JAK Family Members Research Articles

Abstract A179: NMS-P113, a novel orally available JAK2 selective inhibitor

Abstract The Janus Kinases (JAK1, JAK2, JAK3, TYK2) are non-receptor tyrosine kinases that play important roles in hematopoiesis and immune response. In particular, gene ablation of JAK1 or JAK2 in the mouse is incompatible with life, due to neurological defects/immunodeficency and lack of erythropoiesis, respectively, whereas that of JAK3 or TYK2 is associated with severe immunodeficiency. Activating mutations of JAKs are found in association with malignant transformation. The best characterized gain-of-function mutation, JAK2-V617F in the pseudo-kinase domain of JAK2, is present in hematopoietic cells of patients with myeloproliferative disorders (MPD). In particular, the JAK2-V617F mutation is found in >95% of patients with polycythemia vera (PV), circa 50% of patients with essential thrombocythemia (ET), and circa 50% of myelofibrosis (MF) patients. Recently, a central role of JAK2 has been described in upregulation of the immune checkpoint component PD-L1 mediated by IFN-γ or by chromosome 9p24.1 amplification, suggesting that its inhibition might provide a new strategy to increase immune-mediated tumor inhibition in specific contexts. Although JAK inhibitors have been approved in oncological and autoimmune settings (e.g. the JAK1/JAK2 inhibitor ruxolitinib in MF and the pan-JAK inhibitor tofacitinib in rheumatoid arthritis) and multiple agents are in clinical testing, JAK2 selective compounds might be provide an advantage for long-term MPD therapy or in association with immunotherapy, given that inhibition of other JAK family members leads to immunosuppressive effects. Due to high homology amongst JAK family kinases within the ATP binding pocket, discovery of selective JAK2 inhibitors has represented a significant challenge. Here, we report the characterization of NMS-P113, a potent and selective JAK2 inhibitor. Screening of the NMS compound collection led to the identification of a pyrrole series with promising activity against JAK2. An optimization program led to identification of the potent and orally bioavailable JAK2 inhibitor NMS-P113. In biochemical assay this compound possesses low nanomolar potency against JAK2 (IC50 3 nM), with preferential activity over other JAK family members and high selectivity against a panel of 60 further kinases. In cellular assay, NMS-P113 potently inhibits proliferation of the JAK2 dependent SET-2 human megakaryoblastic leukemia line, derived from an ET patient and which harbors the JAK2-V617F mutation, as well as of Ba/F3 cells engineered to express constitutively activated JAK2 (IC50s circa 200 nM). NMS-P113 is 10-fold less active against the DERL-7 T-cell lymphoma cell line (which is dependent upon JAK1/JAK3) and has poor antiproliferative activity in JAK independent lines. Oral administration of NMS-P113 results in dose-related normalization of spleen weight and erythrocyte precursors count in an erythropoietin-induced model of PV in the mouse. Mechanism of action of NMS-P113, as assessed by inhibition of P-STAT5, was confirmed in vitro and in vivo. A favorable ADME profile with high oral bioavailability, together with permissive therapeutic safety margins in test species, indicate that NMS-P113 is suitable for further development, affording the possibility of reduced undesirable immunosuppressive activities compared to inhibitors that target multiple members of the JAK family. Citation Format: Paola Gnocchi, Maria Gabriella Brasca, Nadia Amboldi, Nilla Avanzi, Simona Bindi, Giulia Canevari, Daniele Casero, Roberta Ceruti, Marina Ciomei, Sabrina Cribioli, Cinzia Cristiani, Marcella Nesi, Wilma Pastori, Veronica Patton, Cinzia Pellizzoni, Gemma Texido, Elena Ardini, Eduard R. Felder, Antonella Isacchi, Daniele Donati, Arturo Galvani. NMS-P113, a novel orally available JAK2 selective inhibitor. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A179.

Identification of azabenzimidazoles as potent JAK1 selective inhibitors.

We have identified a class of azabenzimidazoles as potent and selective JAK1 inhibitors. Investigations into the SAR are presented along with the structural features required to achieve selectivity for JAK1 versus other JAK family members. An example from the series demonstrated highly selective inhibition of JAK1 versus JAK2 and JAK3, along with inhibition of pSTAT3 in vivo, enabling it to serve as a JAK1 selective tool compound to further probe the biology of JAK1 selective inhibitors.

Abstract A23: Type II JAK2 inhibitor NVP-CHZ868 has potent activity in JAK2-dependent B-cell acute lymphoblastic leukemias (B-ALLs) in vivo.

Abstract Approximately 10% of B-ALLs harbor CRLF2 rearrangements and have a poor prognosis. Although these leukemias are addicted to JAK2 signaling, ATP-competitive type I JAK2 inhibitors have limited activity (Weigert et al. J Exp Med 2012). This may result from heterodimerization of JAK2 with other JAK family members (Koppikar et al. Nature 2012). Type II inhibitors bind JAK2 in the inactive conformation and may have non-cross resistance with type I inhibitors. In Ba/F3 cells dependent on CRLF2 and the gain-of-function allele JAK2 R683G, the type II JAK2 inhibitor NVP-CHZ868 was more potent (IC50 21nM) than the type I inhibitors NVP-BSK805 (IC50 443nM) and NVP-BVB808 (IC50 111nM). Unlike type I inhibitors, CHZ868 completely abrogated JAK2 and STAT5 phosphorylation. In addition, the JAK2 Y931C allele that confers 4-6-fold resistance to BSK805 and BVB808 did not affect the IC50 of CHZ868. We assessed in vivo efficacy of CHZ868 in mice transplanted with transgenic (CRLF2/JAK2 R683G/Cdkn2a-/- or CRLF2/JAK2 R683G/Pax5+/-/Ts1Rhr) or primary human CRLF2-rearranged B-ALLs. Mice treated for 5-6 days with CHZ868 (30mg/kg/day PO) had significant reductions in spleen size compared to control mice and complete loss of phospho-STAT5 in residual leukemia cells. In both murine leukemias and human xenografts, CHZ868 prolonged survival compared to controls (p<0.001), but all mice ultimately became moribund from B-ALL. To study mechanisms of resistance to type II JAK2 inhibitors, we screened a randomly mutagenized JAK2 R683G library expressed in Ba/F3-CRLF2 cells for clones resistant to the type II inhibitor NVP-BBT594. All (>30) clones sequenced harbored the same JAK2 L884P mutation. Ba/F3 cells expressing CRLF2 with JAK2 R683G/L884P had 14-fold resistance to CHZ868 (R683G IC50 16nM; R683G/L884P IC50 231nM). JAK2 L884P is homologous to an EGFR L747P activating mutation (He et al. Clin Cancer Res 2012), which destabilizes the P-loop and C-helix portion of the kinase domain. Next-generation sequencing of JAK2 from splenocytes of mice that progressed on CHZ868 treatment did not identify L884P or other missense mutations at >1% frequency, suggesting in vivo treatment failure was not due to JAK2 mutation. To improve CHZ868 efficacy, we tested for synergy with multiple chemotherapy agents in MHH-CALL4 cells, which harbor a CRLF2/IGH rearrangement and JAK2 I682F mutation. Among the tested agents, dexamethasone was highly synergistic with CHZ868. In mice transplanted with CRLF2/JAK2 R683G/Pax5+/-/Ts1Rhr murine B-ALL, a 14-day course of CHZ868 prolonged survival compared to vehicle (p<0.0001) or dexamethasone (p<0.01), and the combination prolonged survival beyond CHZ868 monotherapy (p<0.0001). In summary, the type II JAK2 inhibitor CHZ868 potently kills JAK2-dependent B-ALL, overcomes genetic resistance to type I inhibitors, and synergizes with dexamethasone in vitro and in vivo. Thus, combination strategies using dexamethasone with type II JAK2 inhibitors merit testing in patients with relapsed/refractory, JAK2-dependent B-ALL. Citation Format: Loretta S. Li, Nadja Kopp, Shuo-Chieh Wu, Jordy Van Der Zwet, Jacob V. Layer, Oliver Weigert, Amanda L. Christie, Alexandra N. Christodoulou, Huiyun Liu, Akinori Yoda, Thomas Radimerski, David M. Weinstock. Type II JAK2 inhibitor NVP-CHZ868 has potent activity in JAK2-dependent B-cell acute lymphoblastic leukemias (B-ALLs) in vivo. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr A23.

Characteristics and Prognostic Significance of CRLF2 High Expression and the Co-Existence with JAK1 Mutations and Ikaros Deletion in Adult Acute Lymphoblastic Leukemia

Objective Cytokine receptor-like factor 2 (CRLF2) play an important role in differentiation and proliferation of lymphoid precursor cells through activation of JAK signaling pathway. Increased CRLF2 expression associates with mutations in JAK2, a combination that transforms hematopoietic cells, suggesting that mutants in JAK family members and CRLF2 may cooperate to contribute to acute lymphoblastic leukemia (ALL) pathogenesis. Moreover, the Ikaros deletion is also associated with the development of T-/B-cell ALL with poor outcome and relapse of high-risk leukemia. The aim of this study was to determine the clinical characterization and prognostic values of CRLF2 high expression and its concomitant expression with JAK1 mutations and Ikaros deletion in adult ALL patients.Methods Quantitative PCR (qPCR) was performed to detect the expression of CRLF2 in 133 newly diagnosed adult patients with ALL. Genomic DNA was amplified to detect the mutations of the exon 13, 14, 16, 18 and 19 of JAK1, and IKZF1 exons 4 through 7 deletions (△4–7) by direct sequencing or sequencing after cloning. The CD34, CD13, CD33 and other markers were detected on the leukemia cells from bone marrow of the patients by flow cytometry, and the correlations of the CRLF2 high expression with the clinical features, survival, and with co-expression of JAK1 mutations and Ikaros deletion were statistically analyzed with Pearson’s chi-square test or Fisher’s exact test and Kaplan–Meier curves analysis.ResultsCRLF2 high expression was detected in 22.8% of newly diagnosed adult ALL. The patients with CRLF2 high expression has significantly higher percentage of CD34, CD13 or CD33 positive than those with low expression（91.3% vs 62%, P=0.008; 76.2% vs 46.3%, P=0.016; 80.0% vs 37.9%, P=0.001), higher frequency of splenomegaly（60.0% vs 32.0%, P=0.040) in the adult ALL and shorter overall survival and event-free survival（9.5 months vs 16 months, P=0.029; 3 months vs 9 months, P=0.030）in the Philadelphia chromosome negative ALL. Moreover, the 4 JAK1 point mutations with amino acid changes were detected in the patients, which had significant CRLF2 high expression compared to that without mutation（75% vs 21.3%, P=0.037). The co-existence of CRLF2 high expression and IKZF1 exons 4-7 deletion (isoform Ik6) was found in 4 of 10 patients.ConclusionCRLF2 high expression predicts poor survival, and significantly co-exists with JAK1 mutation and Ikaros deletion in adult ALL patients. Our result also suggested that CRLF2, JAK1 and IKZF1 could be integrated in future prognostic model of adult ALL as possible markers for high-risk leukemia. DisclosuresNo relevant conflicts of interest to declare.

Functional Analysis of Kinase-Activating Fusions in Ph-like Acute Lymphoblastic Leukemia

Introduction: Ph-like or BCR-ABL1-like B-progenitor acute lymphoblastic leukemia (ALL) is a high-risk subtype characterized by a gene expression profile similar to BCR-ABL1 ALL. The prevalence of Ph-like ALL rises from 10% in standard risk childhood ALL to over 25% in young adults. Next-generation sequencing of Ph-like ALL identified a variety of alterations involving kinase or cytokine receptor genes, including rearrangement, sequence mutation and copy number alterations. Chromosomal rearrangements in about one-third of Ph-like ALL cases create fusion genes of a variety of 5’ partners that involve ABL1-class genes (ABL1, ABL2, CSF1R and PDGFRB) or activate JAK family members (JAK2, TYK2, IL2RB) that are potentially amenable to treatment with ABL1-class or JAK-class tyrosine kinase inhibitors (TKIs). Notably, ABL2 (Abelson-related gene, ARG), a homolog of ABL1, has rarely been identified as a rearrangement partner in ALL. CSF1R (encoding the macrophage colony stimulating receptor) regulates the differentiation of macrophages, and is not normally expressed in lymphocytes. Likewise, rearrangements involving the JAK family member TYK2, the beta chain of the interleukin 2 cytokine receptor (IL2RB), and the neurotrophic tyrosine kinase receptor type 3 (NTRK3), have not been previously described in leukemia. The goals of this study were to assess the role of these kinase alterations in leukemogenesis, to determine the activation of signaling pathways, and to investigate the efficacy of TKIs.Methods: Kinase fusions were expressed in interleukin-3 dependent Ba/F3 cells, and co-expressed with the dominant negative isoform of IKAROS (IK6) in interleukin-7 dependent Arf-/- mouse pre-B cells. Xenograft models of 10 Ph-like ALL tumors - ETV6-ABL1, RANBP2-ABL1, PAG1-ABL2, RCSD1-ABL2, SSBP2-CSF1R, IGH-EPOR, ETV6-NTRK3, ATF7IP-JAK2, PAX5-JAK2 and ZEB2-PDGFRB - were generated by engrafting primary human leukemia cells into NOD-SCID IL2R gamma null (NSG) mice. Activation of kinase signaling was performed using phosphoflow cytometry analysis, and sensitivity to TKIs was assessed ex vivo and in vivo.Results: All kinase fusions (PAG1-ABL2, MYH9-IL2RB, ATF7IP-JAK2, ETV6-NTRK3 or MYB-TYK2) induced cytokine-independent proliferation of Ba/F3 cells. Mice transplanted with Arf-/- pre-B cells co-expressing IK6 and either RCSD1-ABL2 or SSBP2-CSF1R developed pre-B ALL (CD43+, B220+, CD19+, BP-1+ and IgM-) with a median latency of 36 and 40 days respectively, providing evidence that ABL2 and CSF1R fusions contribute to leukemogenesis.In human leukemic cells harvested from xenograft mice we observed distinct patterns of kinase signaling activation and TKI sensitivity for the different fusions. Xenograft cells expressing ABL1-class kinase fusions showed activation of STAT5 that was inhibited with imatinib or dasatinib. Phosphorylation of CRKL, a known target of ABL1 and ABL2, was only observed in cells expressing ABL1/2 fusions. Cells harboring ATF7IP-JAK2, PAX5-JAK2 or IGH-EPOR showed phosphorylation of STAT5 that was attenuated with the JAK2 inhibitor, ruxolitinib. In contrast, cells expressing ETV6-NTRK3 signaled through the MAPK pathway with constitutive pERK1/2 that was inhibited with the ALK-inhibitor, crizotinib. This TKI response profile was confirmed by cytotoxicity assays in xenograft cells, with ABL1-class fusions being sensitive to dasatinib (IC50 range 1-2nM), whilst cases harboring ATF7IP-JAK2 or EPOR rearrangement uniquely responded to ruxolitinib with IC50 values of 500nM and 850nM respectively. Interestingly, in human leukemic cells harboring the ETV6-NTRK3 fusion we observed selective inhibition with both crizotinib and the FLT3 inhibitor, lestaurtinib. Pre-clinical studies on three xenograft models of Ph-like ALL - ETV6-ABL1, RCSD1-ABL2 and SSBP2-CSF1R – showed significantly reduced leukemic burden in dasatinib treated mice (20mg/kg/day p.o) compared to vehicle treated mice.Conclusions: These data provide important insight on new targets of rearrangement in ALL and describe the first engineered mouse models of Ph-like B-ALL. Functional modeling of these alterations is essential to improve the clinical management of Ph-like ALL by identifying patients with specific genomic lesions at diagnosis and directing them to treatment with appropriate TKIs combined with chemotherapy, analogous to current treatment for BCR-ABL1 B-ALL. DisclosuresHunger:Bristol Myers Squibb: Consultancy.

Type II JAK2 Inhibitor NVP-CHZ868 Is Active in Vivo Against JAK2-Dependent B-Cell Acute Lymphoblastic Leukemias (B-ALLs)

Approximately 10% of B-ALLs harbor CRLF2 rearrangements, which may portend a poor prognosis. Although these leukemias are addicted to JAK2 signaling, ATP-competitive type I JAK2 inhibitors have limited activity in vitro or in vivo (Weigert et al. J Exp Med 2012). This may result from heterodimerization of JAK2 with other JAK family members (Koppikar et al. Nature 2012). Type II inhibitors bind JAK2 in the inactive conformation, which may overcome this resistance. When assayed in MHH-CALL4 cells harboring a CRLF2/IGH rearrangement and JAK2 I682F mutation, the type II JAK2 inhibitors NVP-BBT594 and NVP-CHZ868 were 10-35-fold more potent than the type I JAK2 inhibitors NVP-BSK805 and NVP-BVB808. Similarly, in Ba/F3 cells dependent on CRLF2 and the gain-of-function allele JAK2 R683G, the IC50 for CHZ868 was 5-20-fold lower than the IC50s for BSK805 and BVB808. Unlike type I inhibitors, which induce paradoxical hyperphosphorylation of JAK2, CHZ868 completely blocks JAK2 and STAT5 phosphorylation. In addition, the JAK2 Y931C allele that confers 4-6-fold resistance to BSK805 and BVB808 did not alter sensitivity to CHZ868. CHZ868 abrogates STAT5 phosphorylation in Ba/F3 cells expressing CRLF2 with JAK2 R683G/Y931C while BVB808 does not. CHZ868 is the first type II JAK2 inhibitor amenable to in vivo use. We assessed its efficacy in mice transplanted with transgenic (CRLF2/JAK2 R683G/Cdkn2a-/- or CRLF2/JAK2 R683G/Pax5+/-/Ts1Rhr) or primary human CRLF2-rearranged B-ALLs. Splenocytes from patient-derived xenografts (PDXs) treated with CHZ868 in vivo for 3 days are more primed for apoptosis as demonstrated by a 2-6-fold EC50 reduction for PUMA permeabilizing activity compared to vehicle. Transcriptional profiling of splenocytes from CHZ868-treated PDXs revealed downregulation of critical survival pathways including E2F1, STAT3, and AKT-mediated signaling. Of note, 2 of the most downregulated genes are STAT targets, PIM1 and Myc. Mice treated for 5-6 days with CHZ868 had significant reductions in spleen size and complete loss of phospho-STAT5 in residual leukemia cells. In both murine leukemias and human xenografts, CHZ868 prolonged survival compared to controls (p<0.001). BH3 profiling of splenocytes from PDXs treated until moribund showed a 2-4-fold increase in the EC50 for BIM compared to vehicle, consistent with decreased priming for apoptosis in the relapsed setting. To study mechanisms of resistance to type II JAK2 inhibitors, we screened a randomly mutagenized JAK2 R683G library expressed in Ba/F3-CRLF2 cells for clones resistant to BBT594. All >30 clones sequenced harbored the same JAK2 L884P mutation. Ba/F3 cells expressing CRLF2 with JAK2 R683G/L884P displayed cross-resistance to CHZ868, while sensitivity to type I inhibitors was not affected. Structural modeling of the JAK2 JH1 domain suggested that L884P alters the binding pocket for type II inhibitors. JAK2 L884P is homologous to an EGFR L747P activating mutation, which destabilizes the P-loop and C-helix portion of the kinase domain (He et al. Clin Cancer Res 2012). The fact that L884P was reported in two B-ALL patients lacking additional JAK2 mutations (Torra et al. Blood (ASH Annual Meeting Abstracts) 2010) raised the possibility it was also an activating mutation. We confirmed L884P is an activating allele, as Ba/F3 cells expressing CRLF2, IL7R, and JAK2 L884P proliferated in the absence of TSLP ligand. To improve CHZ868 efficacy, we tested for synergy with multiple chemotherapy agents currently used in B-ALL treatment. Dexamethasone was the most highly synergistic with CHZ868 in MHH-CALL4 cells. To assess the combination in vivo, we treated mice transplanted with CRLF2/JAK2 R683G/Pax5+/-/Ts1Rhr murine B-ALL with vehicle, CHZ868, dexamethasone, or CHZ868 + dexamethasone for 14 days post engraftment. CHZ868 treatment prolonged survival compared to vehicle (p<0.0001) or dexamethasone (p<0.01), and the combination prolonged survival beyond CHZ868 monotherapy (p<0.0001). In summary, the type II JAK2 inhibitor CHZ868 potently kills JAK2-dependent B-ALL and overcomes genetic resistance to type I inhibitors. CHZ868 prolongs survival in murine transgenic and human xenograft models and synergizes with dexamethasone in vivo. Thus, combination strategies using dexamethasone with type II JAK2 inhibitors merit testing in patients with relapsed or refractory JAK2-dependent B-ALL. DisclosuresHofmann:Novartis Institutes for BioMedical Research: Employment. Baffert:Novartis: Employment. Vangrevelinghe:Novartis Institutes for BioMedical Research: Employment. Gaul:Novartis: Employment. Radimerski:Novartis: Employment. Weinstock:Novartis: Consultancy, Research Funding.

Inhibition of TYK2 and JAK1 ameliorates imiquimod-induced psoriasis-like dermatitis by inhibiting IL-22 and the IL-23/IL-17 axis.

Psoriasis is a chronic autoimmune disease affecting the skin and characterized by aberrant keratinocyte proliferation and function. Immune cells infiltrate the skin and release proinflammatory cytokines that play important roles in psoriasis. The Th17 network, including IL-23 and IL-22, has recently emerged as a critical component in the pathogenesis of psoriasis. IL-22 and IL-23 signaling is dependent on the JAK family of protein tyrosine kinases, making JAK inhibition an appealing strategy for the treatment of psoriasis. In this study, we report the activity of SAR-20347, a small molecule inhibitor with specificity for JAK1 and tyrosine kinase 2 (TYK2) over other JAK family members. In cellular assays, SAR-20347 dose dependently (1 nM-10 μM) inhibited JAK1- and/or TYK2-dependent signaling from the IL-12/IL-23, IL-22, and IFN-α receptors. In vivo, TYK2 mutant mice or treatment of wild-type mice with SAR-20347 significantly reduced IL-12-induced IFN-γ production and IL-22-dependent serum amyloid A to similar extents, indicating that, in these models, SAR-20347 is probably acting through inhibition of TYK2. In an imiquimod-induced psoriasis model, the administration of SAR-20347 led to a striking decrease in disease pathology, including reduced activation of keratinocytes and proinflammatory cytokine levels compared with both TYK2 mutant mice and wild-type controls. Taken together, these data indicate that targeting both JAK1- and TYK2-mediated cytokine signaling is more effective than TYK2 inhibition alone in reducing psoriasis pathogenesis.

Small-molecule inhibitors of spleen tyrosine kinase as therapeutic agents for immune disorders: will promise meet expectations?

Following on the heels of the US FDA approval of tofacitinib (Xeljanz, Pfizer, USA), an inhibitor of the JAK family members, and ibrutinib (Imbruvica, Janssen, Belgium), an inhibitor of BTK, for the treatment of rheumatoid arthritis and chronic lymphocytic leukemia, respectively, there is now renewed interest in the biopharmaceutical industry in the development of orally active small-molecule agents targeting key protein kinases implicated in immune regulation. One such 'immunokinase' target is SYK, a non-receptor tyrosine protein kinase critical for transducing intracellular signaling cascades for various immune recognition receptors, such as the B-cell receptor and the Fc receptor. Here, we review and discuss the progress and challenges in the development of small-molecule inhibitors of SYK and their potential as a new class of disease-modifying immunosuppressive agents for certain inflammatory and autoimmune disorders.

ATP-mediated kinome selectivity: the missing link in understanding the contribution of individual JAK Kinase isoforms to cellular signaling.

Kinases constitute an important class of therapeutic targets being explored both by academia and the pharmaceutical industry. The major focus of this effort was directed toward the identification of ATP competitive inhibitors. Although it has long been recognized that the intracellular concentration of ATP is very different from the concentrations utilized in biochemical enzyme assays, little thought has been devoted to incorporating this discrepancy into our understanding of translation from enzyme inhibition to cellular function. Significant work has been dedicated to the discovery of JAK kinase inhibitors; however, a disconnect between enzyme and cellular function is prominently displayed in the literature for this class of inhibitors. Herein, we demonstrate utilizing the four JAK family members that the difference in the ATP Km of each individual kinase has a significant impact on the enzyme to cell inhibition translation. We evaluated a large number of JAK inhibitors in enzymatic assays utilizing either 1 mM ATP or Km ATP for the four isoforms as well as in primary cell assays. This data set provided the opportunity to examine individual kinase contributions to the heterodimeric kinase complexes mediating cellular signaling. In contrast to a recent study, we demonstrate that for IL-15 cytokine signaling it is sufficient to inhibit either JAK1 or JAK3 to fully inhibit downstream STAT5 phosphorylation. This additional data thus provides a critical piece of information explaining why JAK1 has incorrectly been thought to have a dominant role over JAK3. Beyond enabling a deeper understanding of JAK signaling, conducting similar analyses for other kinases by taking into account potency at high ATP rather than Km ATP may provide crucial insights into a compound's activity and selectivity in cellular contexts.

Structural and functional characterization of salmon STAT1, STAT2 and IRF9 homologs sheds light on interferon signaling in teleosts

Mammalian IRF9 and STAT2, together with STAT1, form the ISGF3 transcription factor complex, which is critical for type I interferon (IFN)-induced signaling, while IFNγ stimulation is mediated by homodimeric STAT1 protein. Teleost fish are known to possess most JAK and STAT family members, however, description of their functional activity in lower vertebrates is still scarce. In the present study we have identified two different STAT2 homologs and one IRF9 homolog from Atlantic salmon (Salmo salar). Both proteins have domain-like structures with functional motifs that are similar to higher vertebrates, suggesting that they are orthologs to mammalian STAT2 and IRF9. The two identified salmon STAT2s, named STAT2a and STAT2b, showed high sequence identity but were divergent in their transactivation domain (TAD). Like STAT1, ectopically expressed STAT2a and b were shown to be tyrosine phosphorylated by type I IFNs and, interestingly, also by IFNγ. Microscopy analyses demonstrated that STAT2 co-localized with STAT1a in the cytoplasm of unstimulated cells, while IFNa1 and IFNγ stimulation seemed to favor their nuclear localization. Overexpression of STAT2a or STAT2b together with STAT1a activated a GAS-containing reporter gene construct in IFNγ-stimulated cells. The highest induction of GAS promoter activation was found in IFNγ-stimulated cells transfected with IRF9 alone. Taken together, these data suggest that salmon STAT2 and IRF9 may have a role in IFNγ-induced signaling and promote the expression of GAS-driven genes in bony fish. Since mammalian STAT2 is primarily an ISGF3 component and not involved in IFNγ signaling, our finding features a novel role for STAT2 in fish.

Guest editorial: efficacy of and resistance to molecularly targeted therapy for myeloid malignancies

The introduction of all-trans retinoic acid (ATRA) and the ABL kinase inhibitors has dramatically improved the prognosis of patients with acute promyelocytic leukemia (APL) and chronic myeloid leukemia (CML), respectively. These remarkable clinical successes have proved the principle of molecularly targeted therapy, raising expectations that therapeutic interventions targeting altered molecules will improve outcomes in patients with cancers and hematologic malignancies. A number of targeted agents, such as small molecular compounds and monoclonal antibodies, have already been approved and evaluated in clinical development; however, the accumulation of clinical information has also revealed cases of treatment failure due to resistance. Several mechanisms of resistance to targeted agents, such as mutation or amplification of the targeted gene, up-regulation of P-glycoprotein, activation of other pathways, and pharmacokinetic variability, have been observed in the clinical setting. The present issue of IJH includes four review articles focusing on the efficacy of and resistance to molecularly targeted therapy for myeloid malignancies. Myeloproliferative neoplasms (MPN) are clonal hematopoietic stem cell disorders characterized by proliferation of one or more of the myeloid lineages. Although the diagnosis of CML is confirmed by the presence of the Philadelphia chromosome and/or BCR-ABL1 fusion gene, other MPN subtypes are generally diagnosed based on their clinical and laboratory features. With the identification of the JAK2 mutation in nearly all patients with polycythemia vera (PV) and about half of those with essential thrombocythemia (ET) and primary myelofibrosis (PMF), the mutational status of the JAK2 gene is now considered among the diagnostic criteria for PV, ET, and PMF. Mutant JAK2 is constitutively activated and introduces the activation of downstream molecules, such as the STAT, MAPK, and PI3K pathways, promoting the transformation and proliferation of hematopoietic progenitors. Several JAK2 inhibitors, therefore, have been evaluated in clinical development. Neha Bhagwat, Ross L. Levine, and Priya Koppikar summarize recent advances in the development of JAK2 inhibitors, and clearly show that chronic JAK inhibition leads to JAK inhibitor persistence via transphosphorylation of JAK2 through other JAK family members, a crucial observation for the development of therapeutic strategies using JAK inhibitors in MPN. The FLT3 protein is a member of the class III receptor tyrosine kinases (RTK), which also includes KIT, FMS and PDGFR. Mutations in FLT3 represent the most frequently reported genetic alteration in AML, and are involved in the signaling pathway associated with autonomous proliferation and differentiation block in leukemia cells. The FLT3ITD mutation is strongly associated with leukocytosis and poor prognosis. The development of FLT3 kinase inhibitors may thus yield more efficacious strategies for leukemia therapy. Numerous FLT3 inhibitors have been tested in phase 1 and 2 clinical trials, but their efficacy in the treatment of AML has been unimpressive, and have been plagued by adverse effects and the development of resistance. In their review, Michael R. Grunwald and Mark J. Levis summarize the clinical efficacy of FLT3 inhibitors. They also clearly show several resistance mechanisms, as revealed by clinical trials, and suggest new treatment strategies in combination with FLT3 inhibitors for AML patients harboring FLT3 mutations. H. Kiyoi (&) Department of Hematology and Oncology, Nagoya University Graduate School of Medicine, Tsurumai-cho 65, Showa-ku, Nagoya 466-8550, Japan e-mail: kiyoi@med.nagoya-u.ac.jp

Abstract 931: Highly selective JAK1 kinase inhibitors suppress Y705-mediated STAT3 activation in a wide range of tumor types, with reduced hematological toxicity.

Abstract Constitutively activated pY705-STAT3 is oncogenic and prevalent in many tumor types, and has been shown to be driven in large part by constitutive autocrine and paracrine cytokine signaling. JAK kinases are critical mediators of cytokine signaling, and JAK1 has been shown to play an essential role downstream of IL-6 family cytokines, the upregulation of which has been documented in many tumor types. A JAK1 selective inhibitor should provide a therapeutic means of suppressing Y705 phosphorylation of STAT3 without incurring the hematological toxicity associated with inhibition of JAK2 and JAK3 activity, allowing for more intensive dosing and greater combinability with other agents, particularly those with hematological toxicities. We have developed highly selective inhibitors of JAK1 and used them to address the hypothesis that JAK1 is the primary driver of STAT3 mediated signaling in cancer cells. The compounds are highly selective in enzyme assays, with 1000-fold selectivity for JAK1 vs. other JAK family members, and minimal activity against other kinases at 1uM. In cell lines, they are over 500-fold selective for JAK1 vs. JAK2, measured using assays specific for JAK1-mediated STAT3 vs. JAK2-mediated STAT5 phosphorylation. In vivo, the compounds are also highly selective for JAK1 mediated signaling, measured by comparing inhibition of STAT3 phosphorylation in H1975 subcutaneous xenografts vs. inhibition of STAT5 phosphorylation in a disseminated TEL-JAK2 transformed BaF3 model. At doses where inhibition of STAT3 phosphorylation was greater than 90%, only minimal inhibition of STAT5 phosphorylation was observed. In a wide variety of human tumor lines (CRC, NSCLC, pancreatic, DLBCL) with a variety of genetic backgrounds (K-ras, b-raf, PI3K, EGFR mutant, PTEN null), complete knockdown of pSTAT3 (Y705) was observed with JAK1 selective compounds, at concentrations achievable in vivo. These effects are consistent with experiments where inhibition of Y705 phosphorylation of STAT3 was achieved with a JAK1-specific siRNA. At doses where in vivo activity was observed, the compounds had only minimal effect in an erythropoietin-driven mouse model of polycythemia and extramedullary erythropoiesis. These data support the hypotheses that in a variety of tumor types, pSTAT3-mediated signaling is dependent on JAK1 kinase activity, and that a selective JAK1 inhibitor can suppress STAT3 activation at doses that avoid the hematological toxicity associated with JAK2 and JAK3 inhibition. Citation Format: Richard Woessner, Geraldine Bebernitz, Kirsten Bell, Robert Caccese, Nakpangi Johnson, Jason Kettle, Caroline Rivard, Patricia Schroeder, Qibin Su, Haixia Wang, Minwei Ye, Weijia Zheng, Michael Zinda, Paul Lyne, Melissa Vasbinder, Dennis Huszar, Claudio Chuaqui. Highly selective JAK1 kinase inhibitors suppress Y705-mediated STAT3 activation in a wide range of tumor types, with reduced hematological toxicity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 931. doi:10.1158/1538-7445.AM2013-931

Structure-guided studies of the SHP-1/JAK1 interaction provide new insights into phosphatase catalytic domain substrate recognition.

SHP-1 (PTPN6) is a member of the SHP sub-family of protein tyrosine phosphatases and plays a critical role in the regulation of the JAK/STAT signaling pathway. Previous studies suggested that SHP-1 contains a PTP1B-like second phosphotyrosine pocket that allows for binding of tandem phosphotyrosine residues, such as those found in the activation loop of JAK kinases. To discover the structural nature of the interaction between SHP-1 and the JAK family member, JAK1, we determined the 1.8Å co-crystal structure of the SHP-1 catalytic domain and a JAK1-derived substrate peptide. This structure reveals electron density for only one bound phosphotyrosine residue. To investigate the role of the predicted second site pocket we determined the structures of SHP-1 in complex with phosphate and sulfate to 1.37Å and 1.7Å, respectively, and performed anomalous scattering experiments for a selenate-soaked crystal. These crystallographic data suggest that SHP-1 does not contain a PTP1B-like second site pocket. This conclusion is further supported by analysis of the relative dephosphorylation and binding affinities of mono- and tandem-phosphorylated peptide substrates. The crystal structures instead indicate that SHP-1 contains an extended C-terminal helix α2' incompatible with the predicted second phosphotyrosine binding site. This study suggests that SHP-1 defines a new category of PTP1B-like protein tyrosine phosphatases with a hindered second phosphotyrosine pocket.

Signaling network of Oncostatin M pathway.

Oncostatin M (OSM), belonging to the IL-6 family of cytokines (Heinrich et al. 2003), was first reported and purified from U937 monocytic cells (Zarling et al. 1986; Ensoli et al. 1999; Hasegawa et al. 1999). In normal physiological condition, OSM is associated with multiple biological processes and cellular responses including growth, differentiation, and inflammation However, anti-proliferative activity of OSM against breast cancer cell line generated the interest of biomedical community on this molecule (Douglas et al. 1997, 1998). OSM was also found associated with pathological conditions such as proliferation of ovarian cancer cells (Taga and Kishimoto 1997), prostate cancer 22Rv1 cells (Hoffman et al. 1996), up-regulation of the ER chaperone Grp78/BiP in the liver cells, atherosclerotic lesions, ischemic heart disease and rheumatoid arthritis (Linsley et al. 1990; Dunham et al. 1999). The dual role of OSM in either inducing or inhibiting the proliferation of various types of cells called upon the scientific community to investigate role of OSM in various physiological and experimental contexts in detail. However, diverse molecular level information pertaining to OSM signaling is not available in a centralized resource. Therefore, we have systematically gathered and curated molecular information from literature and created a public resource for OSM induced signaling events. We integrated OSM signaling pathway into NetPath (Kandasamy et al. 2010), which is a public resource of human signaling pathways. OSM is known to mediate its biological effects by binding to two distinct heterodimers of gp130 with either leukemia inhibiting factor receptor (LIFR) or OSM receptor-beta (OSMR-beta) (Thoma et al. 1994). Former heterodimer between gp130 and LIFR is called type I receptor complex and the latter between gp130 and OSMR-beta is called type II receptor complex. Type I receptor can bind to either OSM or leukemia inhibiting factor, whereas type II receptor has more affinity towards OSM (O’Hara et al. 2003). The binding of OSM to either gp130/OSMR-beta or gp130/LIFR induces the activation of Janus Kinase family members through tyrosine phosphorylation (Tanaka and Miyajima 2003). The activated JAK family members in turn induce the activation of Signal Transduction and Activator of Transcription (STAT) proteins (Schaefer et al. 2000). Alternatively, the activated receptors can also activate mitogen-activated protein kinase (MAPK) pathway (Van Wagoner et al. 2000) and PI3K/AKT pathways (Arita et al. 2008). It was also reported that OSM bring about ligand-induced receptor degradation of gp130, OSMR-beta, and LIFR before enhancing the synthesis of the receptor subunits (Blanchard et al. 2001).

Myeloproliferative Neoplasm with BCR-JAK2 Fusion Gene As the Result of t(9;22)(p24,11.2) in a Brazilian Patient

AbstractAbstract 4808 IntroductionChronic Myeloid Leukemia (CML) is the most well described disease result of t(9;22)(q34,q11.2). This chromosomal rearrangement leads to well-know BCR-ABL fusion that promotes tyrosine kinase activity. There are others oncogenic BCR fusion found such as PDGFRA (4q12), FGFR1(8p12) that causes myeloproliferative disorders (MD).JAK2 gene is one of the 4 genes members of JAK family. The JAK2 V617F mutation which results from a G –>T transversion at nucleotide 1849 in exon 14 of the JAK2 gene, the consequence of which is substitution of valine by phenylalanine at codon 617 is associated with MD and it is a major diagnosis criterion for Primary Myeloficrosis, Polycythaemia vera and Essential thrombocytemia. There are described a lot chromosomal translocations involving the JAK2 locus.We report an extremely rare case with BCR-JAK2 fusion gene as the result of t(9;22)(p24,q11.2) for the first time in Brazilian people, and it is the 6thcase all of the world. Case ReportIn April 2010, a 54 years old male patient presented fatigue, abdominal pain and splenomegaly. A blood count revealed leukocytosis 93.380/mm3 with a predominance of neutrophils and left shift. Conventional cytogenetic analysis was performed and it was evidenced 46,XY, t(9;22)(p24;q11.2) in 90% metaphases examined, due to expected association it was promoted BCR-ABL1 fusion gene and it was not detected by using RT-PCR. He was treated with imatinib 400mg/day because the involvement of BCR gene. After three months he presented weight loss, progressive splenomegaly without hematologic response and it was modified to Dasatinib 150mg/day plus hydroxyureia 3g/day. In August 2011, due to not hematologic response, it was stopped Dasatinib treatment and nowadays patient has been treating with hydroxyureia 1.5g/day. His last follow up in May 2012, blood count was abnormal Hb 16.8g/dl leukocytes 7730/mm3 and low platelets count 32.000/mm3. The differential count showed 65.3% segmented granulocytes, 13.6% eosinophlis, 1.6% basophil, 2.6% monocytes, 16.9% lymphocytes.It was repeated conventional Karyotyping and it was evidenced 46,XY, t(9;22)(p24;q11.2) in all of metaphases examined. The presence of BCR-ABL rearrangement was excluded by using the fluorescence in situ hybridization (FISH) using a BCR-ABL probe. In addition, it was not evidenced FIP1L1-PDGFRa fusion gene and JAK2 V617F mutation by using RT-PCR. DiscussionWe have described a male patient with MD with t(9;22)(p24;q11.2) wich leads to the BCR-JAK2 fusion and it was not evidenced BCR-ABL1, FIP1L1-PDGFRa fusion genes and JAK2 V617F mutation by using RT-PCR. Moreover, patient has not been achieved hematologic response with tyrosine kinase inhibitors: imatinib and dasatinib. In the five cases reported three presented MD, one Acute Myeloid Leukemia and one Acute Lymphoblastic Leukemia. Only in one case report it was prescribed imatinib and the patient lost the follow up (Table1). The BCR-JAK2 fusion protein contain the coiled-coil dimerization domain of BCR and the protein tyrosine Kinase domain (JH1) of JAK2. It was not possible to define what would be the best therapy, because tyrosine kinase inhibitors may not be effective to the BCR-JAK2 fusion. Maybe in MD presentation, we could return to pre- tyrosine kinase inhibitors era based on treatments with hydroxyureia, subcutaneous cytarabine and interferon for patients that were not potential candidates for allogeneic transplant.ReferencesAgesextranslocationClinical presentationTreatmentFollow upGriesing F et al63Ft(9;22)(p24;q11.2)MDHy; Cy; MitDeathCirmena G et al67Ft(9;22)(p24;q11)AMLHD + allo BMTDeathLane SW et al44Mt(9;22)(p24;q11.2)MDNot describedNot describedElnaggar MM et al84Mt(9;22)(p24;q11.2)MDHy; ImaLost follow upTirado CA et al14Mt(9;22)(p24;q11.2)ALLPolychemotherapyRemissionHy: Hydroxyurea; Cy: Cytarabine; Mit: Mitoxantrone; HD: High dose chemotherapy; allo BMT allogeneic Bone Marrow Transplantation; Ima: imatinib; ALL: Acute lymphoblastic leukemia. Disclosures:No relevant conflicts of interest to declare.

Editorial - Signal Transduction Pathways in Chronic Inflammatory Rheumatic Diseases

Editorial - Signal Transduction Pathways in Chronic Inflammatory Rheumatic Diseases

Molecular Mechanism of Hepcidin-Mediated Ferroportin Internalization Requires Ferroportin Lysines, Not Tyrosines or JAK-STAT

Ferroportin is the primary means of cellular iron efflux and a key component of iron metabolism. Hepcidin regulates Fpn activity by inducing its internalization and degradation. The mechanism of internalization is reported to require JAK2 activation, phosphorylation of Fpn tyrosine residues 302 and 303, and initiation of transcription through STAT3 phosphorylation. These findings suggest Fpn may be a target for therapeutic intervention through JAK2 modulation. To evaluate the proposed mechanism, Fpn internalization was assessed using several techniques combined with reagents that specifically recognized cell-surface Fpn. Invitro results demonstrated that Hepc-induced Fpn internalization did not require JAK2 or phosphorylation of Fpn residues 302 and 303, nor did it induce JAK-STAT signaling. Invivo, inhibition of JAK2 had no effect on Hepc-induced hypoferremia. However, internalization was delayed by mutation of two Fpn lysine residues that may be targets of ubiquitination.

Abstract 2810: NSC 743380 induces apoptosis in sensitive cell lines through activation of tyrosine kinase 2 (Tyk2)

Abstract Several indole-3-carbinol analogs are under preclinical evaluation at the National Cancer Institute. One analog, NSC743380 (1-[(3-chlorophenyl)-methyl]-1H-indole-3-carbinol), is selectively toxic to a subset of NCI 60 cell lines in vitro and has undergone extensive in vivo testing. In vivo, NSC743380 produced complete regressions in A498 renal xenograft models at doses as low as 45 mg/kg when administered intraperitoneally. Additional studies demonstrated that NSC743380 is orally bioavailable. To extend knowledge regarding the mechanism of action, two pairs of resistant and sensitive cell lines [A498/ACHN (renal) and NCI-H226/A549 (NSCLC), sensitive/resistant, respectively] were used. Results showed that 5-10 min following NSC743380 treatment, phosphorylation of p38 and JNK were enhanced in sensitive but not resistant lines. Two hrs of treatment resulted in an inhibition of transcription and translation (45% and 75% inhibition, respectively) and by 4 hrs NSC743380 induced caspase-dependent apoptosis with loss of c-FLIP. Pathway-specific inhibitors were then used to gain mechanistic insight. Diverse antioxidants and NSAIDs, inhibitors of JNK, RAS/RAF, PPAR, lipid 2nd messenger signaling, transcription and actin polymerization were shown to completely inhibit NSC743380 activity. Microarray analysis of three sensitive cell lines using Affymetrix U133 Plus 2 chipset identified several trends in the transcriptome notably the upregulation of diverse immediate-early genes (IEGs) including; transcriptional regulators [EGR1, FOS/FOSB, HES1, MAFF and SOX9], secreted factors [HBEGF, IL-8 and GDF-15] and several general IEGs [ARC, ERRFI1, GADD45A/B, GEM and IER2]. A second noteworthy trend involved increased expression of the IL-6 family members, IL-6, IL-11 and LIF. These data led to an exploration as to whether enhanced JAK/STAT signaling was responsible for the above effects. Short time course lysates from 3 sensitive (A498, CAKI-1 and NCI-H226) cell lines and 1 resistant (A549) cell line showed that in sensitive lines only, NSC743380 rapidly (5 minutes) enhanced phosphorylation of the JAK family member Tyk2 at Y1054/55 and STAT3 at Y705. Additionally several phosphostate changes were observed in a subset of sensitive cell lines such as JAK1 phosphorylation in A498 cells and transient increase in pSTAT1 in A498 and CAKI-1 cell lines. Furthermore, two inhibitors of JAK/STAT signaling, AG490 and resveratrol, completely inhibited NSC743380 activity and p38/JNK phosphorylation. Subsequent siRNA knockdown experiments showed that Tyk2 siRNA inhibited NSC743380 activity whereas STAT3 and JAK1 siRNA along with scrambled siRNA had no effect. These data suggest that signaling through the JAK family member Tyk2 contributes towards NSC743380 activity and the data are being evaluated to determine how best to move forward. Funded by NCI Contract No. HHSN261200800001E. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2810. doi:1538-7445.AM2012-2810

Potency and Selectivity Assessment of Small Molecules Against Janus Kinase (JAK) 2: Widely Used AG490 Inhibitor Is Neither Potent Nor Selective for JAK2

Abstract 4780Janus 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.

New Insights on Assessing Intra-Family Selectivity for Jak2 Inhibitors

Abstract 5150Essential 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. [Display omitted] 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.