JAK Kinase Inhibitors Research Articles

Experience in treating multifocal alopecia areata in a child with Jak kinase inhibitors

Alopecia Areata is an autoimmune disease characterized by hair loss mediated by CD8 + T cells. There are no proven effective treatments for alopecia. Recent research on the immunology of hair follicles and recent developments in the field of immunopatogenesis, as well as common ways of developing the disease with other autoimmune disorders have led to research on new treatments, targeted at specific immunological pathways. The so-called JAK-STAT pathway is responsible for transmitting signal from different cytokines to the nucleus of the cell, suggesting that blocking this pathway may be effective in the treatment of alopecia.Study purpose. Presentation of Clinical Case of Multi-focal Alopecia Treatment in Child Oral Tofacitinib.Materials and methods. The study was conducted on the basis of the dermatology department with the laser surgery group of the Center for Pediatric Dermatology and Laboratory of Skin Pathology in children FGAU «National Medical Research Center for Children’s Health» of the Ministry of Health of Russia from April 2021 to November 2022. Severity Alopecia Tool (SALT) was used to determine the severity of the disease in participants. The SALT scale takes into account the percentage of affected hair and divides it into 6 subclasses (S 0 %, S1 <25 %, S2 25–49 %, S3 50–74 %, S4 75–99 % and C5100 %).Results. When oral tofacitinib was administered for 6 months in a 9-year-old patient, full recovery of hair growth on the scalp was observed. No significant abnormalities based on clinical and biochemical blood results were found for the period of treatment and for a year after withdrawal. There was no recurrence of hair loss after the withdrawal of the drug for 12 months.Conclusion. JAK inhibitors can be effective new treatments for alopecia in children. Additional data from randomized controlled trials are required for safe use of JAK inhibitors.

Staphyloccocus aureus biofilm, in absence of planktonic bacteria, produces factors that activate counterbalancing inflammatory and immune-suppressive genes in human monocytes.

Staphyloccocus aureus (S. aureus) is a major bacterial pathogen in orthopedic periprosthetic joint infection (PJI). S. aureus forms biofilms that promote persistent infection by shielding bacteria from immune cells and inducing an antibiotic-tolerant metabolic state. We developed an in vitro system to study S. aureus biofilm interactions with primary human monocytes in the absence of planktonic bacteria. In line with previous in vivo data, S. aureus biofilm induced expression of inflammatory genes such as TNF and IL1B, and their anti-inflammatory counter-regulator IL10. S. aureus biofilm also activated expression of PD-1 ligands, and IL-1RA, molecules that have the potential to suppress T cell function or differentiation of protective Th17 cells. Gene induction did not require monocyte:biofilm contact and was mediated by a soluble factor(s) produced by biofilm-encased bacteria that was heat resistant and >3 kD in size. Activation of suppressive genes by biofilm was sensitive to suppression by Jak kinase inhibition. These results support an evolving paradigm that biofilm plays an active role in modulating immune responses, and suggest this occurs via production of a soluble vita-pathogen-associated molecular pattern, a molecule that signals microbial viability. Induction of T cell suppressive genes by S. aureus biofilm provides insights into mechanisms that can suppress T cell immunity in PJI.

P1162 Upadacitinib in Crohn’s Disease Patients: for what type of patients in real world setting ?

Abstract Background The oral selective JAK kinase inhibitor, Upadacitinib (UPA; Rinvoq®) has been shown to induce and maintain clinical response and remission in Crohn’s disease (CD), but also in rheumatic and skin diseases. We report here on the largest Swiss experience in "off label" use of this drug. Methods A chart review of the Crohn’s and Colitis Center Beaulieu Lausanne and Gastroenterology Center Bulle of CD patients was conducted. Results Eighty-nine IBD patients were exposed to UPA treatment. After exclusion of 22 patients (UC and unknown consent status) we analyzed patients’ characteristics of 67 CD patients (72% women) treated between April 2019 and Nov. 2023. All patients had previously failed or were intolerant to, at least, one anti-TNF agent, 75% had been exposed to 4 or more biologics/ small molecules and two-third had received all classes of biologics. Disease location and behavior were mostly ileo-colonic and inflammatory (L3B1) or ileal only and stricturing (L1B2). Half of the patients had co-existing extraintestinal manifestations (mostly spondylarthritis and arthralgias). Advanced combination therapy was used in 27% of cases with anti-TNF agents (n=10), vedolizumab (n=6) and ustekinumab (n=2). UPA was prescribed after a median disease duration of 14 years (range 1-68) for a mean treatment duration of 14 months (mo) (+/- 9.1 SD; range 1-37). Forty-four CD patients (66%) were still on UPA at the time of study completion. A partial or complete response was observed in 51/67 patients (76%) with a mean decrease of CRP of 10.4 mg/l (+/- 17.6 SD) and calprotectin of 610 mcg/g (+/- 1165 SD). Adverse events were mostly viral infections, acnea, hypercholesterolemia, fatigue and headaches. Conclusion Upadacitinib is a good treatment option in difficult to treat CD patients with aggressive disease behavior, alone or in combination in case of concomitant rheumatic and gastrointestinal indications.

Features of gynecological status and concomitant morbidity in menopausal women with resistant rheumatoid arthritis and obesity

Oftentimes, women with RA experience remission during pregnancy. The beneficial effect of pregnancy on the RA course is associated with a specific hormonal status, including high levels of estriol and progesterone that have known anti-inflammatory effects. After childbirth, decreased endogenous production of steroids can trigger the onset or exacerbation of RA.Objective. Studying of the characteristics of the gynecological status and concomitant extragenital diseases in postmenopausal women with rheumatoid arthritis (RA) depending on the presence of obesity.Materials and methods. 394 postmenopausal women with RA (diagnosed according to ACR/EULAR 2010 criteria) were included. By decision of the medical commission, all patients were approved for therapy with genetically engineered biological drugs and/or JAK kinase inhibitors. Before inclusion in the study, all patients had a detailed medical history, an allergic history, registered comorbidities, and a physical and laboratory-instrumental examination. Depending on the BMI values, the study participants were divided into groups. Group A included 103 obese patients of any severity (BMI ≥ 30 kg/m2). Group B included 291 women with BMI <30 kg/m2. Patients with an inflammatory process were found to have groups of inflammation by age and duration of RA (p > 0.05).Results. In patients of group A, more often than in patients of group B, comorbidities were detected: hypertension (p < 0.0001), coronary heart disease (p = 0.03), history of “vascular accidents” (p = 0.0009), especially acute cerebrovascular accident (p = 0.004), type 2 diabetes mellitus (p = 0.0005), which manifest themselves over the age of 45, that is, during the perimenopause. When correcting the gynecological history data, he draws attention to the fact that group A patients are more likely to operate surgically and on the pelvic organs: more had the uterus removed (p = 0.002) or ovarian resection was performed (p < 0.0001), and the frequency of surgical menopause in group A was higher than in group B (p = 0.0009). The debut of RA on average chronologically coincided with the onset of menopause (p = 0.01). At the same time, in the average age of the onset of menopause, it is rare (p > 0.05). The number of women who had a history of pregnancy, childbirth, miscarriages and abortions in the groups were repeated (p > 0.05). But in group A, the average number of pregnancies (p = 0.003) and abortions (p = 0.004) per woman was higher than in group B.Conclusion. The results of the study revealed the identification of clinical manifestations of inflammation and the severity of rheumatoid arthritis, depending on the presence of concomitant obesity in postmenopausal women, as well as a rarer occurrence of osteoporosis in women with RA and concomitant obesity.

Опыт применения ингибитора янус-киназ упадацитиниба у детей с ревматическими болезнями

The efficacy of JAK-kinase inhibitors in autoimmune diseases is due to the peculiarities of their pathogenesis, the involvement of various cytokines that trigger the cascade of inflammatory reactions, enhance the transcriptional response of disease-induced or disease-associated genes and potentiate the production of proinflammatory cytokines. Only tofacitinib is currently approved for use in pediatric rheumatic diseases. Given the literature data on the efficacy of other JAK-kinase inhibitors in adult rheumatic diseases, we assume the possibility of their use in pediatric patients. Objective. To evaluate the efficacy and safety of selective JAK-kinase inhibitor upadacitinib in children with rheumatic diseases. Patients and methods. Data on 85 pediatric patients with various variants of juvenile idiopathic arthritis (JIA) (n = 64) and systemic connective tissue lesions (SCTL) (n = 21) treated with upadacitinib were included in this retrospective study. Initiation of therapy was carried out on the basis of the rheumatology department of the Research Institute of Pediatric Rheumatology of the Federal State Autonomous Institution "Children's Health Research Center" of the Ministry of Health of Russia (Moscow) in the period from May 2022 to July 2023. Descriptive statistics methods were used to assess the efficacy of upadacitinib therapy, comparative analysis was performed using the Wilcoxon criterion. Therapy outcomes in the group of patients with JIA were evaluated by the percentage of improvement according to pediatric criteria of the American College of Rheumatology, achievement of inactive disease stage according to C.Wallace criteria and JADAS-71 activity index, in the group of patients with SCTL – by the dynamics of clinical and laboratory activity parameters, as well as steroid-saving effect. Results. In patients with JIA without systemic manifestations treated with upadacitinib for 3 months (15/54), 30% improvement in ACR Pedi criteria was recorded in 13/15 (87%), 50% in 12/15 (80%), 70% in 11/15 (73%), and 90% in 10/15 (66%) patients; after 6 months. – 30th; 50th; 70th; 90% improvement in 14/15 (93%), 14/15 (93%), 13/15 (87%), 13/15 (87%) patients, respectively. Inactive disease stage according to C.Wallace criteria after 3 months was achieved in 5/15 (33.3%) patients, after 6 months. – у 11/15 (73,3%). In patients with systemic JIA the efficacy of therapy after 3 months was evaluated in 3/10 patients: 2/3 achieved JADAS remission (1 of them – stage of inactive disease according to C.Wallace criteria), in 1 patient the disease activity increased against the background of hip arthroplasty. After 6 months (assessment of outcomes is available in 3/10 patients): 1 patient achieved JADAS remission, 1 patient achieved 50% improvement according to ACR Pedi criteria, 1 patient showed increased disease activity when attempting to reduce the dose of glucocorticoids (GCS). In the group of patients with SCTL, the majority of patients were able to reduce clinical and laboratory disease activity and the demand for oral GCS. Conclusion. The use of upadacitinib in children in rheumatic diseases is highly effective and safe. In patients with refractory course of the disease it is possible to achieve a decrease in disease activity and remission, to reduce the dose of GCS. Key words: systemic connective tissue lesions, systemic juvenile idiopathic arthritis, upadacitinib, juvenile idiopathic arthritis, Janus kinases

Type II mode of JAK2 inhibition and destabilization are potential therapeutic approaches against the ruxolitinib resistance driven myeloproliferative neoplasms.

Ruxolitinib has been approved by the US FDA for the treatment of myeloproliferative neoplasms such as polycythemia vera and primary myelofibrosis. Ruxolitinib will remain a main stay in the treatment of MPN patients due to its effective therapeutic benefits. However, there have been instances of ruxolitinib resistance in MPN patients. As JAK2 is a direct target of ruxolitinib, we generated ruxolitinib-resistant clones to find out the mechanism of resistance. Cell-based screening strategy was used to detect the ruxolitinib-resistant mutations in JAK2. The Sanger sequencing method was used to detect the point mutations in JAK2. Mutations were re-introduced using the site-directed mutagenesis method and stably expressed in Ba/F3 cells. Drug sensitivities against the JAK2 inhibitors were measured using an MTS-based assay. JAK2 and STAT5 activation levels and total proteins were measured using immunoblotting. Computational docking studies were performed using the Glide module of Schrodinger Maestro software. In this study, we have recovered seven residues in the kinase domain of JAK2 that affect ruxolitinib sensitivity. All these mutations confer cross-resistance across the panel of JAK2 kinase inhibitors except JAK2-L983F. JAK2-L983F reduces the sensitivity towards ruxolitinib. However, it is sensitive towards fedratinib indicating that our screen identifies the drug-specific resistance profiles. All the ruxolitinib-resistant JAK2 variants displayed sensitivity towards type II JAK2 inhibitor CHZ-868. In this study, we also found that JAK1-L1010F (homologous JAK2-L983F) is highly resistant towards ruxolitinib suggesting the possibility of JAK1 escape mutations in JAK2-driven MPNs and JAK1 mutated ALL. Finally, our study also shows that HSP90 inhibitors are potent against ruxolitinib-resistant variants through the JAK2 degradation and provides the rationale for clinical evaluation of potent HSP90 inhibitors in genetic resistance driven by JAK2 inhibitors. Our study identifies JAK1 and JAK2 resistance variants against the type I JAK2 inhibitors ruxolitinib, fedratinib, and lestaurtinib. The sensitivity of these resistant variants towards the type II JAK2 inhibitor CHZ-868 indicates that this mode of type II JAK2 inhibition is a potential therapeutic approach against ruxolitinib refractory leukemia. This also proposes the development of potent and specific type II JAK2 inhibitors using ruxolitinib-resistance variants as a prototype.

A newly identified 45-kDa JAK2 variant with an altered kinase domain structure represents a novel mode of JAK2 kinase inhibitor resistance.

Tyrosine-protein kinase (janus kinase; JAK)-signal transducer and activator of transcription (STAT) signaling plays a pivotal role in the development of myeloproliferative neoplasms (MPNs). Treatment with the potent JAK1/JAK2-specific inhibitor, ruxolitinib, significantly reduces tumor burden; however, ruxolitinib treatment does not fully eradicate the malignant clone. As the molecular basis for the disease persistence is not well understood, we set out to gain new insights by generating ruxolitinib-resistant cell lines. Surprisingly, these cells harbor a 45 kDa JAK2 variant (FERM-JAK2) consisting of the N-terminal FERM domain directly fused to the C-terminal kinase domain in 80% of sublines resistant to ruxolitinib. At the molecular level, FERM-JAK2 is able to directly bind and activate STAT5 in the absence of cytokine receptors. Furthermore, phosphorylation of activation-loop tyrosines is dispensable for FERM-JAK2-mediated STAT5 activation and cellular transformation, in contrast to JAK2-V617F. As a result, FERM-JAK2 is highly resistant to several ATP-competitive JAK2 inhibitors, whereas it is particularly sensitive to HSP90 inhibition. A murine model of FERM-JAK2 leukemogenesis showed an accelerated MPN phenotype with pronounced splenomegaly. Notably, most current protocols for the monitoring of emerging JAK variants are unable to detect FERM-JAK2, highlighting the urgent need for implementing next-generation sequencing approaches in MPN patients receiving ruxolitinib.

Elucidating the Morphogenic and Signaling Roles of Defined Growth Factors Controlling Human Endothelial Cell Lumen Formation Versus Sprouting Behavior

Five growth factors [ie, insulin, fibroblast growth factor-2 (FGF-2), stem cell factor, IL-3, and stromal-derived factor 1α] in combination are necessary for human endothelial cells (ECs) to undergo tube morphogenesis, a process requiring both lumen formation and sprouting behavior. This study investigated why these factors are required by subdividing the factors into 4 separate groups: insulin-only, insulinand FGF-2, no FGF-2 (all factors but without FGF-2), and all factors. The study found that the insulin-only condition failed to support EC morphogenesis or survival, the insulinand FGF-2 condition supported primarily EC lumen formation, and the no FGF-2 condition supported EC sprouting behavior. By comparison, the all-factors condition more strongly stimulated both EC lumen formation and sprouting behavior, and signaling analysis revealed prolonged stimulation of multiple promorphogenic signals coupled with inhibition of proregressive signals. Pharmacologic inhibition of Jak kinases more selectively blocked EC sprouting behavior, whereas inhibition of Raf, phosphatidylinositol 3-kinase, and Akt kinases showed selective blockade of lumen formation. Inhibition of Src family kinases and Notch led to increased sprouting coupled to decreased lumen formation, whereas inhibition of Pak, Mek, and mammalian target of rapamycin kinases blocked both sprouting and lumen formation. These findings reveal novel downstream biological and signaling activities of defined factors that are required for the assembly of human EC-lined capillary tube networks.

Modulation of human thrombopoietin receptor conformations uncouples JAK2 V617F-driven activation from cytokine-induced stimulation

AbstractThe thrombopoietin receptor (TpoR) plays a central role in myeloproliferative neoplasms (MPNs). Mutations in JAK2, calreticulin, or TpoR itself drive the constitutive activation of TpoR and uncontrolled proliferation and differentiation of hematopoietic stem cells and progenitors. The JAK2 V617F mutation is responsible for most MPNs, and all driver mutants induce pathologic TpoR activation. Existing therapeutic strategies have focused on JAK2 kinase inhibitors that are unable to differentiate between the mutated MPN clone and healthy cells. Surprisingly, the targeting of TpoR itself has remained poorly explored despite its central role in pathology. Here, we performed a comprehensive characterization of human TpoR activation under physiological and pathological conditions, focusing on the JAK2 V617F mutant. Using a system of controlled dimerization of the transmembrane and cytosolic domains of TpoR, we discovered that human TpoR (hTpoR) adopts different dimeric conformations upon Tpo-induced vs JAK2 V617F–mediated activation. We identified the amino acids and specific dimeric conformation of hTpoR responsible for activation in complex with JAK2 V617F and confirmed our findings in the full-length receptor context in hematopoietic cell lines and primary bone marrow cells. Remarkably, we found that the modulation of hTpoR conformations by point mutations allowed for specific inhibition of JAK2 V617F–driven activation without affecting Tpo-induced signaling. Our results demonstrate that modulation of the hTpoR conformation is a viable therapeutic strategy for JAK2 V617F–positive MPNs and set the path for novel drug development by identifying precise residues of hTpoR involved in JAK2 V617F–specific activation.

MAPK-negative feedback regulation confers dependence to JAK2V617F signaling.

Despite significant advances in developing selective JAK2 inhibitors, JAK2 kinase inhibitor (TKI) therapy is ineffective in suppressing the disease. Reactivation of compensatory MEK-ERK and PI3K survival pathways sustained by inflammatory cytokine signaling causes treatment failure. Concomitant inhibition of MAPK pathway and JAK2 signaling showed improved in vivo efficacy compared to JAK2 inhibition alone but lacked clonal selectivity. We hypothesized that cytokine signaling in JAK2V617F induced MPNs increases theapoptotic threshold that causes TKI persistence or refractoriness. Here, we show that JAK2V617F and cytokine signaling converge to induce MAPK negative regulator, DUSP1. Enhanced DUSP1 expression blocks p38 mediated p53 stabilization. Deletion of Dusp1 increases p53 levels in the context of JAK2V617F signaling that causes synthetic lethality to Jak2V617F expressing cells. However, inhibition of Dusp1 by a small molecule inhibitor (BCI) failed to impart Jak2V617F clonal selectivity due to pErk1/2 rebound caused by off-target inhibition of Dusp6. Ectopic expression of Dusp6 and BCI treatment restored clonal selectively and eradicated the Jak2V617F cells. Our study shows that inflammatory cytokines and JAK2V617F signaling converge to induceDUSP1, which downregulates p53 and establishes a higher apoptotic threshold. These data suggest that selectively targeting DUSP1 may provide a curative response in JAK2V617F-driven MPN.

Targeting the JAK2-STAT3 pathway to inhibit cGAS-STING activation improves neuronal senescence after ischemic stroke

Neuroinflammation after cerebral ischemia is a key event in progressive brain injury after ischemic stroke. The JAK2/STAT3 pathway is pivotal for neuroinflammation; however, its role in brain senescence after ischemic stroke is unclear. Here, we report that inflammation is increased in the brains of C57BL/6 stroke mice. Treatment of ischemic stroke in adult mice with a JAK kinase inhibitor (AG490) alleviated neurobehavioral defects, reduced brain infarct volume, reduced expression of pro-inflammatory cytokines, and decreased activation of pro-inflammatory microglia. Moreover, AG490 treatment reduced oxidative DNA damage and cellular senescence in the brains of mice following ischemic stroke. Cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING) were associated with inflammation and senescence. Furthermore, AG490 blocked cGAS/STING/NF-κBp65 expression. Overall, our results indicate that inhibition of JAK2/STAT3 can alleviate the negative neurological consequences of ischemic stroke, likely due to repression of cGAS/STING/NF-κB p65, leading to reduced neuroinflammation and neuronal senescence. Therefore, JAK2/STAT3 may represent a viable therapeutic target for preventing senescence after ischemic stroke.

Ulcerative colitis: Addressing the manifestations, the role of fecal microbiota transplantation as a novel treatment option and other therapeutic updates

The prevalence and incidence of Ulcerative Colitis (UC), a recurrent and remitting inflammatory condition, are rising. Any part of the colon may be affected, beginning with inflammation of the mucosa in the rectum and continuing proximally continuously. Bloody diarrhea, tenesmus, fecal urgency, and stomach pain are typical presenting symptoms. Many patients present with extraintestinal manifestations (EIMs) including musculoskeletal, ocular, renal, hepatobiliary, and dermatological presentation, among others. Most cases are treated with pharmacological therapy including mesalazine and glucocorticoids. Fecal microbiota transplantation (FMT) is a novel procedure that is increasingly being used to treat UC, however, its use yet remains controversial because of uncertain efficacy. FMT can lower gut permeability and consequently disease severity by boosting short-chain fatty acids production, helping in epithelial barrier integrity preservation. Upadacitinib (JAK Kinase inhibitor) is another newer treatment option, which is an FDA-approved drug that is being used to treat UC. This review article provides a comprehensive review of the EIMs of UC, the role of FMT along with various recent clinical trials pertaining to FMT as well as other diagnostic and therapeutic updates.

Overview on vitiligo and its treatment

Vitiligo, is a skin disorder, in which the skin loses its pigment due to the loss of melanocytes. The exact cause is still under research but the disorder is linked to autoimmune disease, oxidative stress, genetics and environmental variables. The disease being progressive in nature often spreads all over the body. Globally, Vitiligo affects nearly about 0.5% to 1% of people. In 2012, the most recent and approved classification for Vitiligo was given at Vitiligo Global Issues Consensus Conference (VGICC). Understanding the biological mediators and molecular mechanisms that result in metabolic problems, melanocyte degradation, and autoimmunity is crucial in order to identify innovative therapy targets and drugs that may be able to stop the progression of the condition or even cure vitiligo. Treatment options range from oral antibiotics, topical immunosuppressants, melanocyte promoters, oral immunosuppressants, JAK kinase inhibitors, physical therapy, to surgical intervention. Systemic biological therapies that target cytokines have shown promising results in the treatment of diseases like vitiligo and psoriasis.

Эффективность и безопасность упадацитиниба в лечении псориатического артрита

Psoratic arthritis (PsA) is a common disorder affecting every fifth patient with psoriasis. Typical clinical manifestations of PsA include asymmetric mono/oligoathritis or polyarthritis, spondyloarthritis, enthesitis and dactylitis. More rarely PsA is associated with uveitis and inflammatory bowel disease. Upadacitinib is a selective JAK-kinase inhibitor approved for treatment of PsA. Its efficacy and favorable safety profile in PsA patients were established in two randomized placebocontrolled phase 3 clinical trials (SELECT-PsA 1 and 2). In both studies, upadacitinib was significantly more effective than placebo and improved activity of PsA, psoriatic lesions, enthesitis and dactylitis. Moreover, upadacitinib is approved for treatment of axial spondylitis and, therefore, can be used in patients with any manifestations of PsA. The authors present clinical cases of PsA and discuss indications for upadacitinib administration in such patients.

Growth Failure due to JAK Inhibition Overcome by Recombinant IGF-I

Introduction: Normal linear growth requires an intact GH/IGF-I axis, wherein GH from the pituitary induces the synthesis and release of IGF-I from the hepatocyte followed by IGF-I-mediated chondrocyte division at the growth plate. At the hepatocyte, GH binding to its cognate receptor results in a conformational change in the intracellular portion of the receptor that recruits and activates the tyrosine kinase JAK2. JAK2 then phosphorylates the transcription factor Stat5b which allows it to bind to the IGF-I promotor and increase IGF-I production. JAK kinase inhibitors are used for the treatment of inflammatory disorders such as rheumatoid arthritis, and theoretically would be expected to interfere with linear growth by impairing IGF-I production. We present a case of growth failure due to JAK2 inhibition and successful treatment with recombinant IGF-I therapy.

Synthesis and evaluation of hydrazinyl-containing pyrrolo[2,3-d]pyrimidine series as potent, selective and oral JAK1 inhibitors for the treatment of rheumatoid arthritis

Selective inhibition of JAK kinases within the JAK family has been a desired goal of research in order to maximize efficacy while reducing undesired off target effect. Aiming to minimize adverse effects such as anemia, a promising new class of pyrrolo[2,3-d]pyrimidine series containing a hydrazinyl moiety were synthesized and profiled. Among them compound 8m and 8o showed the best enzymatic activity against JAK1 with IC50 value of 0.16 nM and 0.3 nM respectively, and with selectivity over JAK2 by 40.6 and 10 folds respectively. In addition, 8o had an improved PK profile and demonstrated better in vivo efficacy than Tofacitinib in CIA model.

P1004: EFFECT AND MOLECULAR MECHANISM OF TQ05105, A NOVEL SMALL MOLECULE INHIBITOR OF JAK2 IN MYELOPROLIFERATIVE NEOPLASM

Background: The JAK2V617F gene mutation leads to upregulation of the JAK/STAT signaling pathway, which causes excessive bone marrow cell proliferation, elevated inflammatory cytokines, constitutional symptoms and shortened survival in myeloproliferative neoplasms (MPN) patients. The JAK1/2 kinase inhibitor ruxolitinib can dramatically improve disease-related symptoms, but side effect such as anemia and thrombocytopenia limit its use in a substantial proportion of patients. Several JAK2 kinase inhibitor III Clinical trials have yielded unsatisfactory outcomes recently. TQ05105 is a new small molecule inhibitor that targets JAK2 kinase. Aims: We aim to investigate the effect and molecular mechanism of TQ05105 on MPN cell lines and mouse models. Methods: We systematically measured the function of TQ05105 in MPN cell lines, including UKE1 and SET2 cells, by performing proliferation, apoptosis and colony-forming assays. RT-PCR and inflammatory factor multi-assay kit was used to detect the secretion of inflammatory factors such as TNF-α, IL-1β, MCP-1 in mRNA level and protein level respectively. CFU-E and BFU-E assays were performed on MPN cell lines and primary cells. In vivo, TQ05105 was evaluated in JAK2V617F Jak2V617F transplant mouse model and MPL515 retroviral mouse model. Mice were orally administrated with placebo, 25 mg/kg (bid) and 50 mg/kg (bid) every day for 4 weeks. Flow cytometry analysis was performed to determine the proportion of different hematopoietic progenitor cell populations. In addition, HE staining and Gomori staining was used to detect the changes in the spleen and bone marrow. Results: In UKE1 and SET2 cell lines, TQ05105 can inhibit the proliferation and induced cell apoptosis, notably reduce the expression of cytokines, including TNF-α, IL-1 and MCP-1. According to Western blot results, the phosphorylation of JAK/STAT signaling pathway associated proteins JAK2, STAT3 and STAT5 were significantly suppressed by TQ05105. Furthermore, compared to the control group, TQ05105 can strongly limit the ability of UKE1 cell lines, SET2 cell lines and primary cells from MPN patients (PMF, PV, ET) to form BFU-E and CFU-GM. TQ05105 can striking reduce mouse spleen volume, along with favorable impact on leukocyte and erythrocyte counts in Jak2V617F transplant mouse model. The proportion of stem and progenitor cells, especially CMPs and MEPs, was considerably reduced in mice after treatment with TQ05105. HE staining showed remarkably Spleen response such as recovering structure, reduced infiltration of erythrocytes and suppressed proliferation of megakaryocytes. MPLW515L retroviral mouse model recapitulated the feature of myelofibrosis, application of TQ05105 in this model resulted in reduction of the spleen volume, extramedullary hematopoiesis, leukocyte and platelets count, bone marrow fibrosis and decreased fraction of CMPs and MEPs. Summary/Conclusion: TQ05105, as a novel small molecule JAK2 inhibitor, can inhibit cell proliferation, induce cell apoptosis and decrease inflammatory cytokines by affecting the JAK/STAT signaling pathway. In vivo experiments further proved that TQ05105 can reduce spleen size and improve disease-related symptoms of MPN, indicating that it is a promising therapeutic drug for MPN.

GC-MS, Phytochemical Analysis and In Silico Approaches of a Medicinal Plant Acalypha indica

The goal of this study is to look into the preliminary phytochemical and GC-MS analyses of a methanol extract of the leaves of Acalypha indica. Alkaloids, tannins, steroids, saponins, flavonoids, glycosides, and phenolic substances are found in leaf extract after phytochemical analysis. A. indica is a plant that grows in the southern Indian state of Tamil Nadu. It has medicinal characteristics and is used as a diuretic, anthelmintic, and respiratory disorders like bronchitis, asthma, and pneumonia. The present work is designed to investigate GC-MS. Thirty compounds are found in the leaf extract. The structure-based biological activities study supported the Pass online database findings from the current study support that the use of this plant compound responsible for the anticancer activity is based on structure-based drug development. Based on in-silico results, it was shown that the interactions of hydrogen bonds, binding affinity with JAK2 kinase inhibitors, tumor suppressor proteins pRB Retinoblastoma protein, and COX-2 target proteins were compared with known values. It can be considered an increase in the possibility of conniving potential anticancer drugs as an anti-inflammatory agent.

Dual JAK2/Aurora kinase A inhibition prevents human skin graft rejection by allo-inactivation and ILC2-mediated tissue repair

Prevention of allograft rejection often requires lifelong immune suppression, risking broad impairment of host immunity. Nonselective inhibition of host T cell function increases recipient risk of opportunistic infections and secondary malignancies. Here we demonstrate that AJI-100, a dual inhibitor of JAK2 and Aurora kinase A, ameliorates skin graft rejection by human T cells and provides durable allo-inactivation. AJI-100significantly reduces the frequency of skin-homing CLA+ donor T cells, limiting allograft invasion and tissue destruction by T effectors. AJI-100 also suppresses pathogenic Th1 and Th17 cells in the spleen yet spares beneficial regulatory T cells. We show dual JAK2/Aurora kinase A blockade enhances human type 2 innate lymphoid cell (ILC2) responses, which are capable of tissue repair. ILC2 differentiation mediated by GATA3 requires STAT5 phosphorylation (pSTAT5) but is opposed by STAT3. Further, we demonstrate that Aurora kinase A activation correlates with low pSTAT5 in ILC2s. Importantly, AJI-100 maintains pSTAT5 levels in ILC2s by blocking Aurora kinase A and reduces interference by STAT3. Therefore, combined JAK2/Aurora kinase A inhibition is an innovative strategy to merge immune suppression with tissue repair after transplantation.

JAK and mTOR inhibitors prevent cytokine release while retaining T cell bispecific antibody in vivo efficacy

BackgroundT cell engaging therapies, like chimeric antigen receptor T cells and T cell bispecific antibodies (TCBs), efficiently redirect T cells towards tumor cells, facilitating the formation of a cytotoxic synapse...