Auswahl der wissenschaftlichen Literatur zum Thema „JAK1-JAK2 inhibitors“

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

La maladie cœliaque réfractaire de type 2 (MCR2) est un lymphome intraépithélial de bas grade compliquant la maladie cœliaque (MC), et une première étape fréquente vers un lymphome invasif, le lymphome T associé à une entéropathie (EATL). Les cellules de MCR2 sont issues d'une petite sous-population de lymphocytes intraépithéliaux (LIE) appelée LIE iCD3+ innés, présents dans l'intestin normal. Ces cellules, dépourvues de CD3 à leur surface (sCD3-), combinent des caractéristiques de cellules T et NK et se différencient dans l'intestin à partir d'un précurseur hématopoïétique en réponse à un signal NOTCH et à l'IL-15. La MCR2 se caractérise par la transformation maligne et l'accumulation de LIE sCD3-iCD3+ contenant de nombreux de variants somatiques. Les plus récurrents (>80%) sont notamment un variant de JAK1 en position 1097 ou des variants du domaine SH2 de STAT3 qui augmentent leur réponse aux cytokines inflammatoires, notamment à l'IL-15, surexprimée dans l'intestin cœliaque. Ces variants et d'autres évènements génétiques somatiques co-récurrents sont aussi présents dans les EATL, qu'ils compliquent une MCR2 ou surviennent de novo chez des patients cœliaques, témoignant d'un mécanisme commun de lymphomagenèse. Un premier objectif de la thèse était d'évaluer le caractère pilote dans la lymphomagénèse des mutations GdF JAK1 p.G1096D (analogue à p.G1097D chez l'homme). ou STAT3 p.D661V dans le contexte d'une surexpression de l'IL-15. J'ai montré que ces mutations confèrent un avantage sélectif à des cellules iCD3+ innées murines différenciées in vitro en présence d'IL-15. Le transfert adoptif de cellules sCD3-iCD3+ portant la mutation JAK1 p.G1096D chez des souris immunodéficientes surexprimant l'IL-15, n'a pas induit de lymphoprolifération, suggérant l'importance d'autres évènements génétiques. Cependant, ce transfert a induit un syndrome hyperéosinophilique rappelant celui associé chez l'homme à des lymphoproliférations sanguines de lymphocytes sCD3-CD4+. Un second objectif était d'évaluer, à l'aide d'un modèle de xénogreffe, l'efficacité du ruxolitinib (inhibiteur de JAK1 et JAK2) pour traiter la MCR2. Le traitement de 21 jours, débuté 14 jours après le transfert d'une lignée issue de LIE de MCR2, a permis de diminuer l'expansion tumorale mais celle-ci a rapidement repris à l'arrêt du traitement. Les données générées in vitro ont montré l'hétérogénéité génomique de la lignée MCR2, ce qui a permis de dériver à partir de cette lignée, 6 lignées résistantes au ruxolitinib, qui présentaient de nouvelles mutations dont une mutation commune dans le gène immunosuppresseur de tumeur CDK13. Ces résultats suggèrent un risque de sélection de cellules résistantes au ruxolitinib
Refractory celiac disease type 2 (RCD2) is a low-grade intraepithelial lymphoma complicating celiac disease (CD) and is a frequent initial step toward invasive lymphoma, specifically enteropathy-associated T-cell lymphoma (EATL). RCD2 cells originate from a small subpopulation of intraepithelial lymphocytes (IELs) called innate iCD3+ IELs, which are present in normal intestine. These cells, lacking CD3 on their surface (sCD3-), display characteristics of both T and NK cells and differentiate in the intestine from a hematopoietic precursor in response to a NOTCH signals and IL-15. RCD2 is characterized by the malignant transformation and accumulation of sCD3-iCD3+ IELs that harbor numerous somatic mutations. The most recurrent (>80%) include a JAK1 variant at position 1097 or variants in the SH2 domain of STAT3, which increase their response to inflammatory cytokines, as IL-15, which is overexpressed in the celiac intestine. These variants and other co-recurrent somatic genetic events are also present in EATL, whether they complicate RCD2 or occur de novo in celiac patients, indicating a shared mechanism of lymphomagenesis. One primary objective of this thesis was to evaluate the driver role, in lymphomagenesis, of the GdF JAK1 p.G1096D mutations (analogous to p.G1097D in humans) or STAT3 p.D661V in the context of IL-15 overexpression. I demonstrated that these mutations confer a selective advantage to murine innate iCD3+ cells differentiated in vitro in the presence of IL-15. Adoptive transfer of sCD3-iCD3+ cells carrying the JAK1 p.G1096D mutation into IL-15-overexpressing immunodeficient mice did not induce lymphoproliferation, suggesting the importance of additional genetic events. However, this transfer induced a hypereosinophilic syndrome (HSE) mimicing one of HSE discribed in humans with blood lymphoproliferative disorders of sCD3-CD4+ lymphocytes. A second objective was to assess, using a xenograft model, the efficacy of ruxolitinib (a JAK1 and JAK2 inhibitor) in treating RCD2. A 21-day treatment, initiated 14 days after the transfer of a cell line derived from RCD2 IELs, reduced tumor expansion, but this quickly reexpanded when the treatment was stopped. Data generated in vitro shown the genomic heterogeneity of the RCD2 cell line, allowing for the derivation of 6 ruxolitinib-resistant lines, which exhibited new mutations, including a common mutation in the tumor suppressor gene CDK13. These results suggest a risk of selecting ruxolitinib-resistant cells

Oral, small-molecule signalling pathway inhibitors, including ones that inhibit the JAK and SyK pathways, are currently in development for the treatment of rheumatoid arthritis (RA). Tofacitinib is an orally administered small-molecule inhibitor that targets the intracellular Janus kinase 3 and 1 (JAK1/3) molecules to a greater extent than JAK2 while baricitinib (formerly INCB028050) predominantly inhibits JAK1/2. Many of the proinflammatory cytokines implicated in the pathogenesis of RA utilize cell signalling that involves the JAK-STAT pathways and therefore inhibition of JAK-STAT signalling, by targeting multiple RA-associated cytokine pathways, has the potential to simultaneously reduce inflammation, cellular activation, and proliferation of key immune cells. Fostamatinib disodium is an orally available inhibitor of spleen tyrosine kinase (SyK), which is a cytoplasmic tyrosine kinase that is an important mediator of immunoreceptor signalling in mast cells, macrophages, neutrophils, and B cells. Interruption of SyK signalling may interrupt production of tumour necrosis factor (TNF) and metalloproteinase and therefore affect RA disease activity. Tofacitinib has been investigated in multiple phase 2 and phase 3 trials which have investigated its efficacy (clinical, functional, and radiographic) and safety in patients who have failed disease-modifying anti-inflammatory drugs (DMARDs) as monotherapy or in combination with DMARDs, compared to an inhibitor of tumour necrosis factor alpha (TNFα‎) and in patients who have failed TNFα‎ inhibitors. The efficacy of fostamatinib and baricitinib has been investigated in phase 2 trials; both are in large phase 3 clinical programmes. Each of these medications has demonstrated efficacy; their safety profile has been shown to be different from each other and from currently approved biological agents. This chapter discusses what is currently known and understood about their efficacy and safety.

Oral, small-molecule signalling pathway inhibitors, including ones that inhibit the JAK and other pathways, are currently in development for the treatment of rheumatoid arthritis (RA). Many of the pro-inflammatory cytokines implicated in the pathogenesis of RA utilize cell signalling that involves the JAK-STAT pathways and therefore inhibition of JAK-STAT signalling, by targeting multiple RA-associated cytokine pathways, has the potential to simultaneously reduce inflammation, cellular activation, and proliferation of key immune cells. Spleen tyrosine kinase (SyK) is a cytoplasmic tyrosine kinase that is an important mediator of immunoreceptor signalling in mast cells, macrophages, neutrophils, and B cells. Interruption of SyK signalling should interrupt production of tumour necrosis factor (TNF) and metalloproteinase and therefore affect RA disease activity. Tofacitinib, approved in many countries for the treatment of RA, is an orally administered small-molecule inhibitor that targets the intracellular Janus kinase 3 and 1 (JAK1/3) molecules to a greater extent than JAK2; there are other JAK inhibitors in development which are purported to be more specific for JAK3 (Vertex 509), specific for JAK1/2 (baricitinib) or more specific for JAK1 (Galapagos and INCYTE) where clinical data has been reported. Tofacitinib has been investigated in multiple clinical trials which have investigated its efficacy (clinical, functional, and radiographic) and safety in patients who have failed disease-modifying anti-inflammatory drugs (DMARDs) as monotherapy or in combination with DMARDs, compared to an inhibitor of tumour necrosis factor alpha (TNFα‎‎) and in patients who have failed TNFα‎‎ inhibitors. Vertex 509 has been investigated as monotherapy or in combination with MTX in DMARD failures while baricitinib, GLPG0634 (Galapagos), and INCB039110 (Incyte) have been investigated in phase 1 and 2 clinical trials in combination with MTX. Each of these medications has demonstrated efficacy; their safety profile has been shown to be generally similar although with some differences from each other and some differences from most of the currently approved biological agents. Fostamatinib disodium is an orally available inhibitor of SyK which was investigated in multiple phase 3 clinical trials in RA but was found to be generally ineffective with significant safety signals. This chapter discusses what is currently known and understood about the efficacy and safety of these oral, small-molecule DMARDs.

Polycythaemia vera (PV) is a clonal stem cell disorder characterized by erythrocytosis and associated with burdensome symptoms, risk of thrombohaemorrhagic complications, and transformation to myelofibrosis and acute myeloid leukaemia. Diagnostic criteria are very recently revised by the World Health Organization (WHO) based on haemoglobin and haematocrit levels, bone marrow morphology consistent with trilineage proliferation and presence of the JAK2 V617 mutation. Cytoreductive therapy is indicated in patients at increased risk of thrombosis. Hydroxyurea (HU) remains the most commonly used first-line cytoreductive therapy and interferon (IFN) is used either at failure of HU or in selected patients as first-line therapy. A recent phase 3 trial has shown the superiority of the JAK1/2 inhibitor ruxolitinib in comparison to best available treatment in HU-intolerant or resistant patients.