TG101209, a small molecule JAK2-selective kinase inhibitor potently inhibits myeloproliferative disorder-associated JAK2V617F and MPLW515L/K mutations

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1 ORIGINAL ARTICLE (27), 1 11 & 27 Nature Pulishing Group All rights reserved /7 $3. TG1129, a small moleule JAK2-seletive kinase inhiitor potently inhiits myeloproliferative disorder-assoiated JAK2V617F and MPLW515L/K mutations A Pardanani 1, J Hood 2, T Lasho 1, RL Levine 3, MB Martin 2, G Noronha 2, C Finke 1, CC Mak 2, R Mesa 1, H Zhu 2, R Soll 2, DG Gilliland 3,4 and A Tefferi 1 1 Division of Hematology, Mayo Clini, Rohester, MN, USA; 2 Researh and Development, TargeGen In., San Diego, CA, USA; 3 Brigham and Women s Hospital and the Dana-Farer Caner Institute, Harvard Medial Shool, Boston, MA, USA and 4 Howard Hughes Medial Institute, Harvard Medial Shool, Boston, MA, USA JAK2V617F and MPLW515L/K represent reently identified mutations in myeloproliferative disorders (MPD) that ause dysregulated JAK-STAT signaling, whih is impliated in MPD pathogenesis. We developed TG1129, an orally ioavailale small moleule that potently inhiits JAK2 (IC 5 ¼ 6nM), FLT3 (IC 5 ¼ 25 nm) and RET (IC 5 ¼ 17 nm) kinases, with signifiantly less ativity against other tyrosine kinases inluding JAK3 (IC 5 ¼ 169 nm). TG1129 inhiited growth of Ba/F3 ells expressing JAK2V617F or MPLW515L mutations with an IC 5 of B2 nm. In a human JAK2V617F-expressing aute myeloid leukemia ell line, TG1129-indued ell yle arrest and apoptosis, and inhiited phosphorylation of JAK2V617F, STAT5 and STAT3. Therapeuti effiay of TG1129 was demonstrated in a nude mouse model. Furthermore, TG1129 suppressed growth of hematopoieti olonies from primary progenitor ells haroring JAK2V617F or MPL515 mutations. advane online puliation, 31 May 27; doi:1.138/sj.leu Keywords: myeloproliferative disorder; mutation; JAK2V617F; kinase inhiitor Introdution Aquisition of somati mutations suh as JAK2V617F 1 results in onstitutive ativation of JAK-STAT signaling, whih is thought to play a primary role in the pathogenesis of myeloproliferative disorders (MPD) inluding polyythemia vera (PV), essential thromoythemia (ET) and primary myelofirosis (PMF). In normal hematopoiesis, ligand-indued ativation of a spetrum of hematopoieti ytokine reeptors, inluding reeptors for erythropoietin, thromopoietin (MPL) and granuloyte olony stimulating fator, onverges upon Janus kinase 2 (JAK2). The importane of JAK2 in hematopoiesis has een demonstrated in mie that are genetially defiient in JAK2, and have severe defets in erythropoiesis. 2 Dysregulated JAK-STAT signaling may e important in JAK2V617F-negative MPD as well, in that other ativating JAK2 alleles have een identified in these patients, inluding JAK2 exon 12 mutations, JAK2D62E, JAK2DIREED, in addition to ativating mutations in MPL at position W Expression of JAK2V617F in vivo in a murine one marrow transplant assay results in a phenotype resemling PV; 7,8 in ontrast, MPLW515L expression in a similar assay results in a PMF-like phenotype. 9 Inhiition of JAK2 with small moleule tool ompounds that lak potential for linial development, and that are not seletive among JAK family memers, indue apoptoti ell death in hematopoieti ell lines transformed Correspondene: Professor A Tefferi, Division of Hematology, Mayo Clini, 2 First Street SW, Rohester, MN 5595, USA. tefferi.ayalew@mayo.edu Reeived 12 April 27; aepted 18 April 27 either with JAK2V617F 1 or with MPLW515L, 9 and, similarly, may derease the hematorit in mouse models of JAK2V617Findued disease. 8 MPD are urrently not aptured in the surveillane, epidemiology and end results (SEER) dataase or other aner registries, ut inidene of PV, ET and PMF has een estimated in the 1 5/1 per year range. 11 Beause of relatively long survival after diagnosis, it has een estimated that the prevalene of MPD is on the order of 8 1 ases in the United States, signifiantly higher than that of BCR-ABLpositive hroni myeloid leukemia (CML). Although the MPD are relatively indolent, most patients ultimately develop one or more ompliations related to their disease, inluding thromosis, hemorrhage, massive splenomegaly, or progression to aute leukemia. Current therapy is empirially derived, and not without attendant side effets; most patients are not andidates for stem ell transplantation given their advaned age at the time of diagnosis. We developed TG1129, a small moleule, seletive JAK2 kinase inhiitor using struture-ased drug design. We present results from pre-linial studies showing that TG1129 is a potent inhiitor of JAK2V617F and MPLW515L mutations, oth in vitro and in vivo. Materials and methods Reagents TG1129 (N-tert-utyl-3-(5-methyl-2-[4-(4-methyl-piperazin-1-yl)- phenylamino]-pyrimidin-4-ylamino)-enzenesulfonamide) was synthesized y TargeGen In. (San Diego, CA, USA). Stok solutions were made in dimethylsulfoxide (DMSO), and susequently diluted in RPMI-164 medium for use. Anti-phospho- Jak2 [Tyr17/18] (polylonal), anti-jak2 [24B11] (monolonal), anti-phospho-stat5 [Tyr694] (polylonal), anti-stat5 (polylonal), anti-phospho-stat3 [Tyr75] (monolonal) and anti-stat3 [124H6] (monolonal) were purhased from Cell Signaling (Beverly, MA, USA), and anti--atin (monolonal) from Novus Biologials In. (Littleton, CO, USA). Moleular Modeling See Supplementary Information. Cell lines Human erythroleukemia (HEL), Ba/F3, CTLL-2 and normal human dermal firolasts (NHDF) ells were purhased from Amerian Type Culture Colletion (Rokville, MD, USA). Ba/F3 ells expressing JAK2V617F (Ba/F3-EpoR-V617F) 12 and

2 2 TG1129 inhiits JAK2V617F and MPLW515L/K MPLW515L (Ba/F3-W515L) 9 mutants, and the human leukemia ell lines HEL, K562, CHRF and CMK were ultured in RPMI-164 medium (Gio BRL, Gaithesurg, MD, USA), supplemented with 1% fetal ovine serum (FBS), peniillin, streptomyin and L-glutamine at 371C and 5% CO 2. CTLL-2 and Ba/F3 ells were grown in the same media further supplemented with 2 U/ml reominant mouse IL-2 and 1 ng/ml IL-3, respetively (Hoffmann LaRohe, Nutley, NJ, USA). Green fluoresent protein (GFP) was introdued into Ba/F3-V617F ells y lentiviral transdution using plenti6-gfp (Invitrogen, Carlsad, CA, USA), followed y seletion with lastiidin and onfirmation of GFP expression using flow ytometry analysis (Ba/F3-V617F-GFP). Cell-free kinase ativity assays (IC 5 determinations) see Supplementary Information. XTT assay for ell proliferation In rief, approximately ells were plated into mirotiterplate wells in 1 ml RPMI-164 growth media with indiated onentrations of inhiitor. The relative growth of ells was quantified at 24-h intervals using Cell Proliferation Kit II (XTT) as per manufaturer s guidelines (Rohe, Indianapolis, IN, USA). After inuation, 2 ml of XTT was added to the wells and allowed to inuate for 4 6 h. The olored formazan produt was measured spetrophotometrially at 45 nm with orretion at 65 nm, and IC 5 values were determined using the GraphPad Prism 4. software. Data were sujeted to a non-linear regression-fit analysis and IC 5 values were determined as the onentration, that inhiited proliferation y 5%. All experiments were done in tripliate and the results normalized to growth of untreated ells. Cell yle and apoptosis assays Cell yle was analyzed with the FITC BrdU Kit (BD Pharmingen, San Diego, CA, USA), as per manufaturer s instrutions. Cells were inuated with indiated onentrations of inhiitor for 24 h, and susequently pulsed with BrdU for 3 min at 371C. The ells were washed in staining uffer (1 Duleo s phosphate-uffered saline (DPBS) þ 3% FBS), fixed/ permeailized with Cytofix/Cytoperm uffer and washed with Perm/Wash uffer, with all inuations arried out on ie. After permeailization, ells were treated with 3 mg DNAse for 1 h at 371C, and then stained with FITC-onjugated anti-brdu antiody and 7-AAD efore flow ytometri analysis. DNA ontents were analyzed with the use of a FACS Canto flow ytometer with FACS Diva software (BD Biosienes, San Jose, CA, USA). Indution of apoptosis of HEL and K562 ells was determined y analyzing the inding of Annexin V and the inorporation of PI, following inuation with indiated onentrations of inhiitor for, 24, 48 and 72 h. The ells were washed with 1 PBS, resuspended in apoptosis uffer (1 mm HEPES/NaOH, ph, 7.4, 14 mm NaCl and 2.5 mm CaCl 2 ) and stained with 5 ml a JAK2 FLT3 RET JAK3 IC 5 (nm) Figure 1 Moleular struture of TG1129 and its seletive inhiition of JAK2 kinase. (a) Chemial struture of TG1129 (moleular formula, C 26 H 35 N 7 O 2 S; moleular weight, 59.7; melting point 2431C). () Moleular model depiting doking of TG1129 in the JAK2 ATP poket. The shaded surfae illustrates hydrophoi (red) and hydrophili (lue) portions of the protein. Key inhiitor protein interations, inluding the hydrogen ond with the hinge Leu932 residue, the hydrophoi ontats in the shallow angular poket lined y residues Met929, the methylene groups of Lys882, and the initial portion of the DFG (AspPheGly) (Asp994 shown) ativation loop, as well as the hydrogen ond with the NH of the sulfonamide from TG1129 with Asn981 are shown. () TG1129 inhiitory ativity (IC 5 ) against selet kinases in the in vitro kinase assay.

3 Annexin V APC (BD Biosienes, San Diego, CA, USA) for 15 min at room temperature. Cells were resuspended in Apoptosis Buffer and 5 ml of PI was added. Samples were analyzed on a FACSaliur ytometer (BD Biosienes, San Jose, CA, USA). Western lot analysis Cells were treated with inhiitor for 18 h in RPMI-164 efore harvesting in 1 Cell Lysis Buffer (Cell Signaling, Beverly, MA, USA), supplemented with 1 mm PMSF, and omplete protease inhiitor oktail talets (Rohe). Lysates were larified y entrifugation and protein was quantified with the Piere Biotehnology BCA assay (Rokford, IL, USA). Equal amounts of protein were omined with Laemmli sample uffer (Bio-Rad Laoratories, Herules, CA, USA) plus -meraptoethanol, oiled for 5 min, and loaded onto 4 15% Tris HCl gradient eletrophoresis gels (Bio-Rad Laoratories). Gels were transferred to.45 mm trans-lot nitroellulose memrane (Bio-Rad), the memrane loked in 5% nonfat dry milk, and lotted overnight with primary antiodies diluted in either loking solution or 5% ovine serum alumin. After washing, the primary antiodies were revealed using the appropriate horseradish peroxidase (HRP)-onjugated seondary antiody (Cell Signaling) and deteted y the Phototope hemiluminesene kit (Cell Signaling). In onjuntion, lots were proed with anti--atin antiody from Novus Biologials In. to onfirm equal loading of protein. Murine tumor model and drug treatment effet Severe omined immunodefiieny (SCID) mie (Harlan, Indianapolis, IN, USA) were intravenously injeted with sorted GFP-positive BaF/3 ells expressing JAK2V617F (Ba/F3-V617F-GFP). TG1129 was administered y oral gavage at the indiated doses eginning day þ 3 after tumor ell infusion and ending on day þ 2. On day þ 11 following TG1129 inhiits JAK2V617F and MPLW515L/K tumor ell injetion, 1 ml lood was olleted y terminal ardia leeding from the mouse that reeived vehile, and.1 ml of lood was olleted y non-lethal retro-orital olletion from eah of the three six-mouse groups dosed with 1, 3 or 1 mg/kg.i.d. (twie daily) of TG1129, and samples pooled within the dose groups. Blood mononulear ells were isolated y a Fioll (Sigma-Aldrih, St Louis, MO, USA) ushion entrifugation method (6 RCF and 3 min). The isolated ells were sujeted to FACS analysis to determine the perentage of GFP-positive tumor ells (that is, Ba/F3-V617F-GFP ells). In a parallel study, two additional animals were treated as desried aove with the exeption that they were given a single 1 mg/kg dose of drug on day 11 and humanely killed 7 h later for analysis of STAT5 phosphorylation in the tumor-earing spleen. Spleens were homogenized in a FastPrep mahine (Qiogen, Irvine, CA, USA). Susequently, 1 mg of eah spleen homogenate was sujeted to eletrophoreti separation and the protein lot was proed with an antiphospho-stat5 (Tyr694/699) (Upstate), as well as with an anti- STAT5 antiody (Cell Signaling), and visualized y the enhaned hemoluminesene method. Patient arual and sample olletion The urrent study was approved y the Mayo Clini Institutional Review Board. All patients provided veral and written informed onsent, and researh was arried out aording to the priniples of the Delaration of Helsinki. Clonogeni assays/single-olony analysis CD34 þ ells, peripheral lood mononulear ells (PBMC), and granuloytes were isolated as desried previously 13 Flow ytometry analysis has shown CD34 þ ell purity to e at least 95% in our hands. Freshly isolated CD34 þ ells were plated in a 3 a % Control BAF3 jak2v617f d % Control TG1129 (M) HEL K TG1129 (M) % Control e % Control BAF3 mplw515l TG1129 (M) CMK CHRF TG1129 (M) % growth relative to ontrol TG1129(uM) CTLL-2 Figure 2 TG1129 inhiits JAK2V617F- and MPLW515L-dependent ell proliferation, and seletively inhiits JAK2 versus JAK3 kinasedependent ell proliferation. Dose response urves of (a) Ba/F3 ells staly expressing JAK2V617F (IC 5 ¼ 17 nm), () MPLW515L (IC 5 ¼ 22 nm), or () CTLL-2 ells (IC 5 ¼ 34 nm). The perentage of growth, relative to the growth of ells in the asene of drug, is plotted for inreasing onentrations of TG1129 ( nm). (d) Dose response urves omparing HEL ells ( ) and K562 ells (m) with inreasing onentrations of TG1129 (IC 5 ¼ 152 and 2 nm for HEL and K562 ells, respetively). (e) Dose response urves omparing CHRF ells (JAK2T875N-positive) (m) and CMK ells (JAK3A572V-positive) ( ) with inreasing onentrations of TG1129 (IC 5 ¼ 16 and 42 nm for CHRF and CMK ells, respetively).

4 4 methylellulose-ased, semisolid medium (MethoCult media, Stem Cell Tehnologies, Vanouver, BC). Clonogeni assays were set up in dupliate or tripliate (atalog# H4435), as per manufaturer s guidelines; CD34 þ ells were plated for ytokinesupported olony growth. The plates were inuated at 371C in a 5% aron dioxide and 95% air mixture for 1 12 days, and olonies were sored using standard morphologial riteria. In the presene of TG1129, only appreialy hemogloinized olonies were sored as erythroid. Individual well-separated olonies were harvested for DNA sequening as desried previously. 14 Genotyping of mutant alleles was performed y diret DNA sequening as desried previously for JAK2V617F 15 and MPL Results TG1129 inhiits JAK2V617F and MPLW515L/K TG1129 is a seletive JAK2 kinase inhiitor in vitro We utilized rational struture-ased tehniques to design and optimize a new series of pyrimidine-ased inhiitors to target JAK2. We started from a B5 mm IC 5 hit, and made use of two rystal strutures availale in the literature (2B7A.pd for JAK2 and 1YVJ.pd for JAK3) 17,18 to design moleules guided y moleular modeling. With this approah, the series was rapidly optimized to yield low-nanomolar (nm) IC 5 onentration inhiitors, and TG1129, whih is one suh JAK2-seletive inhiitor, was hosen for further haraterization. The moleular struture of the small moleule inhiitor TG1129 is shown in Figure 1a. A moleular model showing TG1129 doked and minimized in the ATP poket of JAK2 kinase, highlighting the key interations is shown in Figure 1. The speifiity of TG1129 was tested against a wide range of kinases using purified proteins. TG1129 inhiited JAK2, FLT3, RET and JAK3 kinases with an IC 5 of nm in in vitro kinase assays (Figure 1). TG1129 was most ative against JAK2 (IC 5 ¼ 6nM), and exhiited seletivity for JAK2 relative to JAK3 (28 greater inhiition of JAK2) in this assay. We assessed TG1129 ativity against a total of 63 kinases at 5 nm. Of these, only six tyrosine kinases, inluding the four aforementioned kinases, were inhiited y X8% (Supplementary Tale 1). TG1129 inhiits JAK2V617F- and MPLW515Ldependent ell proliferation, and seletively inhiits JAK2 versus JAK3 kinase-dependent ell proliferation We next tested the aility of TG1129 to inhiit the interleukin (IL)-3-independent growth of Ba/F3 ells that were staly a BaF3:JAK2 V617F NHDF DMSO 1 µm 3 µm 1 µm DMSO 1 µm 3 µm 1 µm Figure 3 TG1129 indues apoptosis in JAK2V617F-expressing HEL and Ba/F3 ells. HEL or K562 ells were ultured in growth medium in the presene of 6 nm TG1129. Cells were harvested daily and stained with Annexin V-FITC and propidium iodide (PI) followed y flow ytometri analysis. Shown are representative data from two independent experiments. TG1129 treatment inreased the perentage of Annexin V positive HEL ells from a akground level of 6 46% (24 h), 44% (48 h) and 68% (72 h) (a), and from 5 to 8% (24 h), 6% (48 h) and 8% (72 h) for K562 ells (). Effet of TG1129 in induing apoptosis of Ba/F3-V617F ells or normal human dermal firolasts (NHDF) after a 24-h inuation at the indiated onentrations. The extent of DNA fragmentation as a measure of apoptosis is measured y agarose gel eletrophoresis ().

5 transdued with onstitutively ativated JAK2V617F or MPLW515L mutants. Ba/F3 ells expressing eah mutant were grown in the presene of inreasing onentrations of inhiitor ( nm) (Figure 2a and ). TG1129 inhiited the growth of Ba/F3-V617F and Ba/F3-W515L ells with an IC 5 of 17 and 22 nm, respetively. As expeted, parental Ba/F3 ells, whih require IL-3 for growth, were also inhiited y TG1129 (IC 5 ¼ 47; data not shown), refleting their dependene on signaling y wild type JAK2. In ontrast, TG1129 had minimal effets on the growth of CTLL-2 ells, a lone of ytotoxi T ells that is dependent upon IL-2 for growth (IC 5 ¼ 34 nm) (Figure 2). Sine IL-2 reeptor signaling is known to e mediated y JAK1 and JAK3 kinases, ut not JAK2, 19 the less potent inhiitory effet on CTLL-2 ell growth is onsistent with the ompound s relatively seletive inhiition of JAK2 kinase previously seen in the in vitro kinase assay. Furthermore, TG1129 had negligile inhiitory effets on the growth of NHDF (IC5 ¼ 65 nm; data not shown), onfirming that the growth inhiition of the transformed Ba/F3 ells was not due to non-speifi toxiity. To extend these studies in a more linially relevant system, TG1129 ativity on the growth and survival of selet human leukemia ell lines was examined. TG1129 ativity was tested in HEL ells that are homozygous for the JAK2V617F allele. 1 TG1129 inhiits JAK2V617F and MPLW515L/K Inuation with inreasing onentrations of TG1129 ( nm) resulted in dose-dependent inhiition of HEL ell growth (IC 5 ¼ 152 nm) (Figure 2d). In ontrol experiments, TG1129 inhiited BCR-ABL-positive K562 ell growth with an IC 5 of 2 nm (Figure 2d). The relative speifiity of TG1129 for inhiiting JAK2 versus JAK3 kinase was further studied y investigating the effets of TG1129 on the growth of CHRF and CMK ells, whih are aute megakaryolasti leukemia (AMKL) ell lines that arry the JAK2T875N 2 and JAK3A572V 21 mutations, respetively, with inreasing onentrations of TG1129 (Figure 2e). TG1129 was 26 more potent at inhiiting proliferation of CHRF ells (IC 5 ¼ 16 nm) as ompared to CMK ells (IC 5 ¼ 42 nm), thus onfirming the speifiity of TG1129 for JAK2 versus JAK3 in ell-ased assays. TG1129 indues apoptosis in JAK2V617F-expressing human erythroleukemia and Ba/F3 ells To determine whether inhiition of proliferation of JAK2V617Fharoring HEL ells was aompanied y an inrease in apoptosis, we assessed Annexin V inding y flow ytometry. Treatment of HEL ells with 6 nm TG1129 resulted in an inrease in the perentage of Annexin V-positive ells from a 5 Figure 4 TG1129 indues ell yle arrest of JAK2V617F-expressing HEL ells. Cells were analyzed for ell-yle effets y BrdU/7-AAD staining, after TG1129 treatment for 24 h. Data shown are representative of two independent experiments (a) HEL ells without inhiitor (left panel), or with 12 nm TG1129 (right panel). G /G 1 fration inreased from a akground level of 15 5%. () K562 ells either without inhiitor (left panel), or with 12 nm TG1129 (right panel). G /G 1 fration inreased from a akground level of 45 5%.

6 TG1129 inhiits JAK2V617F and MPLW515L/K 6 akground level of 6 46% at 24 h, 44% at 48 h and 68% at 72 h (Figure 3a). As expeted, TG1129 treatment was less potent in induing apoptosis in BCR-ABL-positive K562 ells in ontrol experiments; the perentage of Annexin V-positive ells remained relatively onstant from to 72 h (B8%) (Figure 3). TG1129 also indued apoptosis of Ba/F3-V617F ells as assessed y a DNA laddering assay (Figure 3). A progressive inrease in DNA fragmentation indiating apoptosis was oserved at 24 h for Ba/F3-V617F ells treated with inreasing onentrations of TG1129. In ontrast, no DNA fragmentation was oserved in ontrol ells (dermal firolasts) even at the highest TG1129 onentration used (1 mm), thus demonstrating that indution of apoptosis in Ba/F3-V617F ells was due to JAK2V617F inhiition. a TG1129 (nm) pjak Jak TG1129 (nm) TG1129 indues ell yle arrest of JAK2V617F-expressing human erythroleukemia ells We next assessed whether TG1129 indued ell yle arrest in hematopoieti ells transformed y JAK2V617F. HEL or K562 ells were treated with indiated onentrations of inhiitor, followed y pulse laeling with romodeoxyuridine (BrdU) and staining with 7-AAD. Treatment of HEL ells with 12 nm TG1129 resulted in an inrease in the perentage of G /G 1 fration ells from a akground level of 15 5% at 24 h, thus indiating G /G 1 arrest (Figure 4a). As expeted, TG1129 treatment of BCR-ABL-positive K562 ells had a onsideraly less effet the G /G 1 fration inreased from 45 to 5% (Figure 4). STATS (p-tyr694) - STAT5 - p-stat3 - STAT3 - TG1129 (nm) TG1129 inhiits phosphorylation of JAK2V617F, STAT5 and STAT3 JAK2V617F has een previously shown to e onstitutively autophosphorylated and expression of JAK2V617F kinase results in onstitutive phosphorylation of downstream signaling pathways. 1 We next examined whether TG1129 ould inhiit phosphorylation of JAK2V617F, as well as of its downstream signaling effetors, STAT5 and STAT3. A dose-dependent derease in phosphorylation was seen, with IC 5 of B3 nm for phospho-jak2v617f inhiition and 3 6 nm for phospho-stat5 and phospho-stat3 inhiition (Figure 5a, and ). TG1129 also inhiited STAT5 phosphorylation in the Ba/F3- V617F ell line (IC 5 B3 nm) (Figure 5d). These data suggest that the asis for JAK2V617F-speifi effet on ell growth is the pharmaologi inhiition of this kinase y TG1129, with resulting downregulation of key downstream signaling intermediates. As reported previously, 1 we did not find appreiale levels of phospho-jak2 in K562 ells (data not shown), further indiating that these ells are not dependent on onstitutive JAK2 signaling for their growth. TG1129 effetively treats JAK2V617F-indued hematopoieti disease in a nude mouse model To test the in vivo effiay of TG1129 in inhiiting JAK2V617F, we used a mouse model of JAK2V617F-indued hematopoieti disease. Essentially, Ba/F3-V617F-GFP ells were injeted into immunodefiient SCID mie. The animals develop a rapidly fatal (median lateny 11 days), fully penetrant hematopoieti disease haraterized y peripheral lood leukoytosis, and splenomegaly (mean spleen weight ¼.85 g versus.28 g in plaeo-treated animals) (Figure 6a). Flow ytometri analysis of peripheral lood mononulear ells from the affeted animals (day 11) onfirmed that most ells were derived from the d β-atin- P-STAT5 Total STAT5 3 TG1129 (nm) DMSO JAK2V617F-earing lone (that is, GFP-positive) (Figure 6). Next, the reipient mie were divided into four groups and treated with TG1129 y oral gavage at the following doses: mg/day (vehile ontrol), or 2 mg/kg/day, 6 mg/kg/day, or 2 mg/kg/day, eah in two divided doses (.i.d.), from days þ nm 12 TG1129 Figure 5 TG1129 inhiits phosphorylation of JAK2V617F, STAT5 and STAT3. Effet of inreasing onentration of TG1129 on JAK2V617F phosphorylation (a), STAT5 phosphorylation () and STAT3 phosphorylation () in HEL ells. Western lot was performed using whole ell lysates from HEL ells, and were visualized with antiodies that detet the phosphorylated isoform, as well as the total amount (loading ontrol) of eah protein. -atin served as an additional loading ontrol in these experiments. TG1129 inhiits STAT5 phosphorylation in Ba/F3-V617F ells (d). 9 nm 27 nm 2-45

7 to þ 2 following tumor ell injetion. In the plaeo arm, all the animals died due to disease progression y day 11. In striking ontrast, TG1129 at the highest dose level (1 mg/kg.i.d.) was effetive in treating JAK2V617F-indued disease as there was a statistially signifiant prolongation of survival in this group (1 days; Po.2), and the animals in this group were still alive at the previously defined study end point of 1 days past the time of death of the final plaeo-treated animal (Figure 6a). The animals at the lower dose levels (that is, 1 or 3 mg/kg.i.d.) developed disease with the same lateny and penetrane as the plaeo-treated animals, without evidene of prolongation of survival (Figure 6a). Compared with plaeotreated animals, TG1129-treated animals exhiited a statistially signifiant, dose-dependent redution in the irulating tumor ell urden at day þ 11 (75% GFP þ ells in plaeotreated versus 15% GFP þ ells in 1 mg/kg.i.d. TG1129- treated animals; Po.2) (Figure 6). The linial enefit of TG1129 in this model orrelated with inhiition of JAK2V617F ativity in vivo, evident in the marked derease TG1129 inhiits JAK2V617F and MPLW515L/K in STAT-5 phosphorylation demonstrale in spleni tumors, as early as 7 h after administration of a single dose of TG1129 (1 mg/kg) to the affeted mie (Figure 6). TG1129 inhiits hematopoieti olony formation in vitro omparing effets on progenitor ells from normal ontrols versus those haroring JAK2V617F or MPLW515L/K Sine ell line studies may not aurately represent the true sensitivity of lonal one marrow-derived ells, experiments were performed with primary ells haroring JAK2V617F (n ¼ 5) or MPLW515L/K (n ¼ 3) mutations, or neither mutation (n ¼ 1) (Tale 1). The lonogeni assay readout inluded oserved hanges in (i) erythroid and myeloid olony numer, (ii) olony size/morphology and (iii) mutant olony urden, with TG1129 treatment. On the asis of ell line experiments (aove), we used, 3 and 6 nm as the TG1129 working onentrations for this assay. 7 a 12 Survival * Vehile 1 mg/kg TG1129, PO, id 3 mg/kg TG1129, PO, id 1 mg/kg TG1129, PO, id 2 Day 11 Cirulating Tumor Burden (% GFP positive ells) *Aspiration death during gavage Days Vehile 1 mg/kg 3 mg/kg 1 mg/kg TG1129 P-Stat5 Total Stat5 Vehile 1 mg/kg TG1129 Figure 6 TG1129 effetively treats JAK2V617F-indued hematopoieti disease in a nude mouse model. (a) Kaplan Meier plot of SCID mie injeted with Ba/F3 ells expressing JAK2V617F and green fluoresent protein (Ba/F3-V617F-GFP), and treated with either plaeo or TG1129 at indiated doses. There was a signifiant differene in survival etween the ohort treated with 1 mg/kg.i.d. TG1129 versus either plaeotreated ohort, or lower-dose ohorts (1 or 3 mg/kg.i.d. TG1129). The asterisk indiates the single animal in the 1 mg/kg.i.d. ohort that died from a disease-unrelated ause (traheal aspiration during oral gavage). () Flow ytometry analysis of peripheral lood mononulear ells (PBMC) from affeted mie at day þ 11, indiating perentage of GFP-positive tumor ells in the plaeo- and TG1129-treated animal ohorts. () TG1129 inhiits STAT5 phosphorylation in vivo. Western lot was performed using lysates of spleens harvested 7 h after the administering either plaeo or a single dose of TG1129 (1 mg/kg) with antiodies against phosphorylated STAT5 and total STAT5 (loading ontrol). Results from two independent experiments are shown.

8 8 TG1129 inhiits JAK2V617F and MPLW515L/K CD34 þ ells purified from the uffy oat of healthy donor phleotomy units were used as ontrols for this assay. TG1129 inhiited ytokine-supported olony growth from normal CD34 þ ells, with IC 5 of 1 nm (erythroid olonies) and 6 nm (myeloid olonies) (Tale 1). In terms of effet on olony size, urst forming unit-erythroid (BFU-E) olonies were smaller with TG1129 treatment, although hemogloinization was relatively preserved even at 6 nm TG1129 (Figure 7a). The oserved inhiitory effet on olony growth from normal CD34 þ ells was predited, given the known importane of wild type JAK signaling during normal hematopoiesis. 2 The effet of TG1129 on hematopoieti olony growth was next studied in five PV patients (all JAK2V617F-positive; PV1- PV5) (Tale 1). Inuation with TG1129 produed fewer olonies in PV patients, relative to normal ontrols (IC 5 of B6 nm and 3 6 nm for erythroid and myeloid olonies, respetively). Similarly, TG1129 had a greater dose-dependent inhiitory effet on olony size of erythroid olonies in PV patients, as ompared to normal ontrols (ompare Figure 7a and ). Genotyping of individual olonies revealed a seletive suppression of JAK2V617F haroring olonies in the presene of TG1129 for three of five PV patients (Tale 1). This effet was more pronouned in some patients (for example, PV1; 5% versus 9% JAK2V617F-positive olonies in the presene and asene of inhiitor, respetively), ut not others (PV5) (Tale 1 and Figure 7d). The effet of TG1129 on olony growth was also studied in four PMF patients (3 MPLW515L/K-positive; PMF1-PMF3). Inuation with TG1129 resulted in fewer olonies (IC 5 ¼ 3 6 nm for erythroid and myeloid olonies), as ompared to PV patients, as well as normal ontrols (Tale 1). Correspondingly, a greater dose-dependent inhiitory effet on olony size was also seen (ompare Figure 7 with Figure 7a and ). Singleolony genotyping studies showed that, for two of the three PMF patients (PMF1 and PMF2), growth of olonies haroring MPL515 mutations was seletively suppressed after inuation with TG1129 (Tale 1 and Figure 7e). We also studied TG1129 effets on olony growth in the asene of ytokines for three PV patients (PV2, PV4 and PV5). Here, erythropoietin-independent erythroid olony (EEC) growth was more sensitive to TG1129 as ompared to ytokinesupported olonies (IC 5 oo3 nm) (data not shown). Charaterization of EEC otained in the presene of TG1129 (eyond olony numer) was diffiult given the dysmorphi nature of these olonies. Furthermore, genotyping of these olonies was frequently unsuessful, presumaly eause most ells in these olonies are apoptoti. Disussion JAK2V617F has a primary role in the pathogenesis of PV, PMF, ET and potentially, that of other MPD as well. JAK2V617F is highly prevalent in the former three disorders, and onfers ytokine hypersensitivity, and promotes endogenous erythroid olony formation, whih are salient in vitro harateristis of PV-derived progenitor ells. 14 Furthermore, JAK2V617F expression in murine disease models produes a MPD mimiking PV. 7,8 Finally, genotype phenotype orrelation studies in MPD patients point to a signifiant assoiation etween JAK2V617F Tale 1 TG1129 effet on ytokine-supported hematopoieti olony growth from MPD patients haroring JAK2V617F or MPLW515 Cytokine-supported olonies mutation pattern as assessed y genotyping of 575 individual olonies Patient Mutation IC 5 (nm) Total no. of olonies genotyped Mutation pattern without inhiitor (% olonies genotyped) Mutation pattern with inhiitor (% olonies genotyped) % mutationpositive olonies without inhiitor % mutationpositive olonies with inhiitor Erythroid Myeloid Erythroid Myeloid Erythroid Myeloid Normal WT 1 6 n/a n/a n/a n/a PV1 JAK2V617F B HET/WT (85/15) HET/WT (57/43) PV2 JAK2V617F B6 B3 111 HET/WT (65/35) HET/WT (6/4) PV3 JAK2V617F NA 59 HOM/WT (23/77) HOM/HET/WT (8/8/84) PV4 JAK2V617F B6 B6 61 HOM/HET/WT HOM/HET/WT (8/11/81) (4/4/92) PV5 JAK2V617F B HOM/HET/WT (74/4/22) HOM/HET/WT (92/4/4) PMF1 MPLW515K o3 B3 59 HET/WT (93/7) HET/WT (17/83) PMF2 MPLW515K 3 6 B3 86 HET/WT (9/1) HET/WT (39/61) PMF3 MPLW515L HOM/HET (91/9) HOM/HET/WT (71/22/7) PMF4 WT n/a n/a n/a n/a Areviations: HET, heterozygous; HOM, homozygous; IC 5, inhiitor (TG1129) onentration that suppresses olony numer y 5%; MPD, myeloproliferative disorders; n/a, not appliale; NA, not availale; PMF, primary myelofirosis; PV indiates polyythemia vera; WT, wild-type. Figure 7 TG1129 inhiits the in vitro growth of hematopoieti olonies derived from progenitor ells haroring JAK2V617F or MPLW515 K mutations. Representative photomirographs of olonies derived from CD34 þ ells plated in methylellulose supplemented with ytokines are shown. CD34 þ ells were otained either from healthy ontrols (a), or patients haroring JAK2V617F (), or MPLW515 K () mutations and plated either in the asene of inhiitor (left panels), or presene of 3 nm TG1129 (middle panels), or 6 nm TG1129 (right panels). Bar graph omparing numer of olonies (total, erythroid or myeloid) otained either without inhiitor, or with 3 or 6 nm TG1129, for a representative patient arrying the JAK2V617F (d; left panel) or MPLW515 K mutation (e; left panel). Genotyping of individual olonies shows that TG1129 seletively suppresses growth of olonies haroring JAK2V617F (d; right panel) or MPLW515 K (e; right panel) mutations.

9 a TG1129 inhiits JAK2V617F and MPLW515L/K 9 d 35 # of Colonies TOTAL ERY MYE e ND 3nM 6nM TOTAL ERY MYE # of Colonies ND 3nM 6nM

10 1 TG1129 inhiits JAK2V617F and MPLW515L/K and speifi linial features, inluding higher hemogloin and leukoyte ounts, inreased inidene of venous thromosis, and potentially, inferior survival. 22,23 These data therefore provide a strong rationale for targeting JAK2V617F as a therapeuti strategy in MPD. In this report, we desrie a potent, small moleule JAK2- seletive kinase inhiitor that was developed rationally, using struture-ased drug design. TG1129 is orally ioavailale (pharmaokinetis data are presented as Supplementary Information), and has signifiant in vitro ativity against JAK2V617F. Correspondingly, TG1129 inhiited proliferation of leukemi ells arrying mutant JAK2 alleles through ell yle effets and indution of apoptosis. Furthermore, TG1129 effetively treated an aggressive JAK2V617F-indued hematopoieti disease in mie, an effet that was orrelated with inhiition of STAT5 phosphorylation in vivo. Finally, TG1129 suppressed growth of hematopoieti olonies from primary progenitor ells haroring JAK2V617F or MPL515 mutations. A key question pertains to preisely whih MPD patients will enefit from therapies targeting JAK2 signaling from a pathogeneti standpoint, as well as from the standpoint of safety of suh therapies. While JAK2V617F is the predominant disease assoiated allele in MPD, it is eoming lear that other alleles ontriute to MPD phenotype. In general, the novel allele appears to sustitute for JAK2V617F (for example, JAK2 exon 12 mutations, JAK2DIREED), 4,6 although in some ases, suh alleles at in onjuntion with JAK2V617F (for example, JAK2D62E, MPLW515L/K). 3,16 These mutant alleles effetively onverge upon JAK2, leading to onstitutive autophosphorylation of this kinase. These oservations raise the possiility that most, if not all, MPD patients haror mutations (some yet to e identified) that potentially serve as a moleular target for a seletive JAK2 inhiitor. Indeed, our data revealed that in vitro growth of hematopoieti olonies from a PMF patient (PMF4; Tale 1) without identifiale mutations was inhiited at least as effetively as that from patients haroring JAK2V617F or MPLW515 mutations. Given that most MPD patients have indolent disease and may require only simple therapeuti interventions (for example, intermittent phleotomy plus aspirin in a young low-risk PV patient), it may e that JAK2 targeted therapy is appropriate, at least initially, only for high-risk patients, where the disease is poorly ontrolled with onventional therapies (for example, the advaned PMF patient with signifiant ytopenias, where use of myelosuppressive agents is prolemati). The extent to whih a JAK2-seletive inhiitor will e myelosuppressive and/or immunosuppressive in the setting of MPD therapy remains to e estalished. JAK3 kinase is predominantly expressed in hematopoieti ells, and assoiates with the ommon g hain (g) shared y IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21 reeptors, and thus plays a key role in development and homeostasis of the immune system. These data raise theoretial onerns for immunologi toxiities that may potentially e seen with offtarget JAK3 kinase inhiition. More reently, however, a novel inhiitor, MK-457 or VX-68, that targets Aurora kinases, as well as BCR-ABL and JAK2 kinases, was used to treat three patients with CML or aute lympholasti leukemia with the T315I BCR-ABL mutation. 24 Here, MK-457 given y a 5-day ontinuous i.v. infusion shedule, led to linial responses. Enouragingly, there was no signifiant extramedullary toxiity seen with MK-457 therapy in this preliminary study, whih, y extrapolation, supports feasiility of TG1129 use in MPD patients from a drug safety standpoint. Furthermore, given that TG1129 more seletively targets JAK2 kinase, relative to JAK3 (28 ), this ought to, in theory, limit its potential immunosuppressive/immunomodulatory effets. Whether this partiular harateristi translates into a more favorale linial safety profile, however remains to e demonstrated. In summary, we have shown that a novel small moleule kinase inhiitor, TG1129, is effetive oth in vitro and in vivo against JAK2V617F and MPLW515L/K tyrosine kinases, and provides survival enefit in a mouse disease model. Thus, TG1129, the prototype of a new lass of JAK2 kinase inhiitors, opens the prospet for effetive moleularly-targeted therapy in MPD, and possily other malignanies aused y dysregulated JAK-STAT signaling, regardless of whether the ausative mutations our at the ytokine reeptor level or involve JAK2 itself. Aknowledgements This work was partially supported y researh grants from the Myeloproliferative Disorders Foundation, (Chiago, IL, USA) and from TargeGen In., (San Diego, CA, USA). Referenes 1 Tefferi A, Gilliland DG. Onogenes in myeloproliferative disorders. Cell Cyle 27; 6: Parganas E, Wang D, Stravopodis D, Topham DJ, Marine JC, Teglund S et al. Jak2 is essential for signaling through a variety of ytokine reeptors. Cell 1998; 93: Gruneah F, Bross-Bah U, Kanz L, Brossart P. Detetion of a new JAK2 D62E mutation in addition to V617F in a patient with polyythemia vera. 26; 2: Malinge S, Ben-Adelali R, Settegrana C, Radford-Weiss I, Dere M, Beldjord K et al. A novel ativating JAK2 mutation in a Down Syndrome patient with B-ell aute lympholasti leukemia. Blood 27; 19: Pikman Y, Lee BH, Merher T, MDowell E, Eert BL, Gozo M et al. MPLW515L is a novel somati ativating mutation in myelofirosis with myeloid metaplasia. 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