Cooperative effects of Janus kinase (Jak) and non Jak-targeting kinase inhibitors in cells expressing constitutively active Jak-mutants.

Transcription

1 Cooperative effects of Janus kinase (Jak) and non Jak-targeting kinase inhibitors in cells expressing constitutively active Jak-mutants.

2 Cytokine signaling CBL Signaling pathways Receptor Ras Raf-1 MEK-1 Jak SOS Grb2 Gab1 I3K ERK AKT TF STAT STAT C Negative regulation of signaling pathways T SOCS feedback inhibitors C SOCS Internalisation rotein degradation Cytokines ( 4) Cytokine receptors (min. 36) 4 Jak : Janus kinase (Jak1/2/3, Tyk2) 7 STAT : Signal transducer and activator of transcription (STAT1-4/5A/5B/6) 8 SOCS: Suppressor of cytokine signaling (CIS, SOCS1-7) Additional pathways are activated to different extent (e.g. MAK-, I3K/Akt pathway) TF TF T Additional negative regulators are activated (hosphatases, IAS, CBL, LNK,...)

3 Janus kinase (Jak) structure/function Jak JH7 JH6 JH5 JH4 JH3 JH2 JH1 (F1) (F2) (F3) FERM-domain SH2 seudokinase domain Kinase domain divergent FERM domain non classical SH2 domain Haan et al. (21), J. Cell. Mol. Med. 14: (Review)

4 Wild type Jak activation at the receptor Conformational Jak activation upon cytokine binding (independent of phosphorylation). Activation by phosphorylation (stabilisation of the activated state). Activation by phosphorylation in «Trans» (Jak1 phosphorylates Jak3; IL2 1a Jak3 phosphorylates Jak1). IL2 IL2 Jak1 Jak3 Jak1 Jak3 3a Jak1 Jak3 3b Jak1 Jak3 1b 2 4 : inactive Jak : active Jak (phosphorylation independant) : fully active phosphorylated Jak red arrows: phosphorylation event blue arrows: conformational event STAT5 STAT5 Haan et al. (211), Chem. Biol., 18(3):

5 Constitutively activated Jaks Need a cytokine receptor to have transforming potential (except the «Jak-fusionkinases»(Tel-Jak)) Conformational activation upon cytokine binding still increases downstream signaling. Activation by phosphorylation in «Trans». IL2 IL2 IL2 IL2 Jak1 V658F Jak3 Jak1 V658F Jak3 Jak1 V658F Jak3 A572V Jak1 V658F Jak3 A572V Increased STAT Increased STAT STAT5 STAT5 + Jak hosph. STAT5 STAT5 + Jak hosph. : constitutively active Jak : fully active phosphorylated Jak WT red arrows: phosphorylation event blue arrows: conformational event Haan et al. (211), Chem. Biol., 18(3):

6 Jak mutations in human disease Example: Jak2 More and more Jak inhibitors are developed for different indications Haan et al. (21), J. Cell. Mol. Med. 14: (Review)

7 Jak inhibitors Myeloproliferative neoplasms 2 pyrrolopyrimidine compounds are approved by thefda: Leukemia (myeloid, lymphoid) Transplant rejection Rheumatoid arthritis siorasis C69,55; Tofacitinib; eljanz (Rheumatoid arthritis) INCB18424; Ruxolitinib, Jakafi (Myelofibrosis)

8 Cyt. Family Cytokine Jak1 Jak2 Jak3 Tyk2 IL6 x x IL11 x x LIF x x IL6/IL12 CNTF x x superfamily CLC x x of cytokines CT1 x x OSM x x IL31 x x IL27 x x IL35 x x IL12 IL23 GCSF Leptin rolactin Homodimeric TO receptor EO using cytokines GH IL2 IL4 IL7 IL2 family IL9 of cytokines IL15 IL21 TSL IL13, IL4 IFNa (b,d,e,k,t,w) IFNg IFNl 1-3 (IL28 A/B /29) IL1 family of IL1 cytokines IL19 IL2 IL22 IL24 IL26 common l chain IL3 using cytokines IL5 GM-CSF Cytokines, Jaks and Jak inhibitors Specific Jak inhibitors are sought for a number of clinical applications. Thus it is important to know: which Jak to inhibit. which other cytokine pathways this will affect (side effects!) What is the hierarchy of the different Jaks in signaling through a given cytokine (Jaks in heterodimeric systems are not necessarily of equal importance!)

9 Cyt. Family Cytokine Jak1 Jak2 Jak3 Tyk2 IL6 x x IL11 x x LIF x x IL6/IL12 CNTF x x superfamily CLC x x of cytokines CT1 x x OSM x x IL31 x x IL27 x x IL35 x x IL12 IL23 GCSF Leptin rolactin Homodimeric TO receptor EO using cytokines GH IL2 IL4 IL7 IL2 family IL9 of cytokines IL15 IL21 TSL IL13, IL4 IFNa (b,d,e,k,t,w) IFNg IFNl 1-3 (IL28 A/B /29) IL1 family of IL1 cytokines IL19 IL2 IL22 IL24 IL26 common l chain IL3 using cytokines IL5 GM-CSF Jak1 and Jak3 in IL2 signal transduction Jak1 IL2 Jak3 STAT5 Jak3mut Jak3 KO Severe combined Immunodeficiency (SCID) Jak3, a target for immunosuppressive therapy? Jak3 only expressed in haematopoietic cells Only involved in IL2 type cytokine signaling IL2 type cytokines regulate lymphopoiesis

10 A Jak3-specific inhibitor inefficiently inhibits IL2 signal transduction In vitro kinase assays: Jak inhibitor 1 C69,55 NIBR349 Jak1 Jak2 Jak3 Tyk Legend: IC5 < 1 nm 1 nm < IC5 < 2 nm 2 nm < IC5 < 5 nm 1 nm < IC5 IC 5 of STAT hosph: Jak inhibitor 1 C69,55 NIBR349 Jak3mut. (CMK) IL2 (Kit225) IL2 (CTLL2) IL2 (BL) IL4 (BL) IL15 (BL) IL15 (Mo7e) IC 5 (nm) 2-5-fold higher concentrations of NIBR349 needed to suppress IL2 signaling compared with pan-jak inhibitors, despite similar potencies in inhibition of Jak3 kinase activity, What is the relative contribution of Jak1 and Jak3 to signal transduction of gc cytokines? Haan et al. (211), Chem. Biol., 18(3):

11 -STAT5 (% vs. scr.) WT-Jaks Jak3 inactivation is insufficient to abrogate IL2 signaling Knock-down approach Jak1 KO Jak3 KO Dominant negative approach Jak1 DN Jak3 DN Analogue sensitive approach AS-Jak1 AS-Jak3 Jak1 Jak3 Jak1DN Jak3 Jak1 Jak3DN as-jak1 Jak3 Jak1 as-jak3 1% % % % 55% 1% 1% Kit 225 (IL2) sirna: scr. Jak1 Jak3 scrambled Jak1 Jak3 Summary: Jak3 Knock down: abrogates signaling Kinase deficient Jak3: reduced but efficient signaling harmacological inhibition of Jak3: ineffective in reducing signaling Jak1AS 1NM-1: IL-2: STAT5 STAT5 -Jak1 -Jak3 Jak1 Jak3 Jak3AS Haan et al. (211), Chem. Biol., 18(3):

12 Cyt. Family Cytokine Jak1 Jak2 Jak3 Tyk2 IL6 x x IL11 x x LIF x x IL6/IL12 CNTF x x superfamily CLC x x of cytokines CT1 x x OSM x x IL31 x x IL27 x x IL35 x x IL12 IL23 GCSF Leptin rolactin Homodimeric TO receptor EO using cytokines GH IL2 IL4 IL7 IL2 family IL9 of cytokines IL15 IL21 TSL IL13, IL4 IFNa (b,d,e,k,t,w) IFNg IFNl 1-3 (IL28 A/B /29) IL1 family of IL1 cytokines IL19 IL2 IL22 IL24 IL26 common l chain IL3 using cytokines IL5 GM-CSF Jak1 and Jak2 are crutial for IFNg signal transduction Jak1 IFNg Jak2 STAT1 Both Jak1 and Jak2 are essential in: The Knock down approach The dominant negative Jak approach The analog sensitive Jak approach

13 Cyt. Family Cytokine Jak1 Jak2 Jak3 Tyk2 IL6 x x IL11 x x LIF x x IL6/IL12 CNTF x x superfamily CLC x x of cytokines CT1 x x OSM x x IL31 x x IL27 x x IL35 x x IL12 IL23 GCSF Leptin rolactin Homodimeric TO receptor EO using cytokines GH IL2 IL4 IL7 IL2 family IL9 of cytokines IL15 IL21 TSL IL13, IL4 IFNa (b,d,e,k,t,w) IFNg IFNl 1-3 (IL28 A/B /29) IL1 family of IL1 cytokines IL19 IL2 IL22 IL24 IL26 common l chain IL3 using cytokines IL5 GM-CSF Cytokines, Jaks and Jak inhibitors Specific Jak inhibitors are sought for a number of clinical applications. The different hierarchy of Jaks signaling as heterodimers might lead to less «off-cytokine-target» effects than initially expected

14 Jak2V617F in Myeloproliferative neoplasms (MN) Jak2 V617F hematopoietic stem cell common myeloid progenitor common lymphoid progenitor granulocytes erythrocyte thrombocytes monocyte T cell B cell NK cell olycythemia Essential rimary vera 1% 4% thrombocythemia myelofibrosis Jak2V617F 95% 35% 5% 5% 55% 5% 5% 25% 1% 55% Jak2 exon 12 TpoR exon 1? LNK CBL Gäbler et al. (213), JakSTAT, in print (Review)

15 Characterisation of a small panel of proposed Jak targetting inhibitors Aims: 1. Characterise the described Jak inhibitors in cellular assays (hosphorylation of signaling components (Jaks, STATs), Growth assay) 2. Identify compounds which do not target Jak2 and might be combined with Jak inhibitors How do we try to achieve these aims? 1. Define adequate «postive controls» to quantitate Jak inhibitory activity 2. Develop assays which measure Jak activation: - robustly - quickly (without lengthy treatments)(fluorescent readout) - in intact cells - and which are medium to high throughput compatible

16 Defining adequate positive controls in cellular assays ossible signals to monitor Jak activation: 1. Jak phosphorylation 2. STAT phosphorylation ossible assay formats to monitor Jak activation: 1. SOCS protein membrane recruitment 2. STAT transcriptional activity (Reporter gene assay) 3. STAT translocation into the nucleus upon phosphorylation

17 HEL (Jak2V617F) AT9283 CE71 TG1129 JI1 TG11348 CT387 M Jak2 Jak2 -STAT5 STAT5 Jak hyper -phosphorylation upon inhibition with AT competitive inhibitors -STAT5 C69, CMK (Jak3A572V) Jak inhib. I C69,55 TG1129 AZ96 M Jak3 Jak3 -STAT5 STAT5 All tested Jak inhibitors induce hyper -phosphorylation of Jak2V617F upon inhibition This effect also occurs upon inhibition of Jak3A572V and is thus not Jak2 specific Gäbler et al. (213), JCMM, 17(2): Rolvering C. and Kaczor J., unpublished

18 Jak hyper -phosphorylation upon inhibition with AT competitive inhibitors hyper -phosphorylation is stable over time effect reproducible in all cells types expressing Jak2V617F effect is also observed for exon 12 mutations (Jak2K539L) I: Jak2: Time dependance JI1 (h) Jak2 -Jak2 Jak2 - Immunoprecipited Jak shows hyperphosphorylation too Other groups also describe the effect: Grandage et al 26; Hart et al 211; Andraos et al 212 Andraos et al., 212: Type I-Jak inhibitors induce hyperphosphorylation Type II-Jak inhibitors do not induce hyperphosphorylation Gäbler et al. (213), JCMM, 17(2): Haan et al. (211), Chem. Biol., 18(3):

19 Defining adequate positive controls in cellular assays for the establishment of the screening assays ossible signals to monitor Jak activation: 1. Jak phosphorylation (due to Jak hyperphosphorylation upon Type I inhibitor binding) 2. STAT phosphorylation ossible assay formats to monitor Jak activation: 1. SOCS protein membrane recruitment (SOCS1/3 bind -Jak2) 2. STAT transcriptional activity (Reporter gene assay) 3. STAT translocation into the nucleus upon phosphorylation SOCS 1-F translocation assay Jak2V617F membrane localisation Jak2V617F dependant SOCS1-F membrane translocation Exogenous SOCS1-F Jak2 V617F Haan et al. (29), Oncogene. 28(34):

20 Assay I: ZsGreen reporter gene assay Jak2 V617F Cells stably transfected with reporter gene construct Cells stably transfected with doxycycline-inducible Jak2V617F Jak2 V617F STAT3 Jak inhibitor STAT3 ZsGreen ZsGreen ZsGreen ZsGreen TL TL 12 x STAT-binding site ZsGreen gene ZsGreen transcription 12 x STAT-binding site ZsGreen gene Gäbler et al. (213), JCMM, 17(2):

21 Assay I: STAT-ZsGreen reporter gene assay counts % ZsGreen-positive cells 2 possible assay formats: harvesting flow cytometry inhibition curves determination of IC 5 values ZsGreen-positive cells JI1 TG1129 TG11348 log(intensity) 5 cell lysis fluorescence measurement in plate reader log(concentration) Advantages: Live cell assay (FACS version) No complex and lenghthy treatments Fast measurement Good reproducibility Allows detection of Type II and allosteric inhibtiors Gäbler et al. (213), JCMM, 17(2):

22 Assay II: STAT3-F translocation assay Jak2 V617F STAT3- F Stably transfected cell line (Jak2V617F + STAT3-F) Doxycycline-inducible expression of Jak2V617F and STAT3-F Jak inhibitor Jak2 V617F STAT3-F nuclear translocation 1 m 1 m STAT3-F STAT3-F nucleus 1 m 1 m nucleus Hoechst Hoechst Gäbler et al. (213), JCMM, 17(2):

23 2 nm 437 nm Assay II: STAT3-F translocation assay relative fluorescence Measurement: staining of nuclei confocal microscopy analysis of images by software (e.g. Cell rofiler) overlay of F signal with nuclei calculation of amount of STAT3 localized in nuclei TG1129 treatment Advantages: Live cell assay No complex and lenghthy treatments Fast measurement Allows detection of Type II and allosteric inhibtiors JI1 TG1129 TG11348 nuclear staining STAT3-F 1.2 nuclear staining STAT3-F log(concentration) Gäbler et al. (213), JCMM, 17(2):

24 Characterisation of the panel of Jak inhibitors: 6 out of the 12 compounds do not inhibit Jak(2) 12 compounds which are claimed to target Jak(2) and/or MN patient cells small-molecule drugs (supposed to be) AT-competitive Jak2 V617F model cell line: human erythroleukemia (HEL) cells positive for Jak2V617F mutation constitutively activated STAT5 STAT5 phosphorylation state of STAT5 capacity of compounds to inhibit Jak2(V617F) M -STAT5 STAT5 AT CE TG JI TG CT Sunitinib V W AG49 LFM- A13 JNJ Jak-targeting inhibitors No Jak inhibitors Gäbler et al. (213), JCMM, 17(2):

25 % proliferation Some of the proposed Jak inhibitors do not target Jak2 but suppress cell growth. roliferation of HEL cells Jak-targeting inhibitors No Jak inhibitors 125 AT 9283 CE 71 TG 1129 JI1 TG CT 387 Sunitinib V68 W 166 AG49 LFM- A13 JNJ no growth inhibition M V68 efficiently reduces cell proliferation although not inhibiting Jak2 IC 5 s of all inhibitors in HEL growth assays were determined. Gäbler et al. (213), JCMM, 17(2):

26 % STAT activation % STAT activation % STAT activation Quantitation of STAT5-hosphorylation by Western blot served as positive control % STAT activation % STAT activation % STAT activation JI1 1 TG1129 Inhibitor (nm) 5 5 -STAT5 STAT5 TG log(conc) log(conc) -STAT5 STAT5 CE71 1 TG CE71 -STAT5 STAT5 JI STAT5 STAT5 AT log(conc) log(conc) -STAT5 STAT5 TG CT387 1 AT9283 -STAT5 STAT5 CT log(conc) log(conc) Gäbler et al. (213), JCMM, 17(2):

27 IC 5 values Detailed analysis & comparison not determined IC 5 values not determined IC 5 values IC 5 values not determined Determination of IC 5 values for the Jak targeting inhibitors in: - the novel assays, - WB detection of -STAT5 (HEL) - proliferation assays (HEL) 1 STAT3-F translocation 1 STAT reporter gene 1 Western Blot -STAT Conclusions: Compounds with no Jak inhibitory potential are detected by a combination of both the reporter gene assay and the F-STAT translocation assay proliferation The differences in IC5s of specific Jak inhibitors are reproducibly detectable in all assays. 5 Deviations could interestingly be attributed to additional activities (AT9283, CE71) Gäbler et al. (213), JCMM, 17(2):

28 Does CE71 have Aurora kinase inhibitory potential? STAT3-F translocation assay Aurora kinase regulation of mitosis & cytokinesis AT9283 V68 CE71 TG1129 Aurora borealis Aurora kinase A 2 nm 18 nm 162 nm increase of nuclei size 486 nm Aurora borealis = polar light Aurora kinase during mitosis known to also target Aurora kinases CE71??? inhibition of Aurora kinase suppression of cell proliferation Gäbler et al. (213), JCMM, 17(2):

29 CE71 has Aurora kinase inhibitory potential. Cell cycle analysis Confirmation in various assays increase of nuclear size endoreduplication demonstrated in cell cycle analysis validation as target in in vitro kinase assays reduction of histone H3 phosphorylation Western Blot: hospho-histone H3 AT9283 V68 CE71 TG1129 M HisH3 HisH3 CE71 also targets Aurora kinases in cellular assays Gäbler et al. (213), JCMM, 17(2):

30 Summary of important compound characteristics V68 inhibition of Jak2 inhibition of Aurora kinase! AT9283 CE71! <!!! TG1129! Gäbler et al. (213), JCMM, 17(2):

31 % colonies % colonies % colonies Jak inhibitors preferentially suppress erythroid colonies. Aurora inhibitors preferentially suppress granulomonocytic colonies colony forming cell (CFC) assays with CD34 + cells isolated from MN patients JI1 Janus kinase inhibitors Aurora kinase inhibitors TG1129 TG11348 CE71 AT9283 V concentration (nm) AT in detail V CE71 suppresses erythroid and myeloid colonies equally well concentration (nm) concentration (nm) Gäbler et al. (213), JCMM, 17(2):

32 % colonies Jak and Aurora kinase inhibitors cooperate to suppress growth of Jak2V617F-expressing cells. % colonies mimic effect of CE71 (dual function) combination of single compounds targeting Jak2 (TG1129/TG11348) and Aurora kinase (V68) TG1129 (nm) nm V68 5 nm V68 1 nm V TG (nm) nm V68 1 nm V68 Erythroid colonies Granulo-monocytic colonies 2 different types of inhibitors conserve their function when applied in combination combination of both inhibitor types suppressed growth of colonies more efficiently Gäbler et al. (213), JCMM, 17(2):

33 Jak and Aurora kinase inhibitors synergise to suppress growth of Jak2V617F-expressing cells. % proliferaton % % proliferation proliferaton Growth of BAF3-EpoR-Jak2V617F cells TG ( M) V68 ( M) CI values TG1129 V68 TG V68 Gäbler et al. (213), JCMM, 17(2):

34 Summary detailed, quantitative & comparative analysis of Jak2 inhibitors development of 2 new cellular assays to screen for Jak2 inhibitory activity 6 out of 12 compounds showed no inhibitory activity against Jak2V617F AT9283 (which inhibits Jak2) has much higher activity against Aurora kinases and the cellular effects are unlikely due to Jak2 inhibition. CE71 displays equipotent inhibitory activity against Jaks and Aurora kinases Combined application of Janus and Aurora kinase inhibitor novel option to test in further studies Gäbler et al. (213), JCMM, 17(2):

35 THANK OU Signal Transduction uni.lu Collaborators: rof. Iris Berhmann Dr. Stephanie Kreis Dr. Christiane Wurth-Margue Demetra hilippidou Susanne Reinsbach Martina Schmitt Andreas Zimmer Funding: Dr. Karoline Gäbler Catherine Rolvering Dr. Jakub Kaczor Dr. Guy Berchem Dr. Valérie alissot Audrey Lemasson Dr. Hans Günter Zerwes Dr. Gebhard Thoma Dr. Friedrich Raulf Dr. eter Drückes Dr. Manela Kapp rof. Fred Schaper Alexandra Wolf Dr. René Eulenfeld