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Danese, Silvio, Marjorie Argollo, Catherine Le Berre, and Laurent Peyrin-Biroulet. "JAK selectivity for inflammatory bowel disease treatment: does it clinically matter?" Gut 68, no. 10 (June 21, 2019): 1893–99. http://dx.doi.org/10.1136/gutjnl-2019-318448.
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The two major forms of inflammatory bowel disease (IBD), encompassing Crohn’s disease (CD) and ulcerative colitis (UC), are chronic immune-mediated conditions characterised by an increased production of pro-inflammatory cytokines that act as critical drivers of intestinal inflammation. Anti-cytokine therapy has been shown to improve clinical outcomes in IBD. Janus kinases (JAKs) are tyrosine kinases that bind different intracellular cytokine receptors, leading to phosphorylation of signal transducer and activation of transcription molecules implicated on targeted gene transcription. Four isoforms of JAKs have been described: JAK1, JAK2, JAK3 and TYK2. Oral JAK inhibitors (JAKi) have been developed as synergic anti-cytokine therapy in IBD, showing different selectivity towards JAK isoforms. Tofacitinib, a pan-JAK inhibitor, has been recently approved for the treatment of moderate-to-severe UC. With the aim of improving the benefit: risk ratio of this drug class, several second-generation subtype-selective JAKi are under development. However, whether selective inhibition of JAK isoforms is associated with an increased clinical efficacy and/or a better safety profile remains debatable. The aim of this review is to critically review the preclinical and clinical data for the differential selectivity of JAK inhibitors and to summarise the potential clinical implications of the selective JAK inhibitors under development for UC and CD.
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Constantinescu, Stefan N., Emilie Leroy, Vitalina Gryshkova, Christian Pecquet, and Alexandra Dusa. "Activating Janus kinase pseudokinase domain mutations in myeloproliferative and other blood cancers." Biochemical Society Transactions 41, no. 4 (July 18, 2013): 1048–54. http://dx.doi.org/10.1042/bst20130084.
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The discovery of the highly prevalent activating JAK (Janus kinase) 2 V617F mutation in myeloproliferative neoplasms, and of other pseudokinase domain-activating mutations in JAK2, JAK1 and JAK3 in blood cancers, prompted great interest in understanding how pseudokinase domains regulate kinase domains in JAKs. Recent functional and mutagenesis studies identified residues required for the V617F mutation to induce activation. Several X-ray crystal structures of either kinase or pseudokinase domains including the V617F mutant of JAK2 pseudokinase domains are now available, and a picture has emerged whereby the V617F mutation induces a defined conformational change around helix C of JH (JAK homology) 2. Effects of mutations on JAK2 can be extrapolated to JAK1 and TYK2 (tyrosine kinase 2), whereas JAK3 appears to be different. More structural information of the full-length JAK coupled to cytokine receptors might be required in order to define the structural basis of JH1 activation by JH2 mutants and eventually obtain mutant-specific inhibitors.
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Raivola, Juuli, Teemu Haikarainen, Bobin George Abraham, and Olli Silvennoinen. "Janus Kinases in Leukemia." Cancers 13, no. 4 (February 14, 2021): 800. http://dx.doi.org/10.3390/cancers13040800.
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Janus kinases (JAKs) transduce signals from dozens of extracellular cytokines and function as critical regulators of cell growth, differentiation, gene expression, and immune responses. Deregulation of JAK/STAT signaling is a central component in several human diseases including various types of leukemia and other malignancies and autoimmune diseases. Different types of leukemia harbor genomic aberrations in all four JAKs (JAK1, JAK2, JAK3, and TYK2), most of which are activating somatic mutations and less frequently translocations resulting in constitutively active JAK fusion proteins. JAKs have become important therapeutic targets and currently, six JAK inhibitors have been approved by the FDA for the treatment of both autoimmune diseases and hematological malignancies. However, the efficacy of the current drugs is not optimal and the full potential of JAK modulators in leukemia is yet to be harnessed. This review discusses the deregulation of JAK-STAT signaling that underlie the pathogenesis of leukemia, i.e., mutations and other mechanisms causing hyperactive cytokine signaling, as well as JAK inhibitors used in clinic and under clinical development.
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Taldaev, Amir, Vladimir R. Rudnev, Kirill S. Nikolsky, Liudmila I. Kulikova, and Anna L. Kaysheva. "Molecular Modeling Insights into Upadacitinib Selectivity upon Binding to JAK Protein Family." Pharmaceuticals 15, no. 1 (December 25, 2021): 30. http://dx.doi.org/10.3390/ph15010030.
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Rheumatoid arthritis (RA) is a chronic disease characterized by bone joint damage and incapacitation. The mechanism underlying RA pathogenesis is autoimmunity in the connective tissue. Cytokines play an important role in the human immune system for signal transduction and in the development of inflammatory responses. Janus kinases (JAK) participate in the JAK/STAT pathway, which mediates cytokine effects, in particular interleukin 6 and IFNγ. The discovery of small molecule inhibitors of the JAK protein family has led to a revolution in RA therapy. The novel JAK inhibitor upadacitinib (RinvoqTM) has a higher selectivity for JAK1 compared to JAK2 and JAK3 in vivo. Currently, details on the molecular recognition of JAK1 by upadacitinib are not available. We found that characteristics of hydrogen bond formation with the glycine loop and hinge in JAKs define the selectivity. Our molecular modeling study could provide insight into the drug action mechanism and pharmacophore model differences in JAK isoforms.
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Montresor, Alessio, Matteo Bolomini-Vittori, Lara Toffali, Barbara Rossi, Gabriela Constantin, and Carlo Laudanna. "JAK tyrosine kinases promote hierarchical activation of Rho and Rap modules of integrin activation." Journal of Cell Biology 203, no. 6 (December 23, 2013): 1003–19. http://dx.doi.org/10.1083/jcb.201303067.
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Lymphocyte recruitment is regulated by signaling modules based on the activity of Rho and Rap small guanosine triphosphatases that control integrin activation by chemokines. We show that Janus kinase (JAK) protein tyrosine kinases control chemokine-induced LFA-1– and VLA-4–mediated adhesion as well as human T lymphocyte homing to secondary lymphoid organs. JAK2 and JAK3 isoforms, but not JAK1, mediate CXCL12-induced LFA-1 triggering to a high affinity state. Signal transduction analysis showed that chemokine-induced activation of the Rho module of LFA-1 affinity triggering is dependent on JAK activity, with VAV1 mediating Rho activation by JAKs in a Gαi-independent manner. Furthermore, activation of Rap1A by chemokines is also dependent on JAK2 and JAK3 activity. Importantly, activation of Rap1A by JAKs is mediated by RhoA and PLD1, thus establishing Rap1A as a downstream effector of the Rho module. Thus, JAK tyrosine kinases control integrin activation and dependent lymphocyte trafficking by bridging chemokine receptors to the concurrent and hierarchical activation of the Rho and Rap modules of integrin activation.
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Gonzalez-Traves, P., B. Murray, F. Campigotto, A. Meng, and J. A. DI Paolo. "THU0067 JAK SELECTIVITY AND THE IMPACT ON CYTOKINE SIGNALING INHIBITION AT CLINICAL RHEUMATOID ARTHRITIS DOSES." Annals of the Rheumatic Diseases 79, Suppl 1 (June 2020): 246.1–246. http://dx.doi.org/10.1136/annrheumdis-2020-eular.2074.
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Background:Janus kinase 1 (JAK1) inhibitors are efficacious in rheumatoid arthritis (RA). Despite having similar efficacy, in vitro studies have shown differences in JAK selectivity profiles for the small-molecule JAK inhibitors (JAKi) baricitinib (BARI), tofacitinib (TOFA), and upadacitinib (UPA).1For example, BARI and UPA are JAK1/JAK2 selective, while TOFA is JAK1/JAK3 selective, but each JAKi has some activity against other JAKs. As JAKs form signaling pairs, differences in selectivity could lead to distinct pharmacologic profiles that may impact clinical efficacy and safety.Objectives:As a first step to understand the basis of potential differences at therapeutic doses, we compared the selectivity and potency of filgotinib (FIL) and its major metabolite (MET) to those of BARI, TOFA, and UPA in cytokine-stimulated peripheral blood mononuclear cells (PBMCs) and whole blood (WB).Methods:PBMCs and WB from healthy donors were incubated in vitro with 8 doses of each JAKi, and levels of signal transducer and activator of transcription phosphorylation (pSTAT) were measured following cytokine stimulation. Half maximal inhibitory concentration (IC50) values were calculated in phenotypically sorted leukocyte populations by flow cytometry. Therapeutic dose relevance of the in vitro analyses was assessed using calculated mean concentration-time profiles from JAKi population pharmacokinetic data in RA subjects. For each JAKi, the time above IC50and average daily pSTAT inhibition were calculated for each cytokine/STAT pair in B cells, CD4+ T cells, CD8+ T cells, monocytes, and/or NK cells.Results:Cellular assays in PBMCs and WB showed dose-dependent inhibition of cytokine-induced pSTATs with all JAKi (correlation between the protein-adjusted IC50values from PBMCs and IC50values from WB, r2=0.98). Among the most potently inhibited pathways were JAK1/TYK2-dependent cytokine, interferon alpha (IFNα), and the JAK1/2-dependent cytokine, interleukin (IL)-6. FIL and MET had weaker potencies against JAK2/TYK2 (G-CSF/pSTAT3), JAK1/2 (IFNƴ/pSTAT1), and JAK2/2 (granulocyte-macrophage colony-stimulating factor [GM-CSF])-dependent pathways compared to JAK1/TYK2 (IFNα/pSTAT5). FIL and MET showed the greatest selectivity vs the JAK2/2 pathway (GM-CSF/pSTAT3) in monocytes.The mean concentration-time profiles and time above IC50over 24 hr for each cytokine/STAT pathway showed that JAK1/2 (IL-6/pSTAT1) and JAK1/TYK2 (IFNα/pSTAT1) pathways were strongly modulated with all tested JAKi. FIL (200 mg) showed similar activity in average target coverage and time above IC50to the approved low doses of TOFA (5 mg) and UPA (15 mg); conversely, FIL had reduced mean average inhibition and time above IC50levels against JAK1/2 (IFNƴ/pSTAT1), JAK1/3-dependent cytokines (IL-2, -4, and -15), JAK2/TYK2 (G-CSF/pSTAT3), and JAK2/2 (GM-CSF/pSTAT5)-dependent pathways compared to TOFA and UPA, and in certain cases to BARI (2 mg).Conclusion:Different JAKi modulate distinct cytokine pathways to varying degrees, and no agent potently and continuously inhibited an individual cytokine signaling pathway throughout the dosing interval. FIL (200 mg) showed a similar inhibition profile to TOFA, BARI, and UPA against the JAK1/TYK2- (IFNα/pSTAT1) or JAK1/2-dependent (IL-6/pSTAT1) responses, consistent with the role of these pathways in clinical efficacy.2However, FIL displayed a differentiated pharmacologic profile from the other JAKi, showing biologically reduced activity on the JAK1/2 (IFNγ)-, JAK1/3 (IL-2, -4 and -15)-, JAK2/TYK2 (G-CSF)-, and JAK2/2 (GM-CSF)-dependent pathways, which play important roles in hematopoiesis and immune function. These data suggest that FIL (200 mg) may have less impact on a subset of homeostatic immune functions signaling via JAK2 and JAK3 than those observed at the clinically approved doses of TOFA (5 mg and 10 mg), UPA (15 mg), and BARI (4 mg).References:[1]McInnes IB, et al. Arthritis Res Ther. 2019;21:183.[2]Banerjee S, et al. Drugs. 2017;77:521-546.Disclosure of Interests:Paqui Gonzalez-Traves Employee of: Gilead, Bernard Murray Employee of: Gilead, Federico Campigotto Employee of: Gilead, Amy Meng Shareholder of: Gilead Sciences, Employee of: Gilead, Julie A. Di Paolo Employee of: Gilead
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Conzelmann, Michael, Elena Rodionova, Michael Hess, Thomas Giese, Anthony D. Ho, Peter Dreger, and Thomas Luft. "Complementary JAK/STAT Signalling Is Required for the Pro-Inflammatory Effects of CD40 Ligation: Differential Effects in Human Myeloid and B Cells." Blood 110, no. 11 (November 16, 2007): 2413. http://dx.doi.org/10.1182/blood.v110.11.2413.2413.
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Abstract CD40L represents a strong endogenous danger signal that induces pro-inflammatory activation of CD40-expressing cells such as dendritic cells (DC), monocytes, and B cells. However, since CD40 activation alone is insufficient to induce pro-inflammatory cytokines such as IL-12p70, we studied whether CD40-mediated pro-inflammatory activity might be dependent on co-signalling pathways involving JAK/STAT. Using quantitative Western blotting, we demonstrate that JAK/STAT signalling is induced by cytokines such as IL-4, GM-CSF and IFNg, whereas CD40 activation mediates NFkB signalling. CD40L-induced IL-12p70 and IL-10 secretion in human DC, monocytes, B cells, and chronic lymphocytic leukemia (CLL) cells was measured upon complementary JAK/STAT activation by IL-4, GM-CSF and IFNg in the presence and absence of specific inhibitors of JAK2, JAK3, and pan-JAK. Whereas IL-12p70 could not be induced by CD40 ligation or by cytokines alone, IL-12p70 secretion and suppression of IL-10 was reproducibly observed after co-stimulation of CD40L with IL-4, GM-CSF, or IFNg. This effect could be completely reversed by pan-JAK inhibition. Persistence of IL-4/GM-CSF/IFNg-mediated JAK/STAT signalling as late as 12 hours following cellular activation via CD40 was required for IL-12p70 secretion as shown by the effects of delayed JAK inhibition. Similarly, persistence between 12 and 24 hours of IL-12p35 and p40 mRNA expression correlated best with the level of IL-12p70 secretion. Specific inhibition of JAK2 and JAK3 further revealed a context-dependent action of the distinct JAK family members: JAK2 showed a strong co-dominant effect in the setting of IL-4-induced JAK/STAT activity. Both, JAK2 and JAK3 were required for IL-12p70 secretion, whereas JAK2 alone was sufficient to modulate IL-10 secretion. However, in the context of IFNg-induced JAK/STAT signalling in DC, neither JAK2 nor JAK3 inhibition had effects on IL-12p70. Here, only inhibition by the pan-JAK inhibitor involving JAK1 abrogated IL-12p70 secretion, indicating that in IFNg-dependent signalling, JAK2 is apparently sub-dominant to JAK1 and had only a small enhancing effect on IL-10. This context dependence markedly differed in myeloid cells and B cells, as normal and malignant (CLL) B cells maintain a co-dominant JAK2 activity in the context of IFNg-induced JAK/STAT-signalling. In conclusion, complementary JAK/STAT signalling is required for the pro-inflammatory effects of CD40 ligation in humans, with different JAK subset predominance in myeloid and B cells. These results may open new ways of lineage-specific interfering with CD40 signals by modulating JAK/STAT activity using tyrosine kinase inhibitors.
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Rodriguez Moncivais, Omar J., Stephanie A. Chavez, Victor H. Estrada Jimenez, Shengjie Sun, Lin Li, Robert A. Kirken, and Georgialina Rodriguez. "Structural Analysis of Janus Tyrosine Kinase Variants in Hematological Malignancies: Implications for Drug Development and Opportunities for Novel Therapeutic Strategies." International Journal of Molecular Sciences 24, no. 19 (September 26, 2023): 14573. http://dx.doi.org/10.3390/ijms241914573.
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Janus tyrosine kinase (JAK) variants are known drivers for hematological disorders. With the full-length structure of mouse JAK1 being recently resolved, new observations on the localization of variants within closed, open, and dimerized JAK structures are possible. Full-length homology models of human wild-type JAK family members were developed using the Glassman et al. reported mouse JAK1 containing the V658F structure as a template. Many mutational sites related to proliferative hematological disorders reside in the JH2 pseudokinase domains facing the region important in dimerization of JAKs in both closed and open states. More than half of all JAK gain of function (GoF) variants are changes in polarity, while only 1.2% are associated with a change in charge. Within a JAK1-JAK3 homodimer model, IFNLR1 (PDB ID7T6F) and the IL-2 common gamma chain subunit (IL2Rγc) were aligned with the respective dimer implementing SWISS-MODEL coupled with ChimeraX. JAK3 variants were observed to encircle the catalytic site of the kinase domain, while mutations in the pseudokinase domain align along the JAK-JAK dimerization axis. FERM domains of JAK1 and JAK3 are identified as a hot spot for hematologic malignancies. Herein, we propose new allosteric surfaces for targeting hyperactive JAK dimers.
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Musumeci, Francesca, Chiara Greco, Ilaria Giacchello, Anna Lucia Fallacara, Munjed M. Ibrahim, Giancarlo Grossi, Chiara Brullo, and Silvia Schenone. "An Update on JAK Inhibitors." Current Medicinal Chemistry 26, no. 10 (June 20, 2019): 1806–32. http://dx.doi.org/10.2174/0929867325666180327093502.
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Janus kinases (JAKs) are a family of non-receptor tyrosine kinases, composed by four members, JAK1, JAK2, JAK3 and TYK2. JAKs are involved in different inflammatory and autoimmune diseases, as well as in malignancies, through the activation of the JAK/STAT signalling pathway. Furthermore, the V617F mutation in JAK2 was identified in patients affected by myeloproliferative neoplasms. This knowledge prompted researchers from academia and pharmaceutical companies to investigate this field in order to discover small molecule JAK inhibitors. These efforts recently afforded to the market approval of four JAK inhibitors. Despite the fact that all these drugs are pyrrolo[2,3-d]pyrimidine derivatives, many compounds endowed with different heterocyclic scaffolds have been reported in the literature as selective or multi-JAK inhibitors, and a number of them is currently being evaluated in clinical trials. In this review we will report many representative compounds that have been published in articles or patents in the last five years (period 2013-2017). The inhibitors will be classified on the basis of their chemical structure, focusing, when possible, on their structure activity relationships, selectivity and biological activity. For every class of derivatives, compounds disclosed before 2013 that have entered clinical trials will also be briefly reported, to underline the importance of a particular chemical scaffold in the search for new inhibitors.
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Raivola, Juuli, Teemu Haikarainen, and Olli Silvennoinen. "Characterization of JAK1 Pseudokinase Domain in Cytokine Signaling." Cancers 12, no. 1 (December 27, 2019): 78. http://dx.doi.org/10.3390/cancers12010078.
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The Janus kinase-signal transducer and activator of transcription protein (JAK-STAT) pathway mediates essential biological functions from immune responses to haematopoiesis. Deregulated JAK-STAT signaling causes myeloproliferative neoplasms, leukaemia, and lymphomas, as well as autoimmune diseases. Thereby JAKs have gained significant relevance as therapeutic targets. However, there is still a clinical need for better JAK inhibitors and novel strategies targeting regions outside the conserved kinase domain have gained interest. In-depth knowledge about the molecular details of JAK activation is required. For example, whether the function and regulation between receptors is conserved remains an open question. We used JAK-deficient cell-lines and structure-based mutagenesis to study the function of JAK1 and its pseudokinase domain (JH2) in cytokine signaling pathways that employ JAK1 with different JAK heterodimerization partner. In interleukin-2 (IL-2)-induced STAT5 activation JAK1 was dominant over JAK3 but in interferon-γ (IFNγ) and interferon-α (IFNα) signaling both JAK1 and heteromeric partner JAK2 or TYK2 were both indispensable for STAT1 activation. Moreover, IL-2 signaling was strictly dependent on both JAK1 JH1 and JH2 but in IFNγ signaling JAK1 JH2 rather than kinase activity was required for STAT1 activation. To investigate the regulatory function, we focused on two allosteric regions in JAK1 JH2, the ATP-binding pocket and the αC-helix. Mutating L633 at the αC reduced basal and cytokine induced activation of STAT in both JAK1 wild-type (WT) and constitutively activated mutant backgrounds. Moreover, biochemical characterization and comparison of JH2s let us depict differences in the JH2 ATP-binding and strengthen the hypothesis that de-stabilization of the domain disturbs the regulatory JH1-JH2 interaction. Collectively, our results bring mechanistic understanding about the function of JAK1 in different receptor complexes that likely have relevance for the design of specific JAK modulators.
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Hashemi, David, and Neal Bhatia. "The JAK-Cytokine Interface – A Review and Update on Prospective Clinical Considerations." SKIN The Journal of Cutaneous Medicine 7, no. 4 (July 17, 2023): 932–35. http://dx.doi.org/10.25251/skin.7.4.16.
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Janus kinases (JAKs) are non-receptor tyrosine kinases that work together with signal transducers and activators of transcription (STAT) proteins to form the JAK/STAT pathway. Together, this pathway is responsible for mediating a wide range of downstream cytokines and growth factors, and inhibition of various components of this pathway has been a major area of research focus in recent years. Each of the major enzymes of the family – which include JAK1, JAK2, JAK3, and Tyrosine Kinase 2 (TYK2) – or combinations of JAKs is responsible for its own set of most strongly-associated inflammatory mediators, and inhibition of specific JAKs or combination of JAKs can therefore also potentially allow for modulation of specific inflammatory factors and their associated conditions. To date, JAK inhibitors have particularly been studied in the treatment of atopic dermatitis (felt to be primarily driven by IL-4, IL-13, and IL-5), psoriasis (IL-12/IL-23), alopecia areata (IL-2, IL-15, and IFN-γ), and vitiligo (IL-15 and IFN-γ), given that these factors can all be found downstream of specific JAK/STAT pathways as shown in Figure 1. By providing a concise review of the inflammatory factors affected by each JAK, this article aims to support clinicians as they engage in the ever-growing body of research around the use of JAK inhibitors for potential treatment of dermatologic conditions.
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Avouac, Jérôme. "Janus Kinase Inhibitor Selectivity in Rheumatoid Arthritis: Where Do We Stand?" Rheumatology 1, no. 1 (2022): 5. http://dx.doi.org/10.17925/rmd.2022.1.1.5.
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The selectivity of Janus kinase inhibitors (JAKis) is still a matter of debate, as no JAKi is specific for only one Janus kinase (JAK) isoform. Currently approved JAKis in rheumatoid arthritis (RA) all inhibit JAK1, which is an effective therapeutic target in RA. Although selective JAK1 inhibition seems not to decrease drug efficacy, JAKi selectivity may modify the safety profile of this class. Indeed, the balance of benefit and risk of inhibiting JAK2, JAK3 and tyrosine kinase 2 is not certain and should be carefully evaluated in the future.
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Canté-Barrett, Kirsten, Joost CM Uitdehaag, Jessica GCAM Buijs-Gladdines, Wilco K. Smits, Rogier C. Buijsman, Guido JR Zaman, Rob Pieters, and Jules PP Meijerink. "3D Modeling of Novel Transforming JAK Mutations in T-Cell Acute Lymphoblastic Leukemia Reveals Altered Pseudokinase-Kinase Domain Interactions That Result in Constitutive JAK Kinase Activity." Blood 126, no. 23 (December 3, 2015): 869. http://dx.doi.org/10.1182/blood.v126.23.869.869.
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Abstract Background: Many pediatric T-cell acute lymphoblastic leukemia patients harbor mutations in IL7Ra or downstream molecules encoded by JAK1, JAK3, N-RAS, K-RAS, NF1, AKT, and PTEN. These mutated signaling molecules can contribute to leukemia by disturbing a multitude of cellular processes such as the cell cycle, epigenetics, apoptosis, or affecting other important signal transduction pathways. Aims: We aimed to determine the overall incidence of JAK family mutations in a large cohort of T-ALL patients. We also aimed to generate a 3D JAK1 model including known and newly identified JAK mutations in order to better understand how these contribute to JAK kinase activity and the transformation of cells. Methods: We screened 146 pediatric T-ALL patient samples for mutations in the FERM, pseudokinase and kinase domains of the Janus kinase gene family (JAK1-3, TYK2). To establish a 3D JAK1 model, we superimposed individual pseudokinase and kinase crystallographic structures on the homologous TYK2 pseudokinase-kinase structure. We visualized JAK mutations and their effects on the 3D structure. We modified the IL3-dependent Ba/F3 cell line to express JAK mutant or wild type genes upon induction by doxycycline. We tested these Ba/F3 derivative lines for transforming ability, signaling, and resistance to various inhibitors in the absence of IL3. Results: JAK1 or JAK3 mutations were found in 10 patients; no mutations were found in JAK2 or TYK2. We found JAK1 and JAK3 mutations as previously reported, but also identified amino acid substitutions as a result of novel JAK1 mutations including V427M, L624YPILKV, E668Q, P815S, and T901G. Our novel 3D model of JAK1 places most mutations in one of two crucial pseudokinase-kinase interaction sites, which can weaken the interaction and facilitate constitutive kinase activity. One interaction is between the hinge region of the pseudokinase domain and the loop in the kinase domain, which is supported by four salt bridges. Mutations in T-ALL disrupting these salt bridges include E668Q, R724H and its JAK3 equivalent R657Q, and T901G. The second interaction with the kinase domain is formed by a helical domain in the pseudokinase domain, located just upstream of the conserved F575-F636-V658 triad. This F-F-V triad is predicted to act as a structural switch that controls the catalytic activity of JAK kinases. Various mutations occur in the direct vicinity and can affect the function of this switch. V658F in T-ALL and its JAK2 equivalent V617F in polycythemia vera patients are mutations in this triad. The frequent JAK3 mutation M511I in T-ALL flanks the F513 residue (equivalent of JAK1 F575) and also affects the F-F-V triad. The L624YPILKV insertion mutation is located in a loop near the helical domain, which may also subtly compromise the F-F-V triad structural switch leading to derepression of the kinase domain. Expression of mutant JAK genes-in contrast to the wild type genes-transforms Ba/F3 cells by supporting IL3-independent growth, and by activating downstream RAS-MEK-ERK and PI3K-AKT pathways. This pathway activation as a result of ligand-independent mutant JAK kinase activity was confirmed by measuring phospho-proteins including p-MEK, p-ERK, p-AKT, p-mTOR, and p-p70S6K, and can be blocked by JAK inhibitors. Notably, JAK3 mutants signal significantly weaker than JAK1 mutants, possibly due to different dependence on (endogenous) receptors that normally mediate wild type JAK signaling. Summary/Conclusion: In a 3D model, we show that JAK mutations are located in critical interface regions between the pseudokinase and kinase domains, maintaining the kinase in an open, active confirmation. The inducible Ba/F3 model system confirms the transforming capacity of JAK mutations, reveals constitutive active downstream signaling, and is also suitable to test the effect of various inhibitors. The visualization of various JAK mutations in a 3D model and how these contribute to kinase activity provides insight in how mutant JAK could be inhibited, helping guide the development of new small molecule inhibitors of mutant JAKs. Disclosures Buijsman: Netherlands Translational Research Center B.V.: Equity Ownership, Other: founder and shareholder. Zaman:Netherlands Translational Research Center B.V.: Equity Ownership, Other: founder and shareholder.
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Ahluwalia, Manmeet Singh, Yasmeen Rauf, Glen Stevens, and David M. Peereboom. "Phase 1 trial of ruxolitinib, temozolomide, and radiation in high-grade gliomas." Journal of Clinical Oncology 40, no. 16_suppl (June 1, 2022): 2061. http://dx.doi.org/10.1200/jco.2022.40.16_suppl.2061.
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2061 Background: Ruxolitinib is a novel, selective inhibitor of JAK1 (Janus kinase 1) and JAK2 and JAK3. JAK signaling involves recruitment of signal transducers and activators of transcription (STATs) to cytokine receptors, activation, and subsequent localization of STATs to the nucleus leading to modulation of gene expression. Dysregulation of the JAK/STAT pathway has been associated with several types of cancer and increased proliferation and survival of malignant cells. Preclinical evidence supports inhibition of JAJK STAT pathway arrogated glioma growth. Methods: Ruxolitinib is a novel, selective inhibitor of JAK1 (Janus kinase 1) and JAK2 and JAK3. JAK signaling involves recruitment of signal transducers and activators of transcription (STATs) to cytokine receptors, activation, and subsequent localization of STATs to the nucleus leading to modulation of gene expression. Dysregulation of the JAK/STAT pathway has been associated with several types of cancer and increased proliferation and survival of malignant cells. Preclinical evidence supports inhibition of JAJK STAT pathway arrogated glioma growth. Results: 60 patients were treated on the study and there were no dose limiting toxicity seen on the protocol. Survival data was calculated for GBM. The OS for arm 1 was 18.17 (10.15, NA) and was not reached for arm 2. The 1 year OS was 0.62 for arm 1 and 0.93 for arm 2. Patients that received ruxolitinib + radiation x 60 Gy + daily temozolomide at 75 mg/m2 for 6 weeks over 6 weeks (Arm 2) had significantly better PFS and OS than those that received ruxolitinib + radiation x 60 Gy alone. Conclusions: Dose of 20 mg twice daily of ruxolitinib is safe with radiation and temozolomide. Preliminary survival data appears promising compared to the historical benchmarks and randomized phase 2 trial is planned. Clinical trial information: NCT03514069. [Table: see text]
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Sakamoto, Hiroshi, Hideo Yasukawa, Masaaki Masuhara, Shyu Tanimura, Atsuo Sasaki, Kentaro Yuge, Motoaki Ohtsubo, et al. "A Janus Kinase Inhibitor, JAB, Is an Interferon-γ–Inducible Gene and Confers Resistance to Interferons." Blood 92, no. 5 (September 1, 1998): 1668–76. http://dx.doi.org/10.1182/blood.v92.5.1668.
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Abstract It has been shown that interferons (IFNs) exert their signals through receptor-associated Janus kinases (JAKs) and signal transducers and activators of transcription (STATs). However, molecular mechanism of regulation of IFN signaling has not been fully understood. We have reported novel cytokine-inducible SH2 protein (CIS) and JAK binding protein (JAB) family genes that can potentially modulate cytokine signaling. Here we report that JAB is strongly induced by IFN-γ but not by IFN-β in mouse myeloid leukemia M1 cells and NIH-3T3 fibroblasts. NIH-3T3 cells ectopically expressing JAB but not CIS3 lost responsiveness to the antiviral effect of IFN-β and IFN-γ. M1 leukemic cells stably expressing JAB were also resistant to IFN-γ and IFN-β–induced growth arrest. In both NIH-3T3 and M1 transformants expressing JAB, IFN-γ did not induce tyrosine phosphorylation and DNA binding activity of STAT1. Moreover, IFN-γ–induced activation of JAK1 and JAK2 and IFN-β–induced JAK1 and Tyk2 activation were inhibited in NIH-3T3 JAB transformants. These results suggest that JAB inhibits IFN signaling by blocking JAK activity. We also found that IFN-resistant clones derived from LoVo cells and Daudi cells expressed high levels of JAB without stimulation. In IFN-resistant Daudi cells, IFN-induced STAT1 and JAK phosphorylation was partially reduced. Therefore, overexpression of JAB could be, at least in part, a mechanism of IFN resistance. © 1998 by The American Society of Hematology.
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Sakamoto, Hiroshi, Hideo Yasukawa, Masaaki Masuhara, Shyu Tanimura, Atsuo Sasaki, Kentaro Yuge, Motoaki Ohtsubo, et al. "A Janus Kinase Inhibitor, JAB, Is an Interferon-γ–Inducible Gene and Confers Resistance to Interferons." Blood 92, no. 5 (September 1, 1998): 1668–76. http://dx.doi.org/10.1182/blood.v92.5.1668.417k09_1668_1676.
Full textAbstract:
It has been shown that interferons (IFNs) exert their signals through receptor-associated Janus kinases (JAKs) and signal transducers and activators of transcription (STATs). However, molecular mechanism of regulation of IFN signaling has not been fully understood. We have reported novel cytokine-inducible SH2 protein (CIS) and JAK binding protein (JAB) family genes that can potentially modulate cytokine signaling. Here we report that JAB is strongly induced by IFN-γ but not by IFN-β in mouse myeloid leukemia M1 cells and NIH-3T3 fibroblasts. NIH-3T3 cells ectopically expressing JAB but not CIS3 lost responsiveness to the antiviral effect of IFN-β and IFN-γ. M1 leukemic cells stably expressing JAB were also resistant to IFN-γ and IFN-β–induced growth arrest. In both NIH-3T3 and M1 transformants expressing JAB, IFN-γ did not induce tyrosine phosphorylation and DNA binding activity of STAT1. Moreover, IFN-γ–induced activation of JAK1 and JAK2 and IFN-β–induced JAK1 and Tyk2 activation were inhibited in NIH-3T3 JAB transformants. These results suggest that JAB inhibits IFN signaling by blocking JAK activity. We also found that IFN-resistant clones derived from LoVo cells and Daudi cells expressed high levels of JAB without stimulation. In IFN-resistant Daudi cells, IFN-induced STAT1 and JAK phosphorylation was partially reduced. Therefore, overexpression of JAB could be, at least in part, a mechanism of IFN resistance. © 1998 by The American Society of Hematology.
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Liu, Liqin, Violeta Yu, Jeanne Pistillo, Josie Lee, Laurie B. Schenkel, Stephanie Geuns-Meyer, Ivonne Archibeque, Angus Sinclair, Renee Emkey, and Graham Molineux. "New Insights on Assessing Intra-Family Selectivity for Jak2 Inhibitors." Blood 118, no. 21 (November 18, 2011): 5150. http://dx.doi.org/10.1182/blood.v118.21.5150.5150.
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Abstract Abstract 5150 Essential thrombocythemia (ET), polycythemia vera (PV) and myelofibrosis (MF) are myeloproliferative disorders (MPDs) characterized by a chronic over-production of cells of one or more blood cell lineages and/or bone marrow fibrosis which may, on occasion, progress to acute myeloid leukemia. The V617F gain of function mutation in the pseudokinase domain of Jak2, which results in constitutive activation of Jak2, is the most frequent mutation associated with MPD. Constitutively activated Jak2 can lead to dysregulated downstream signaling pathways (STAT, MAP kinase, and PI3 kinase) which in turn trigger abnormal growth, survival and differentiation of hematopoietic progenitors. Therefore, inhibition of constitutively activated Jak2 may offer therapeutic potential. Designing a Jak2V617F specific inhibitor encounters challenges due to the lack of enzymatic activity of the pseudokinase domain of Jak2. In lieu of a Jak2V617F mutant selective inhibitor, a highly selective inhibitor of Jak2 is likely an attainable goal. Jak2 is a member of the Jak family of kinases including Jak1, Jak3, and Tyk2. Highly selective Jak2 inhibitors may provide a better safety margin in chronic dosing settings in ET and PV patients since inhibiting other Jak family members could cause side-effects such as immunosuppression. Attaining the desired selectivity of Jak2 inhibition versus the other family members has been challenging and few compounds have been reported to date that have the desired Jak2 selectivity. This can be attributed to the high homology of the ATP binding pocket among Jak family members, but is also hampered by a lack of assays capable of distinguishing the Jak-selectivity profile in a physiologically relevant setting. We compared the potency and selectivity of compounds tested in a pSTAT5 AlphaScreen® assay panel consisting of isogenic Ba/F3 cell lines individually expressing translocated ETS leukemia (TEL) fusions of each Jak-family member (Ba/F3-TEL-Jak) with data from corresponding Jak enzyme assays. Here we report that the selectivity of inhibitor compounds illustrated in enzyme assays did not correlate with the selectivity profile in cell lines due to different shifts in potency for each family member between enzyme and cells (Figure 1). As a consequence the selectivity of compounds for Jak2 against Jak1 observed in enzyme assays may be reduced or reversed in cellular assays. On the other hand, Jak2 selectivity over Jak3 seen in the enzyme assays was conserved in the cellular assay. Thus, we propose that compounds that exhibit greater potency on Jak2 compared to Jak1 in the enzyme assays are needed and should be the main focus of medicinal chemistry efforts in order to attain Jak2 selectivity over Jak1 in a cellular context. We also compared the potency and selectivity of compounds in the isogenic Ba/F3-TEL-Jak cell lines with data obtained with cytokine stimulated peripheral blood mononuclear cells (PBMCs). The potency and selectivity of compounds in PBMCs are determined by measuring the inhibition of phosphorylation of STAT5 in TPO or GM-CSF stimulated platelets or monocytes (mediated by Jak2) and in IL-2 stimulated lymphocytes (mediated by Jak1 and Jak3). We found that potency correlated well between PBMCs and Ba/F3-TEL-Jak2 cells, and the rank order of compounds based on IC50 values obtained with Ba/F3-TEL-Jak cell lines were conserved well in PBMCs; the compound selectivity profiles derived from the Ba/F3-TEL-Jak cell assays were predictive of Jak2 selectivity profiles obtained in the PBMC assays. Therefore, inclusion of Ba/F3-TEL-Jak pSTAT5 cellular assays may be useful for Jak family inhibitor development. Our results also suggest that relying solely on enzyme potency and selectivity data can be misleading, and that evaluating cellular selectivity in a biologically relevant context may provide a more meaningful understanding of selectivity and lead to the development of more selective Jak2 compounds. Disclosures: Liu: Amgen, Inc: Employment. Yu:Amgen: Employment. Pistillo:Amgen: Employment. Lee:Amgen: Employment. Schenkel:Amgen: Employment. Geuns-Meyer:Amgen: Employment. Archibeque:Amgen: Employment. Sinclair:Amgen: Employment. Emkey:Amgen: Employment. Molineux:Amgen: Employment.
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Vainchenker, William, Emilie Leroy, Laure Gilles, Caroline Marty, Isabelle Plo, and Stefan N. Constantinescu. "JAK inhibitors for the treatment of myeloproliferative neoplasms and other disorders." F1000Research 7 (January 17, 2018): 82. http://dx.doi.org/10.12688/f1000research.13167.1.
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JAK inhibitors have been developed following the discovery of theJAK2V617F in 2005 as the driver mutation of the majority of non-BCR-ABL1myeloproliferative neoplasms (MPNs). Subsequently, the search for JAK2 inhibitors continued with the discovery that the other driver mutations (CALRandMPL) also exhibited persistent JAK2 activation. Several type I ATP-competitive JAK inhibitors with different specificities were assessed in clinical trials and exhibited minimal hematologic toxicity. Interestingly, these JAK inhibitors display potent anti-inflammatory activity. Thus, JAK inhibitors targeting preferentially JAK1 and JAK3 have been developed to treat inflammation, autoimmune diseases, and graft-versus-host disease. Ten years after the beginning of clinical trials, only two drugs have been approved by the US Food and Drug Administration: one JAK2/JAK1 inhibitor (ruxolitinib) in intermediate-2 and high-risk myelofibrosis and hydroxyurea-resistant or -intolerant polycythemia vera and one JAK1/JAK3 inhibitor (tofacitinib) in methotrexate-resistant rheumatoid arthritis. The non-approved compounds exhibited many off-target effects leading to neurological and gastrointestinal toxicities, as seen in clinical trials for MPNs. Ruxolitinib is a well-tolerated drug with mostly anti-inflammatory properties. Despite a weak effect on the cause of the disease itself in MPNs, it improves the clinical state of patients and increases survival in myelofibrosis. This limited effect is related to the fact that ruxolitinib, like the other type I JAK2 inhibitors, inhibits equally mutated and wild-type JAK2 (JAK2WT) and also the JAK2 oncogenic activation. Thus, other approaches need to be developed and could be based on either (1) the development of new inhibitors specifically targetingJAK2V617F or (2) the combination of the actual JAK2 inhibitors with other therapies, in particular with molecules targeting pathways downstream of JAK2 activation or the stability of JAK2 molecule. In contrast, the strong anti-inflammatory effects of the JAK inhibitors appear as a very promising therapeutic approach for many inflammatory and auto-immune diseases.
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Spinelli, Francesca Romana, Robert A. Colbert, and Massimo Gadina. "JAK1: Number one in the family; number one in inflammation?" Rheumatology 60, Supplement_2 (May 1, 2021): ii3—ii10. http://dx.doi.org/10.1093/rheumatology/keab024.
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Abstract 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 CreditsTM upon successful completion of the assessment.
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Zhong, Haizhen A., and Suliman Almahmoud. "Docking and Selectivity Studies of Covalently Bound Janus Kinase 3 Inhibitors." International Journal of Molecular Sciences 24, no. 7 (March 23, 2023): 6023. http://dx.doi.org/10.3390/ijms24076023.
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The Janus kinases (JAKs) are a family of non-receptor cytosolic protein kinases critical for immune signaling. Many covalently bound ligands of JAK3 inhibitors have been reported. To help design selective JAK inhibitors, in this paper, we used five model proteins to study the subtype selectivity of and the mutational effects on inhibitor binding. We also compared the Covalent Dock programs from the Schrodinger software suite and the MOE software suite to determine which method to use for the drug design of covalent inhibitors. Our results showed that the docking affinity from 4Z16 (JAK3 wild-type model), 4E4N (JAK1), 4D1S (JAK2), and 7UYT (TYK2) from the Schrödinger software suite agreed well with the experimentally derived binding free energies with small predicted mean errors. However, the data from the mutant 5TTV model using the Schrödinger software suite yielded relatively large mean errors, whereas the MOE Covalent Dock program gave small mean errors in both the wild-type and mutant models for our model proteins. The docking data revealed that Leu905 of JAK3 and the hydrophobic residue at the same position in different subtypes (Leu959 of JAK1, Leu932 of JAK2, and Val981 of TYK2) is important for ligand binding to the JAK proteins. Arg911 and Asp912 of JAK3, Asp939 of JAK2, and Asp988 of TYK2 can be used for selective binding over JAK1, which contains Lys965 and Glu966 at the respective positions. Asp1021, Asp1039, and Asp1042 can be utilized for JAK1-selective ligand design, whereas Arg901 and Val981 may help guide TYK2-selective molecule design.
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Shawky, Ahmed M., Faisal A. Almalki, Ashraf N. Abdalla, Ahmed H. Abdelazeem, and Ahmed M. Gouda. "A Comprehensive Overview of Globally Approved JAK Inhibitors." Pharmaceutics 14, no. 5 (May 6, 2022): 1001. http://dx.doi.org/10.3390/pharmaceutics14051001.
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Janus kinase (JAK) is a family of cytoplasmic non-receptor tyrosine kinases that includes four members, namely JAK1, JAK2, JAK3, and TYK2. The JAKs transduce cytokine signaling through the JAK-STAT pathway, which regulates the transcription of several genes involved in inflammatory, immune, and cancer conditions. Targeting the JAK family kinases with small-molecule inhibitors has proved to be effective in the treatment of different types of diseases. In the current review, eleven of the JAK inhibitors that received approval for clinical use have been discussed. These drugs are abrocitinib, baricitinib, delgocitinib, fedratinib, filgotinib, oclacitinib, pacritinib, peficitinib, ruxolitinib, tofacitinib, and upadacitinib. The aim of the current review was to provide an integrated overview of the chemical and pharmacological data of the globally approved JAK inhibitors. The synthetic routes of the eleven drugs were described. In addition, their inhibitory activities against different kinases and their pharmacological uses have also been explained. Moreover, their crystal structures with different kinases were summarized, with a primary focus on their binding modes and interactions. The proposed metabolic pathways and metabolites of these drugs were also illustrated. To sum up, the data in the current review could help in the design of new JAK inhibitors with potential therapeutic benefits in inflammatory and autoimmune diseases.
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Malemud, Charles J. "The role of the JAK/STAT signal pathway in rheumatoid arthritis." Therapeutic Advances in Musculoskeletal Disease 10, no. 5-6 (May 19, 2018): 117–27. http://dx.doi.org/10.1177/1759720x18776224.
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Proinflammatory cytokine activation of the Janus kinase/signal transducers and activators of transcription (JAK/STAT) signal transduction pathway is a critical event in the pathogenesis and progression of rheumatoid arthritis. Under normal conditions, JAK/STAT signaling reflects the influence of negative regulators of JAK/STAT, exemplified by the suppressor of cytokine signaling and protein inhibitor of activated STAT. However, in rheumatoid arthritis (RA) both of these regulators are dysfunctional. Thus, continuous activation of JAK/STAT signaling in RA synovial joints results in the elevated level of matrix metalloproteinase gene expression, increased frequency of apoptotic chondrocytes and most prominently ‘apoptosis resistance’ in the inflamed synovial tissue. Tofacitinib, a JAK small molecule inhibitor, with selectivity for JAK2/JAK3 was approved by the United States Food and Drug Administration (US FDA) for the therapy of RA. Importantly, tofacitinib has demonstrated significant clinical efficacy for RA in the post-US FDA-approval surveillance period. Of note, the success of tofacitinib has spurred the development of JAK1, JAK2 and other JAK3-selective small molecule inhibitors, some of which have also entered the clinical setting, whereas other JAK inhibitors are currently being evaluated in RA clinical trials.
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Levy, Gabriel, Paola Guglielmelli, Peter Langmuir, and Stefan Constantinescu. "JAK inhibitors and COVID-19." Journal for ImmunoTherapy of Cancer 10, no. 4 (April 2022): e002838. http://dx.doi.org/10.1136/jitc-2021-002838.
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During SARS-CoV-2 infection, the innate immune response can be inhibited or delayed, and the subsequent persistent viral replication can induce emergency signals that may culminate in a cytokine storm contributing to the severe evolution of COVID-19. Cytokines are key regulators of the immune response and virus clearance, and, as such, are linked to the—possibly altered—response to the SARS-CoV-2. They act via a family of more than 40 transmembrane receptors that are coupled to one or several of the 4 Janus kinases (JAKs) coded by the human genome, namely JAK1, JAK2, JAK3, and TYK2. Once activated, JAKs act on pathways for either survival, proliferation, differentiation, immune regulation or, in the case of type I interferons, antiviral and antiproliferative effects. Studies of graft-versus-host and systemic rheumatic diseases indicated that JAK inhibitors (JAKi) exert immunosuppressive effects that are non-redundant with those of corticotherapy. Therefore, they hold the potential to cut-off pathological reactions in COVID-19. Significant clinical experience already exists with several JAKi in COVID-19, such as baricitinib, ruxolitinib, tofacitinib, and nezulcitinib, which were suggested by a meta-analysis (Patoulias et al.) to exert a benefit in terms of risk reduction concerning major outcomes when added to standard of care in patients with COVID-19. Yet, only baricitinib is recommended in first line for severe COVID-19 treatment by the WHO, as it is the only JAKi that has proven efficient to reduce mortality in individual randomized clinical trials (RCT), especially the Adaptive COVID-19 Treatment Trial (ACTT-2) and COV-BARRIER phase 3 trials. As for secondary effects of JAKi treatment, the main caution with baricitinib consists in the induced immunosuppression as long-term side effects should not be an issue in patients treated for COVID-19.We discuss whether a class effect of JAKi may be emerging in COVID-19 treatment, although at the moment the convincing data are for baricitinib only. Given the key role of JAK1 in both type I IFN action and signaling by cytokines involved in pathogenic effects, establishing the precise timing of treatment will be very important in future trials, along with the control of viral replication by associating antiviral molecules.
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Perner, Florian, Felix C. Saalfeld, Tina M. Schnoeder, Denise Wolleschak, Corinna Fahldieck, Satish Ranjan, Berend H. Isermann, et al. "Specificity of JAK-Kinase Inhibition Determines Impact on T-Cell Function." Blood 124, no. 21 (December 6, 2014): 1410. http://dx.doi.org/10.1182/blood.v124.21.1410.1410.
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Abstract Inhibitors of JAK2-kinase (Ruxolitinib, Momelotinib) are already approved or currently investigated in advanced clinical trials for treatment of myeloproliferative neoplasia (MPN). Besides their effect on mutated JAK2-kinase these compounds inhibit wildtype JAK and thereby impair JAK-STAT-signaling, which is an important pathway for proliferation and activation of other cell types such as human T-cells. Accumulating evidence suggests that they may also exert substantial immunosuppressive activity. Very recent reports highlighting hepatitis B reactivation complemented the series of severe infections in ruxolitinib-treated patients among which cryptococcus neoformans pneumonia, toxoplasmosis retinitis, disseminated tuberculosis, and progressive multifocal leukencephalopathy are the most alarming. We hypothesized that JAK-kinase inhibitors may act as immunosuppressant drugs by impairment of T-cell responses through inhibition of T-cell signaling (JAK-STAT pathway) and that specificity of JAK-kinase inhibition may be of major importance for the degree of T-cell inhibition. Therefore we investigated the effects of pharmacological JAK-kinase inhibition on healthy donor (HD-) and MPN patient T-cells. Selective inhibitors of JAK2-kinase (BSK805) and JAK3-kinase (BQM245) as well as clinically relevant inhibitors of JAK1/2-kinases (Ruxolitinib and Momelotinib) were used for pharmacologic inhibition. The SRC-kinase inhibitor Dasatinib served as a positive control for T-cell inhibition. Knockdown of specific JAK-kinases by RNAi was used to control for target specificity. In regard to T-cell receptor (TCR)-mediated signaling we investigated bona fide signaling molecules downstream of the TCR by Western Blotting. Besides SRC-kinases like LCK also ZAP70, PLCG1 and the MAPK/ERK pathway have been described to play a pivotal role in T-cell activation. In our data set, selectivity of JAK-kinase inhibition (JAK2, JAK3 or JAK1/2) influenced TCR-signaling in regard to overall tyrosine phosphorylation but also in regard to downstream effectors such as ERK. As activation and proliferation of primary T-cells is a critical step in immune responses against viral and tumor antigens we aimed to investigate the influence of JAK-kinase inhibition on activation and proliferation of human T-cells. T-cells from healthy donors were stimulated using either PHA 0.5% or CD3/CD28 beads to ensure a more T-cell receptor specific stimulation. CD69 expression was used as a marker for T-cell activation and CFSE staining was applied to assess for T-cell proliferation. Using CD3/CD28 stimulation, CD69 expression was almost abrogated following Dasatinib treatment and proliferation was significantly reduced. Applying relevant doses of specific JAK2 and JAK3 inhibitors to isolated T-cells did neither influence CD69 expression nor T-cell proliferation. These findings are confirmed by RNAi. In contrast, clinically relevant doses of JAK1/2 inhibitors Ruxolitinib and Momelotinib, respectively reduced CD69 expression and T-cell proliferation. Likewise, T-cells derived from MPN patients treated with Ruxolitinib revealed decreased CD69 expression and decreased proliferative capacity upon stimulation, compared to untreated patients or HD-controls. In order to investigate T-cell function, we assessed for allo-reactivity in a mixed lymphocyte culture. Human pan-T-cells were co-cultured with allogeneic antigen presenting cells. T-cell reactivity – as measured by 3H-thymidine incorporation – was significantly impaired by Ruxolitinib and Momelotinib. Specific inhibition of JAK2 or JAK3 kinase, however, did not affect T-cell reactivity. These effects could be confirmed using T-cells derived from Ruxolitinib-treated MPN patients. Investigation of leukemia- and virus-antigen-specific T-cell responses are currently under way to gain deeper insight regarding this clinically relevant scenario. Taken together, specificity of JAK-kinase inhibition influences the inhibitory potential on T-cell function. JAK1 kinase seems to play an important role in T-cell activation, as unspecific inhibitors of JAK1 & JAK2 Kinase inhibit T-cell function while selective inactivation of JAK2 kinase leaves T-cell function almost unaffected. Heterogeneity in T-cell function of Ruxolitinib-treated patients is an important finding that deserves detailed investigation. Disclosures Heidel: Novartis: Consultancy.
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Hammarén, Henrik M., Daniela Ungureanu, Jean Grisouard, Radek C. Skoda, Stevan R. Hubbard, and Olli Silvennoinen. "ATP binding to the pseudokinase domain of JAK2 is critical for pathogenic activation." Proceedings of the National Academy of Sciences 112, no. 15 (March 30, 2015): 4642–47. http://dx.doi.org/10.1073/pnas.1423201112.
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Pseudokinases lack conserved motifs typically required for kinase activity. Nearly half of pseudokinases bind ATP, but only few retain phosphotransfer activity, leaving the functional role of nucleotide binding in most cases unknown. Janus kinases (JAKs) are nonreceptor tyrosine kinases with a tandem pseudokinase–kinase domain configuration, where the pseudokinase domain (JAK homology 2, JH2) has important regulatory functions and harbors mutations underlying hematological and immunological diseases. JH2 of JAK1, JAK2, and TYK2 all bind ATP, but the significance of this is unclear. We characterize the role of nucleotide binding in normal and pathogenic JAK signaling using comprehensive structure-based mutagenesis. Disruption of JH2 ATP binding in wild-type JAK2 has only minor effects, and in the presence of type I cytokine receptors, the mutations do not affect JAK2 activation. However, JH2 mutants devoid of ATP binding ameliorate the hyperactivation of JAK2 V617F. Disrupting ATP binding in JH2 also inhibits the hyperactivity of other pathogenic JAK2 mutants, as well as of JAK1 V658F, and prevents induction of erythrocytosis in a JAK2 V617F myeloproliferative neoplasm mouse model. Molecular dynamic simulations and thermal-shift analysis indicate that ATP binding stabilizes JH2, with a pronounced effect on the C helix region, which plays a critical role in pathogenic activation of JAK2. Taken together, our results suggest that ATP binding to JH2 serves a structural role in JAKs, which is required for aberrant activity of pathogenic JAK mutants. The inhibitory effect of abrogating JH2 ATP binding in pathogenic JAK mutants may warrant novel therapeutic approaches.
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Vignais, M. L., H. B. Sadowski, D. Watling, N. C. Rogers, and M. Gilman. "Platelet-derived growth factor induces phosphorylation of multiple JAK family kinases and STAT proteins." Molecular and Cellular Biology 16, no. 4 (April 1996): 1759–69. http://dx.doi.org/10.1128/mcb.16.4.1759.
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Receptors for interferons and other cytokines signal through the action of associated protein tyrosine kinases of the JAK family and latent cytoplasmic transcription factors of the STAT family. Genetic and biochemical analysis of interferon signaling indicates that activation of STATs by interferons requires two distinct JAK family kinases. Loss of either of the required JAKs prevents activation of the other JAK and extinguishes STAT activation. These observations suggest that JAKs provide interferon receptors with a critical catalytic signaling function and that at least two JAKs must be incorporated into an active receptor complex. JAK and STAT proteins are also activated by ligands such as platelet-derived growth factor (PDGF), which act through receptors that possess intrinsic protein tyrosine kinase activity, raising questions about the role of JAKs in signal transduction by this class of receptors. Here, we show that all three of the ubiquitously expressed JAKs--JAK1, JAK2, and Tyk2--become phosphorylated on tyrosine in both mouse BALB/c 3T3 cells and human fibroblasts engineered to express the PDGF-beta receptor. All three proteins are also associated with the activated receptor. Through the use of cell lines each lacking an individual JAK, we find that in contrast to interferon signaling, PDGF-induced JAK phosphorylation and activation of STAT1 and STAT3 is independent of the presence of any other single JAK but does require receptor tyrosine kinase activity. These results suggests that the mechanism of JAK activation and JAK function in signaling differs between receptor tyrosine kinases and interferon receptors.
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Alunno, Alessia, Ivan Padjen, Antonis Fanouriakis, and Dimitrios T. Boumpas. "Pathogenic and Therapeutic Relevance of JAK/STAT Signaling in Systemic Lupus Erythematosus: Integration of Distinct Inflammatory Pathways and the Prospect of Their Inhibition with an Oral Agent." Cells 8, no. 8 (August 15, 2019): 898. http://dx.doi.org/10.3390/cells8080898.
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Four Janus kinases (JAKs) (JAK1, JAK2, JAK3, TYK2) and seven signal transducers and activators of transcription (STATs) (STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, STAT6) mediate the signal transduction of more than 50 cytokines and growth factors in many different cell types. Located intracellularly and downstream of cytokine receptors, JAKs integrate and balance the actions of various signaling pathways. With distinct panels of STAT-sensitive genes in different tissues, this highly heterogeneous system has broad in vivo functions playing a crucial role in the immune system. Thus, the JAK/STAT pathway is critical for resisting infection, maintaining immune tolerance, and enforcing barrier functions and immune surveillance against cancer. Breakdowns of this system and/or increased signal transduction may lead to autoimmunity and other diseases. Accordingly, the recent development and approval of the first small synthetic molecules targeting JAK molecules have opened new therapeutic avenues of potentially broad therapeutic relevance. Extensive data are now available regarding the JAK/STAT pathway in rheumatoid arthritis. Dysregulation of the cytokines is also a hallmark of systemic lupus erythematosus (SLE), and targeting the JAK/STAT proteins allows simultaneous suppression of multiple cytokines. Evidence from in vitro studies and animal models supports a pivotal role also in the pathogenesis of cutaneous lupus and SLE. This has important therapeutic implications, given the current paucity of targeted therapies especially in the latter. Herein, we summarize the currently available literature in experimental SLE, which has led to the recent promising Phase II clinical trial of a JAK inhibitor.
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Kumar, Narendra, Longxiang Kuang, Ryan Villa, Priyam Kumar, and Jayshree Mishra. "Mucosal Epithelial Jak Kinases in Health and Diseases." Mediators of Inflammation 2021 (March 16, 2021): 1–17. http://dx.doi.org/10.1155/2021/6618924.
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Janus kinases (Jaks) are a family of nonreceptor tyrosine kinase that include four different members, viz., Jak1, Jak2, Jak3, and Tyk2. Jaks play critical roles in immune cells functions; however, recent studies suggest they also play essential roles in nonimmune cell physiology. This review highlights the significance of epithelial Jaks in understanding the molecular basis of some of the diseases through regulation of epithelial-mesenchymal transition, cell survival, cell growth, development, and differentiation. Growth factors and cytokines produced by the cells of hematopoietic origin use Jak kinases for signal transduction in both immune and nonimmune cells. Among Jaks, Jak3 is widely expressed in both immune cells and in intestinal epithelial cells (IECs) of both humans and mice. Mutations that abrogate Jak3 functions cause an autosomal severe combined immunodeficiency disease (SCID) while activating Jak3 mutations lead to the development of hematologic and epithelial cancers. A selective Jak3 inhibitor CP-690550 (Xeljanz) approved by the FDA for certain chronic inflammatory conditions demonstrates immunosuppressive activity in rheumatoid arthritis, psoriasis, and organ transplant rejection. Here, we also focus on the consequences of Jak3-directed drugs on adverse effects in light of recent discoveries in mucosal epithelial functions of Jak3 with some information on other Jaks. Lastly, we brief on structural implications of Jak3 domains beyond the immune cells. As information about the roles of Jak3 in gastrointestinal functions and associated diseases are only just emerging, in the review, we summarize its implications in gastrointestinal wound repair, inflammatory bowel disease, obesity-associated metabolic syndrome, and epithelial cancers. Lastly, we shed lights on identifying potential novel targets in developing therapeutic interventions of diseases associated with dysfunctional IEC.
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Cacciapaglia, F., V. Venerito, S. del Vescovo, S. Stano, R. Bizzoca, D. Natuzzi, N. Lacarpia, M. Fornaro, and F. Iannone. "AB0070 INHIBITION OF STAT3 IN PBMCs FROM RHEUMATOID ARTHRITIS PATIENTS: CLUES TO UNDERSTAND SELECTIVITY OF JANUS KINASE INHIBITORS." Annals of the Rheumatic Diseases 81, Suppl 1 (May 23, 2022): 1167.2–1168. http://dx.doi.org/10.1136/annrheumdis-2022-eular.1997.
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BackgroundThe Janus kinase (Jak) - signal transducer and activator of transcription (STAT) pathway has 4 Jak proteins and 7 STAT factors that mediate intracellular downstream of cytokine receptors. Targeted small-molecule therapies with different bond affinity to Jak proteins have been demonstrated effective in rheumatoid arthritis (RA) treatment, but the clinical significance of selective inhibition remains unclear.ObjectivesTo explore the effect of selective inhibition of Jak-STAT pathway in peripheral blood mononuclear cells (PBMC) from RA patients compared to healthy donors (HD).MethodsIn vitro Jak inhibition of the subunit 3 of phosphorylated (p) than activated STAT was measured by flow cytometry in peripheral blood mononuclear cells (PBMC) from RA patients with active disease (DAS28>5.1) naïve to any DMARDs (n.5) and HD (n.5), following recombinant human 0.1 ng/ml IL-6 (Peprotech – NJ, USA) stimulation. After blood separation, PBMC were overnight incubated with IC50 concentrations of selective Jak1-, Jak2-, Jak3- and Tyk2-inhibitors (Biovision Inc. – CA, USA) with or without IL-6 stimulation. Mean fold-increase of pSTAT3 was then compared in presence of different compounds stimulation.ResultsMean pSTAT3 activity after overnight incubation was significantly higher in RA patients compared to HD (37%; 95CI 8.2-56.7 vs 17.9%; 95CI 4.6-21 – p=0.01). After IL-6 stimulation, a 2-fold and a 1.4-fold increase in pSTAT3 levels was observed in PBMC from RA patients and HD, respectively. In unstimulated PBMC from HD Jak-inhibitors didn’t significantly reduced pSTAT3 activity. In CD14+ cells from RA patients, pSTAT3 activity was reduced with no differences between all four selective Jak-inhibitors, while in CD4+ cells only Jak1-inhibition was able to reduce by 40% pSTAT3 activity. After IL-6 stimulation, the co-culture with Jak1- or JaK3- selective inhibitors was able to significantly reduce pSTAT3 levels in CD4+ lymphocytes, by an average of 20%. While in CD14+ monocytes Jak1-, Jak2- and Jak3- selective inhibitors were able to reduce pSTAT3 activity by a mean of 30%. Tyk-2 selective inhibitor did not interfere with STAT3 activation by IL-6 stimulation of PBMC from RA patients and HD.ConclusionJak/STAT3 activity of PBMC from RA patients with active disease may be differently modulated by specific inhibitors. Selectivity of Jak-inhibitors seems more relevant in lymphocytes after IL-6 stimulation. These preliminary findings may explain discrepancies in effectiveness of selective Jak-inhibitors and pave the way for different choices in clinical practice.References[1]Tanaka Y, et al. Nat Rev Rheumatol. 2022 Jan 5:1–13.[2]Traves PG, et al. Ann Rheum Dis. 2021 Jul;80(7):865-875.[3]Choy EH. Rheumatology (Oxford). 2019 Jun 1;58(6):953-962.Disclosure of InterestsNone declared
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Wang, H. Y., J. Zamorano, J. L. Yoerkie, W. E. Paul, and A. D. Keegan. "The IL-4-induced tyrosine phosphorylation of the insulin receptor substrate is dependent on JAK1 expression in human fibrosarcoma cells." Journal of Immunology 158, no. 3 (February 1, 1997): 1037–40. http://dx.doi.org/10.4049/jimmunol.158.3.1037.
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Abstract It has been shown that IL-4 induces the tyrosine phosphorylation of JAK1 and JAK3 in the majority of hemopoietic cell types, and JAK2 and TYK2 in several other types. However, the significance of this tyrosine phosphorylation in regulating IL-4 signaling has not been shown. To determine whether JAKs play a role in activating a signal transduction pathway different from the classical JAK/STAT pathway, we analyzed the ability of huIL-4 to stimulate the tyrosine phosphorylation of one of its major cellular substrates, the insulin receptor substrate (IRS). Using human fibrosarcoma cell lines with mutations in JAK1, JAK2, and TYK2, we found that expression of functional JAK1, but not TYK2 or JAK2, is essential for IL-4-induced tyrosine phosphorylation of IRS. We also provide evidence that the IRS pathway is independent of STAT-6, showing that JAK1 is essential for activating a STAT-independent pathway.
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Dai, Jun, LiXi Yang, and Glynn Addison. "Current Status in the Discovery of Covalent Janus Kinase 3 (JAK3) Inhibitors." Mini-Reviews in Medicinal Chemistry 19, no. 18 (November 29, 2019): 1531–43. http://dx.doi.org/10.2174/1389557519666190617152011.
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The search for inhibitors of the Janus kinase family (JAK1, JAK2, JAK3 and TYK2) has been ongoing for several decades and has resulted in a number of JAK inhibitors being approved for use in patients, such as tofacitinib for the treatment of autoimmune diseases such as Rheumatoid Arthritis (RA). Although initially thought to be a JAK3 selective inhibitor, tofacitinib was subsequently found to possess significant activity to inhibit JAK1 and JAK2 which has contributed to some adverse side effects. A selective JAK3 inhibitor should only have an effect within the immune system since JAK3 is solely expressed in lymphoid tissue; this makes JAK3 a target of interest in the search for treatments of autoimmune diseases. A method to obtain selectivity for JAK3 over the other JAK family members, which has attracted more scientific attention recently, is the targeting of the active site cysteine residue, unique in JAK3 within the JAK family, with compounds containing electrophilic warheads which can form a covalent bond with the nucleophilic thiol of the cysteine residue. This review encompasses the historical search for a covalent JAK3 inhibitor and the most recently published research which hasn’t been reviewed to date. The most important compounds from the publications reviewed the activity and selectivity of these compounds together with some of the more important biological results are condensed in to an easily digested form that should prove useful for those interested in the field.
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Babon, Jeffrey J., Isabelle S. Lucet, James M. Murphy, Nicos A. Nicola, and Leila N. Varghese. "The molecular regulation of Janus kinase (JAK) activation." Biochemical Journal 462, no. 1 (July 24, 2014): 1–13. http://dx.doi.org/10.1042/bj20140712.
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The JAK (Janus kinase) family members serve essential roles as the intracellular signalling effectors of cytokine receptors. This family, comprising JAK1, JAK2, JAK3 and TYK2 (tyrosine kinase 2), was first described more than 20 years ago, but the complexities underlying their activation, regulation and pleiotropic signalling functions are still being explored. Here, we review the current knowledge of their physiological functions and the causative role of activating and inactivating JAK mutations in human diseases, including haemopoietic malignancies, immunodeficiency and inflammatory diseases. At the molecular level, recent studies have greatly advanced our knowledge of the structures and organization of the component FERM (4.1/ezrin/radixin/moesin)-SH2 (Src homology 2), pseudokinase and kinase domains within the JAKs, the mechanism of JAK activation and, in particular, the role of the pseudokinase domain as a suppressor of the adjacent tyrosine kinase domain's catalytic activity. We also review recent advances in our understanding of the mechanisms of negative regulation exerted by the SH2 domain-containing proteins, SOCS (suppressors of cytokine signalling) proteins and LNK. These recent studies highlight the diversity of regulatory mechanisms utilized by the JAK family to maintain signalling fidelity, and suggest alternative therapeutic strategies to complement existing ATP-competitive kinase inhibitors.
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Grant, Alice H., Alejandro C. Rodriguez, Omar J. Rodriguez Moncivais, Shengjie Sun, Lin Li, Jonathon E. Mohl, Ming-Ying Leung, Robert A. Kirken, and Georgialina Rodriguez. "JAK1 Pseudokinase V666G Mutant Dominantly Impairs JAK3 Phosphorylation and IL-2 Signaling." International Journal of Molecular Sciences 24, no. 7 (April 6, 2023): 6805. http://dx.doi.org/10.3390/ijms24076805.
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Overactive Janus kinases (JAKs) are known to drive leukemia, making them well-suited targets for treatment. We sought to identify new JAK-activating mutations and instead found a JAK1-inactivating pseudokinase mutation, V666G. In contrast to other pseudokinase mutations that canonically lead to an active kinase, the JAK1 V666G mutation led to under-activation seen by reduced phosphorylation. To understand the functional role of JAK1 V666G in modifying kinase activity we investigated its influence on other JAK kinases and within the Interleukin-2 pathway. JAK1 V666G not only inhibited its own activity, but its presence could inhibit other JAK kinases. These findings provide new insights into the potential of JAK1 pseudokinase to modulate its own activity, as well as of other JAK kinases. Thus, the features of the JAK1 V666 region in modifying JAK kinases can be exploited to allosterically inhibit overactive JAKs.
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Cacciapaglia, F., S. Perniola, S. del Vescovo, S. Stano, R. Bizzoca, D. Natuzzi, M. Fornaro, and F. Iannone. "AB0134 IN-VITRO STUDY ON THE EFFECT OF SELECTIVE Jak-INHIBITORS ON PBMCs STAT3 PHOSPHORYLATION FROM SYSTEMIC SCLEROSIS PATIENTS." Annals of the Rheumatic Diseases 81, Suppl 1 (May 23, 2022): 1196.3–1197. http://dx.doi.org/10.1136/annrheumdis-2022-eular.2625.
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BackgroundSystemic sclerosis (SSc) is a rare autoimmune connective tissue disease characterized by autoimmunity-driven damage and vasculopathy leading to fibrosis of the skin and internal organs (1). The Janus kinase (Jak) - signal transducer and activator of transcription (STAT) pathway has been evidenced markedly activated in SSc patients (2, 3), and its inhibition has been proved in preclinical and clinical trials (4), but no data on Jak selective inhibition are available.ObjectivesTo explore the effect of selective inhibition of Jak/STAT pathway in peripheral blood mononuclear cells (PBMC) from SSc patients.MethodsIn vitro Jak inhibition of the subunit 3 of phosphorylated (p) than activated STAT was measured by flow cytometry in peripheral blood mononuclear cells (PBMC) from SSc patients naïve to any immunosuppressive and/or corticosteroids (n.5). pSTAT3 activity was also assessed after stimulation with recombinant human 0.1 ng/ml IL-6 (Peprotech – NJ, USA). The PBMC were overnight incubated with IC50 concentrations of selective Jak1-, Jak2-, Jak3- and Tyk2-inhibitors (Biovision Inc. – CA, USA). Percentages of pSTAT3 positive cells were compared in presence of different compounds stimulation.ResultsAfter overnight incubation, percentage of pSTAT3 positive cells was significantly higher in CD14pos compared to CD4pos (16.3%; 95CI 10-22 vs 10.7%; 95CI 4--18, – p=0.02). pSTAT3posCD14pos cells were halved only by selective Jak3-inhibitor, while pSTAT3posCD4pos cells were reduced by 36% by selective Jak1-inhibitor. Selective Jak2- or Tyk2-inhibitors did not interfere with STAT3 phosphorylation in PBMC from SSc patients. After IL-6 stimulation, we observed a 2- and a 1.5-fold increase in percentage of pSTAT3posCD4pos and pSTAT3posCD14pos cells, respectively. pSTAT3posCD14pos cells were reduced in the PBMC co-culture with IL-6 and Jak-selective inhibitors, in contrast no effects were found in CD4pos cells. Specifically, selective Jak1- and Jak3-inhibitors reduced pSTAT3posCD14pos cells by an average of 37% and 25%, respectively. No effects were observed after co-culture with IL-6 and selective Jak2- or Tyk2-inhibitors.ConclusionJak/STAT3 pathway of PBMC from SSc patients with active disease may be differently modulated by specific inhibitors. Selectivity of Jak1- and Jak3-inhibitors seems more relevant, especially in CD14pos monocytes after IL-6 stimulation. These preliminary findings highlight some evidence for effectiveness of selective Jak-inhibitors in SSc treatment.References[1]Benfaremo D, et al. Systemic Sclerosis: From Pathophysiology to Novel Therapeutic Approaches. Biomedicines. 2022;10(1):163.[2]Talotta R. The rationale for targeting the JAK/STAT pathway in scleroderma-associated interstitial lung disease. Immunotherapy. 2021;13(3):241-256.[3]Cacciapaglia F, et al. Phosphorylated signal transducer and activator of transcription 3 (pSTAT3) is highly expressed in CD14+ circulating cells of scleroderma patients. Rheumatology (Oxford). 2020;59(6):1442-1444.[4]Karalilova RV, et al. Tofacitinib in the treatment of skin and musculoskeletal involvement in patients with systemic sclerosis, evaluated by ultrasound. Rheumatol Int. 2021;41(10):1743-1753.Disclosure of InterestsNone declared
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Purandare, Ashok V., Animesh Pardanani, Theresa McDevitt, Marco Gottardis, Terra Lasho, Dan You, Louis Lombardo, et al. "Characterization of BMS-911543, a Functionally Selective Small Molecule Inhibitor of JAK2." Blood 116, no. 21 (November 19, 2010): 4112. http://dx.doi.org/10.1182/blood.v116.21.4112.4112.
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Abstract Abstract 4112 We report the characterization of BMS-911543, a potent and functionally selective small molecule inhibitor of the Janus kinase family (JAK) member, JAK2. BMS-911543 is a reversible inhibitor of JAK2 with a biochemical IC50 of 0.001 μ M and Ki of 0.48 nM. It has over 74- and 350-fold selectivity against the other JAK family members, JAK3 and JAK1, respectively. Further, examination of > 450 other kinases did not reveal significant inhibitory activity for this JAK2 inhibitor. Functionally, BMS-911543 displayed potent anti-proliferative and pharmacodynamic (PD) effects in mutated JAK2-expressing cell lines dependent upon JAK2-STAT signaling and had little activity in cell types dependent upon other pathways such as JAK1 and JAK3. BMS-911543 was evaluated in colony growth assays using primary progenitor cells isolated from patients with JAK2V617F-positive myeloproliferative disease (MPD) and resulted in an increased anti-proliferative response in MPD cells as compared with those from healthy volunteers. Similar to these in vitro observations, BMS-911543 was also highly active in in vivo models of JAK2-pSTAT signaling in multiple species (mouse, rat, dog and monkey) with sustained pathway suppression being observed after a single oral dose. Additionally, BMS-911543 was evaluated for effects in a JAK2V617F-expressing SET-2 xenograft model system and displayed a minimally effective dose of <2 mg/kg on pSTAT5 pathway suppression, which lasted up to 8 hours. BMS-911543 was also compared to pan-JAK inhibitors in a mouse model of immunosuppression. At low dose levels active in JAK2-dependent PD models, no effects were observed on antigen-induced IgG and IgM production whereas a pan-JAK family inhibitor showed pronounced effects at all dose levels tested. The mechanistic selectivity of BMS-911543 to pan-JAK family inhibitors was extended through comparative analysis of these inhibitors in whole genome gene expression profiling experiments performed in sensitive cell types. In this comparison, BMS-911543 modulated a distinct subset of transcriptional changes as compared to pan-JAK inhibitors, thereby defining a minimal set of transcriptional changes underlying the pharmacologic effects of JAK2 inhibition. Collectively these results define the mechanistic basis for a differential therapeutic index between selective JAK2 and pan-JAK family inhibition pre-clinically and suggest a therapeutic rationale for the further characterization of BMS-911543 in patients with MPD and in other disorders characterized by constitutively active JAK2 signaling. Disclosures: Purandare: Bristol-Myers Squibb: Employment. McDevitt:Bristol-Myers Squibb: Employment. Gottardis:Bristol-Myers Squibb: Employment. You:Bristol-Myers Squibb: Employment. Lombardo:Bristol_Myers Squibb: Employment. Penhallow:Bristol-Myers Squibb: Employment. Vuppugalla:Bristol-Myers Squibb: Employment. Trainor:Bristol-Myers Squibb: Employment. Lorenzi:Bristol-Myers Squibb: Employment.
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Meyer, Sara C., Matthew D. Keller, Priya Koppikar, Olga A. Guryanova, Maria Kleppe, Anna Sophia McKenney, William R. Sellers, et al. "Type II Inhibition of JAK2 with NVP-CHZ868 Reverses Type I JAK Inhibitor Persistence and Demonstrates Increased Efficacy in MPN Models." Blood 124, no. 21 (December 6, 2014): 160. http://dx.doi.org/10.1182/blood.v124.21.160.160.
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Abstract The identification of JAK2 mutations in patients with myeloproliferative neoplasms (MPN) led to the clinical development of JAK2 inhibitors, and the JAK1/2 inhibitor ruxolitinib has been approved for the treatment of myelofibrosis (MF). Although clinically tested JAK inhibitors improve MPN-associated splenomegaly and systemic symptoms, they do not significantly reduce the MPN clone in most MPN patients.We previously demonstrated that MPN cells can acquire persistence to ruxolitinib and other type I JAK inhibitors which bind the active conformation of JAK2, and that JAK2 inhibitor persistence is associated with reactivation of JAK-STAT signaling and with heterodimerization between activated JAK2 and JAK1/TYK2, consistent with activation of JAK2 in trans by other JAK kinases. We have now extended our studies to other type I JAK inhibitors in clinical development, including CYT387, BMS911543 and SAR302503. In each case we see the same mechanism of persistence as observed with ruxolitinib, with transactivation of JAK2 by other JAK kinases. Most importantly, we found that MPN cells which were persistent to one JAK inhibitor were insensitive to the other JAK inhibitors, suggesting that the mechanisms which limit overall efficacy of ruxolitinib will limit the efficacy of other JAK inhibitors in clinical development. All JAK inhibitors in clinical development are type I inhibitors that interact with and inhibit the active confirmation of the JAK2 kinase. We hypothesized that novel, type II JAK inhibitors that interact with and inhibit JAK2 in the inactive conformation might retain activity in JAK inhibitor persistent cells and show increased efficacy in murine MPN models. We therefore characterized the efficacy of NVP-CHZ868, a novel type II JAK inhibitor, in MPN cells and in murine MPN models. CHZ868 potently inhibited proliferation of cells expressing the JAK2V617F mutation or the TEL-JAK2 fusion. We found that JAK2/MPL-mutant cell lines were universally sensitive to NVP-CHZ868. CHZ868 treatment of JAK2-mutant SET2 cells induced a higher degree of apoptosis compared to ruxolitinib. Signaling studies demonstrated that CHZ868 more potently attenuated JAK-STAT signaling in JAK2/MPL-mutant cells, with suppression of JAK2 phosphorylation consistent with a type II mechanism of kinase inhibition. We next investigated the ability of CHZ868 to inhibit the proliferation and signaling of MPN cells that had acquired persistence to type I JAK inhibitors. Type II inhibition with CHZ868 completely suppressed JAK-STAT signaling in type I JAK inhibitor-persistent cells, and prevented heterodimeric activation of JAK2 by JAK1 and TYK2. Most importantly, JAK2/MPL-mutant cells which were insensitive to type I JAK inhibitors remained highly sensitive to CHZ868, demonstrating that type I JAK inhibitor persistence does not confer resistance to type II inhibitors. We next evaluated the efficacy of CHZ868 in murine models of JAK2/MPL-mutant MPN. CHZ868 showed significant activity in conditional knock-in and bone marrow transplant (BMT) models of Jak2V617F-induced polycythemia vera, with normalization of hematocrit, reversal of stem/progenitor expansion, normalization of splenomegaly/splenic architecture, and reversal of bone marrow fibrosis. CHZ868 demonstrated similar activity in the MPLW515L BMT model of MF, with normalization of blood counts, stem/progenitor expansion, spleen weights, and extramedullary hematopoiesis in vivo. Most importantly, CHZ868 resulted in significant reductions of mutant allele burden (mean allele burden reduction 49%) in the Jak2V617F model. We observed analogous reductions in allele burden in the Jak2V617F and MPLW515L BMT models, consistent with disease modifying activity. Taken together, our data demonstrate that a spectrum of type I JAK inhibitors induce JAK inhibitor persistence, by a similar mechanism of JAK2 transactivation as observed with ruxolitinib. By contrast, type II JAK inhibition with CHZ868 remains highly active in JAK inhibitor persistent cells, and shows increased activity in murine MPN models. These data demonstrate that novel JAK inhibitors can increase target inhibition and therapeutic efficacy and should be pursued as an approach to improve outcomes for MPN patients. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures Koppikar: Amgen: Employment. Sellers:Novartis: Employment. Hofmann:Novartis: Employment. Baffert:Novartis: Employment. Gaul:Novartis: Employment. Radimerski:Novartis: Employment. Levine:Novartis: Consultancy, Grant support Other.
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Frede, N., J. Hueppe, R. Lorenzetti, A. Troilo, M. T. Schleyer, R. Voll, J. Thiel, N. Venhoff, and M. Rizzi. "THU0030 DISTINCT EFFECTS OF FIVE JAK INHIBITORS IN THE MODULATION OF HUMAN B CELL ACTIVATION." Annals of the Rheumatic Diseases 79, Suppl 1 (June 2020): 228.2–229. http://dx.doi.org/10.1136/annrheumdis-2020-eular.5763.
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Background:JAK inhibitors have been successfully introduced in the treatment of rheumatoid arthritis (RA) and psoriatic arthritis and are in clinical trials for numerous other autoimmune diseases. JAK inhibition effectively reduces cytokine-mediated activation and survival of pathology-driving immune cells by targeting signaling downstream of cytokine receptors. The outcome of such immunomodulation hence will largely depend on the intrinsic expression of the four different JAKs, the cytokine environment and the targeted cell type. Comparative studies investigating the effect on B cells are lacking. In light of the use of JAK inhibitor treatment in autoantibody mediated diseases, the study of the B cell compartment represents a milestone to assess their potential.Objectives:We thus aimed to study the B cell compartment as well as B cell function under JAK inhibition in RA patients and to compare the specific effect the JAK inhibitors tofacitinib (pan-JAK), baricitinib (JAK1/2), ruxolitinib (JAK1/2), upadacitinib and filgotinib (selective JAK1) on in vitro B cell activation, differentiation, proliferation, and class switch.Methods:B cell subpopulations in RA patients treated with baricitinib or tofacitinib was assessed by flow cytometric analysis of peripheral blood mononuclear cells. For in vitro studies, magnetically isolated total B cells from healthy donors were stimulated T-cell -independently with CpG and treated with scalar doses of the JAK inhibitors tofacitinib, baricitinib, ruxolitinib, upadacitinib and filgotinib. Flow cytometric analysis was performed on days 0, 3 and 6. Cytokine secretion was measured by Cytokine Multiplex Assay.Results:B cell phenotyping of RA patients treated with JAK inhibitors baricitinib or tofacitinib showed an increase in marginal zone (MZ) B cells. To investigate this further, we turned to an in vitro model of T-cell-independent B cell activation with CpG via TLR9, known to support MZ B cell expansion. Here, JAK1/2 and selective JAK1 inhibitor treatment led to a dose-dependent decrease of total B cell numbers. When assessing B cell-subpopulations, we observed an altered B cell differentiation with a significant increase in MZ-like B cells under JAK inhibition, which led to a subsequent increase in plasmablast differentiation in the first days. This effect was more pronounced upon pan-JAK inhibitor treatment than JAK1 or JAK1/2 inhibition, indicating that broader JAK inhibition is associated with a stronger effect (tofa > ruxo > bari > upa > filgo).Notably, we further detected a significant dose-dependent reduction of switched memory formation, strongest with JAK1/2 inhibition (upa > ruxo > bari > tofa > filgo). Consistent with this finding, we observed decreased AID expression under JAK inhibition. Concomitantly, induction of STAT3 expression and STAT3 phosphorylation were reduced under JAK inhibition, suggesting that downstream signalling was abrogated.To assess the role of autocrine signaling in this system, we measured cytokine secretion upon JAK inhibition and found that JAK2 inhibition led to reduced IL10 secretion. This in turn resulted in an increase of inflammatory cytokines such as IL6, TNF, highlighting the importance of B cell as cytokine-secreting cell type.Conclusion:In a T-independent in vitro B cell model JAK inhibition led to a reduced total B cell number as well as reduced switched memory development, whereas MZ-like B cells were increased. Especially JAK2 inhibition strongly impaired switched memory formation. JAK inhibition does not only impact cytokine signalling but also leads to changes in cytokine secretion dynamics and amounts, potentially impacting other cell types.In conclusion, JAK inhibition has a major effect on B cell activation and maturation, with differential outcomes between JAK inhibitors hinting towards distinct and unique effects on B cell homeostasis.Disclosure of Interests:None declared
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Zheng, Ying, Hongwei Qin, Stuart J. Frank, Luqin Deng, David W. Litchfield, Ayalew Tefferi, Animesh Pardanani, et al. "A CK2-dependent mechanism for activation of the JAK-STAT signaling pathway." Blood 118, no. 1 (July 7, 2011): 156–66. http://dx.doi.org/10.1182/blood-2010-01-266320.
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Abstract JAK-STAT signaling is involved in the regulation of cell survival, proliferation, and differentiation. JAK tyrosine kinases can be transiently activated by cytokines or growth factors in normal cells, whereas they become constitutively activated as a result of mutations that affect their function in tumors. Specifically, the JAK2V617F mutation is present in the majority of patients with myeloproliferative disorders (MPDs) and is implicated in the pathogenesis of these diseases. In the present study, we report that the kinase CK2 is a novel interaction partner of JAKs and is essential for JAK-STAT activation. We demonstrate that cytokine-induced activation of JAKs and STATs and the expression of suppressor of cytokine signaling 3 (SOCS-3), a downstream target, are inhibited by CK2 small interfering RNAs or pharmacologic inhibitors. Endogenous CK2 is associated with JAK2 and JAK1 and phosphorylates JAK2 in vitro. To extend these findings, we demonstrate that CK2 interacts with JAK2V617F and that CK2 inhibitors suppress JAK2V617F autophosphorylation and downstream signaling in HEL92.1.7 cells (HEL) and primary cells from polycythemia vera (PV) patients. Furthermore, CK2 inhibitors potently induce apoptosis of HEL cells and PV cells. Our data provide evidence for novel cross-talk between CK2 and JAK-STAT signaling, with implications for therapeutic intervention in JAK2V617F-positive MPDs.
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Suryani, Santi, Keith CS Sia, Lauryn Bracken, Hernan Carol, Kathryn Evans, Raushan Kurmasheva, Peter J. Houghton, Malcolm A. Smith, and Richard B. Lock. "Dual Inhibition of JAK/STAT and MAPK Pathways Results in Synergistic Cell Killing of JAK-Mutated Pediatric Acute Lymphoblastic Leukemia." Blood 120, no. 21 (November 16, 2012): 3562. http://dx.doi.org/10.1182/blood.v120.21.3562.3562.
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Abstract Abstract 3562 Relapsed/refractory pediatric acute lymphoblastic leukemia (ALL) remains a continuing challenge to treat with currently available therapies, and new treatments are urgently required for the management of these high-risk cases. Activating mutations in the pseudokinase or kinase domains of Janus kinases (JAKs) 1, 2 and 3 are present in approximately 10% of high-risk pediatric ALL and are associated with high expression of cytokine receptor-like factor 2 (CRLF2) and poor outcome. These mutations can lead to continuous activation of JAKs, resulting in constitutive activation by phosphorylation of downstream signaling, including the signal transducer and activator of transcription (STAT), AKT, and mitogen-activated protein kinase (MAPK) pathways. The availability of specific JAK inhibitors, developed primarily for the treatment of JAK-mutated myeloproliferative diseases (MPDs), represents an opportunity to improve the treatment options for JAK-mutated pediatric ALL. AZD1480, a potent ATP-competitive small-molecule JAK2 inhibitor that also exhibits inhibitory activity against JAK1, is in solid tumor clinical trials. The purpose of this study was to gain a greater understanding of a potential role for AZD1480 in the treatment of JAK-mutated pediatric ALL either as a single agent or in rational drug combinations, using a preclinical model of xenografts established in immune-deficient mice from direct patient explants. As part of the Pediatric Preclinical Testing Program (PPTP) we previously showed that AZD1480 administered at 10 mg/kg twice daily × 5 then at 15mg/kg once daily × 2 via oral gavage for an intended three weeks significantly delayed the progression of only one in five JAK-mutated xenografts, with no tumor regressions observed. We now show that the relative insensitivity of JAK-mutated ALL xenografts to AZD1480 is a cell-intrinsic phenomenon, since 6/7 JAK1- or JAK2-mutated xenografts exhibited ex vivo IC50 values >2 μM following 72 h drug exposures, as assessed by mitochondrial function cell viability (MTT) assay. In order to gain a greater understanding of the underlying mechanisms for the lack of AZD1480 single-agent efficacy against JAK-mutated xenografts we analyzed intracellular signaling pathways and their responses to AZD1480 treatment. In contrast with “Typical” B-cell precursor (BCP)-ALL xenograft cells, JAK-mutated xenografts exhibited constitutive JAK pathway activation, as assessed by increased levels of phospho-JAK1 (pJAK1), pJAK2, pSTAT1/3/5, pAKT, pMAP2K1/2 (MEK1/2) and phospho-extracellular signal-regulated kinase 1/2 (pERK1/2). Ex vivo exposure of JAK-mutated xenografts to 1 μM AZD1480 caused rapid (within 1 h) and sustained (up to 24 h) decreases in pSTATs, but minimal reduction in pMEK1/2 and pERK1/2. These results indicate that AZD1480 alone selectively inhibits JAK downstream signaling pathways, which may be insufficient to delay leukemia progression in vivo or induce cell death ex vivo. Moreover, they provide a rationale for dual targeting of the JAK and MAPK pathways to elicit synergistic anti-leukemic cell killing in JAK-mutated ALL. Ex vivo exposure of two JAK2-mutated xenografts to 1 μM of the MEK1/2 inhibitor AZD6244 (selumetinib) caused a profound decrease in pERK1/2, and the combination of 1 μM each of AZD1480 and AZD6244 resulted in reductions of both pSTATs and pERK1/2. Moreover, fixed-ratio MTT cytotoxicity assays using these two JAK2-mutated xenografts demonstrated very strong synergy between AZD1480 and AZD6244, with Combination Indices for each xenograft of 0.36 and 0.098 at the ED50; 0.23 and 0.015 at the ED75; and 0.15 and 0.002 at the ED90. This strong synergistic effect was observed despite AZD1480 and AZD6244 exerting minimal cell killing activity against the xenograft cells when used as single agents. In conclusion, our data indicate that AZD1480 is unlikely to exert significant single-agent activity in the treatment of JAK-mutated pediatric ALL, and that future efforts focusing on dual targeting of the JAK/STAT and MAPK offer a potential pathway to achieving clinical efficacy. Disclosures: No relevant conflicts of interest to declare.
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Bhagwat, Neha, Priya Koppikar, Outi Kilpivaara, Taghi Manshouri, Mazhar Adli, Ann Mullally, Omar Abdel-Wahab, et al. "Heterodimeric JAK-STAT Activation As a Mechanism of Persistence to JAK2 Inhibitor Therapy." Blood 118, no. 21 (November 18, 2011): 122. http://dx.doi.org/10.1182/blood.v118.21.122.122.
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Abstract Abstract 122 Although JAK2 inhibitor therapy improves MPN-associated splenomegaly and systemic symptoms, JAK2 inhibitor treatment does not significantly reduce or eliminate the MPN clone in most MPN patients. We therefore sought to characterize mechanisms by which MPN cells can persist despite chronic JAK2 inhibition. We performed saturation mutagenesis followed by next-generation sequencing in JAK2 mutant cells exposed to two different JAK2 inhibitors, INCB18424, a dual JAK1/JAK2 inhibitor, and JAK Inhibitor I, a pan-JAK inhibitor. Although we were able to identify candidate resistance alleles, these alleles were present in less than 50% of the total population. These data and the clinical experience with JAK2 inhibitors suggest that the failure of JAK2 inhibitors to reduce disease burden is not due to acquired drug resistance but rather due to persistent growth and signaling in the setting of chronic JAK2 kinase inhibition. We therefore generated JAK2/MPL mutant JAK2-inhibitor persistent (JAKper) cell lines (SET-2, UKE-1, Ba/F3-MPLW515L). JAKper cell lines are able to survive and proliferate in the presence of JAK2 inhibitors including JAK Inhibitor I, INCB18424 and TG101348 without acquiring second-site resistance alleles and are also insensitive to other JAK inhibitors. Signaling studies revealed JAK-STAT signaling was reactivated in persistent cells at concentrations of inhibitor that completely abrogated signaling in naïve cells, and JAK2 phosphorylation was reactivated in JAK inhibitor persistent cells consistent with reactivation of the JAK-STAT pathway in JAKper cells despite inhibitor exposure. We hypothesized that JAK2 may be activated in trans by other JAK kinases, and found an increased association between activated JAK2 and JAK1/TYK2 consistent with activation of JAK2 in trans by other JAK kinases in JAKper cells. We next assessed whether JAK inhibitor persistence was reversible. Withdrawal of JAK2 inhibitors from JAKper cells for 2 weeks led to resensitization such that JAKper resensitized cells were now sensitive to different JAK2 inhibitors regardless of previous exposure. Resensitization was associated with reversal of heterodimerization and loss of transactivation of JAK2 by JAK1 and TYK2. The reversible nature of JAK inhibitor persistence led us to hypothesize epigenetic alterations are responsible for JAK inhibitor insensitivity in JAKper cells; we observed increased expression of JAK2 at the mRNA and protein level in JAK2 inhibitor persistent cells compared to parental as well as resensitized cells. ChIP-PCR analysis of the JAK2 locus revealed a significant increase in H3K4-trimethylation and a reduction in H3K9 trimethylation in persistent cells compared to parental cells consistent with a change to a more active chromatin state at the JAK2 locus and increased JAK2 mRNA expression in persistent cells. We next assessed whether the same phenomenon of JAK2 inhibitor persistence was observed in vivo. In a MPLW515L-mutant murine bone marrow transplant model of primary myelofibrosis, we observed increased JAK2 expression, increased JAK2 phosphorylation and JAK-inhibitor induced association between JAK1 and JAK2 in hematopoietic cells from INCB18424 treated mice. We next extended our findings to samples from patients treated with INCB18424. We identified 5 patients who had a significant clinical response and 5 patients without a significant clinical response as assessed by spleen size and JAK2V617F allele burden responses and measured JAK2 granulocyte mRNA expression before and during INCB18424 treatment. We found that JAK2 mRNA levels significantly increased in INCB18424 nonresponders compared to responders (p=0.05) suggesting this phenomenon is observed in cell lines, mouse models and primary samples. Finally, we investigated whether JAKper cells remain JAK2 dependent. Studies with shRNA targeting JAK2 and pharmacologic studies using Hsp90 inhibitors that degrade JAK2 protein demonstrate that JAK2 inhibitor persistent cells remain dependent on JAK2 protein expression. Our data indicate that JAK2/MPL mutant cells persist in the presence of JAK2 kinase inhibitors through epigenetic alterations which reactivate signaling in persistent cells, and that therapies which lead to JAK2 degradation can be used to inhibit signaling and improve outcomes in patients with persistent disease despite chronic JAK2 inhibition. Disclosures: Verstovsek: Incyte Corporation: Research Funding.
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Vian, Laura, Mimi Lee, Giuseppe Sciumè, Nathalia Gazaniga, Stefania Dell'Orso, Stephen Brooks, and Massimo Gadina. "Elucidating the role of cytokine signaling in the homeostasis of innate immune cells with JAK inhibitors." Journal of Immunology 202, no. 1_Supplement (May 1, 2019): 181.27. http://dx.doi.org/10.4049/jimmunol.202.supp.181.27.
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Abstract The role of the JAK/STAT pathway in the function on innate lymphoid cells (ILCs) has been mostly investigated using genetically modified animals. While elegant, these studies present with the limitation of a complete loss of ILC populations. Therefore, pharmacological manipulation of this signaling cascade with JAK inhibitors (jakinibs) is an attractive alternative strategy. Here we investigated the effect of pan- and JAK-selective inhibitors on the development and functions of murine, IFN-γ producing, ILCs and iNKT cells after oral administration of tofacitinib (JAK1; JAK3 and JAK2), ruxolitinib (JAK1; JAK2), or PF-06651600 (JAK3). We observed a significant reduction in number and function (IFN-γ production) of both splenic and hepatic NKp46+ cells with either tofacitinib or PF- 006651600. The reduction appears to correlate with the number of proliferating Ki67+ NKp46+ cells. In addition, using an iNKT-dependent mouse model of acute liver inflammation, we observed a significant decreased production of IFN-γ by liver iNKT upon tofacitinib administration. Notably, we found that PF-06651600 selectively impairs the thymic development and the proliferation of IFN-γ-producing iNKT1 cells. Overall our data suggest that the transient inhibitory activity with jakinibs has strong impact on proliferation and activity of mouse IFN- γ producing innate cells. These preliminary findings bring us one step closer to elucidating mechanisms underlying innate cells homeostasis.
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Traves, Paqui G., Bernard Murray, Federico Campigotto, René Galien, Amy Meng, and Julie A. Di Paolo. "JAK selectivity and the implications for clinical inhibition of pharmacodynamic cytokine signalling by filgotinib, upadacitinib, tofacitinib and baricitinib." Annals of the Rheumatic Diseases 80, no. 7 (March 19, 2021): 865–75. http://dx.doi.org/10.1136/annrheumdis-2020-219012.
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ObjectiveJanus kinase inhibitors (JAKinibs) are efficacious in rheumatoid arthritis (RA) with variable reported rates of adverse events, potentially related to differential JAK family member selectivity. Filgotinib was compared with baricitinib, tofacitinib and upadacitinib to elucidate the pharmacological basis underlying its clinical efficacy and safety.MethodsIn vitro JAKinib inhibition of signal transducer and activator of transcription phosphorylation (pSTAT) was measured by flow cytometry in peripheral blood mononuclear cells and whole blood from healthy donors and patients with RA following cytokine stimulation of distinct JAK/STAT pathways. The average daily pSTAT and time above 50% inhibition were calculated at clinical plasma drug exposures in immune cells. The translation of these measures was evaluated in ex vivo-stimulated assays in phase 1 healthy volunteers.ResultsJAKinib potencies depended on cytokine stimulus, pSTAT readout and cell type. JAK1-dependent pathways (interferon (IFN)α/pSTAT5, interleukin (IL)-6/pSTAT1) were among the most potently inhibited by all JAKinibs in healthy and RA blood, with filgotinib exhibiting the greatest selectivity for JAK1 pathways. Filgotinib (200 mg once daily) had calculated average daily target inhibition for IFNα/pSTAT5 and IL-6/pSTAT1 that was equivalent to tofacitinib (5 mg two times per day), upadacitinib (15 mg once daily) and baricitinib (4 mg once daily), with the least average daily inhibition for the JAK2-dependent and JAK3-dependent pathways including IL-2, IL-15, IL-4 (JAK1/JAK3), IFNγ (JAK1/JAK2), granulocyte colony stimulating factor, IL-12, IL-23 (JAK2/tyrosine kinase 2) and granulocyte-macrophage colony-stimulating factor (JAK2/JAK2). Ex vivo pharmacodynamic data from phase 1 healthy volunteers clinically confirmed JAK1 selectivity of filgotinib.ConclusionFilgotinib inhibited JAK1-mediated signalling similarly to other JAKinibs, but with less inhibition of JAK2-dependent and JAK3-dependent pathways, providing a mechanistic rationale for its apparently differentiated efficacy:safety profile.
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Bellucci, Roberto, Allison Martin, Marc Buren, Hong-Nam Nguyen, Davide Bommarito, and Jerome Ritz. "JAK1 and JAK2 Modulate Myeloma Cell Susceptibility to NK Cells Through the Interferon Gamma (IFN-γ) Pathway,." Blood 118, no. 21 (November 18, 2011): 3960. http://dx.doi.org/10.1182/blood.v118.21.3960.3960.
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Abstract Abstract 3960 Multiple myeloma (MM) is a B cell neoplasm characterized by clonal expansion of malignant plasma cells in the bone marrow. Despite the use of new drugs such as lenalidomide and bortezomib, MM remains an incurable disease. Successful treatment of MM with allogeneic stem cell transplantation suggests that MM is susceptible to immunologic approaches. NK cells are the primary effectors of the innate immune response against infectious pathogens and malignant transformation. Unlike T and B cells, NK cells do not recognize antigens in the context of classical major histocompatibility complex (MHC) but lyse target cells without specific antigen recognition. Nevertheless, MM cells have developed mechanisms to evade innate immune surveillance and the molecular basis for target resistance to NK cell-mediated lysis is not well understood. To identify novel pathways that modulate MM cell resistance to the immune system, we previously developed a genetic screen to detect cell-cell interactions using a large lentiviral shRNA library containing a total of 6,144 shRNAs targeting more than 1,000 human genes. Using this approach we found that silencing JAK1 and JAK2 results in significantly increased MM cell susceptibility to NK cell lysis. This effect was not noted when JAK3 and TYK2 were targeted. JAK1, JAK2 JAK3 and TYK2 are members of a family of tyrosine kinases that are constitutively associated with many membrane cytokine receptors. After activation, JAK proteins regulate phosphorylation/activation of STAT proteins, which subsequently initiate gene transcription. To understand JAK1 and JAK2 involvement in MM resistance to NK cells, we undertook a series of experiments to analyze the JAK signaling pathway in MM cells. We first analyzed the activation status of STAT proteins in a series of MM cell lines (IM-9, KM12BM, RPMI 8226, U266) in which JAK1 and JAK2 expression was reduced by specific shRNAs. Constitutive activation of STAT proteins was not affected by JAK1 or JAK2 gene silencing suggesting that these kinases were not activated in the absence of cytokine receptor-mediated signaling. Since JAK1 and JAK2 are associated with the IFN-γ receptor and we previously showed that JAK1 and JAK2 silencing induces increased secretion of IFN-γ from NK cells, we pre incubated MM cell lines with NK activated supernatant or recombinant IFN-γ and tested them for STAT activation. 15 min incubation was sufficient to initiate phosphorylation of STAT1 but no other STATs were activated. Silencing of JAK1 or JAK2 with specific shRNAs prevented STAT1 activation. To validate this finding, we tested primary MM cells treated with different concentrations of Jak inhibitor 1 (0 nM, 10 nM, 30 nM and 40 nM). These cells had a similar STAT profile at their basal level when compared with the previously tested MM cell lines. Pre-incubation with NK activated supernatant or IFN-γ also induced rapid activation of STAT1, which was completely inhibited when cells were pre-treated with Jak inhibitor 1. Treatment of MM cells with 10, 30 and 40 nM of Jak inhibitor enhanced killing by NK cells by 46.6%, 51% and 53%, compared to untreated cells (p=0.0036, p=0.0011 and p=0.0010 respectively). These findings demonstrate that IFN-γ signals rapidly enhance resistance of MM cells to NK cells but inhibition of this pathway at the level of JAK1 and JAK2 reverses this effect and induces susceptibility to NK cell mediated lysis. Disclosures: No relevant conflicts of interest to declare.
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Miguel González, J. A., P. Pavlidis, and U. M. Marigorta. "P105 Mendelian Randomization screening for JAK inhibitor related potential serious adverse events." Journal of Crohn's and Colitis 18, Supplement_1 (January 1, 2024): i388. http://dx.doi.org/10.1093/ecco-jcc/jjad212.0235.
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Abstract Background JAK inhibitors (JAKi) suppress the JAK/STAT signalling pathway through inhibition of the four Janus kinase proteins JAK1, JAK2, JAK3 and TYK2. JAKi are licensed for the treatment of a wide range of immune mediated inflammatory diseases (IMIDs) including inflammatory bowel disease. Concerns have been raised for the potential of this class of medication to associate with serious adverse events, particularly in those older than 65 years of age or with previous cardiovascular comorbidities. Mendelian randomization (MR) uses genetic variants as instrumental variables (IVs) to examine the causal effects of an exposure (e.g. inhibition of a drug target) on an outcome (e.g. the risk of developing a disease and/or potential adverse effects due to alteration of gene activity). We carried out a comprehensive MR evaluation to explore the therapeutic potential of JAKi for a range of IMIDs, and to study the unintended drug effects that might be caused by this therapeutic strategy. Methods The basis of MR relies on the use of genetic variants as IVs that are reliably related to the risk factor and that are not susceptible of reverse causation and confounding. We used genetic variants (SNPs) associated with protein levels and gene expression (pQTLs and eQTLs, respectively) as IVs to study the effects of inhibiting the genes in the JAK/STAT signalling pathway. The underlying assumption is that these IVs serve as proxies of the effects of JAKi. As a source of the effect estimates on the selected outcomes, we used summary statistics from a compiled list of genome-wide association studies. Results We used the MR approach to chart the range of possible outcomes associated with JAKi treatment. First, we explored the therapeutic potential of JAKi for IMIDs. Second, we evaluated a range of potential adverse events associated with JAKi, focusing particularly on cardiovascular, infectious and cancer-related malignancies. Our MR results confirm a general tendency towards JAK/STAT signalling pathway involvement in IMIDs risk, implying the suitability of JAKi for IMIDs. Regarding potential adverse outcomes, we carried out stratification-based analyses to evaluate whether potential adverse events are enhanced in individuals at particular high risk. The MR analyses highlighted potential risk of JAKi for non-IMIDs traits, particularly when targeting JAK2. This includes already known associations with cardiovascular disease and specific cancer types. Conclusion We performed an outcome-wide exploration based on MR to characterize the therapeutic potential and range of adverse effects expected for JAKi. We recapitulate the known potential of these drugs to treat IMIDs, while uncovering specific severe adverse effects associated with this therapeutic target.
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Hsu, Leeyen, and April W. Armstrong. "JAK Inhibitors: Treatment Efficacy and Safety Profile in Patients with Psoriasis." Journal of Immunology Research 2014 (2014): 1–7. http://dx.doi.org/10.1155/2014/283617.
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Janus kinase (JAK) pathways are key mediators in the immunopathogenesis of psoriasis. Psoriasis treatment has evolved with the advent of targeted therapies, which inhibit specific components of the psoriasis proinflammatory cascade. JAK inhibitors have been studied in early phase trials for psoriasis patients, and the data are promising for these agents as potential treatment options. Tofacitinib, an oral or topically administered JAK1 and JAK3 inhibitor, and ruxolitinib, a topical JAK1 and JAK2 inhibitor, have been most extensively studied in psoriasis, and both improved clinical symptoms of psoriasis. Additional JAK1 or JAK3 inhibitors are being studied in clinical trials. In phase III trials for rheumatoid arthritis, tofacitinib was efficacious in patients with inadequate responses to tumor necrosis factor inhibitors, methotrexate monotherapy, or disease-modifying antirheumatic drugs. The results of phase III trials are pending for these therapies in psoriasis, and these agents may represent important alternatives for patients with inadequate responses to currently available agents. Further investigations with long-term clinical trials are necessary to verify their utility in psoriasis treatment and assess their safety in this patient population.
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Sinclair, Angus, Ivonne Archibeque, Jinghui Zhan, Liqin Liu, Renee Emkey, Elizabeth Doherty, and C. Glenn Begley. "Potency and Selectivity Assessment of Small Molecules Against Janus Kinase (JAK) 2: Widely Used AG490 Inhibitor Is Neither Potent Nor Selective for JAK2." Blood 118, no. 21 (November 18, 2011): 4780. http://dx.doi.org/10.1182/blood.v118.21.4780.4780.
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Abstract Abstract 4780 Janus kinases (JAK) are the most proximal signaling components of multiple cytokine receptors and have four family members JAK1, JAK2, JAK3 and TYK2. JAK2 is essential for the development of normal erythroid and myeloid lineages by mediating signaling though the erythropoietin receptor (EPOR), thrombopoietin receptor (TPOR) and the β-common chain of GM-CSF, IL-3 and IL-5. Recently, JAK2 has been the focus of considerable research due to the discovery that patients with myeloproliferative disorders (MPDs) essential thrombocythemia, polycythemia vera and myelofibrosis contain somatically derived inactivating mutations in the JAK2 pseudokinase repressor domain. The deregulated expansion of erythro/myeloid cells in MPDs is thought to be due to the sustained signaling though JAK2 and downstream STAT, PI3K and MAPK signaling pathways to enhance the proliferation, survival and differentiation of progenitor cells. As a consequence, the discovery and development of small molecule inhibitors for JAK2 has been a focus for potential therapeutic intervention and has provided tools to examine cytokine networks. In order to discover small molecule JAK2 inhibitors we evaluated a number of benchmark and commercially available inhibitors as well as new inhibitors we generated. Tyrphostin AG490 has been widely used in the literature as a “JAK2” inhibitor in EPOR signaling and MPD research. However, AG490 has also been reported as an inhibitor of JAK3, EGFR, HER2, guanylyl cyclase C and BCR-ABL. In JAK2 enzyme assays, our new JAK inhibitors AMG-Jak2-02 and AMG-Jak2-03 were found to have transit IC50 <0.005 μM. However, AG490 was considered inactive in JAK2 enzyme assays with an IC50 >125 μM. When profiled against other JAK family members in enzyme assays, AG490 was also considered inactive on JAK1 (IC50 >125 μM), JAK3 (IC50 >80 μM) and TYK2 (IC50 >80 μM) whereas the IC50 of AMG-Jak2-02 and AMG-Jak2-03 were between 0.02 – 1.1 μM in JAK enzyme assays. Due to the lack of inhibitory activity of AG490 in JAK family enzyme assays, we performed a broader kinase screen on AG490. AG490 was profiled at 1 μM against 48 kinases. To our surprise, it was inactive on all kinases tested (most potent was SGK1 with ∼20% inhibition). In a broader binding screen of 441 lipid and protein kinases, 25 μM of AG490 was considered active on 4 kinases: STK17A, STK17B, PDGFRA and PDGFRB with a >70% inhibition. AG490 was inactive on all JAK family members. To investigate the potential for AG490 to inhibit JAK2 in a cellular context we examined the phosphorylation (p) of downstream molecules STAT5, AKT and ERK1/2 in an EPO dependent cell line UT-7/EPO. UT-7/EPO cells were incubated with AG490 (dose range up to 100 μM) or other JAK inhibitors (doses up to 33 or 100 μM) and phosphorylation of downstream molecules was assessed using Western immunoblot analysis. The EPO-EPOR induced downstream generation of pSTAT5, pAKT and pERK1/2 was suppressed by pan-JAK inhibitor I (Calbiochem) with an IC50 ∼ 0.1 μM, by AMG-Jak2-02 with an IC50 ∼ 10 μM and by AMG-Jak2-03 with an IC50 ∼ 0.1 μM. However, AG490 at 100 μM was unable to suppress the EPO-EPOR induced generation of pERK1/2 or pAKT but had modest effects on suppressing the generation of pSTAT5 (IC50 between 50–100 μM). We also investigated the potential for AG490 to inhibit the viability of JAK2 dependent (UT-7/EPO) and JAK2-independent (γ2A JAK2 null) cells. Pan JAK inhibitor I (Calbiochem), AMG-Jak2-01 and AMG-Jak2-02 were > 10 fold more potent at reducing JAK2 dependent cell viability (UT-7/EPO cells) compared with the viability of JAK2 independent cell line (γ2A cells). However, AG490 was found to be equipotent at inhibiting the viability of JAK2 dependent and independent cell lines. Similar results were obtained when these studies were repeated multiple times using multiple lots of compound (confirmed to be structurally correct based on NMR analysis). Taken together, we have identified that the widely used “JAK2” inhibitor AG490 is neither potent nor selective for JAK2. Thus, published data generated with AG490 should be interpreted with caution. Careful validation of JAK2 compounds for future research and assay development purposes should be taken into consideration. Disclosures: Sinclair: Amgen: Employment, Stock and Options. Archibeque:Amgen: Employment, Stock and Options. Zhan:Amgen, Inc: Employment, Stock and Options. Liu:Amgen, Inc: Employment, Stock and Options. Emkey:Amgen, Inc: Employment, Stock and Options. Doherty:Amgen, Inc: Employment, Stock and Options. Begley:Amgen, Inc: Employment, Stock and Options.
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Ávila-Mendoza, José, Karen Delgado-Rueda, Valeria A. Urban-Sosa, Martha Carranza, Maricela Luna, Carlos G. Martínez-Moreno, and Carlos Arámburo. "KLF13 Regulates the Activity of the GH-Induced JAK/STAT Signaling by Targeting Genes Involved in the Pathway." International Journal of Molecular Sciences 24, no. 13 (July 7, 2023): 11187. http://dx.doi.org/10.3390/ijms241311187.
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The Krüppel-like factor 13 (KLF13) has emerged as an important transcription factor involved in essential processes of the central nervous system (CNS). It predominantly functions as a transcriptional repressor, impacting the activity of several signaling pathways with essential roles in the CNS, including the JAK/STAT pathway, which is the canonical mediator of growth hormone (GH) signaling. It is now recognized that GH has important actions as a neurotrophic factor. Therefore, we analyzed the effects of KLF13 on the activity of the JAK/STAT signaling pathway in the hippocampus-derived cell line HT22. Results showed that KLF13 directly regulates the expression of several genes involved in the JAK-STAT pathway, including Jak1, Jak2, Jak3, and Socs1, by associating with their proximal gene promoters. In addition, it was found that in KLF13-deficient HT22 neurons, the expression of Jak1, Stat3, Socs1, Socs3, and Igf1 was dysregulated, exhibiting mRNA levels that went up to 7-fold higher than the control cell line. KLF13 displayed a differential effect on the GH-induced JAK/STAT pathway activity, decreasing the STAT3 branch while enhancing the STAT5 branch. In KLF13-deficient HT22 cells, the activity of the STAT3 branch was enhanced, mediating the GH-dependent augmented expression of the JAK/STAT output genes Socs1, Socs3, Igf1, and Bdnf. Furthermore, GH treatment increased both the nuclear content of KLF13 and Klf13 mRNA levels, suggesting that KLF13 could be part of the mechanisms that maintain the homeostatic state of this pathway. These findings support the notion that KLF13 is a regulator of JAK/STAT activity.
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Ki, S. Y., H. Shin, Y. Lee, H. R. Bak, H. Yu, S. C. Kim, J. Lee, D. Kim, D. H. Ko, and D. Kim. "AB0095 PRECLINICAL CHARACTERIZATION OF CJ-15314, A HIGHLY SELECTIVE JAK1 INHIBITOR, FOR THE TREATMENT OF AUTOIMMUNE DISEASES." Annals of the Rheumatic Diseases 79, Suppl 1 (June 2020): 1347.2–1347. http://dx.doi.org/10.1136/annrheumdis-2020-eular.650.
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Background:Janus kinases (JAK1, JAK2, JAK3, and TYK2) play critical roles in mediating various cytokine signaling, and has been developed as a target for autoimmune diseases such as RA. Tofacitinib, oral Pan-JAK inhibitor, demonstrated efficacy in RA patients, but its widespread use is limited by safety issues. Baricitinib, JAK1/2 inhibitor, is also known to interfere with the hematopoiesis system, such as anemia and thrombocytopenia associated with suppression of JAK2 signals. Therefore, it is necessary to develop a new potent compound that selectively inhibits JAK1 over JAK2, 3Objectives:To identify the pharmacological characteristic based on efficacy of CJ-15314 as potent and selective JAK1 inhibitor for treatment of autoimmune disease.Methods:In vitro, cell-based, kinase panel, Kd value and human whole blood assay were performed to determine the inhibition potency and selectivity for JAK subfamily kinases. In vivo therapeutic potential was evaluated by RA model including rat Adjuvant-Induced Arthritis (AIA) and collagen-induced arthritic (CIA). To confirm the possibility of further expansion into the autoimmune disease, BioMAP® Diversity PLUS® Panel was performed by discoverX.Results:In vitro assay, CJ-15314 inhibited JAK kinase family in a concentration-dependent manner with IC50 values of 3.8 nM against JAK1, Selectivity for JAK1 over JAK2, 3 was approximately 18, 83 fold greater for CJ-15314. In 1mM ATP condition, CJ-15314 has been confirmed to have the highest JAK1 selectivity over competing drugs, under 1 mM ATP condition that reflects the physiological environment in the body. Similarly, Kd values has also confirmed the selectivity of JAK1, which is 10 fold higher than JAK2, 3. Accordingly, in human whole blood assays, CJ-15314 is 11 fold more potent against IL-6 induced pSTAT1 inhibition through JAK1 (IC50 value: 70 nM) than GM-CSF-induced pSTAT5 inhibition (JAK2) whereas baricitinib and filgotinib exhibited only 2 fold and 7 fold respectively.In vivo efficacy model, CJ-15314 inhibited disease severity scores in a dose dependent manner. In the rat AIA model, CJ-15314 at 30 mg/kg dose showed 95.3% decrease in arthritis activity score, 51.2% in figotinib at 30 mg/kg, 97.7% showed baricitinib at 10 mg/kg. CJ-15314 showed superior anti-arthritic efficacy than filgotinib. CJ-15314 also minimally affected anemia-related parameters but not bricitinib end of the 2-week treatment. In the rat CIA model, like 10 mg/kg of bricitinib, 30 mg/kg of CJ-15314 also has a similar effect, with a significant reduction in histopathological scores.In biomap diversity panel, CJ-15314 inhibited the expression of genes such as MCP-1, VCAM-1, IP-10, IL-8, IL-1, sTNF-α and HLA-DR confirming the possibility of expansion into other diseases beyond arthritis.Conclusion:CJ-15314 is a highly selective JAK1 inhibitor, demonstrates robust efficacy in RA animal model and is good candidate for further development for inflammatory diseases.* CJ-15314 is currently conducting a phase I trial in south Korea.References:[1]Clark JD et al. Discovery and development of Janus kinase (JAK) inhibitors for inflammatory diseases. J Med Chem. 2014; 57(12):5023-38.[2]Burmester GR et al. Emerging cell and cytokine targets in rheumatoid arthritis. Nat Rev Rheumatol. 2014; 10(2):77-88[3]Jean-Baptiste Telliez et al. Discovery of a JAK3-selective inhibitor: functional differentiation of JAK3-selective inhibition over pan-JAK or JAK1-selective inhibition. ACS Chem. Biol., 2016; 11 (12):3442-3451Disclosure of Interests:so young Ki Employee of: CJ healthcare, hyunwoo shin Employee of: CJ healthcare, yelim lee Employee of: CJ healthcare, Hyoung rok Bak Employee of: CJ healthcare, hana yu Employee of: CJ healthcare, Seung Chan Kim Employee of: CJ healthcare, juhyun lee Employee of: CJ healthcare, donghyun kim Employee of: CJ healthcare, Dong-hyun Ko Employee of: CJ Healthcare, dongkyu kim Employee of: CJ healthcare
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Clarke, A., J. Di Paolo, B. Downie, A. Meng, N. Mollova, Y. Yu, and P. Han. "P460 Evaluation of potential mechanisms underlying the safety observations of filgotinib in clinical studies in rheumatoid arthritis." Journal of Crohn's and Colitis 14, Supplement_1 (January 2020): S409. http://dx.doi.org/10.1093/ecco-jcc/jjz203.589.
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Abstract Background Inhibitors of the Janus kinase-signal transducers and activators of transcription (JAK-STAT) pathway have demonstrated efficacy in the treatment of rheumatoid arthritis (RA) and inflammatory bowel disease (IBD). Differences in selectivity of JAK inhibitors for JAK1, JAK2, JAK3 and TYK2 may influence their respective safety profiles, and the mechanisms responsible are not currently known. Filgotinib (FIL), a JAK1 inhibitor, did not negatively impact haemoglobin, LDL:HDL ratios or natural killer (NK) cell counts in clinical trials. Here, we compare the in vitro mechanistic profiles of four JAK inhibitors at clinically relevant doses. Methods JAK inhibitors (FIL, FIL metabolite [GS-829845], baricitinib [BARI], tofacitinib [TOFA], and upadacitinib [UPA]) were evaluated in vitro in human-cell-based assays. Growth of erythroid progenitors from human cord blood CD34+ cells was assessed using a HemaTox™ liquid expansion assay, NK cell proliferation was induced by IL-15 and LXR agonist-induced cholesteryl ester transfer protein (CETP) expression was assessed in the hepatic cell line, HepG2. Using assay-generated IC50 values and the reported human plasma concentrations from clinical studies, we calculated the target coverage for each JAK inhibitor at clinically relevant doses. The activity of FIL in humans was based on PK/PD modelling of FIL + GS-829845. Results Inhibition of cellular activity was calculated for each JAK inhibitor based on in vitro dose-response data, human exposure data and modelled PK/PD relationships. At clinically relevant doses, FIL resulted in lower calculated inhibition of NK cell proliferation compared with other JAK inhibitors. FIL 100 mg and 200 mg also reduced CETP expression, whereas other JAK inhibitors had no effect. There was no difference in the effect of FIL vs. other JAK inhibitors on erythroid progenitor cell differentiation or maturation. Conclusion FIL, a JAK1 inhibitor, resulted in less inhibition of NK cell proliferation compared with BARI, TOFA, and UPA. FIL also reduced LXR agonist-induced CETP expression, while the other inhibitors did not alter these levels. These results provide a potential mechanistic link between the observed reduction of CETP concentration following FIL treatment and the previously observed reduction in the LDL:HDL ratio in RA patients.
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Gorre, M., I. Jilani, H. Kantarjian, F. Giles, A. Hannah, and M. Albitar. "Novel Quantitative Flow Cytometry-Based Signaling Assays Reveal a Potential Role for HSP90 Inhibitors in the Treatment of JAK2 Mutant-Positive Diseases." Blood 106, no. 11 (November 16, 2005): 3526. http://dx.doi.org/10.1182/blood.v106.11.3526.3526.
Full textAbstract:
Abstract The V617F mutation in the JAK2 tyrosine kinase, recently described in a majority of patients with myeloproliferative disorders (MPDs), confers growth factor independence in vitro and oncogenicity in mice. Therefore, targeted inhibition of mutant JAK2 kinase activity may be an effective strategy for treatment of MPD patients with this mutation. The ability to measure the activation status of JAK2 in patient samples will thus be of substantial value for monitoring therapeutic efficacy. We have developed quantitative flow cytometry-based assays for rapid and reproducible measurement of intracellular total and phosphorylated proteins of the canonical JAK/STAT pathway, as well as heat shock proteins (HSPs). In this study we examined the ability of these assays to detect altered levels of total and phosphorylated JAK/STAT signaling pathway components and HSP in a cell line (HEL) that is homozygous for the V617F JAK2 mutant. HEL cell cultures were incubated with 17AAG, a geldanamycin analog with clinical utility in a broad range of diseases. 17AAG exerts its inhibitory effect by binding to heat shock protein 90 (HSP90), preventing its chaperone association with client oncoproteins. AKT is among these client proteins and a component of the JAK/STAT pathway, representing a potential therapeutic target. 17AAG exposure reduced total AKT protein levels by 42%. 17AAG also inhibited mutant JAK2 activity by 66% and had a smaller effect (17%) on total JAK2 levels, suggesting that mutant JAK2 activation may rely on HSP90, either directly or through dependence on other client proteins. Exposure to 17AAG also reduced levels of P-STAT5 (50%) and, to a lesser extent, total STAT5 (27%). 17AAG-treated cells showed a 55% reduction in HSP90 levels and a 14% increase in HSP70 protein levels. JAK Inhibitor I (Calbiochem), a potent pan-JAK Inhibitor that blocks JAK1, JAK2, and JAK3 activity, caused reductions in P-JAK2 and P-STAT5 levels (29% and 26% decreases, respectively). However, the combining of JAK Inhibitor I with 17AAG did not result in an enhanced effect beyond what was observed with 17AAG treatment alone. Similar results were seen with AG490, a potent and selective JAK2 inhibitor. 17AAG caused a 40% decrease in viable cells after 18 hrs of treatment, compared with a 35% reduction for the pan-JAK inhibitor and a 20% decrease for AG490. Combining 17AAG with the pan-JAK inhibitor or AG490 caused only minor enhancement of these cytotoxic effects (46% and 41% reduction in cell viability, respectively). Our data support the potential utility of HSP90 inhibitors such as 17AAG in the development of small-molecule therapy for mutant JAK2 kinase-positive MPD. These results also show that flow cytometry-based assays for JAK/STAT signaling components and HSPs can be used to quantitatively monitor drug efficacy at the protein level in intact cells. These tests are likely to have broad clinical utility given the spectrum of diseases in which a pathogenic role for mutant JAK2 kinase is implicated.