Journal articles: 'JAK1-JAK2 inhibitors'

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Zhang, Yun, Ruifang Liang, Chih-Wei Chen, Tatjana Mallano, Clara Dees, Alfiya Distler, Adam Reich, et al. "JAK1-dependent transphosphorylation of JAK2 limits the antifibrotic effects of selective JAK2 inhibitors on long-term treatment." Annals of the Rheumatic Diseases 76, no. 8 (May 6, 2017): 1467–75. http://dx.doi.org/10.1136/annrheumdis-2016-210911.

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ObjectivesJanus kinase 2 (JAK2) has recently been described as a novel downstream mediator of the pro-fibrotic effects of transforming growth factor-β. Although JAK2 inhibitors are in clinical use for myelodysplastic syndromes, patients often rapidly develop resistance. Tumour cells can escape the therapeutic effects of selective JAK2 inhibitors by mutation-independent transactivation of JAK2 by JAK1. Here, we used selective JAK2 inhibition as a model to test the hypothesis that chronic treatment may provoke resistance by facilitating non-physiological signalling pathways in fibroblasts.MethodsThe antifibrotic effects of long-term treatment with selective JAK2 inhibitors and reactivation of JAK2 signalling by JAK1-dependent transphosphorylation was analysed in cultured fibroblasts and experimental dermal and pulmonary fibrosis. Combined JAK1/JAK2 inhibition and co-treatment with an HSP90 inhibitor were evaluated as strategies to overcome resistance.ResultsThe antifibrotic effects of selective JAK2 inhibitors on fibroblasts decreased with prolonged treatment as JAK2 signalling was reactivated by JAK1-dependent transphosphorylation of JAK2. This reactivation could be prevented by HSP90 inhibition, which destabilised JAK2 protein, or with combined JAK1/JAK2 inhibitors. Treatment with combined JAK1/JAK2 inhibitors or with JAK2 inhibitors in combination with HSP90 inhibitors was more effective than monotherapy with JAK2 inhibitors in bleomycin-induced pulmonary fibrosis and in adTBR-induced dermal fibrosis.ConclusionFibroblasts can develop resistance to chronic treatment with JAK2 inhibitors by induction of non-physiological JAK1-dependent transactivation of JAK2 and that inhibition of this compensatory signalling pathway, for example, by co-inhibition of JAK1 or HSP90 is important to maintain the antifibrotic effects of JAK2 inhibition with long-term treatment.

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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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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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Jarocha, Danuta Jadwiga, Paul Gadue, Wei Tong, Robert C. Newton, and Mortimer Poncz. "Janus Kinase (Jak) 1 Inhibition Affects Both Megakaryopoiesis and Thrombopoiesis." Blood 132, Supplement 1 (November 29, 2018): 2559. http://dx.doi.org/10.1182/blood-2018-99-115407.

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Abstract JAK inhibitors are being developed to treat inflammatory, myeloproliferative and neoplastic disorders. Murine and human studies have demonstrated an essential role for JAK2 in the proliferation of hematopoietic stem/progenitor cells (HSPC) and multiple hematopoietic lineages, including erythrocytes and megakaryocytes, while Jak1 murine studies have shown a role in HSPC proliferation and myelopoiesis, but not in megakaryopoiesis. Patients enrolled in clinical studies of INCB052793, which selectively binds to JAK1, have shown thrombocytopenia occurring within 2 weeks. The aim of this study was to elucidate the basis for thrombocytopenia associated with this JAK1 inhibitor, in comparison to INCB026115, which inhibits JAK2 more so than Jak1 (Jak2/1). Knowing the precise mechanism by which Jak inhibitors induce thrombocytopenia may lead to therapeutic strategies limiting side effects, while preserving intended clinical application. We tested a broad concentration range of each of these Jak1 and Jak2/1 inhibitors from IC50 (40 and 30nM, respectively) to IC90 (400 and 300nM) to 10xIC90 (4 and 3 µM) on mobilized progenitor-derived CD34+ cells incubated 12-14 days under semisolid and under liquid conditions, focusing on effects on megakaryocyte (Meg) and platelet production. At IC90, the Jak1-selective inhibitor limited large Meg colony number to 47±8% of untreated control in semisolid growth conditions. Under similar concentrations in liquid growth conditions, the number of Megs seen was 45±8% of the untreated controls, but with a 139±17% higher level of ≥8N Megs. Agonist response of mature Megs to thrombin was not compromised. Total number of healthy, in vitro-released, platelet-like particles (PLPs) collected from Jak1-exposed cultures at Day 12 was reduced to 57±14% of the control, and similar to the decrease in Meg yield. At a similar level of inhibition, the Jak2/1 inhibitor was more robust at inhibiting megakaryopoiesis. At IC90, the Jak2/1 inhibitor fully inhibited development of large Meg colonies and reduced the number of small colonies to 43±14% of untreated control. Under liquid growth conditions, the number of Megs seen at Day 12 was 20±9% of the untreated controls, but with 132±28% higher % of ≥8N Megs. Agonist response of mature Megs was not compromised. Total number of healthy PLPs collected at Day 12 was insignificantly different despite much lower Meg yield. More detailed Jak2/1 inhibitor cultures analysis revealed enhanced Meg apoptosis by 209±61% at Day 7, and accelerated maturation as indicated by a 2-fold and 3-fold mpl receptor level at Days 7 and 11 and 321±217% higher number of Megs >2N at Day 7. As opposite to what might be expected, thrombopoiesis appeared not to be impaired by the Jak2/1 inhibitor. Inhibitor-treated Megs released similar or higher number of platelets per Meg as untreated controls upon their infusion into immunocompromized NSG mice, with similar high levels of young, thiazole orange-positive, low apoptotic, Annexin-V+ platelets. Baseline released platelet CD62p expression and PAC1 binding prior to agonist exposure were similar and increased to the same extent after thrombin (0.1-10U/ml) stimulation. In contrast, Jak1 inhibitor-treated Megs had ~50% lower number of released human platelets upon infusion into NSG mice although the released platelets were healthy and responsive to agonists. In summary, our results shed significant insight into the mechanisms of Jak1 inhibitor-associated thrombocytopenia observed in patients. We show that thrombocytopenia post the Jak2/1 inhibitor INCB026115 is due to impaired megakaryopoiesis with intact thrombopoiesis and functional, released platelets. In contrast, thrombocytopenia post the Jak1 inhibitor INCB052793 is a result of combined impairment of both megakaryopoiesis and thrombopoiesis, although the released platelets appear intact. The exact pathways blocked by the Jak1 inhibitor important for thrombopoiesis remain to be defined. Also, as liver hepatocytes together with bone marrow stromal cells are a source of thrombopoietin (TPO), and Jak1 and Jak2 are known to be involved in regulation of TPO production, studies to check the influence of Jak inhibitors on TPO production from both hepatocytes and marrow stromal cells are needed to fully understand the influence of Jak inhibitors on megakaryopoiesis/thrombopoiesis. Disclosures Jarocha: Incyte Corporation: Consultancy, Research Funding. Gadue:Incyte Corporation: Consultancy, Research Funding. Tong:Incyte Corporation: Consultancy, Research Funding. Newton:Incyte Research Institute: Employment, Equity Ownership. Poncz:Incyte Corporation: Consultancy, Research Funding.

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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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Kopp, Nadja, Jordy C. Van der Zwet, Jacob Layer, Oliver Weigert, Eric Vangrevelinghe, Akinori Yoda, Thomas Radimerski, and David Weinstock. "JAK2 L884P Mutation Confers Resistance To The Type II JAK2 Inhibitor NVP-BBT594 When Co-Occurring With JAK2 R683G But Not JAK2 V617F." Blood 122, no. 21 (November 15, 2013): 1429. http://dx.doi.org/10.1182/blood.v122.21.1429.1429.

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Abstract Approximately 50% of myeloproliferative neoplasms (MPNs) harbor the JAK2 V617F mutation while approximately 50% of B-cell acute lymphoblastic leukemias (B-ALLs) with CRLF2 rearrangements harbor JAK2 exon 16 mutations that primarily involve R683. Multiple enzymatic inhibitors of JAK2 are in clinical development for the treatment of patients with malignant and nonmalignant conditions that depend on constitutive JAK2 signaling. Most of these drugs are ATP-mimetics that block JAK2 signaling in the active conformation (so-called “type I JAK2 inhibitors”). Resistance to type I JAK2 inhibitors can occur through heterodimerization between activated JAK2 and either JAK1 or TYK2 (Koppikar et al. Nature 2012). In addition, E864K, Y931C, and G935R mutations in the kinase domain of JAK2 (JH1 domain) confer resistance to a panel of type I JAK2 inhibitors (including ruxolitinib, tofacitinib, TG101348, JAK inhibitor I) without drastically affecting signaling by JAK2 (Weigert et al. J Exp Med 2012). Resistance caused by these mutations is independent of whether in the context of CRLF2 with JAK2 R683G or EPOR with JAK2 V617F (Weigert et al. J Exp Med 2012). In contrast to type I inhibitors, type II JAK2 inhibitors bind to and stabilize the inactive confirmation of JAK2 and prevent the activation loop from being phosphorylated. Thus, transphosphorylation of JAK2 by JAK1 or TYK2 does not confer resistance to the type II JAK2 inhibitor NVP-BBT594 (BBT594) (Koppikar et al. Nature 2012). In this study we report the first evidence that mutation of JAK2 can also confer resistance to type II Jak2 inhibitors. BBT594 had similar potency to the type I JAK2 inhibitor NVP-BVB808 (BVB808) in murine lymphoblast BaF3 cells dependent on CRLF2 with JAK2 R683G (IC50 8.5nM vs 15.7nM) or EPOR with JAK2 V617F (IC50 29nM vs 10nM). In contrast, the Y931C mutation conferred >3-fold resistance to BVB808 in BaF3 cells expressing CRLF2 with JAK2 R683G but no significant change in sensitivity to BBT594. Thus, type II JAK2 inhibitors can overcome genetic resistance to type I JAK2 inhibitors. We performed a random mutagenesis screen of JAK2 R683G and expressed the mutagenized library in BaF3 cells that express CRLF2. Selecting in the presence of 3uM BBT594 resulted in a large number of clones, of which all screened (n>30) harbored the same JAK2 L884P mutation. Structural modeling of this mutation predicted changes in the JH1 domain that may impact the conformation of the P-loop and helix C, and thereby compromise the sub-pocket required for type II inhibitor binding. In contrast to mutations that confer resistance to type I JAK2 inhibitors, the L884P mutation only conferred resistance to BBT594 in the context of CRLF2/JAK2 R683G (IC50 504nM versus 8.5nM for R683G alone) and not EPOR/JAK2 V617F. To our knowledge, this is the first mechanism of resistance specific to JAK2 R683G. BaF3 cells expressing CRLF2 with JAK2 R683G L884P Y931C remained resistant to BBT594. Transduction of the mutagenized JAK2 R683G library into BaF3 cells expressing CRLF2 followed by selection in both BVB808 and BBT594 did not yield any resistant colonies. In conclusion, mutations that affect the binding of type I JAK2 inhibitors do not affect the potency of the type II JAK2 inhibitor BBT594. The L884P mutation confers resistance to BBT594 when co-occurring with the activating mutation R683G but not with V617F. Thus, combinations of multiple JAK2 inhibitors with distinct mechanisms may be useful in overcoming de novo and acquired resistance to JAK2 inhibitors. Disclosures: Vangrevelinghe: Novartis: Employment. Radimerski:Novartis: Employment. Weinstock:Novartis: Consultancy, Research Funding.

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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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Xue, Chengfeng, Jingjing Wang, Na Xu, Yaqiong Pei, Donghai Chen, Jiaping Sun, Qingyang Gu, and Qiyao Zhang. "Comparative assessment of selective Janus Kinase inhibitors in rheumatoid arthritis mouse model: Insights into immune modulation and therapeutic implications." Journal of Immunology 212, no. 1_Supplement (May 1, 2024): 0434_4828. http://dx.doi.org/10.4049/jimmunol.212.supp.0434.4828.

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Abstract Janus kinase inhibitors (JAKi) are a class of orally available drugs for treating rheumatoid arthritis (RA). With a focus on enhancing safety profiles and therapeutic efficacy, the development of JAKi has transitioned from first-generation non-selective inhibitors to second-generation selective inhibitors. To better understand the roles of distinct JAK isoforms in the pathogenesis and progression of RA, we employed the Collagen-Induced Arthritis (CIA) mouse model to evaluate the effects of four selective JAK inhibitors: Upadacitinib (JAK1 inhibitor), CEP-33779 (JAK2 inhibitor), Ritlecitinib (JAK3 inhibitor), and Deucravacitinib (TYK2 inhibitor). All four JAK inhibitors showed varying degrees of alleviation in paw swelling and histological severity. We utilized flow cytometry to characterize the lymphoid and myeloid compartments within the spleen and Olink technology to analyze cytokines in the plasma. Our findings suggested that JAK1 and JAK3 play an important role in the differentiation and proliferation of T cells, while JAK2 is crucial in the development of myeloid cells. Notably, the TYK2 inhibitor exhibited significant effect in suppressing the expression of inflammatory cytokines. In summary, this study represents a comprehensive comparison of four selective JAKi in the CIA mouse model, offering valuable insights into immune modulation and potential implications for optimizing the benefit-risk profile of selective JAK inhibitors in the treatment of RA.

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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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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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Xu, Lichao, Ding Zhang, Guoqiang Wang, Chao Chen, Ying Wang, Haozhe Huang, and Zhenghua Zhang. "Correlation between JAK1/2 expression and immune-related genes and JAK2 gene variants: A pan-cancer analysis." Journal of Clinical Oncology 38, no. 15_suppl (May 20, 2020): e15057-e15057. http://dx.doi.org/10.1200/jco.2020.38.15_suppl.e15057.

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e15057 Background: Loss of function mutations for Janus kinases 1/2 (JAK1/2) have shown to be the underling mechanism of primary resistance to immune checkpoint inhibitors (ICIs). However, the correlation between JAK1/2 expression and immune-related genes have not been studied. Methods: Survival, mRNA expression and whole-exome sequencing data from 32 pan-cancer atlas studies were obtained from The Cancer Genome Atlas (TCGA). Correlations between JAK1/2 expression and immune-related genes were depicted in heatmaps. We also analyzed the association between JAK2 gene variants and JAK2 expression. Results: In total, 10071 samples with mRNA expression data were included for analysis. Expression of 46 immune-related genes were positively correlated with JAK2 expression in 25 tumors instead of JAK1 expression. Patients with higher expression of JAK2 had better prognosis than patients with lower expression of JAK2 in 13 tumors. Among 10071 patients, 363 (3.60%) patients harbored JAK2 variants, including 8 with frame shift mutations, 44 with nonsense mutations, 142 with missense mutations, 11 with splices, 8 with fusions, 90 with copy-number reduction and 116 with copy-number amplification. There was no difference in JAK2 expression between patients with JAK2 variants and those without JAK2 variants. However, JAK2 fusion (2.20%, 8/363) and amplification (31.96%, 116/363) were associated with higher JAK2 expression. Conclusions: Our pan-cancer analysis found that JAK2 expression was correlated with immune-related genes and the prognosis of cancer patients. JAK2 fusion and amplification increased the expression of JAK2. Altogether, patients with high JAK2 expression may benefit from ICIs.

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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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Tyner, Jeffrey W., Thomas G. Bumm, Jutta Deininger, Lisa Wood, Karl J. Aichberger, Marc M. Loriaux, Brian J. Druker, Christopher J. Burns, Emmanuelle Fantino, and Michael W. Deininger. "CYT387, a novel JAK2 inhibitor, induces hematologic responses and normalizes inflammatory cytokines in murine myeloproliferative neoplasms." Blood 115, no. 25 (June 24, 2010): 5232–40. http://dx.doi.org/10.1182/blood-2009-05-223727.

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Abstract Activating alleles of Janus kinase 2 (JAK2) such as JAK2V617F are central to the pathogenesis of myeloproliferative neoplasms (MPN), suggesting that small molecule inhibitors targeting JAK2 may be therapeutically useful. We have identified an aminopyrimidine derivative (CYT387), which inhibits JAK1, JAK2, and tyrosine kinase 2 (TYK2) at low nanomolar concentrations, with few additional targets. Between 0.5 and 1.5μM CYT387 caused growth suppression and apoptosis in JAK2-dependent hematopoietic cell lines, while nonhematopoietic cell lines were unaffected. In a murine MPN model, CYT387 normalized white cell counts, hematocrit, spleen size, and restored physiologic levels of inflammatory cytokines. Despite the hematologic responses and reduction of the JAK2V617F allele burden, JAK2V617F cells persisted and MPN recurred upon cessation of treatment, suggesting that JAK2 inhibitors may be unable to eliminate JAK2V617F cells, consistent with preliminary results from clinical trials of JAK2 inhibitors in myelofibrosis. While the clinical benefit of JAK2 inhibitors may be substantial, not the least due to reduction of inflammatory cytokines and symptomatic improvement, our data add to increasing evidence that kinase inhibitor monotherapy of malignant disease is not curative, suggesting a need for drug combinations to optimally target the malignant cells.

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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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Rummelt, Christoph, Sivahari P. Gorantla, Michael Sigl, Jana Saenger, Katharina Götze, Christian Peschel, Justus Duyster, and Nikolas von Bubnoff. "FLT3-ITD Interacts with and Phosphorylates IL-3β, and JAK1/2 Dependent IL-3β Activation Bypasses FLT3-ITD in FLT3 Kinase Independent Inhibitor Resistance in Vitro: Evidence for the Significance of IL-3β for FLT3-ITD Dependent Oncogeneic Signaling in AML." Blood 120, no. 21 (November 16, 2012): 2423. http://dx.doi.org/10.1182/blood.v120.21.2423.2423.

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Abstract Abstract 2423 Activating FLT3 mutations are found in 30% of AML patients. Internal tandem duplication (ITD) mutations are most common, and are associated with poor prognosis. FLT3 tyrosine kinase inhibitors (TKI) display limited activity in FLT3 mutant AML. Most patients experience primary or secondary TKI resistance. Only single cases of FLT3 kinase-dependent TKI resistance by secondary FLT3-ITD kinase domain mutations were reported. We therefore examined the mechanism of FLT3-ITD kinase-independent TKI resistance using an in vitro model. FLT3-ITD cell lines resistant to PKC412 or Sorafenib in 50% of the cases did not harbor secondary FLT3-ITD kinase domain mutations. However, 30% of these lines displayed a persistent phosphorylation of JAK2, IL-3βc and STAT5, while FLT3 phosphorylation was suppressed, indicating that these cell lines bypassed FLT3-ITD by activation of IL-3β by JAK2. Strikingly, TKI sensitive FLT3-ITD cells also displayed IL-3β phosphorylation, which in contrast to TKI resistant cells was JAK2 independent and was suppressed upon FLT3 TKI treatment. Expression of FLT3-ITD in IL-3β and JAK2 deficient g2A cells was sufficient for IL-3β phosphorylation. Co-IP experiments indicated a direct interaction of IL-3β and FLT3-ITD in TKI-sensitive and -resistant cells, which did not require interaction of IL-3β with IL-3a. This indicates that FLT3-ITD “physiologically” uses IL-3β as signaling intermediate. Mapping experiments showed that FLT3-ITD binding occurs in the membrane proximal region of IL-3β, independent of 8 tyrosines present in the cytoplasmic part. In search of the kinase bypassing FLT3-ITD in TKI-resistant cell lines, we identified an activating JAK1 mutation V658F in all TKI resistant FLT3-ITD cell lines displaying phosphorylation of JAK2, IL-3β and STAT5. JAK1 V658F is homologous to JAK2 V617F present in PV, phosphorylates IL-3β and STAT5, and confers TKI resistance in FLT3-ITD positive cells, which could be overcome by the JAK1/2 inhibitor Ruxolitinib. In summary, these data indicate that IL-3β not only mediates “physiological” FLT3-ITD dependent STAT5 activation, but also serves as a signaling module mediating JAK1/2-dependent TKI resistance. Thus, JAK inhibitors might represent an attractive therapeutic option in addition to FLT3 inhibitors in FLT3-ITD positive AML. Disclosures: Duyster: Novartis: Honoraria, Speakers Bureau. von Bubnoff:Novartis: Honoraria, Research Funding, Speakers Bureau.

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Watson, Eleanor, Michaela Waibel, Prerak Trivedi, Evan Pappas, Stacey Fynch, Robyn Sutherland, Thomas Kay, and Helen Thomas. "Prevention of Islet Inflammatory Stress with JAK1/JAK2 Inhibitors." Transplantation 102 (July 2018): S370. http://dx.doi.org/10.1097/01.tp.0000543121.88749.46.

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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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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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Liu, X., F. Tan, and C. Liang. "THU0080 PRECLINICAL CHARACTERIZATION OF TLL018, A NOVEL, HIGHLY POTENT AND SELECTIVE JAK1/TYK2 INHIBITOR FOR TREATING AUTOIMMUNE DISEASES." Annals of the Rheumatic Diseases 79, Suppl 1 (June 2020): 252.1–252. http://dx.doi.org/10.1136/annrheumdis-2020-eular.1547.

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Background:Janus kinases (JAKs) are important regulators of intracellular responses triggered by many key proinflammatory cytokines and are clinically validated therapeutic targets for treating various autoimmune diseases. However, current approved JAK inhibitors failed to achieve maximal clinical benefit in part due to their unfavorable selectivity for individual JAKs such as JAK2 and/or JAK3, leading to dose-limiting toxicities or severe toxicities (e.g., thrombosis, anemia, immune suppression). Selective inhibition of JAK1 and/or TYK2 may minimize or avoid some of the toxicities and potentially offer a better therapeutic window for treating autoimmune diseases. No highly selective JAK1/TYK2 inhibitor has been reported to date.Objectives:Discovery of a highly selective JAK1/TYK2 inhibitor that maximally avoids JAK2 and JAK3 inhibition. We described preclinical characterization of a novel, highly potent and selective JAK1/TYK2 inhibitor TLL018 and its potential utility in treating autoimmune diseases such as rheumatoid arthritis (RA).Methods:Using predicting SAR, TLL018 was designed to achieve exquisite selectivity for both JAK1 and TYK2 while sparing JAK2, JAK3 and other human kinases. Its enzyme and cell activities, kinase selectivity, andin vivoefficacy were assessed in a battery of relevant enzyme, cell and whole blood assays, andin vivoarthritis animal models. Additional preclinical DMPK and toxicology studies were conducted to support its clinical development.Results:TLL018 is a highly potent and selective, orally bioavailable JAK1/TYK2 inhibitor against JAK1 (IC50= 4 nM) and TYK2 (IC50= 5 nM) as measured inin vitrokinase assays with ATP concentrations at individual Km. Its potency against JAK2 or JAK3 is greater than 1 µM. Profiling against a panel of over 350 human kinase showed that TLL018 is exclusively selective for JAK1 and TYK2, with ≥ 90-fold selectivity against all other kinases tested. TLL018 exhibited potent cellular activity for JAK1-mediated IL-6 signaling (IC50= 0.6 µM) with greater than 100-fold selectivity against JAK2-mediated cytokine (e.g., TPO) signaling in human whole blood-based assays.Oral administration of TLL018 demonstrated dose-dependent efficacy in commonly studied rat adjuvant-induced arthritis (rAIA) model and mouse collagen-induced arthritis (mCIA) model. Significant inhibition of inflammation, bone resorption, splenomegaly and body weight change was observed in adjuvant-induced disease in rats. In addition, significant inhibition of inflammation, cartilage destruction, bone resorption and histological signs was demonstrated in collagen-induced arthritis in mice. Noticeably, TLL018 exhibited significant anti-inflammation activity at doses that only blocked JAK1 and TYK2 and exerted little inhibition of JAK2 and JAK3.In support of clinical development of TLL018, preclinical ADME and PK studies and IND-enabling toxicology and safety pharmacology studies were completed, confirming that TLL018 possesses excellent ADME and PK properties, and exhibits a clean on-target safety profile.Conclusion:TLL018 is a highly potent and selective JAK1/TYK2 inhibitor that demonstrated excellent efficacy and tolerability in relevant mouse and rat arthritis models. The collective data of its preclinical pharmacology, PK and toxicology showed a favorable pharmaceutical profile, further supporting its development for treating autoimmune diseases including RA. Clinical evaluation of TLL018 is ongoing.Disclosure of Interests:Xiangdong Liu Shareholder of: I own shares of TLL Pharmaceutical LLC, Employee of: I am employed by TLL Pharmaceutical LLC, Fenlai Tan Shareholder of: I own shares of TLL Pharmaceutical LLC, Employee of: I am employed by TLL Pharmaceutical LLC, Chris Liang Shareholder of: I own shares of TLL Pharmaceutical LLC, Employee of: I am employed by TLL Pharmaceutical LLC

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Balko, Justin M., Luis J. Schwarz, Na Luo, Mónica V. Estrada, Jennifer M. Giltnane, Daniel Dávila-González, Kai Wang, et al. "Triple-negative breast cancers with amplification of JAK2 at the 9p24 locus demonstrate JAK2-specific dependence." Science Translational Medicine 8, no. 334 (April 13, 2016): 334ra53. http://dx.doi.org/10.1126/scitranslmed.aad3001.

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Amplifications at 9p24 have been identified in breast cancer and other malignancies, but the genes within this locus causally associated with oncogenicity or tumor progression remain unclear. Targeted next-generation sequencing of postchemotherapy triple-negative breast cancers (TNBCs) identified a group of 9p24-amplified tumors, which contained focal amplification of the Janus kinase 2 (JAK2) gene. These patients had markedly inferior recurrence-free and overall survival compared to patients with TNBC withoutJAK2amplification. Detection ofJAK2/9p24 amplifications was more common in chemotherapy-treated TNBCs than in untreated TNBCs or basal-like cancers, or in other breast cancer subtypes. Similar rates ofJAK2amplification were confirmed in patient-derived TNBC xenografts. In patients for whom longitudinal specimens were available,JAK2amplification was selected for during neoadjuvant chemotherapy and eventual metastatic spread, suggesting a role in tumorigenicity and chemoresistance, phenotypes often attributed to a cancer stem cell–like cell population. In TNBC cell lines withJAK2copy gains or amplification, specific inhibition of JAK2 signaling reduced mammosphere formation and cooperated with chemotherapy in reducing tumor growth in vivo. In these cells, inhibition of JAK1–signal transducer and activator of transcription 3 (STAT3) signaling had little effect or, in some cases, counteracted JAK2-specific inhibition. Collectively, these results suggest that JAK2-specific inhibitors are more efficacious than dual JAK1/2 inhibitors against JAK2-amplified TNBCs. Furthermore,JAK2amplification is a potential biomarker for JAK2 dependence, which, in turn, can be used to select patients for clinical trials with JAK2 inhibitors.

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Ahmed, Emad A., and Salah A. Abdelsalam. "Marine Bioactive Molecules as Inhibitors of the Janus Kinases: A Comparative Molecular Docking and Molecular Dynamics Simulation Approach." Current Issues in Molecular Biology 46, no. 9 (September 23, 2024): 10635–50. http://dx.doi.org/10.3390/cimb46090631.

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A treasure trove of naturally occurring biomolecules can be obtained from sea living organisms to be used as potential antioxidant and anti-inflammatory agents. These bioactive molecules can target signaling molecules involved in the severity of chronic autoimmune diseases such as rheumatoid arthritis (RA). The intracellular tyrosine kinases family, Janus kinases (JAKs, includes JAK1, JAK2, and JAK3), is implicated in the pathogenesis of RA through regulating several cytokines and inflammatory processes. In the present study, we conducted molecular docking and structural analysis investigations to explore the role of a set of bioactive molecules from marine sources that can be used as JAKs’ specific inhibitors. Around 200 antioxidants and anti-inflammatory molecules out of thousands of marine molecules found at the Comprehensive Marine Natural Products Database (CMNPD) website, were used in that analysis. The details of the interacting residues were compared to the recent FDA approved inhibitors tofacitinib and baricitinib for data validation. The shortlisted critical amino acids residues of our pharmacophore-based virtual screening were LYS905, GLU957, LEU959, and ASP1003 at JAK1, GLU930 and LEU932 at JAK2, and GLU905 and CYS909 of JAK3. Interestingly, marine biomolecules such as Sargachromanol G, Isopseudopterosin E, Seco-Pseudopterosin, and CID 10071610 showed specific binding and significantly higher binding energy to JAK1 active/potential sites when being compared with the approved inhibitors. In addition, Zoanthoxanthin and Fuscoside E bind to JAK2′s critical residues, GLU930 and LEU932. Moreover, Phorbaketal and Fuscoside E appear to be potential candidates that can inhibit JAK3 activity. These results were validated using molecular dynamics simulation for the docked complexes, JAK1(6sm8)/SG, JAK2 (3jy9)/ZAX, and JAK3 (6pjc)/Fuscoside E, where stable and lower binding energy were found based on analyzing set of parameters, discussed below (videos are attached). A promising role of these marine bioactive molecules can be confirmed in prospective preclinical/clinical investigations using rheumatoid arthritis models.

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Steeghs, Elisabeth M. P., Isabel S. Jerchel, Willemieke de Goffau-Nobel, Alex Q. Hoogkamer, Judith M. Boer, Aurélie Boeree, Cesca van de Ven, et al. "JAK2 Aberrations in Childhood B-Cell Precursor Acute Lymphoblastic Leukemia." Blood 128, no. 22 (December 2, 2016): 583. http://dx.doi.org/10.1182/blood.v128.22.583.583.

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Abstract Background In high risk pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL) patients, gain of function mutations and translocations affecting JAK2 have been described. These mutations and translocations result in aberrant kinase signaling and may therefore serve as an ideal target for precision medicines. Aim Evaluate the frequency and prognosis of JAK2 lesions among different subtypes of childhood BCP-ALL, and study the efficacy of the JAK1/2 inhibitors momelotinib and ruxolitinib. Methods This study comprised 77 BCR-ABL1-like cases and 76 B-other cases which were screened for JAK2 translocations using RT-PCR. Furthermore a representative pediatric cohort of 461 newly diagnosed BCP-ALL cases was screened for JAK2 mutations using targeted next-generation sequencing. Clinical analyses were performed in 341 BCP-ALL patients. Patient-derived-xenograft (PDX) cells were isolated from NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice, which were injected with primary leukemic cells. Purity of PDX cells was enriched to over 90% and presence or absence of JAK2 lesions was validated. PDX and primary leukemic cells were exposed to a dilution series of momelotinib or ruxolitinib for four days. Where indicated, cells were pre-incubated with 25 ng/ml TSLP for 1 hour. In mono-culture assays, cytotoxicity was quantified using MTT and in co-culture assays flow cytometry was used. Leukemic cells were discriminated from mesenchymal stromal cells (MSCs) using CD19 and viability was assessed by Annexin V and Propidium Iodide. Western blotting was used to study protein expression levels. Results JAK2 translocations were detected in 6.5% of BCR-ABL1-like cases (3 PAX5-JAK2 cases, 1 TERF2-JAK2 case and 1 BCR-JAK2 case), but not in B-other cases. JAK2 mutations were identified in 3.5% of all BCP-ALL cases, which included JAK2 mutations in BCR-ABL1-like (7.6%), B-other (11.9%), and high hyperdiploid cases (1.6%), but not in MLL rearranged, BCR-ABL1-positive, ETV6-RUNX1-positive or TCF3-PBX1-positive cases. Cumulative incidence of relapse in patients harboring JAK2 lesions was as poor as in JAK2 wildtype BCR-ABL1-like and B-other patients. Efficacy of the JAK1/2 inhibitors momelotinib and ruxolitinib was examined in JAK2 lesion positive (primary and PDX) leukemic cells. Inhibitors were cytotoxic in both translocated and mutated cells, although efficacy in JAK2 mutated cells highly depended on CRLF2 activation by TSLP. CRLF2 activation resulted in downstream STAT5 activation and sensitization towards ruxolitinib compared to unstimulated cells (p < 0.05). Cells harboring JAK2 translocations signaled independently of CRLF2. Although momelotinib and ruxolitinib exposure blocked downstream STAT1/5 phosphorylation, both inhibitors also induced accumulation of phosphorylated JAK2Y1007. Consequently, release of the inhibitors resulted in a profound re-activation of JAK2 signaling, observed by upregulation of downstream STAT1/5 signaling. Furthermore, we observed microenvironment-induced resistance. Culturing leukemic cells in the presence of primary bone marrow MSCs induced resistance to ruxolitinib, compared to leukemic cells in single cultures (p < 0.05). A similar trend was observed for momelotinib. In addition, patients harboring JAK2 mutations displayed a heterogeneous leukemic cell population. Mouse xenograft models revealed different outgrowth patterns of leukemic cells, in which the JAK2 mutated clone persisted, decreased or even disappeared, resulting in outgrowth of JAK2 wildtype leukemic cells. Moreover, JAK2 mutations were not mutually exclusive for other pathway mutations (e.g. KRAS). Conclusion JAK2 translocations and mutations were detected in poor prognostic BCP-ALL cases. In ex vivo assays, the JAK1/2 inhibitors momelotinib and ruxolitinib were cytotoxic in JAK2 aberrant cells. Despite these promising findings, we identified certain limitations of these inhibitors. Inhibitors induced accumulation of phosphorylated JAK2Y1007, which resulted in a profound re-activation of JAK2 signaling upon their release. Furthermore, our data suggest that the effect of JAK inhibition may be compromised by mutations in alternative survival pathways and by microenvironment-induced resistance. Taken together, our data yield important directives for the clinical use of JAK inhibitors in pediatric BCP-ALL. Disclosures No relevant conflicts of interest to declare.

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Kim, Sena, Peter Ruminski, Megh Singh, Karl Staser, Kidist Ashami, Julie Ritchey, Sora Lim, John F. DiPersio, and Jaebok Choi. "Novel JAK Inhibitors to Reduce Graft-Versus-Host Disease after Allogeneic Hematopoietic Cell Transplantation in a Preclinical Mouse Model." Molecules 29, no. 8 (April 16, 2024): 1801. http://dx.doi.org/10.3390/molecules29081801.

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Allogeneic hematopoietic cell transplantation (allo-HCT) is a highly effective, well-established treatment for patients with various hematologic malignancies and non-malignant diseases. The therapeutic benefits of allo-HCT are mediated by alloreactive T cells in donor grafts. However, there is a significant risk of graft-versus-host disease (GvHD), in which the donor T cells recognize recipient cells as foreign and attack healthy organs in addition to malignancies. We previously demonstrated that targeting JAK1/JAK2, mediators of interferon-gamma receptor (IFNGR) and IL-6 receptor signaling, in donor T cells using baricitinib and ruxolitinib results in a significant reduction in GvHD after allo-HCT. Furthermore, we showed that balanced inhibition of JAK1/JAK2 while sparing JAK3 is important for the optimal prevention of GvHD. Thus, we have generated novel JAK1/JAK2 inhibitors, termed WU derivatives, by modifying baricitinib. Our results show that WU derivatives have the potential to mitigate GvHD by upregulating regulatory T cells and immune reconstitution while reducing the frequencies of antigen-presenting cells (APCs) and CD80 expression on these APCs in our preclinical mouse model of allo-HCT. In addition, WU derivatives effectively downregulated CXCR3 and T-bet in primary murine T cells. In summary, we have generated novel JAK inhibitors that could serve as alternatives to baricitinib or ruxolitinib.

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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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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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Bose, Prithviraj, and Srdan Verstovsek. "JAK2 inhibitors for myeloproliferative neoplasms: what is next?" Blood 130, no. 2 (July 13, 2017): 115–25. http://dx.doi.org/10.1182/blood-2017-04-742288.

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Abstract Since its approval in 2011, the Janus kinase 1/2 (JAK1/2) inhibitor ruxolitinib has evolved to become the centerpiece of therapy for myelofibrosis (MF), and its use in patients with hydroxyurea resistant or intolerant polycythemia vera (PV) is steadily increasing. Several other JAK2 inhibitors have entered clinical testing, but none have been approved and many have been discontinued. Importantly, the activity of these agents is not restricted to patients with JAK2 V617F or exon 12 mutations. Although JAK2 inhibitors provide substantial clinical benefit, their disease-modifying activity is limited, and rational combinations with other targeted agents are needed, particularly in MF, in which survival is short. Many such combinations are being explored, as are other novel agents, some of which could successfully be combined with JAK2 inhibitors in the future. In addition, new JAK2 inhibitors with the potential for less myelosuppression continue to be investigated. Given the proven safety and efficacy of ruxolitinib, it is likely that ruxolitinib-based combinations will be a major way forward in drug development for MF. If approved, less myelosuppressive JAK2 inhibitors such as pacritinib or NS-018 could prove to be very useful additions to the therapeutic armamentarium in MF. In PV, inhibitors of histone deacetylases and human double minute 2 have activity, but their role, if any, in the future treatment algorithm is uncertain, given the availability of ruxolitinib and renewed interest in interferons. Ruxolitinib is in late-phase clinical trials in essential thrombocythemia, in which it could fill an important void for patients with troublesome symptoms.

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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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Beinhoff, Paul, Lavannya Sabharwal, Vindhya Udhane, Cristina Maranto, Peter S. LaViolette, Kenneth M. Jacobsohn, Susan Tsai, et al. "Second-Generation Jak2 Inhibitors for Advanced Prostate Cancer: Are We Ready for Clinical Development?" Cancers 13, no. 20 (October 17, 2021): 5204. http://dx.doi.org/10.3390/cancers13205204.

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Androgen deprivation therapy (ADT) for metastatic and high-risk prostate cancer (PC) inhibits growth pathways driven by the androgen receptor (AR). Over time, ADT leads to the emergence of lethal castrate-resistant PC (CRPC), which is consistently caused by an acquired ability of tumors to re-activate AR. This has led to the development of second-generation anti-androgens that more effectively antagonize AR, such as enzalutamide (ENZ). However, the resistance of CRPC to ENZ develops rapidly. Studies utilizing preclinical models of PC have established that inhibition of the Jak2-Stat5 signaling leads to extensive PC cell apoptosis and decreased tumor growth. In large clinical cohorts, Jak2-Stat5 activity predicts PC progression and recurrence. Recently, Jak2-Stat5 signaling was demonstrated to induce ENZ-resistant PC growth in preclinical PC models, further emphasizing the importance of Jak2-Stat5 for therapeutic targeting for advanced PC. The discovery of the Jak2V617F somatic mutation in myeloproliferative disorders triggered the rapid development of Jak1/2-specific inhibitors for a variety of myeloproliferative and auto-immune disorders as well as hematological malignancies. Here, we review Jak2 inhibitors targeting the mutated Jak2V617F vs. wild type (WT)-Jak2 that are currently in the development pipeline. Among these 35 compounds with documented Jak2 inhibitory activity, those with potency against WT-Jak2 hold strong potential for advanced PC therapy.

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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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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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Manshouri, Taghi, Alfonso Quintás-Cardama, Zeev Estrov, Liza Knez, Ying Zhang, Hagop Kantarjian, and Srdan Verstovsek. "Bone Marrow Stroma-Mediated Paracrine Inhibition of Ruxolitinib (INCB018424) Induced Apoptosis of JAK2V617F-Mutated Cells." Blood 116, no. 21 (November 19, 2010): 1976. http://dx.doi.org/10.1182/blood.v116.21.1976.1976.

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Abstract Abstract 1976 Janus kinases (JAK) comprise a small family of cytoplasmic protein tyrosine kinases, which play an important role in the initiation of cytokine-triggered signaling events via signal transducer and activator of transcription (STAT) proteins. The activating somatic mutation JAK2V617F is highly prevalent among patients with myeloproliferative neoplasias (MPNs), which prompted the development of JAK2 inhibitors for patients with these malignancies. We have reported ruxolitinib (INCB018424) to be a potent orally bioavailable inhibitor of JAK1 and JAK2. Ruxolitinib inhibits IL-6 signaling and the proliferation of JAK2V617F Ba/F3 cells (IC50 127nM). We have streamlined a co-culture experimental platform designed to interrogate the interplay between JAK2V617F–positive cells and the stromal cells. Ruxolitinib caused marked apoptosis of human JAK2V617F mutant SET2 (IC50 56nM) and human UKE-1 (IC50 329nM) cells without any effect on the proliferation of the wild-type JAK2 human mast cell lines MHC1.1 or MHC1.2 which carry mutant KIT. The proapoptotic effect was markedly attenuated when JAK2V617F mutant cells were co-cultured on either immortalized or primary human marrow stromal cells (MSC: HS5, NK.tert or TM-R1). Co-culturing with each human-MSCs monolayer also hampered the ability of the JAK1/2 inhibitor to dephosphorylate STAT3 and STAT5, and prevented cytochrome c release and the cleavage of PARP protein. This protective effect was maintained in non-contact co-culture assays (cell lines separated by 0.4 μm micropore membranes), suggesting a paracrine effect. Cytokine profiling of supernatants from co-cultures of JAK2V617F–positive cells and human MSC monolayers are being analyzed to determine paracrine signals evoked upon ruxolitinib treatment. Neutralizing antibodies against over-secreted cytokines and downregulation of mRNAs encoding such cytokines with shRNAs will further validate the putative protective effects of such cytokines. Our preliminary results suggest that marrow stroma secreted cytokines play a critical role in protecting JAK2V617F–positive clones from JAK2 inhibitor therapy, thus highlighting the possible significant role of the microenvironment not only in the pathogenesis of MPNs but also in the resistance to JAK2–directed therapies of bone marrow JAK2V617 F-positive cells. Complete results on experiments using the clinically relevant JAK1/2 inhibitor ruxolitinib will be presented. Disclosures: Estrov: Celgene Corporation: Consultancy. Verstovsek:Incyte Corporation: Research Funding.

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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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Hornakova, T., L. Springuel, J. Devreux, A. Dusa, S. N. Constantinescu, L. Knoops, and J. C. Renauld. "Oncogenic JAK1 and JAK2-activating mutations resistant to ATP-competitive inhibitors." Haematologica 96, no. 6 (March 10, 2011): 845–53. http://dx.doi.org/10.3324/haematol.2010.036350.

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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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Porpaczy, Edit, Sabrina Tripolt, Andrea Hoelbl-Kovacic, Bettina Gisslinger, Zsuzsanna Bago-Horvath, Emilio Casanova-Hevia, Emmanuelle Clappier, et al. "Aggressive B-cell lymphomas in patients with myelofibrosis receiving JAK1/2 inhibitor therapy." Blood 132, no. 7 (August 16, 2018): 694–706. http://dx.doi.org/10.1182/blood-2017-10-810739.

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Inhibition of Janus-kinase 1/2 (JAK1/2) is a mainstay to treat myeloproliferative neoplasms (MPN). Sporadic observations reported the co-incidence of B-cell non-Hodgkin lymphomas during treatment of MPN with JAK1/2 inhibitors. We assessed 626 patients with MPN, including 69 with myelofibrosis receiving JAK1/2 inhibitors for lymphoma development. B-cell lymphomas evolved in 4 (5.8%) of 69 patients receiving JAK1/2 inhibition compared with 2 (0.36%) of 557 with conventional treatment (16-fold increased risk). A similar 15-fold increase was observed in an independent cohort of 929 patients with MPN. Considering primary myelofibrosis only (N = 216), 3 lymphomas were observed in 31 inhibitor-treated patients (9.7%) vs 1 (0.54%) of 185 control patients. Lymphomas were of aggressive B-cell type, extranodal, or leukemic with high MYC expression in the absence of JAK2 V617F or other MPN-associated mutations. Median time from initiation of inhibitor therapy to lymphoma diagnosis was 25 months. Clonal immunoglobulin gene rearrangements were already detected in the bone marrow during myelofibrosis in 16.3% of patients. Lymphomas occurring during JAK1/2 inhibitor treatment were preceded by a preexisting B-cell clone in all 3 patients tested. Sequencing verified clonal identity in 2 patients. The effects of JAK1/2 inhibition were mirrored in Stat1−/− mice: 16 of 24 mice developed a spontaneous myeloid hyperplasia with the concomitant presence of aberrant B cells. Transplantations of bone marrow from diseased mice unmasked the outgrowth of a malignant B-cell clone evolving into aggressive B-cell leukemia-lymphoma. We conclude that JAK/STAT1 pathway inhibition in myelofibrosis is associated with an elevated frequency of aggressive B-cell lymphomas. Detection of a preexisting B-cell clone may identify individuals at risk.

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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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Vannucchi, Alessandro M., Costanza Bogani, Niccolò Bartalucci, Lorenzo Tozzi, Serena Martinelli, Paola Guglielmelli, Chiara Paoli, Lisa Pieri, and Alberto Bosi. "Inhibitors of PI3K/Akt and/or mTOR Inhibit the Growth of Cells of Myeloproliferative Neoplasms and Synergize with JAK2 Inhibitor and Interferon,." Blood 118, no. 21 (November 18, 2011): 3835. http://dx.doi.org/10.1182/blood.v118.21.3835.3835.

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Abstract Abstract 3835 Dysregulated JAK/STAT signaling, occurring mainly but not exclusively in cells harboring mutations in JAK2 or other proteins involved in JAK/STAT pathway such as MPL, CBL, or Lnk, represents a pathogenetic event in chronic myeloproliferative neoplasms (MPN). However, activation of other downstream pathways such as the ERK and PI3K/Akt/mTOR pathway has been also documented in JAK2V617F-mutated cells. In this study we explored in-vitro the potential relevance of targeting PI3K/Akt/mTor pathway with specific inhibitors, alone or in combination with JAK2 inhibitor. Indeed, clinical trials have recently documented the effectiveness of JAK1/2 inhibitors (Verstovsek S, NEJM, 2010;363:117; Pardanani A, JCO 2011; 29:789) and RAD001, an mTOR inhibitor (Guglielmelli 2001; Blood, in press), in patients with MPN, mainly with myelofibrosis. The following drugs were used: an allosteric (RAD001) and an ATP (PP242) mTOR competitor; a dual PI3K/mTOR inhibitor (BEZ235); the JAK1/2 kinase ATP competitor AZD1480 and INCB018424. In the BA/F3/EPOR JAK2V617F-mutated cells, cell proliferation was prevented by lower doses of RAD001 (615±50nM, measured as IC50), PP242 (98±5nM) and BEZ235 (87±50nM) compared to BA/F3/EPOR JAK2wild-type (wt) cells (>10,000nM; 5,931±1,000nM; 676±200nM, respectively). In case of JAK inhibitors, IC50 was 313±23nM for AZD1480 and 51±2nM for INCB018424 as compared to 752±30nM and 457±15nM in wt cells, respectively. mTOR inhibitors induced cell cycle arrest in Go but were very poorly inducers of apoptosis (less than 15–20% at maximum); conversely JAK1/2 inhibitors induced dose-dependent increase of Annexin-V +ve cells up to >60% and BEZ235 induced 30–40% apoptosis at highest concentrations. All above drugs were able to prevent short-term cell proliferation and colony formation also in JAK2V617F-mutated HEL and SET2 cells. Western blot analysis demonstrated that, in addition to the expected inhibition of phosphorylation of specific drug targets (mTOR, 4EBP1), all three PI3K/mTOR inhibitors also reduced the degree of phophoSTAT5. siRNA-induced down-regulation of mTOR in SET2, HEL and BA/F3/EPOR JAK2V617F cells resulted in reduced phosphoSTAT5, indicating a direct mTOR-dependent phopshoSTAT5 regulation. Then, the activity of RAD001, BEZ235 and AZD1480 was analyzed in primary cells from MPN patients. All three drugs reduced clonogenic growth of MPN erythroid, myeloid and megakaryocytic progenitors at doses significantly lower (from 5 to 10-fold) than in normal cells, and prevented erythropoietin-independent colony (EEC) formation in the low nM range. Single colony genotyping in JAK2V617F mutated patients showed a median of 30±20% (range, 5–57%) reduction of V617F mutated colonies in favor of wt colonies Overall, these data indicated that inhibitors of PI3K/mTOR prevent proliferation and clonogenic capacity of MPN cells mainly through a cytostatic rather apoptotic effect (as JAK1/2 inhibitors do). To exploit whether simultaneous treatment with PI3K/AKt and JAK1/2 inhibitor displayed synergism we treated SET2 cells with different drug doses and measured their proliferation and apoptotic rate. Synergism was calculated as the combination index (CI) according to Chou and Talalay. Evidence of synergism was obtained for AZD1480 and INCB018424 with RAD001 (CI: 0.13 and 0.20, respectively), PP242 (CI: 0.13 and 0.20, respectively) and BEZ235 (CI: 0.77 and 0.37, respectively). Synergism was similarly demonstrated in BA/F3/EPOR JAK2V617F-mutated cells. Activity of RAD001 with AZD1480 and INCB018242 was also assessed in a EEC assay. We found that addition of RAD001 (5nM) or BEZ235 (50nM) to very low doses of JAK1/2 inhibitors (in the range of 5 to 50 nM) resulted in significant synergism and almost completely prevented EEC formation. In summary, these in vitro data indicate that PI3K/mTOR inhibitors are active against MPN cells and their combination with JAK1/2 inhibitors produced synergism, allowing to use lower doses of each drug; studies in murine models are ongoing to confirm these results in vivo. Thus, concurrent targeting of PI3K/mTOR and JAK/STAT pathway might represent a new therapeutic strategy to optimize efficacy and reduce toxicity in patients with MPN. Disclosures: Vannucchi: Italfarmaco: Consultancy; Novartis: Honoraria.

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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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Refici, Marion, Ziping Yang, Jacob Riehm, Darren Phillips, Andrew Souers, and Jason Harb. "Abstract 5339: BCL2A1 is expressed in myelofibrosis specimens and JAK2-mutated UKE-1 cells, yet does not inhibit synergistic cell killing by BCL-XL inhibitor navitoclax plus JAK1/2 inhibitors, including ruxolitinib." Cancer Research 82, no. 12_Supplement (June 15, 2022): 5339. http://dx.doi.org/10.1158/1538-7445.am2022-5339.

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Abstract Myelofibrosis (MF) is driven by mutually exclusive mutations in JAK2, CALR, and MPL in hematopoietic stem cells, leading to constitutively active JAK-STAT signaling and consequently, increased proliferation and cell survival. The JAK1/2 inhibitor ruxolitinib (Rux) is standard of care in front line patients; however, JAK inhibition alone does not induce long-term remission or disease-modifying effects. The BCL-2 family pro-survival proteins BCL-XL and MCL-1 have been implicated in resistance to Rux, and combined activity between the BCL-XL/BCL-2 inhibitor navitoclax (Nav) and JAK2 inhibition has been described in preclinical models. The combination of Nav + Rux resulted in clinically meaningful responses (e.g., improvements in spleen volume, constitutional symptoms, anemia, and bone marrow fibrosis) in patients with suboptimal response to, or progression while on, Rux (Harrison et al., EHA 2021, EP1087). Nav does not have strong affinity for MCL-1 or BCL2A1. BCL2A1 has garnered recent attention given its potential role in tumor survival and drug resistance; however, its role in MF is unknown. Given the potential of Nav + Rux in MF, we evaluated the expression of BCL-2 pro-survival proteins, including BCL2A1, in MF patient specimens and assessed the ability of BCL2A1 to serve as a resistance factor to Nav + Rux in vitro. DNA/RNA was extracted from PBMCs from healthy donors (n=15), JAK1/2-inhibitor naïve patients with MF (n=18), and suboptimal responders or progressors (R/R MF) currently on stable dose of Rux (n=72). When compared to healthy donors, expression of BCL2L1, MCL1 and BCL2A1, but not BCL2, was higher in naïve and R/R MF patients, with greatest increase observed in BCL2L1. Increase in median expression in naïve patients vs healthy donors for BCL2L1, MCL1, and BCL2A1 was ~4-, 1.5-, and 2-fold, respectively. A similar trend was observed with R/R MF patients vs healthy donors. We next evaluated BCL-2 family expression in in vitro models of MPN. Notably, MCL1 expression was high in UKE-1, SET-2 and HEL cell lines, while high expression of BCL2A1 was only observed in UKE-1. The combination of Nav plus either Rux, fedratinib or momelitinib induced synergistic cell killing in all lines. These data suggest that although MCL1 and BCL2A1 are expressed in JAK2-mutated cells and other malignancies characterized by constitutive JAK/STAT signaling, they do not confer resistance to a Nav + JAK-inhibitor combination. In summary, our data demonstrate dysregulated pro-survival BCL-2 family member expression, including MCL1 and BCL2A1, in MF patient samples and JAK2-mutated cell lines, and further implicate BCL-XL as a major survival factor in both naïve and Rux-treated R/R MF patients. Together these data support a Nav/JAK1/2-inhibitor combination for both JAK1/2-inhibitor naïve patients, as well as those no longer benefiting from Rux therapy. Citation Format: Marion Refici, Ziping Yang, Jacob Riehm, Darren Phillips, Andrew Souers, Jason Harb. BCL2A1 is expressed in myelofibrosis specimens and JAK2-mutated UKE-1 cells, yet does not inhibit synergistic cell killing by BCL-XL inhibitor navitoclax plus JAK1/2 inhibitors, including ruxolitinib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5339.

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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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Gotlib, Jason. "JAK inhibition in the myeloproliferative neoplasms: lessons learned from the bench and bedside." Hematology 2013, no. 1 (December 6, 2013): 529–37. http://dx.doi.org/10.1182/asheducation-2013.1.529.

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AbstractThe discovery of the JAK2 V617F mutation in the classic BCR-ABL1–negative myeloproliferative neoplasms in 2005 catalyzed a burst of research efforts that have culminated in substantial dividends for patients. Beyond JAK2 V617F, a more detailed picture of the pathobiologic basis for activated JAK-STAT signaling has emerged. In some patients with myelofibrosis (MF), next-generation sequencing technologies have revealed a complex clonal architecture affecting both genetic and epigenetic regulators of cell growth and differentiation. Although these bench-top findings have informed the clinical development of JAK inhibitors in MF, they have also provided scientific context for some of their limitations. The JAK1/JAK2 inhibitor ruxolitinib is approved for treatment of MF in North America and Europe and other lead JAK inhibitors discussed herein (fedratinib [SAR302503], momelotinib [CYT387], and pacritinib [SB1518]), have entered advanced phases of trial investigation. Uniformly, these agents share the ability to reduce spleen size and symptom burden. A major challenge for practitioners is how to optimize dosing of these agents to secure clinically relevant and durable benefits while minimizing myelosuppression. Suboptimal responses have spurred a “return to the bench” to characterize the basis for disease persistence and to inform new avenues of drug therapy.

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Gotlib, Jason. "JAK inhibition in the myeloproliferative neoplasms: lessons learned from the bench and bedside." Hematology 2013, no. 1 (December 6, 2013): 529–37. http://dx.doi.org/10.1182/asheducation.v2013.1.529.3847112.

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The discovery of the JAK2 V617F mutation in the classic BCR-ABL1–negative myeloproliferative neoplasms in 2005 catalyzed a burst of research efforts that have culminated in substantial dividends for patients. Beyond JAK2 V617F, a more detailed picture of the pathobiologic basis for activated JAK-STAT signaling has emerged. In some patients with myelofibrosis (MF), next-generation sequencing technologies have revealed a complex clonal architecture affecting both genetic and epigenetic regulators of cell growth and differentiation. Although these bench-top findings have informed the clinical development of JAK inhibitors in MF, they have also provided scientific context for some of their limitations. The JAK1/JAK2 inhibitor ruxolitinib is approved for treatment of MF in North America and Europe and other lead JAK inhibitors discussed herein (fedratinib [SAR302503], momelotinib [CYT387], and pacritinib [SB1518]), have entered advanced phases of trial investigation. Uniformly, these agents share the ability to reduce spleen size and symptom burden. A major challenge for practitioners is how to optimize dosing of these agents to secure clinically relevant and durable benefits while minimizing myelosuppression. Suboptimal responses have spurred a “return to the bench” to characterize the basis for disease persistence and to inform new avenues of drug therapy.

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Shao, Shuai, Lam C. Tsoi, Mrinal K. Sarkar, Xianying Xing, Ke Xue, Ranjitha Uppala, Celine C. Berthier, et al. "IFN-γ enhances cell-mediated cytotoxicity against keratinocytes via JAK2/STAT1 in lichen planus." Science Translational Medicine 11, no. 511 (September 25, 2019): eaav7561. http://dx.doi.org/10.1126/scitranslmed.aav7561.

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Lichen planus (LP) is a chronic debilitating inflammatory disease of unknown etiology affecting the skin, nails, and mucosa with no current FDA-approved treatments. It is histologically characterized by dense infiltration of T cells and epidermal keratinocyte apoptosis. Using global transcriptomic profiling of patient skin samples, we demonstrate that LP is characterized by a type II interferon (IFN) inflammatory response. The type II IFN, IFN-γ, is demonstrated to prime keratinocytes and increase their susceptibility to CD8+ T cell–mediated cytotoxic responses through MHC class I induction in a coculture model. We show that this process is dependent on Janus kinase 2 (JAK2) and signal transducer and activator of transcription 1 (STAT1), but not JAK1 or STAT2 signaling. Last, using drug prediction algorithms, we identify JAK inhibitors as promising therapeutic agents in LP and demonstrate that the JAK1/2 inhibitor baricitinib fully protects keratinocytes against cell-mediated cytotoxic responses in vitro. In summary, this work elucidates the role and mechanisms of IFN-γ in LP pathogenesis and provides evidence for the therapeutic use of JAK inhibitors to limit cell-mediated cytotoxicity in patients with LP.

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de Melo Campos, Paula, Joao Machado-Neto, Adriana Silva Santos Duarte, Rafaela Mendonça, Irene Lorand-Metze, Fernando F. Costa, Sara T. O. Saad, and Fabiola Traina. "IRS2 Associates With JAK2 and May Be Involved In Cell Proliferation Pathways In Chronic Myeloproliferative Neoplasms." Blood 122, no. 21 (November 15, 2013): 1598. http://dx.doi.org/10.1182/blood.v122.21.1598.1598.

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Abstract Background Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF) are BCR-ABL1 negative Chronic Myeloproliferative Neoplasms (MPN) characterized by increased myeloid proliferation, with predominant erythroid, megakaryocytic and megakaryocytic/granulocytic expansion, respectively. The finding of a recurrent mutation in the gene of the tyrosine-kinase Janus kinase 2 (JAK2 V617F) in these diseases has raised the hypothesis that this could be the main cause of their development. However, the evidence that MPN patients have a very similar response to JAK2 inhibitors regardless of JAK2 mutation status, and the knowledge that many receptors and substrates may lead to the activation of JAK/STAT, Ras/Raf/MAP kinases and PI3K/Akt/mTOR pathways, indicate the need to investigate other crucial proteins involved in the physiopathology of these diseases. Insulin receptor substrate 2 (IRS2) mediates mitogenic and antiapoptotic signaling from IR, IGF-IR, EPO-R and TPO-R. Previous studies performed on non-hematological cell lines have shown the association of IRS2 with JAK/STAT, PI3K/Akt/mTOR and Ras/Raf/MAP kinases pathways, giving rise to the hypothesis that IRS2 could participate in the activation of crucial signaling pathways in MPN through direct interaction with JAK2 or through alternative mechanisms. Aims To identify the JAK2/IRS2 protein interaction and to study the effects of pharmacological JAK1/2 inhibition (Ruxolitinib) over IRS2 phosphorylation in leukemia cell lines harboring or not the JAK2 V617F mutation; to characterize IRS2 expression in CD34+ cells from patients with MPN and its correlation with clinical data including JAK2 mutation status. Methods Leukemia cell lines carrying JAK2 V617F mutation (HEL) or not (HL60) were used for immunoprecipitation and immunobloting with IRS2 and JAK2 antibodies. Cells treated or not with JAK1/2 inhibitor Ruxolitinib were also submitted to immunoprecipitation and immunobloting with IRS2 and anti-phosphotyrosine antibodies. Peripheral blood mononuclear cells from 28 healthy donors and 97 patients with MPN (PV=28, ET=38, PMF=31) were included, and CD34+ cells were submitted to quantitative PCR (q-PCR). Relative expression of IRS2 was correlated with clinical data and with JAK2 V617F mutation status. Results Immunoprecipitation analysis showed that IRS2 associates with JAK2 in leukemia cell lines harboring (HEL) or not (HL60) the JAK2 V617F mutation. Furthermore, treatment of HEL cell line with the JAK1/2 selective inhibitor Ruxolitinib resulted in decreased IRS2 tyrosine phosphorylation. IRS2 mRNA expression in CD34+ cells were significantly higher in patients with ET when compared to healthy donors (1.70 [0.42-10.60] versus 0.87 [0.01-11.22], p=0.03). There was no difference in IRS2 mRNA expression in PV or PMF patients when compared to healthy donors. Furthermore, significantly higher levels of IRS2 mRNA expression were observed in patients harboring JAK2 V617F mutation when compared to the wild type JAK2 for ET (2.37 [0.96-10.60], n=14 versus 1.54 [0.42-1.54], n=22; p=0.01); and for PMF (2.27 [0.003-10.59], n=20 versus 0.60 [0.02-2.42], n=11; p=0.02). Although there was also a significant difference in IRS2 mRNA expression in mutated versus non mutated JAK2 in PV (p=0.02), the number of non mutated samples was low (n=2). Conclusions Our data indicate that IRS2 is a binding partner of JAK2 in myeloproliferative neoplasms and suggest that this protein association may be involved in cell proliferation in these diseases. The higher IRS2 expression in mutated samples (JAK2 V617F) might be associated with the constitutive activation of JAK2 in these samples. Disclosures: No relevant conflicts of interest to declare.

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Zhang, Xuekang, Jun Zhou, Qian Hu, Zhengren Liu, Qiuhong Chen, Wenxiang Wang, Huaigen Zhang, Qin Zhang, and Yuanlu Huang. "The Role of Janus Kinase/Signal Transducer and Activator of Transcription Signalling on Preventing Intestinal Ischemia/Reperfusion Injury with Dexmedetomidine." Journal of Nanoscience and Nanotechnology 20, no. 5 (May 1, 2020): 3295–302. http://dx.doi.org/10.1166/jnn.2020.16416.

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Dexmedetomidine (Dex) works as a crucial agent for the treatment of intestinal ischemia/reperfusion (I/R), but its mechanism remains unclear. Recent articles demonstrated the pivotal role of Janus kinase/signal transducer and activator of transcription (JAK2/STAT3) signalling in I/R. Therefore, it is reasonable to explore the associated mechanism of JAK2/STAT3 signalling in Dex treatment. The study purpose was to evaluate the JAK2/STAT3 signalling regulatory mechanisms of Dex in preventing I/R. Anaesthetized rats were subjected to superior mesenteric artery occlusion consisting of 1 h of ischemia and 2 h of reperfusion while served as controls. Animals received subcutaneous administration of 50 μg/kg Dex, JAK1 and JAK2 inhibitor, Ruxolitinib, selective JAK2 inhibitor, 10 mg/kg AG490 or STAT inhibitor and 0.4 mg/kg rapamycin; or Dex-treatment in the presence of α2-adrenoceptor antagonists Atip or Dex-treatment alone after I/R. Injury was scored histologically, apoptosis was detected via the apoptotic mediators caspase-3 and Bcl-2/Bax and the degree of activation of the JAK/STAT pathway was evaluated. Dex inhibited I/R injury by decreasing apoptosis significantly with rescue of cleaved caspase-3 and the Bcl-2/Bax ratio. Furthermore, phosphorylation of JAK2, STAT1 and STAT3 was affected, suggesting the involvement of activated JAK/STAT in response to Dex. Meanwhile, the JAK2 or STAT inhibitors AG490 and rapamycin, but not Ruxolitinib, exhibited a similar but even greater JAK2 and STAT3 regulatory effect, thus leading to a greater benefit. JAK2/STAT3 activation is crucial to the diminishing effect of Dex on mesenteric I/R injury; however, the efficacy and timing of Dex administration should be considered in clinical practice.

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Upadhayaya, Ram S., Raghava Reddy Kethiri, Avanish Vellanki, Jeff Lightfoot, Andrea Local, and William G. Rice. "Discovery of Selective Dual Inhibitors of Bromodomain Protein BRD4 and JAK2 for Treatment of Hematologic Malignancies." Blood 128, no. 22 (December 2, 2016): 5212. http://dx.doi.org/10.1182/blood.v128.22.5212.5212.

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Abstract The Bromodomain and Extra-Terminal (BET) proteins (BRD2, BRD3, BRD4, and BRDT) are functional readers of acetylated lysine residues of histones, and have emerged as potential therapeutic targets in hematologic cancers and solid tumors characterized by dysregulated epigenetic processes. Targeted inhibition of BET proteins has proven to be an effective strategy for transcriptional downregulation of c-MYC, an oncogene that is frequently activated or overexpressed in leukemias, lymphomas, and multiple myeloma. Of the BET family members BRD4 is the most extensively studied for its role in cancer, furthermore C-MYC downregulation by BET inhibitors is attributed to inhibition of enhancer binding by BRD4 (Delmore et al., Cell. 2011. 146:904-17). BRD4 is a critical factor in AML disease maintenance (Zuber et al., Nature. 2011. 478:524-8), and its suppression is the dominant mechanism of BET inhibitor JQ1 activity in AML (Rathert et al., Nature. 2015. 525:543-7). Recent reports have shown that BRD4/BET inhibitors and kinase inhibitors act synergistically in a range of cancer types (Sun et al., Blood. 2015, 126:1565-74; Stratikopoulos et al., Cancer Cell. 2015, 27:837-51). Therefore, optimizing for this synergy by prospectively designing and developing multi-targeting BRD4-kinase inhibitors may prolong therapeutic efficacy and overcome tumor resistance of single-activity BET and oncogenic kinase inhibitors. Dual inhibitors of BET proteins and Janus kinase 2 (JAK2), initially developed by Moffitt Cancer Center (Reuther et al., ASH 2015 Poster, Abstract #2826), demonstrated an opportunity for novel, potent dual inhibitors of BRD4 and JAK for treatment of myeloproliferative neoplasms (MPNs) and other disorders driven by the constitutively active somatic mutation, JAK2-V617F. Although JAK inhibitors such as ruxolitinib show clinical benefits in MPNs, these molecules demonstrate limited ability to induce remissions and are associated with significant toxicities such as myelosuppression. Therefore, selective JAK2 inhibitors that also target BRD4 hold promise as treatments of hematologic malignancies with improved activity and less off-target toxicity. While the dual inhibitor candidates from Moffitt exhibit strong potency, they possess a sub-optimal profile for inhibition of the thiamine transporter and other properties. Herein, we report that next generation, novel dual inhibitors of BRD4 and JAK2 have been discovered by Aptose, in a collaboration with Laxai Avanti Life Sciences (LALS), without observed thiamine transporter inhibition but with favourable drug-like properties. In biochemical assays, the Aptose dual inhibitor compounds exhibit IC50 values ranging from 82-678 nM for BRD4 (BD1) and 0.7-35 nM for JAK2. Compounds exhibited selectivity of 2-7 fold for BRD4 compared to other BRD isoforms, and 3-152 fold selectivity for JAK2 over JAK1 or JAK3. Cellular IC50 values of the compounds in antiproliferative assays ranged from 6.6 - 118 nM in AML (MV4-11) and from 171-721 nM in myeloma (MM1.S) cell lines. Candidate structures show good solubility and metabolic stability in human, rat and mouse liver microsomes. Structure-activity relationship studies are ongoing to improve biochemical and antiproliferative activity and drug like properties. Collectively, we demonstrate that a rational design approach can be used to generate potent molecules with combined BRD4/JAK2 inhibitory activities for treatment of hematologic cancers. Disclosures No relevant conflicts of interest to declare.

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Choi, Jaebok, Matthew L. Cooper, Kiran R. Vij, Bing Wang, Julie Ritchey, Bader Alahmari, Matthew Holt, and John F. DiPersio. "Pharmacologic Co-Blockade of IFNγR and IL6R Pathways to Prevent and Treat GvHD." Blood 128, no. 22 (December 2, 2016): 3353. http://dx.doi.org/10.1182/blood.v128.22.3353.3353.

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Abstract The therapeutic benefits of allogeneic hematopoietic stem cell transplantation (allo-HSCT) for hematologic malignancies are primarily derived from an anti-leukemia effect that is mediated by T cells in donor grafts. Unfortunately, these T cells also mediate graft-versus-host disease (GvHD), the major complication of allo-HSCT. We have previously published that in vivo administration of JAK1/JAK2 inhibitors to murine allo-HSCT recipients of interferon gamma receptor deficient (IFNγR-/-) T cells results in 100% survival in a fully MHC-mismatched B6 to Balb/c allo-HSCT model (Choi et al., 2014, PLoS ONE). Since the infusion of IFNγR-/- T cells alone is associated with only ~70% survival, we hypothesize that JAK1/JAK2 inhibitors have either additional off-target effects or are inhibiting other non-IFNγR signaling pathways which are themselves dependent on JAK1/JAK2. The major other cytokine receptor signaling pathway mediated via both JAK1 and JAK2 is the interleukin 6 receptor (IL6R) signaling pathway. Thus, it is possible that JAK1/JAK2 inhibitors also block signaling through IL6R in addition to IFNγR. In addition, Alam et al. have recently reported that single nucleotide polymorphisms of donor IFNγ and IL6 are closely linked to gastrointestinal GvHD in patients (2015, BMT). Therefore, we examined if blockade of both IFNγR and IL6R signaling results in complete elimination of GvHD after a fully MHC-mismatched allo-HSCT in which B6 (H-2b) T cell-depleted bone marrow cells (5x106) along with B6 pan T cells (5x105) are intravenously injected into lethally irradiated Balb/c mice (H-2d). As shown in Fig. 1, we have found that blocking both IFNγR (IFNγR-/- T cells) and IL6R (α-IL6R Ab) signaling dramatically reduces GvHD and results in >95% survival. In addition, we found that blocking both IFNγR and IL6R signaling significantly increased regulatory T cells (Tregs) in peripheral blood (23.2% Foxp3+ in CD4+ T cells (n=17) vs 2.5% in WT T cell control (n=16) at day 27 after allo-HSCT), suggesting that increase in Tregs might be a potential mechanism underlying the reduced GvHD after dual blockade of IFNγR and IL6R signaling. Baricitinib is a potent and balanced JAK1/JAK2 inhibitor currently being clinically developed by Eli Lilly for the treatment of inflammatory diseases. We hypothesize that baricitinib will optimally block both IFNγR and IL6R signaling pathways and prevent GvHD. We found that that baricitinib is a potent suppressor of GvHD in B6 to Balb/c allo-HSCT models (100% survival), superior to ruxolitinib and similar to blockade of both IFNγR and IL6R signaling (Fig. 2A). Baricitinib increases Tregs in vivo (Fig. 2B) and reduces the ratio of IL5 (Th2 cytokine) to IL2 (cytokine for Treg induction) in plasma (p=0.0046), a potential diagnostic marker for GvHD (Fujii et al., 2006, Int J Mol Med), significantly better than ruxolitinib. Lastly, we found that baricitinib inhibits the expression of T-bet (Fig. 2C), which is the master transcription factor of Th1 cells, that are primary effector T cells in inducing GvHD. These data suggest that the suppression of GvHD by baricitinib results from increased Tregs and decreased Th1 and Th2 cells. We next examined if the prevention of GvHD by baricitinib is dependent on natural regulatory T cells (Tregs) in donor grafts. Tregs were depleted from donor pan T cells before allo-HSCT (B6 to Balb/c). We found that in vivo administration of baricitinib resulted in 70% survival (0% control, p<0.0001; 100% Treg-replete T cells + baricitinib. In addition, based on clinical GvHD score when recipients of Treg-replete T cells were compared with those of Treg-deplete T cells, the beneficial effect of Tregs in the donor grafts for the prevention of GvHD was observed only for the first two weeks after allo-HSCT (p≤0.01). Lastly, we examined whether baricitinib can cure ongoing GvHD by administering baricitinib starting at day 10 after allo-HSCT when GvHD is established (B6 to Balb/c). We found that baricitinib treatment results in a significant reduction of GvHD and 100% survival (10% control, p<0.0001). All of these data suggest that pharmacologic co-blockade of IFNγR and IL6R pathways is a promising therapeutic strategy to prevent and effectively treat established GvHD. The inhibitory effect of baricitinib, ruxolitinib, and blockade of IFNγR and IL6R on JAK-STAT signaling using JAK/STAT phosphorylation antibody arrays is currently being investigated and will be presented. Disclosures DiPersio: Incyte Corporation: Research Funding.

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Markovtsov, Vadim, Elizabeth Tonkin, Shuling Fang, Chiang Liu, Marina Gelman, Wayne Lang, Jason Romero, et al. "In Vitro and in Vivo Inhibition of JAK2 Signaling by Potent and Selective JAK2 Inhibitor." Blood 112, no. 11 (November 16, 2008): 3721. http://dx.doi.org/10.1182/blood.v112.11.3721.3721.

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Abstract Recent advances in understanding the role of JAK2 V617F mutation in Bcr-Abl negative myeloproliferative (MPD) diseases pathogenesis opened up a possibility to develop highly targeted therapies against these debilitating ailments. We used a Ba/F3 cell line expressing the V617F mutant of JAK2 to screen a focused small molecule library for potential inhibitors of JAK2 V617F-dependent proliferation. Further extensive SAR of initial hits resulted in identification of R723, a potent and selective JAK2 inhibitor. This molecule is strongly antiproliferative (IC50 130–200 nM) against mouse BaF3 cells used for initial screening as well as against human UKE1 and SET2 cell lines harboring the same mutation. On the other hand, R723 has only weak activity in IL2-dependent (i.e. JAK1/JAK3-dependent) proliferation assays performed with human primary T (IC50 1300 nM) and mouse T-cell leukaemia CTLL2 cells (IC50 600 nM). A 10 to 20 fold cell-based selectivity of R723 was further confirmed by measuring inhibition of constitutive STAT5 phosphorylation in SET2 and BaF3 cells versus inhibition of IL-2 inducible STAT5 phosphorylation in human primary T and mouse CTLL2 cells using FACS-based approach. Compound R723 has low nonspecific antiproliferative activity against JAK2-independent MOLT4, A549 and H1299 cell lines with an IC50 ranging from 4 to 6 uM. The molecule has been also proven to be potent (IC50 of 2 nM against JAK2 in biochemical assay) and highly selective (window of more than 500 fold over JAK1 and 10 fold over JAK3) inhibitor of JAK2 kinase in vitro. Moreover, when tested in biochemical assay against a panel of more than 200 kinases at a concentration of 20 nM (IC90 for JAK2), R723 inhibited none of them. The selectivity of R723 was further confirmed using a variety of cell-based assays probing T-, B- and mast cell activation. Compound R723 was further evaluated in a stress-induced erythropoiesis mouse model, where kinetics of EPO-dependent hematocrit recovery from phenylhydrazine-induced anemia was assessed. Significant delay in recovery was observed at doses of 75 and 100 mg/kg bid indicating strong compound effect on EPOR signaling in vivo. The result could not be attributed to general toxicity effects as 14 day toxicology study did not identify any abnormalities at doses tested. As a result, R723 could become the basis for next generation of potent and selective compounds targeting JAK2-dependent myeloproliferative 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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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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Journal articles on the topic 'JAK1-JAK2 inhibitors'

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