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Characterisation of the Janus Kinase Inhibitory Activity of Novel Kinase Inhibitor At9283

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Characterisation of the Janus Kinase Inhibitory Activity of Novel Kinase Inhibitor At9283 CHARACTERISATION OF THE JANUS KINASE INHIBITORY ACTIVITY OF NOVEL KINASE INHIBITOR AT9283 Jayne Curry, Kim Lewry, Caroline Richardson, Dominic Tisi and Nicola Wallis. Astex Therapeutics Ltd, 436 Cambridge Science Park, Milton Road, Cambridge, CB4 0QA, UK Introduction The Janus kinase family (JAKs) consists of four intra-cellular tyrosine kinases, JAK1, JAK2, JAK3 and TYK2. The JAK- Two different methods were used to investigate the mechanism of action of AT9283 in JAK2-dependent cell lines. STAT pathway is activated through specific membrane bound receptors e.g. beta common receptor. The receptors are A) The In-Cell WesternTM immunocytochemical assay developed by LI-COR optimised to detect the JAK2 specific activated by ligand induced dimerisation. The proximity of the bound JAKs enables trans-phosphorylation of multiple sites phosphorylation site of STAT5a Tyrosine 694. on the cytoplasmic domain of the receptor. B) CellSensor® irf1-bla HEL cells (Invitrogen) were used containing a beta lactamase reporter gene under control of the STAT5 response element present in the Interferon Regulatory Factor-1 (IRF-1) gene promoter that had been The major substrates of the JAKs are signal transducers and activators of transcription (STATs). These are found in stably integrated into HEL cells. Beta lactamase constitutive expression is inhibited in the presence of a JAK2 monomeric form in the cytoplasm and are recruited to these phosphorylated receptor sites where they are also inhibitor. phosphorylated by the JAKs and homodimerise becoming activated themselves. STAT homodimers translocate to the nucleus, bind to specific regulatory sequences to activate or repress transcription of target genes. Figure 3. Mechanism of action: Inhibition of STAT5 Figure 1. activation a) Reporter gene transcription assay b) In Cell Ligand Extracellular WesternTM. JAK2 JAK2 Cytoplasm P P STAT5 Negative A) B) Ras SHP2 P SOS SOCS GRB2 IC = 140nM 100 IC50= 130nM Shc STAT5 regulator 100 50 RAS-MAPK PATHWAY P P PI3K IRS STAT5 PI-3K/Akt PATHWAY STAT5 dimerisation and Cell In translocation to the nucleus 75 Tyr694 50 50 Control) P70 S6K STAT5 DMSO Control) S6 P STAT5P 25 Nucleus (%Western DMSO pSTAT5 Irf-bla HEL Cell Sensor Transcription Assay (% DNA 0 0 -3 -2 -1 0 1 2 -3 -2 -1 0 1 2 A somatic activating mutation, JAK2 V617F has been identified in a significant proportion of the Myeloproliferative Log [AT9283] (μM) Log10[AT9283] (μM) 10 Disorders (MPD). These are clonal disorders of the multipotent haematopoietic progenitor cells and this mutation was (A) HEL cells were compound treated, fixed, probed for phospho-STAT5a Tyr694 and scanned on an Odyssey found in 95% of patients with Polycythaemia Vera (PV) and 50% of patients with Essential Thrombocythaemia (ET) and scanner. (B) Irf-bla cells were incubated with compound for 16h and then a fluorescent beta-lactamase substrate Idiopathic Myelofibrosis (IMF) (Baxter et al., 2005, James et al., 2005, Kralovics et al., 2005, Levine et al., 2005). The added for 4h. mutation is located in the pseudokinase domain of JAK2 and results in constitutive activation of the JAK-STAT pathway. Subsequent studies have identified additional JAK2 activating mutations that also give rise to MPD including T875N in the kinase domain and JAK2 exon 12 mutations. The IC50s generated for inhibition of STAT5 phosphorylation and STAT5-induced transcription correlate well with the anti-proliferative IC50 of 110nM for HEL cells (Table 2) and indicate that the JAK2 inhibition may be responsible for the JAK3 has traditionally been associated with immune function and as an anti-inflammatory target however there is recent sensitivity of this cell line. evidence to show a role in some leukaemias also including transient myeloproliferative disorder (TMD) as well as in acute megakaryoblastic leukaemia of Down’s syndrome (DS-AMKL). (Malinge et al., 2008). The JAK2 and JAK3 activities of this compound were compared using TF-1 cells stimulated with IL-3 or IL-4 to stimulate through the JAK2 or JAK1 and JAK3 pathways respectively (Malabarba et al., 1995). A time course to AT9283 is a multi-targeted kinase inhibitor with potent activity against Aurora and JAK kinases. Here we show that the investigate the signaling response was used to determine the time at which the maximum downstream signal was activity of AT9283 against the isolated JAK enzymes translates into potent cellular JAK2 and JAK3 inhibition in a number obtained following each stimulation and is shown in Figure 4A, whilst a dose-response after 2h treatment is shown in of systems. Figure 4B. Results Figure 4. AT9283 inhibits JAK2 and JAK3 kinases signaling pathways in cytokine stimulated TF-1 cells. Inhibitory activity of AT9283 against selected isolated kinases is shown in Table 1 and these data show that it is a potent inhibitor of JAK and Aurora kinases. Pyridone 6, a commercially available pan-JAK inhibitor (Calbiochem #420099), is shown for comparison. A pJAK2 Tyr1007/1008 Table 1. In vitro activity of AT9283 against Aurora and JAK kinases. pSTAT5 Tyr694 Protein Inhibition pERK1/2 Ser240/244 Pyridone 6 IC (nM) AT9283 IC (nM) 50 50 ERK1/2 JAK1 0.98 84.0 Ser473 JAK2 1.1 1.2 pAkt JAK3 2.3 0.96 Akt Tyk2 2.3 2.0 pS6 Tyr240/244 Aurora B 27.0 ~3.0 S6 The crystal structure of AT9283 bound in JAK2 (Figure1) shows that AT9283 binds in the ATP site. pSTAT6 Tyr641 Figure 1. X-ray crystal structure of AT9283 complexed - + + + + - - - - - IL-3 5ng/ml with JAK2. - - - - - - + + + + IL-4 10ng/ml 0 5 20 40 60 - 5 20 40 60 Time Post Stimulation (minutes) B pSTAT6 (Tyr641) STAT6 pSTAT5 (Tyr694) (Tyr694) The anti-proliferative activities of AT9283 and Pyridone 6 were profiled against a panel of cell lines reported to have STAT5 varying dependencies on JAK2 including tumour cell lines with activating JAK mutations, stimulation through wild-type JAK-STAT pathways and non-JAK related mutations. Proliferation assays of ≥3 doubling times for each cell line were pERK1/2 (Ser240/244) used. Relative cell numbers were determined using Alamar Blue™ and results are shown in Table 2. ERK1/2 Table 2. Inhibitory Effect of AT9283 on Tumour Cell Proliferation. (Ser240/244) Cell Line Origin Mutation or Receptor Inhibition pS6 stimulation Pyridone 6 IC AT9283IC 50 50 S6 (Ser240/244) HEL * Erythroleukaemia JAK2 V617F mut/mut 300nM 110nM AML M6 MWM - + + + + + + - - - - - - - IL-3 (5ng/ml) SET2 * Essential JAK2 V617F mut/wt 53nM 57nM - - - - - - - - + + + + + + IL-4 (10ng/ml) Thrombocythaemia --1 0.3 0.1 0.03 0.01 --1 0.3 0.1 0.03 0.01 AT9283 (μM) AML M7 Ba/F3 TEL- Murine Constitutively active 180nM 17nM (A) Effects of IL-3 and IL-4 stimulation on TF-1 cells over 1h. (B) Dose response of TF-1 cells treated for 2h with JAK2 haematopoietic wtJAK2 AT9283 and stimulated for 20 minutes. Ba/F3 TEL- Murine Constitutively active 320nM 18nM JAK3 haematopoietic wtJAK3 Stimulation of TF-1 cells with IL-3 resulted in increased levels of phosphorylation of an immediate downstream substrate, STAT5 on Tyrosine 694, as well as activation of the MAPK and Akt pathways shown here by ERK1/2 and S6 ribosomal Ba/F3 wt * Murine IL-3 stimulated through IL- 380nM 25nM protein phosphorylation. These levels were reduced in the presence of 100nM - 1 M AT9283 indicating JAK2 inhibition. haematopoietic 3R and wtJAK2 μ Stimulation of TF-1 cells with IL-4 resulted in increased levels of phosphorylation of STAT6 on Tyrosine 641, but no TF-1 * Erythroleukaemia IL-3 stimulated (JAK2) 79nM 40nM activation of MAPK and Akt pathways. These levels were also reduced in the presence of 100nM - 1μM AT9283 IL-4 stimulated (JAK 1 & 3) 39nM 58nM indicating JAK3 is also inhibited by AT9283 in cells and correlating with inhibition of TF-1 proliferation (Table 2). CMK * AMKL JAK3 A572V mut 30nM 18nM K562 # CML Bcr-Abl 25%I 3uM Polyploid at 30nM Conclusions HL60 # AML M2 Ras, amplified c-Myc, 54%I 10uM Polyploid at 30nM We have established a number of cell based assays characterising the JAK2 and JAK3 * Cell lines purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) inhibitory activity of AT9283. In contrast to the Aurora B inhibited phenotype in non-JAK # Cell lines purchased from European Collection of Cell Cultures (ECACC) dependent cells, in JAK dependent cells the anti-proliferative activity correlates with the Treatment with the pan-JAK inhibitors Pyridone 6 and AT9283 resulted in anti-proliferative IC50 curves in those cell inhibition of JAK2 or JAK3 activity. lines dependent on JAK2 or JAK3 signalling. In the non-JAK dependent K562 and HL60 cell lines Pyridone 6 was relatively AT9283 is currently in Phase I clinical trials for solid and haematological malignancies. insensitive and AT9283 treatment resulted in a biphasic curve and observations These data indicate AT9283 may be beneficial in cancers where JAK2 or JAK3 activation has of polyploid cells at 30nM indicating an Aurora B inhibition phenotype (Ditchfield a role in the disease pathology et al., 2003). These data indicate the JAK activity of these compounds give the dominant cellular phenotype in JAK dependent cell lines. .
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