key downstream IL2R-intermediate JAK 3 using a clinically relevant highly specific JAK3-inhibitor (JAK3i; PF-06651600) that potently inhibited STAT5-phosphorylation in vitro. Whereas pulsed high-dose JAK3i administration inhibited antitumor T-cell effector function, low-dose chronic JAK3i significantly improved T-cell responses and decreased tumor load in mouse models of solid cancer. Low-dose JAK3i combined with cellular and peptide vaccine strategies further decreased tumor load compared with both monotherapies alone. Collectively, these results identify JAK3 as a novel and promising target for combination immunotherapy.

Osimertinib is a third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor used to treat non-small cell lung cancer. However, its off-targets are obscure, and systematic analysis of off-target activities remains to be performed. Here, we identified the off-targets of osimertinib using PharmMapper and DRAR-CPI and analyzed the intersected targets using the GeneMANIA and DAVID servers. A drug-target-pathway network was constructed to visualize the associations. The results showed that osimertinib is associated with 31 off-targets, 40 Kyoto Encyclopedia of Genes and Genomes pathways, and 9 diseases. Network analysis revealed that the targets were involved in cancer and other physiological processes. In addition to EGFR, molecular docking analysis showed that seven proteins, namely Janus kinase 3, peroxisome proliferator-activated receptor alpha, renin, mitogen-activated protein kinases, lymphocyte-specific protein tyrosine kinase, cell division protein kinase 2 and proto-oncogene tyrosine-protein kinase Src, could also be potential targets of osimertinib. In conclusion, osimertinib is predicted to target multiple proteins and pathways, resulting in the formation of an action network via which it exerts systematic pharmacological effects.

Along with the occurrence of cisplatin resistance, treatment on gastric cancer (GC) becomes difficult. Therefore, researches on new therapeutic methods to revert cisplatin resistance are becoming increasingly urgent. qRT-PCR was used to quantify the expression of miR-4486, JAK3 in SGC-7901 or SGC-7901/DDP cell lines. WB was utilized to analyze the expression of JAK3, STAT3 and p-STAT3 in SGC-7901/DDP cell lines. CCK-8 assay was used to determine the IC50 of cisplatin on both cell lines and cell viability of SGC-7901/DDP cell lines. The target relationship between miR-4486 and JAK3 was determined by luciferase assay. MiR-4486 expression on apoptosis of SGC-7901/DDP cell lines was determined by flow cytometry. qRT-PCR testified that miR-4486 decreased in SGC-7901/DDP cells, and the expression of miR-4486 mimic increased significantly compared with miR-4486 NC. By CCK-8 assay, the IC50 of cisplatin on both cell lines were 9 μg/mL and 81.3 μg/mL, and overexpression of miR-4486 decreased the viability of SGC-7901/DDP cells. Compared with DDP group, the expression of miR-4486 accelerated SGC-7901/DDP cells apoptosis. Dual-luciferase assay suggested that JAK3 was the target gene of miR-4486. qRT-PCR and WB proved that miR-4486/JAK3 axis inhibit the activation of JAK3/STAT3 pathway, and JAK3 overexpression can partly reverse this. As shown by CCK-8 and flow cytometry, miR-4486 overexpression decreased viability and stimulated apoptosis of SGC-7901/DDP cells. However, JAK3 overexpression can also partly revert this. miR-4486 overexpression could decrease viability and improve apoptosis of SGC-7901/DDP cells to revert its cisplatin-resistance, and the mechanism may be related to JAK3/STAT3 signalling pathway.

C1q tumor necrosis factor‐related peptide 8 (CTRP8) is the least studied member of the C1Q‐TNF‐related peptide family. We identified CTRP8 as a ligand of the G protein‐coupled receptor relaxin family peptide receptor 1 (RXFP1) in glioblastoma multiforme (GBM). The CTRP8‐RXFP1 ligand–receptor system protects human GBM cells against the DNA‐alkylating damage‐inducing temozolomide (TMZ), the drug of choice for the treatment of patients with GBM. The DNA protective role of CTRP8 was dependent on a functional RXFP1‐STAT3 signaling cascade and targeted the monofunctional glycosylase N‐methylpurine DNA glycosylase (MPG) for more efficient base excision repair of TMZ‐induced DNA‐damaged sites. CTRP8 also improved the survival of GBM cells by upregulating anti‐apoptotic BCl‐2 and BCL‐XL. Here, we have identified Janus‐activated kinase 3 (JAK3) as a novel member of a novel CTRP8‐RXFP1‐JAK3‐STAT3 signaling cascade that caused an increase in cellular protein content and activity of the small Rho GTPase Cdc42. This is associated with significant F‐actin remodeling and increased GBM motility. Cdc42 was critically important for the upregulation of the actin nucleation complex N‐Wiskott–Aldrich syndrome protein/Arp3/4 and actin elongation factor profilin‐1. The activation of the RXFP1‐JAK3‐STAT3‐Cdc42 axis by both RXFP1 agonists, CTRP8 and relaxin‐2, caused extensive filopodia formation. This coincided with enhanced activity of ezrin, a key factor in tethering F‐actin to the plasma membrane, and inhibition of the actin filament severing activity of cofilin. The F‐actin remodeling and pro‐migratory activities promoted by the novel RXFP1‐JAK3‐STAT3‐Cdc42 axis were blocked by JAK3 inhibitor tofacitinib and STAT3 inhibitor STAT3 inhibitor VI. This provides a new rationale for the design of JAK3 and STAT3 inhibitors with better brain permeability for clinical treatment of the pervasive brain invasiveness of GBM., The C1Q‐TNF‐related peptide family member C1q tumor necrosis factor‐related peptide 8 (CTRP8) and its G protein‐coupled receptor relaxin family peptide receptor 1 (RXFP1) are emerging as a novel ligand–receptor system that can protect glioblastoma cells against DNA‐alkylating damage and apoptosis. We describe a novel CTRP8‐RXFP1‐Janus‐activated kinase 3‐STAT3 signaling cascade that enhances cellular protein content and activity of small Rho GTPase Cdc42, which directs F‐actin cytoskeletal remodeling, filopodia formation, and increased glioblastoma multiforme migration.

Several groups have developed in vitro expansion cultures for mouse metanephric nephron progenitor cells (NPCs) using cocktails of small molecules and growth factors including BMP7. However, the detailed mechanisms by which BMP7 acts in the NPC expansion remain to be elucidated. Here, by performing chemical screening for BMP substitutes, we identified a small molecule, TCS21311, that can replace BMP7 and revealed a novel inhibitory role of BMP7 in JAK3-STAT3 signaling in NPC expansion culture. Further, we found that TCS21311 facilitates the proliferation of mouse embryonic NPCs and human induced pluripotent stem cell-derived NPCs when added to the expansion culture. These results will contribute to understanding the mechanisms of action of BMP7 in NPC proliferation in vitro and in vivo and to the stable supply of NPCs for regenerative therapy, disease modeling and drug discovery for kidney diseases.

Thymic epithelial tumors are the most common adult mediastinal tumors; however, their growth mechanism remains relatively unknown. Among the JAK/STAT pathway-related proteins, which control various intracellular events, STAT3 is deeply involved in cell proliferation. Constitutive activation of STAT3 and the resulting overexpression of cyclin D1 have been confirmed in various tumors, but have not been thoroughly investigated in thymic epithelial tumors. In this study, we immunohistochemically examined STAT3 activation, cyclin D1 expression, and JAK3 activation in thymic epithelial tumors and statistically analyzed their correlation with clinicopathological features. Formalin-fixed paraffin-embedded specimens of 94 thymic epithelial tumors surgically resected at Kyorin University Hospital between 2005 and 2018 were included in this study. pSTAT3, cyclin D1, and pJAK3 were immunohistochemically examined, and the correlation with histology, Masaoka stage, and survival time was statistically analyzed. Cyclin D1 was found to be significantly overexpressed in the STAT3-activated group. This phenomenon was associated with histology and Masaoka stage. JAK3 was also activated in thymic epithelial tumors; however, JAK3 and STAT3 activation were not always correlated. Using survival time analysis, the STAT3-activated group, cyclin D1-expressed group, and JAK3-activated group had significantly lower progression-free survival times than those for both the non-activated and non-expressed groups. STAT3 activation may promote cyclin D1 overexpression in thymic epithelial tumors, and intracellular signaling pathways other than JAK3 may be involved in STAT3 activation. STAT3 activation, cyclin D1 overexpression, and JAK3 activation are biomarker candidates that indicate clinically poor prognosis.

Objective: The objective of this study is to determine the role of JAK3 (Janus kinase 3) in reendothelialization after vascular injury. Methods and Results: By using mouse carotid artery wire injury and rat balloon injury model, we found that JAK3 regulates reendothelialization and endothelial cell proliferation after vascular injury. JAK3 and phospho-JAK3 levels were increased in neointimal smooth muscle cells in response to vascular injury in mice. JAK3 deficiency dramatically attenuated the injury-induced intimal hyperplasia in carotid arteries of both male and female mice. Importantly, JAK3 deficiency caused an increased rate of reendothelialization following mechanical injury. Likewise, knockdown of JAK3 in medial smooth muscle cells elicited an accelerated reendothelialization with reduced intimal hyperplasia following balloon injury in rat carotid arteries. Interestingly, knockdown of JAK3 restored the expression of smooth muscle cell contractile protein smooth muscle α-actin in injury-induced intimal smooth muscle cells while increased the proliferating endothelial cells in the intima area. Conclusions: Our results demonstrate a novel role of JAK3 in the regeneration of endothelium after vascular injury, which may provide a new strategy to enhance reendothelialization while suppressing neointimal formation for effective vascular repair from injury.

Background and Aims: A compromise in intestinal mucosal functions is associated with several chronic inflammatory diseases. Previously, we reported that obese humans have a reduced expression of intestinal Janus kinase-3 (Jak3), a non-receptor tyrosine kinase, and a deficiency of Jak3 in mice led to predisposition to obesity-associated metabolic syndrome. Since meta-analyses show cognitive impairment as co-morbidity of obesity, the present study demonstrates the mechanistic role of Jak3 in obesity associated cognitive impairment. Our data show that high-fat diet (HFD) suppresses Jak3 expression both in intestinal mucosa and in the brain of wild-type mice. Methodology: Recapitulating these conditions using global (Jak3-KO) and intestinal epithelial cell-specific conditional (IEC-Jak3-KO) mice and using cognitive testing, western analysis, flow cytometry, immunofluorescence microscopy and 16s rRNA sequencing, we demonstrate that HFD-induced Jak3 deficiency is responsible for cognitive impairments in mice, and these are, in part, specifically due to intestinal epithelial deficiency of Jak3. Results: We reveal that Jak3 deficiency leads to gut dysbiosis, compromised TREM-2-functions-mediated activation of microglial cells, increased TLR-4 expression and HIF1-α-mediated inflammation in the brain. Together, these lead to compromised microglial-functions-mediated increased deposition of β-amyloid (Aβ) and hyperphosphorylated Tau (pTau), which are responsible for cognitive impairments. Collectively, these data illustrate how the drivers of obesity promote cognitive impairment and demonstrate the underlying mechanism where HFD-mediated impact on IEC-Jak3 deficiency is responsible for Jak3 deficiency in the brain, reduced microglial TREM2 expression, microglial activation and compromised clearance of Aβ and pTau as the mechanism during obesity-associated cognitive impairments. Conclusion: Thus, we not only demonstrate the mechanism of obesity-associated cognitive impairments but also characterize the tissue-specific role of Jak3 in such conditions through mucosal tolerance, gut–brain axis and regulation of microglial functions.

Janus kinase 3 (JAK3) is a potential target for the treatment of hematological malignancies. Herein, we report the discovery of a series of new orally bioavailable irreversible JAK3 kinase inhibitors. The representative compound

Janus Kinases (JAKs) have emerged as an important drug target for the treatment of a number of immune disorders due to the central role that they play in cytokine signalling. 4 isoforms of JAKs exist in mammalian cells and the ideal isoform profile of a JAK inhibitor has been the subject of much debate. JAK3 has been proposed as an ideal target due to its expression being largely restricted to the immune system and its requirement for signalling by cytokine receptors using the common γ-chain. Unlike other JAKs, JAK3 possesses a cysteine in its ATP binding pocket and this has allowed the design of isoform selective covalent JAK3 inhibitors targeting this residue. We report here that mutating this cysteine to serine does not prevent JAK3 catalytic activity but does greatly increase the IC50 for covalent JAK3 inhibitors. Mice with a Cys905Ser knockin mutation in the endogenous JAK3 gene are viable and show no apparent welfare issues. Cells from these mice show normal STAT phosphorylation in response to JAK3 dependent cytokines but are resistant to the effects of covalent JAK3 inhibitors. These mice therefore provide a chemical-genetic model to study JAK3 function.

Several cytokines involved in inflammatory pathologies signal via the Janus kinase-signal transducer and activator of transcription pathway. Four JAKs are known: JAK1, JAK2, JAK3 and TYK2. The specific activation of JAKs and STATs determines the biological effects of each cytokine. JAK1 is involved in the signalling of ‘γc’ receptor cytokines (IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21), pro-inflammatory cytokines including IL-6, as well as IFN. The critical position of JAK1 downstream of these cytokines suggests that JAK1-selective inhibitors are comparable to non-selective ones, without the unwanted consequences of JAK2- or JAK3-blockade. JAK inhibition has led to a better understanding of the biology of synovial inflammation and bone homeostasis. Moreover, the efficacy of non-selective JAK inhibitors and novel JAK1-selective drugs in RA supports a role for JAK1 in its pathogenesis. JAK1-selective drugs are also showing promise in axial spondyloarthritis, suggesting that they may target additional regulatory pathways that impact cytokines such as TNF and IL-17A, which do not use JAKs. Additionally, evidence now supports a JAK1 predominance in the signalling of IL-6 and oncostatin M, and indirectly, of TNF in synovial fibroblasts, macrophages and endothelial cells. Notably, bone homeostasis is also dependent on cytokines relying on JAK1 signalling to promote receptor activator of NF-κB ligand expression in osteoblasts and T cells, contributing to osteoclastogenesis. Here, the contribution of JAK1 over other kinases is unclear. While beneficial effects of JAK inhibitors on bone erosion are supported by preclinical and clinical data, effects on new bone formation in axial spondyloarthritis requires additional study. CME: This supplement is CME Accredited. To receive a CME certificate of participation, you should: Read all the papers in the supplement Register or log in at www.paradigmmc.com/962 to complete and submit the post activity assessment. You must answer 70% of the questions correctly to earn credit. You will have unlimited opportunities to successfully complete the assessment. You will receive a maximum of 7.0 AMA PRA Category 1 Credits(TM) upon successful completion of the assessment.

Background Asthma is a chronic inflammatory heterogeneous respiratory disease. Previous studies showed that the lncRNA NEAT1 (nuclear paraspeckle assembly transcript 1) might play an important role...

The Janus tyrosine kinases (JAKs) have shown great promise as therapeutic protein targets in the treatment of cancer and inflammation diseases. This study used pharmacophore modeling to identify potential inhibitors of Janus kinase 3 (JAK3). A pharmacophore model was developed based on a known JAK3 inhibitor (1NX) and was employed to search for potential JAK3 inhibitors against Indonesian herbal compounds. Among 28 hit molecules that were identified and subjected to a molecular docking protocol against JAK3, the three compounds that had the highest affinities toward JAK3 were camelliaside B, 3-O-galloylepicatechin-(4beta-6)-epicatechin-3-O-gallate, and mesuaferrone B. These were then each subjected to a 50-ns molecular dynamics (MD) simulation. Analysis of RMSD and RMSF values indicated that the three compounds reached stability during the MD simulation. Interestingly, all three compounds had lower binding energies than 1NX against JAK3, as predicted by the MM-PBSA binding energy calculation.

Background Janus kinase 3 (JAK3) acts downstream of the interleukin-2 (IL-2) receptor family to play a pivotal role in the regulation of lymphoid cell development. Activating JAK3 mutations are associated with a number of lymphoid and other malignancies, with mutations within the regulatory pseudokinase domain common. Methods The pseudokinase domain mutations A572V and A573V were separately introduced into the highly conserved zebrafish Jak3 and transiently expressed in cell lines and zebrafish embryos to examine their activity and impact on early T cells. Genome editing was subsequently used to introduce the A573V mutation into the zebrafish genome to study the effects of JAK3 activation on lymphoid cells in a physiologically relevant context throughout the life-course. Results Zebrafish Jak3 A573V produced the strongest activation of downstream STAT5 in vitro and elicited a significant increase in T cells in zebrafish embryos. Zebrafish carrying just a single copy of the Jak3 A573V allele displayed elevated embryonic T cells, which continued into adulthood. Hematopoietic precursors and NK cells were also increased, but not B cells. The lymphoproliferative effects of Jak3 A573V in embryos was shown to be dependent on zebrafish IL-2Rγc, JAK1 and STAT5B equivalents, and could be suppressed with the JAK3 inhibitor Tofacitinib. Conclusions This study demonstrates that a single JAK3 A573V allele expressed from the endogenous locus was able to enhance lymphopoiesis throughout the life-course, which was mediated via an IL-2Rγc/JAK1/JAK3/STAT5 signaling pathway and was sensitive to Tofacitinib. This extends our understanding of oncogenic JAK3 mutations and creates a novel model to underpin further translational investigations.

JAK3 differs from other JAK family members in terms of tissue distribution and functional properties, making it a promising target for autoimmune disease treatment. However, due to the high homology of these family members, targeting JAK3 selectively is difficult. As a result, exploiting small changes or selectively boosting affinity within the ATP binding region to produce new tailored inhibitors of JAK3 is extremely beneficial. PubChem CID 137321159 was used as the lead inhibitor in this study to preserve the characteristic structure and to collocate it with the redesigned new parent core structure, from which a series of 1,7-dihydro-dipyrrolo [2,3-b:3′,2′-e] pyridine derivatives were obtained using the backbone growth method. From the proposed compounds, 14 inhibitors of JAK3 were found based on the docking scoring evaluation. The RMSD and MM/PBSA methods of molecular dynamics simulations were also used to confirm the stable nature of this series of complex systems, and the weak protein–ligand interactions during the dynamics were graphically evaluated and further investigated. The results demonstrated that the new parent core structure fully occupied the hydrophobic cavity, enhanced the interactions of residues LEU828, VAL836, LYS855, GLU903, LEU905 and LEU956, and maintained the structural stability. Apart from this, the results of the analysis show that the binding efficiency of the designed inhibitors of JAK3 is mainly achieved by electrostatic and VDW interactions and the order of the binding free energy with JAK3 is: 8 (−70.286 kJ/mol) > 11 (−64.523 kJ/mol) > 6 (−51.225 kJ/mol) > 17 (−42.822 kJ/mol) > 10 (−40.975 kJ/mol) > 19 (−39.754 kJ/mol). This study may provide a valuable reference for the discovery of novel JAK3 inhibitors for those patients with immune diseases.

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

Objectives: This study amied to whether IL-21 promotes osteoblast transdifferentiation of cultured human Valvular interstitial cells (VICs). Methods: We first confirmed that IL-21 alters gene expression between CAVD aortic valve tissue and normal samples by immunohistochemistry, qPCR, and western blotting. VICs were cultured and treated with IL-21. Gene and protein expression levels of the osteoblastic markers ALP and Runx2, which can be blocked by specific JAK3 inhibitors and/or siRNA of STAT3, were measured. Results: IL-21 expression was upregulated in calcified aortic valves and promotes osteogenic differentiation of human VICs. IL-21 accelerated VIC calcification through the JAK3/STAT3 pathway. Conclusion: Our data suggest that IL-21 is a key factor in valve calcification and a promising candidate for targeted therapeutics for CAVD.

Background. Stomach adenocarcinoma (STAD) is one of the most common malignant tumors. The Janus kinases (JAKs) play a significant part in cellular biological process, inflammation, and immunity. The roles of JAKs in STAD are still not systematically described. Methods. A series of bioinformatics tools were used to clarify the role of JAKs in STAD. Results. JAK3/TYK2 levels were significantly increased in STAD during subgroup analyses based on gender, tumor grade, cancer stages, and nodal metastasis status. STAD patients with high levels of JAK3/TYK2 had poor overall survival, postprogression survival, and first progression. Immune infiltration revealed a significant correlation between JAK3/TYK2 expression and the abundance of immune cells as well as immune biomarker expression in STAD. JAK3/TYK2 was associated with the adaptive immune response, chemokine signaling pathway, and JAK-STAT signaling pathway. Conclusions. JAK3 and TYK2 serve as prognostic biomarkers and are associated with immune infiltration in STAD.

Objective To evaluate the efficacy and safety of PF‐06651600 (ritlecitinib), an irreversible inhibitor of JAK3 and the tyrosine kinase expressed in hepatocellular carcinoma (TEC) kinase family, in comparison with placebo in patients with rheumatoid arthritis (RA). Methods An 8‐week, phase II, double‐blind, parallel‐group study was conducted. Seventy patients who were seropositive for anti–citrullinated protein antibodies and/or rheumatoid factor were randomized 3:2 to receive oral PF‐06651600 (200 mg once daily) or placebo for 8 weeks. Eligible patients had an inadequate response to methotrexate, and the study design allowed up to 50% of patients to have previously received 1 tumor necrosis factor inhibitor that was inadequately effective and/or not tolerated. The primary end point was change from baseline in the Simplified Disease Activity Index (SDAI) score at week 8, assessed by Bayesian analysis using an informative prior distribution for placebo response. Results Mean change from baseline in the SDAI score at week 8 was greater in the PF‐06651600 group (−26.1 [95% credible interval −29.7, −22.4]) than in the placebo group (−16.8 [95% credible interval −20.9, −12.7]; P < 0.001). Most adverse events (AEs) were mild in severity, and no treatment‐related serious AEs, severe AEs, or deaths were reported. The most common classes of AE were infections and infestations as well as skin and subcutaneous tissue disorders; there was 1 mild case of herpes simplex in the PF‐06651600 group that was considered to be treatment related, which resolved within 3 days without study treatment discontinuation or antiviral therapy. Conclusion Treatment with the oral JAK3/TEC inhibitor PF‐06651600 (200 mg once daily) was associated with significant improvements in RA disease activity and was generally well‐tolerated in this small 8‐week study.

Summary Objectives Recent studies have shown that tRNA-derived RNA fragments (tRFs) are novel regulators of post-transcriptional gene expression. However, the expression profiles and their role in post-transcriptional gene regulation in chondrocytes is unknown. Here, we determined tRFs expression profile and explored tRF-3003a role in post-transcriptional gene regulation in IL-1β stimulated chondrocytes. Methods We used qPCR arrays to determine tRNAs and tRFs expression in age- and sex-matched primary human OA chondrocytes and TC28/I2 cells stimulated with IL-1β. Chondrocytes were transfected with tRNA-CysGCA overexpression plasmid or tRF-3003a mimic and 3′UTR luciferase reporter plasmids of mRNAs harboring predicted tRF target “seed sequence”. The AGO-RNA-induced silencing complex (AGO-RISC)-dependent repressive activity of tRF-3003a was determined by siRNA-mediated knockdown of AGO2. Results IL-1β increased the expression levels of specific tRNAs and of tRF-3003a, a type 3 tRF produced by the cleavage of tRNA-CysGCA. tRF-3003a “seed sequence” was identified in the 3′UTR of JAK3 mRNA and tRNA-CysGCA overexpression or transfection of a tRF-3003a mimic in chondrocytes downregulated JAK3 expression and significantly reduced the activity of the 3′UTR reporter. RIP assay showed enrichment of tRF-3003a into AGO2/RISC in IL-1β treated chondrocytes. The suppressive effect of tRF-3003a on JAK3 3′UTR reporter was abrogated with siRNA-mediated depletion of AGO2. Conclusions We demonstrate that under pathological conditions chondrocytes display perturbations in the expression profile of specific tRNAs and tRFs. Furthermore, a specific tRF namely tRF-3003a can post-transcriptionally regulate JAK3 expression via AGO/RISC formation in chondrocytes. Identification of this novel mechanism may be of value in the design of precision therapies for OA.

Background/aims Suicidal erythrocyte death (eryptosis) is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface following a Ca2+ entry in the cell. Eryptosis is stimulated by increased cytosolic Ca2+ ([Ca2+]i), oxidative stress, energy depletion, or high osmotic shock. Eryptosis signaling includes p38 mitogen-activated protein kinase (MAPK), caspases, casein kinase 1 (CK1), janus kinase 3 (JAK3), and protein kinase C (PKC). Dog and human erythrocytes have different characteristics, for example, dog erythrocytes lack Na+/K+- ATPase activity. Whether eryptosis occurs in dog erythrocytes in an analogous way as that in humans remains unclear. Eryptosis in dogs has not been investigated. This study aimed to explore which stimulator and signaling molecules are involved in eryptosis in healthy dog erythrocytes. Methods Erythrocytes were isolated from 10 dogs, and eryptosis was stimulated by oxidative stress with tert-butyl hydroperoxide (tBOOH), high osmotic shock with excessive sucrose condition, energy depletion with minus glucose condition, and high [Ca2+]i with ionomycin. Phosphatidylserine exposure was estimated using annexin V binding. Erythrocyte volume and [Ca2+]i were measured by forward scatter and Fluo3-fluorescence, respectively. In addition, the role of certain mediators was assessed using the following inhibitors to determine the detailed mechanisms of eryptosis in dog erythrocytes: p38MAPK, caspase family, CK1, JAK3, and PKC inhibitors. Results All eryptosis-inducing factors resulted in phosphatidylserine exposures, except for ionomycin. In addition, the erythrocyte volume increased with ionomycin and tBOOH but decreased with excessive sucrose and minus glucose condition. All treatments increased [Ca2+]i. Furthermore, WH1-P154 and chelerythrine significantly blunted the increase of annexin V binding erythrocytes following the tBOOH treatment. Conclusion Eryptosis in dogs is triggered by oxidative stress, hyperosmotic shock, and energy depletion. It is suggested that JAK3 and PKC play an important role in eryptosis following an oxidative stress in dog erythrocytes.

Severe combined immunodeficiency (SCID) has a diverse genetic aetiology, where a clinical phenotype, caused by single and/or multiple gene variants, can give rise to multiple presentations. The advent of next-generation sequencing (NGS) has recently enabled rapid identification of the molecular aetiology of SCID, which is crucial for prognosis and treatment strategies. We sought to identify the genetic aetiology of various phenotypes of SCIDs and assessed both clinical and immunologic characteristics associated with gene variants. An amplicon-based targeted NGS panel, which contained 18 most common SCID-related genes, was contumely made to screen the patients (n = 38) with typical SCID, atypical SCID or OMENN syndrome. Allelic segregations were confirmed for the detected gene variants within the families. In total, 24 disease-causing variants (17 known and 7 novel) were identified in 23 patients in 9 different SCID genes: RAG1 (n = 5), RAG2 (n = 2), ADA (n = 3), DCLRE1C (n = 2), NHEJ1 (n = 2), CD3E (n = 2), IL2RG (n = 3), JAK3 (n = 4) and IL7R (n = 1). The overall success rate of our custom-made NGS panel was 60% (39.3% for NK+ SCID and 100% for NK- SCID). Incidence of autosomal-recessive inherited genes is more frequently found in our cohort than the previously reported populations probably due to the high consanguineous marriages in Turkey. In conclusion, the custom-made sequencing panel was able to identify and confirm the previously known and novel disease-causing variants with high accuracy. Istanbul Bilgi UniversityIstanbul Bilgi University [NGYY-2018.01.0006]; Bilimsel Arastirma Projeleri Birimi, Istanbul Universitesi [52575, 20499] Istanbul Bilgi University, Grant/Award Number: NGYY-2018.01.0006; Bilimsel Arastirma Projeleri Birimi, Istanbul Universitesi, Grant/Award Number: 52575 and 20499 WOS:000534741000001 32445296 Q4

With a resurgence in interest in covalent drugs, there is a need to identify new moieties capable of cysteine bond formation that are differentiated from commonly employed systems such as acrylamide. Herein, we report on the discovery of new alkynyl benzoxazine and dihydroquinazoline moieties capable of covalent reaction with cysteine. Their utility as alternative electrophilic warheads for chemical biological probes and drug molecules is demonstrated through site-selective protein modification and incorporation into kinase drug scaffolds. A potent covalent inhibitor of JAK3 kinase was identified with superior selectivity across the kinome and improvements in in vitro pharmacokinetic profile relative to the related acrylamide-based inhibitor. In addition, the use of a novel heterocycle as a cysteine reactive warhead is employed to target Cys788 in c-KIT, where acrylamide has previously failed to form covalent interactions. These new reactive and selective heterocyclic warheads supplement the current repertoire for cysteine covalent modification while avoiding some of the limitations generally associated with established moieties.

TMEM16A is a Ca2+-activated Cl−channel in the apical membrane of biliary epithelial cells, known as cholangiocytes, which contributes importantly to ductular bile formation. Whereas cholangiocyte TMEM16A activity is regulated by extracellular ATP-binding membrane purinergic receptors, channel expression is regulated by interleukin-4 (IL-4) through an unknown mechanism. Therefore, the aim of the present study was to identify the signaling pathways involved in TMEM16A expression and cholangiocyte secretion. Studies were performed in polarized normal rat cholangiocyte monolayers, human Mz-Cha-1 biliary cells, and cholangiocytes isolated from murine liver tissue. The results demonstrate that all the biliary models expressed the IL-4Rα/IL-13Rα1 receptor complex. Incubation of cholangiocytes with either IL-13 or IL-4 increased the expression of TMEM16A protein, which was associated with an increase in the magnitude of Ca2+-activated Cl−currents in response to ATP in single cells and the short-circuit current response in polarized monolayers. The IL-4- and IL-13-mediated increase in TMEM16A expression was also associated with an increase in STAT6 phosphorylation. Specific inhibition of JAK-3 inhibited the increase in TMEM16A expression and the IL-4-mediated increase in ATP-stimulated currents, whereas inhibition of STAT6 inhibited both IL-4- and IL-13-mediated increases in TMEM16A expression and ATP-stimulated secretion. These studies demonstrate that the cytokines IL-13 and IL-4 regulate the expression and function of biliary TMEM16A channels through a signaling pathway involving STAT6. Identification of this regulatory pathway provides new insight into biliary secretion and suggests new targets to enhance bile formation in the treatment of cholestatic liver disorders.NEW & NOTEWORTHY The Ca2+-activated Cl−channel transmembrane member 16A (TMEM16A) has emerged as an important regulator of biliary secretion and hence, ductular bile formation. The present studies represent the initial description of the regulation of TMEM16A expression in biliary epithelium. Identification of this regulatory pathway involving the IL-4 and IL-13 receptor complex and JAK-3 and STAT-6 signaling provides new insight into biliary secretion and suggests new therapeutic targets to enhance bile formation in the treatment of cholestatic liver disorders.

Erman, Baran/0000-0001-9398-8465 Background: Primary immunodeficiencies (PIDs) are a heterogeneous group of congenital disorders characterized by susceptibility to recurrent infections, allergy, malignancies and autoimmunity. The identification of disease-causing genetic defects is critically important for treatment options. In last decade, next-generation sequencing (NGS)-based methods has enabled the rapid genetic screening and the discovery of new genetic defects in PIDs. In this study, we investigated causative mutations in patients with PID by NGS. Methods: We applied whole-exome sequencing in 8 PID patients. Detected mutations by NGS were validated by Sanger sequencing. Results: We made a genetic diagnosis in 5 of 8 (63%) patients, including 3 novel disease-causing variants. The identified mutations were found in RAG1, RAG2, JAK3, RFXANK, and CYBA genes. Conclusions: Our results show that whole-exome sequencing can facilitate the genetic diagnosis of the patients with PID. Can Sucak Candan Biseyler'' Foundation This study was partly supported by the Can Sucak Candan Biseyler'' Foundation. WOS:000517964000001 33406023 Q4

Immune status affects the initiation and progression of clear cell renal cell carcinoma (ccRCC), the most common subtype of renal cell carcinoma. In this study, we identified an immune-related, five-gene signature that improves survival prediction in ccRCC. Patients were classified as high- and low-risk based on the signature risk score. Survival analysis showed differential prognosis, while principal component analysis revealed distinctly different immune phenotypes between the two risk groups. High-risk patients tended to have advanced stage, higher grade disease, and poorer prognoses. Functional enrichment analysis showed that the signature genes were mainly involved in the cytokine-cytokine receptor interaction pathway. Moreover, we found that tumors from high-risk patients had higher relative abundance of T follicular helper cells, regulatory T cells, and M0 macrophages, and higher expression of PD-1, CTLA-4, LAG3, and CD47 than low-risk patients. This suggests our gene signature may not only serve as an indicator of tumor immune status, but may be a promising tool to select high-risk patients who may benefit from immune checkpoint inhibitor therapy. Multivariate Cox regression analysis showed that the signature remained an independent prognostic factor after adjusting for clinicopathological variables, while prognostic accuracy was further improved after integrating clinical parameters into the analysis.

Introduction Tofacitinib is an oral Janus kinase inhibitor for the treatment of psoriatic arthritis (PsA). Objective Our objective was to compare the incidence rates (IRs) of adverse events in tofacitinib clinical trials and real-world observational data for alternative treatments. Methods The tofacitinib “dose-comparison cohort” included months 0–12 of two phase III studies (tofacitinib 5 [n = 238] and 10 [n = 236] mg twice daily [BID]); the “all-tofacitinib comparison cohort” (n = 783) included two phase III and one ongoing long-term extension study (data cutoff May 2016). An “observational comparison cohort” (n = 5799) comprised patients initiating a conventional synthetic disease-modifying antirheumatic drug (DMARD), biologic DMARD, or apremilast in the US Truven MarketScan database from 2010 to 2015. IRs for serious infections (SIEs; requiring hospitalization), herpes zoster (HZ), malignancies (excluding non-melanoma skin cancer [NMSC]), NMSC, and major adverse cardiovascular events (MACE) across cohorts were qualitatively compared. Results IRs (patients with events/100 patient-years) for SIEs were similar between the tofacitinib dose-comparison cohort (5 mg BID: 1.3; 10 mg BID: 2.0) and the observational comparison cohort (1.1–7.9; treatment dependent). The tofacitinib dose-comparison cohort had a higher rate of HZ (5 mg BID: 2.0; 10 mg BID: 2.7) than did the observational comparison cohort (0.8–2.0). IRs for NMSC were generally lower in the all-tofacitinib comparison cohort (0.5) than in the observational comparison cohort (0.4–6.0). IRs for MACE, malignancies excluding NMSC, and NMSC were similar between cohorts. Conclusion In patients with PsA, tofacitinib had a safety profile similar to that of other systemic therapies in real-world settings, except for the risk of HZ, a known risk of tofacitinib. Trial Registration ClinicalTrials.gov: NCT01877668; NCT01882439; NCT01976364. Electronic supplementary material The online version of this article (10.1007/s40264-020-00904-9) contains supplementary material, which is available to authorized users.

The processes leading from disturbed B-cell development to adult B-cell progenitor acute lymphoblastic leukemia (BCP-ALL) remain poorly understood. Here, we describe Irf4−/− mice as prone to developing BCP-ALL with age. Irf4−/− preB-I cells exhibited impaired differentiation but enhanced proliferation in response to IL-7, along with reduced retention in the IL-7 providing bone marrow niche due to decreased CXCL12 responsiveness. Thus selected, preB-I cells acquired Jak3 mutations, probably following irregular AID activity, resulting in malignant transformation. We demonstrate heightened IL-7 sensitivity due to Jak3 mutants, devise a model to explain it, and describe structural and functional similarities to Jak2 mutations often occurring in human Ph-like ALL. Finally, targeting JAK signaling with Ruxolitinib in vivo prolonged survival of mice bearing established Irf4−/− leukemia. Intriguingly, organ infiltration including leukemic meningeosis was selectively reduced without affecting blood blast counts. In this work, we present spontaneous leukemogenesis following IRF4 deficiency with potential implications for high-risk BCP-ALL in adult humans.

The directing group assisted decarboxylative

Aberrantly activated Janus kinase 3 (JAK3) has been constantly detected in various immune disorders and hematopoietic cancers, suggesting its potential of being an attractive therapeutic target for these indications. Clinical benefits of drugs selectively targeting JAK3 versus pan-JAK inhibitors remain unclear. In this study, we report the design and synthesis of a new series of JAK3 covalent inhibitors with a pyrido[2,3-d]pyrimidin-7-one scaffold. After the extensive SAR study, compound 10f emerged to be the most potent JAK3 inhibitor with an IC

The idea of using ranked binding energies of residues and data fusion are presented here for the first time as a valuable tool to classify active and selective inhibitors. Selective inhibitors of JAK3 can inhibit inflammatory cytokine while preventing targeting other subtypes of JAK1 and JAK2. Herein, we report a novel way to identify both active JAK3 and selective JAK1/JAK3 and JAK2/JAK3 inhibitors using the effective activity and selectivity classifications. The most important residues (top 10) responsible for the inhibition mechanism are sorted from high to low energies, which are considered as variables in the classification process. In addition, the ranked energies of ligands' heteroatoms (top 5), ranked energies of hydrogen bonds (top 5) and important molecular descriptors (top 10) were used to construct different data fusion possibilities. It is shown that the proposed data fusion strategy can increase the accuracy of the activity classification to 100% and the selectivity classification to 96.4%. The proposed strategies represented in this paper can help medicinal or pharmaceutical chemist in evaluation of both active and selective inhibitors before synthesizing new pharmaceuticals.

BACKGROUND Osteoporosis is an osteolytic disease resulted from imbalance in bone homeostasis. Studies indicated that N-myc downstream-regulated gene 2 (NDRG2) could affect the osteoclast differentiation. However, the effect of NDRG2 on osteoblastic differentiation and calcification remains unknown. Hence, we aimed to analyze the effect of NDRG2 on the proliferation and differentiation of osteoblasts. MATERIAL AND METHODS The differentiation of bone morphogenetic protein 2 (BMP2) induced MC3T3-E1 cells was observed by the microscope. Real-time quantitative polymerase chain reaction (RT-qPCR) and western blot analysis detected the expression of BMP2, NDRG2, runt-related transcription factor 2 (Runx2), osteoprotegerin (OPG), osterix (OSX), and osteocalcin (OCN). Alkaline phosphatase (ALP) activity assay was detecting the ALP activity and alizarin red staining assay was analyzing intracellular calcium salt deposition. The cell transfection was also verified by RT-qPCR analysis. RESULTS The results demonstrated that BMP2 promoted the osteoblastic differentiation with the increasing expression of Runx2, OPG, OSX, and OCN. NDRG2 expression was upregulated during osteogenic differentiation. NDRG2 overexpression promoted the expression of Runx2, OPG, OSX, and OCN, and increased the ALP activity while NDRG2 inhibition reversed the changes. NDRG2 overexpression increased the intracellular calcium salt deposition and NDRG2 inhibition reversed the changes. The role of NDRG2 in osteoblastic differentiation and calcification was played through the JAK3/STAT3 signal pathway. CONCLUSIONS The presented data indicated that NDRG2 promoted BMP2-induced osteoblastic differentiation and calcification by activating the JAK3/STAT3 signal pathway.

Background Monomorphic epitheliotropic T-cell lymphoma (MEITL) is an aggressive non-Hodgkin lymphoma with a high fatality rate. This study was aimed to explore the clinicopathological and molecular genetic features of MEITL in the Chinese population. Methods A retrospective analysis was performed based on the clinical manifestations and pathological features of 20 Chinese MEITL. 9 cases with paired diseased-normal tissues were also analyzed for molecular information by whole-exome sequencing. Results There were 14 men and 6 women with a median age of 58.5 (28-81) years. 17(17/20) lesions were located in the jejunum or ileum; 13(13/20) cases had ulcers or perforations. Microscopically, except for 1(1/20) case of pleomorphic cells, the monomorphic, middle-sized tumor cells infiltrating into the intestinal epithelial and peripheral intestinal mucosa recess could be seen in the other 19 cases. Immunohistochemistry showed that most of the tumor cells in MEITL were positive for CD3(20/20), CD8(17/20), CD43(19/20), and CD56(15/20), but negative for CD5(20/20). The most frequently mutated genes of these Chinese cases were STAT5B (4/9) and TP53 (4/9), not SETD2(2/9). JAK3 mutations (3/9) were also detected with a high mutated frequency. We demonstrated that mutations of JAK-STAT pathway-related genes and the amplification of Chromosome 9q appeared at the same time in most cases(5/9). Conclusions The clinicopathological features were consistent with that in previous western studies, but a special case with pleomorphic cells was found in this study. The co-occurrence of JAK-STAT pathway-related gene mutations and the amplification of Chr9q is a molecular feature of MEITL.

Alopecia areata is a chronic autoimmune disorder attacking the hair follicle epithelium; hence, causing non-scarring hair loss. It has been found that Janus kinase 3 (JAK3) hyperactivity plays a key role in the pathogenesis of the disease. Tofacitinib is an effective JAK1 and JAK3 inhibitor that can block several cytokines such as IL-2, IL-7, and IL-6. Several studies have demonstrated the efficacy of oral tofacitinib in hair regrowth in alopecia areata patients. With the recent COVID-19 pandemic, it has been advised to withhold JAK inhibitors during the period of active infection due to possible immunosuppression. We herein report two cases of patients with alopecia universalis who continued to use tofacitinib during their active COVID-19 infection and showed no deterioration in their course of illness.