Z3, a Novel Jak2 Tyrosine Kinase Small Molecule Inhibitor that Suppresses Jak2-mediated Pathologic Cell Growth

Abstract

Jak2 tyrosine kinase is essential for animal development and hyper-kinetic Jak2 function has been linked to a host of human diseases. Control of this pathway using Jak2-specific inhibitors would therefore potentially serve as a useful research tool and/or therapeutic agent. Here, we used a high throughput program called DOCK, to predict the ability of 20,000 small molecules to interact with a structural pocket adjacent to the ATP binding site of murine Jak2. One small molecule, 2-methyl-1-phenyl-4-pyridin-2-yl-2-(2-pyridin-2-ylethyl)butan-1-one (herein designated as Z3) bound to Jak2 with a favorable energy score. Z3 inhibited Jak2-V617F and Jak2-WT autophosphorylation in a dose-dependent manner, but was not cytotoxic to cells at concentrations that inhibited kinase activity. Z3 selectively inhibited Jak2 kinase function with no effect of Tyk2 or c-Src kinase function. Z3 significantly inhibited proliferation of the Jak2-V617F-expressing, human erythroleukemia cell line, HEL 92.1.7. The Z3-mediated reduction in cell proliferation correlated with reduced Jak2 and STAT3 tyrosine phosphorylation levels as well as marked cell cycle arrest. Finally, Z3 inhibited the growth of hematopoietic progenitor cells isolated from the bone marrow of an essential thrombocythemia patient harboring the Jak2-V617F mutation and a polycythemia vera patient carrying a Jak2-F537I mutation. Collectively, the data suggest that Z3 is a novel specific inhibitor of Jak2 tyrosine kinase.

INTRODUCTION

Aside from its essential role in embryonic development (1), Jak2 plays an important role in pathophysiology. Jak2 has been linked to several hematological malignancies including acute lymphoid leukemia and chronic myeloid leukemia (2–4). Recently, a Jak2-V617F somatic mutation has been identified in approximately 98% of patients with polycythemia vera (PV) and in a substantial proportion (50%) of patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF) (5). This valine to phenylalanine substitution (V617F) occurs in the JH2 pseudokinase domain of Jak2. These myeloproliferative disorders are characterized by the clonal overproduction of normally differentiated hematopoietic lineages. The V617F substitution leads to constitutive activation of Jak2 and downstream effector signaling pathways including the STAT transcription pathway and the PI3-kinase and ERK signaling networks, which in turn induce inappropriate cytokine-independent proliferation of cells (5, 6). In addition, the Jak2-V617F mutation contributes to myelofibrosis by constitutively phosphorylating STAT3 and diminishing myeloid cell apoptosis (7). Collectively, these results implicate dysregulated Jak2 signaling in various hematological and myeloproliferative disorders.

As components of the Jak-STAT signaling pathway are hyper-activated in some leukemias and myeloproliferative disorders, control of this pathway using pharmacological inhibition is highly desirable. As a research tool, AG490 is by far the most extensively used commercially available Jak2 inhibitor. Although it has been valuable in identifying Jak2 as a therapeutic target, it suffers from a general lack of specificity. For example, AG490 inhibits calf-serum inducible cell growth and DNA synthesis (8) and is a partial blocker of c-Src activity (9). Most importantly, AG490 inhibits epidermal growth factor receptor autophosphorylation more potently than it inhibits Jak2 activity (10, 11). Thus, identification and characterization of novel Jak2-specific inhibitors may serve as useful research tools and/or therapeutic agents.

Here, we utilized a rapid structure-based approach combining molecular docking with cell based functional testing in order to identify a Jak2-selective inhibitor. One small molecule, 2-methyl-1-phenyl-4-pyridin-2-yl-2-(2-pyridin-2-ylethyl)butan-1-one (herein designated as Z3) bound to a pocket that was adjacent to the ATP binding site of Jak2 with a favorable energy score. Our functional testing data suggest that Z3 is a novel Jak2 tyrosine kinase specific, small molecule inhibitor.

MATERIAL AND METHODS

Small Molecule Database

The small molecules were obtained from the National Cancer Institute/Developmental Therapeutics Program (NCI/DTP), which maintains a repository of approximately 140,000 compounds.

Cell Culture

BSC-40 and COS-7 cells were maintained as previously described (12). Human erythroleukemia (HEL) and Burkitt’s lymphoma (Raji) cells were purchased from ATCC (Rockville, MD, USA). HEL and Raji cells were maintained in RPMI-1640 medium (Mediatech, Herndon, VA) containing 10% fetal bovine serum and L-glutamine (2 mM final). All cells were cultured at 37°C in a 5% CO₂ humidified atmosphere.

BSC-40 Cell Transfection/Infection

Jak2 autophosphorylation assays were performed in BSC-40 cells using the vaccinia virus transfection/infection procedure which results in high level Jak2 expression and subsequent tyrosine phosphorylation independent of exogenous ligand addition (13). Specifically, cells were transfected with 10 μg of a plasmid encoding either the wild-type murine Jak2 cDNA (pRC-CMV-Jak2-WT) or V617F mutant murine Jak2 cDNA (pRC-CMV-Jak2-V617F) under the control of the bacteriophage T7 promoter, using Lipofectin per the manufacturer’s instructions (Invitrogen, Carlsbad, CA). After 4 hours of transfection, the cells were infected with the recombinant vaccinia virus, vTF7-3, at a multiplicity of infection (MOI) of 1.0 for 1 hour. The media containing Lipofectin/DNA/vTF7-3 was then removed from the cells, replaced with fresh serum-containing media and incubated overnight.

COS-7 Cell Transient Transfection

COS-7 cells were transfected with 10 μg of plasmid encoding the wild-type human Tyk2 cDNA (pMT2T-Tyk2-WT) for 5 hours using Lipofectin. Following transfection, the cells were grown at 37°C for 48 hours in serum-containing media to allow for high-level Tyk2 tyrosine autophosphorylation. The cells were then treated with DMSO or Z3 for 16 hours prior to lysis.

Cell Lysis and Immunoprecipitation

Cells were washed with two volumes of ice-cold PBS containing 1 mM Na₃VO₄. BSC-40 cells were lysed in 0.8 mL of ice cold RIPA buffer (20 mM Tris pH 7.5, 10% glycerol, 1% Triton X-100, 1% deoxycholic acid, 0.1% SDS, 2.5 mM EDTA, 50 mM NaF, 10 mM Na₄P₂O₇, 4 mM benzamidine, and 10 μg/mL aprotinin) while HEL cells (1.0 × 10⁷) were lysed in 0.8 mL of ice cold Gentle Lysis Buffer (25 mM Tris, pH 7.4, 10% glycerol, 1% IGEPAL and 137 mM NaCl, 4 mM benzamidine, and 10 μg/mL aprotinin). Cleared protein lysates were incubated with 2 μg of antibody and 20 μL of protein A/G plus agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA) for 2 hours at 4°C. Protein complexes were washed three times with wash buffer (25 mM Tris, pH 7.5, 150 mM NaCl, and 0.1% Triton X-100) and resuspended in SDS sample buffer. Bound proteins were boiled, separated by SDS-PAGE, and transferred onto nitrocellulose membranes. The immunoprecipitating anti-Jak2, anti-STAT3 and anti-Tyk2 polyclonal antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA).

Western Blotting

Anti-phosphotyrosine Western blotting was performed using a combination of PY99, PY20, and 4G10 monoclonal antibodies from Santa Cruz Biotechnology, BD Transduction Labs (San Jose, CA), and Millipore (Billerica, MA), respectively. Anti-Jak2 pY1007/pY1008 and phospho-STAT3 Western blotting antibodies were from Santa Cruz Biotechnology. The anti-c-Src pY⁴¹⁸ was from BioSource (Carlsbad, CA). Western blotting for total c-Src protein was performed using a cocktail of polyclonal antibodies from Santa Cruz Biotechnology and BD Transduction Laboratories. The anti-Jak2 Western blotting antibodies consisted of a cocktail of antibodies from Millipore and BioSource. Western blots were visualized using the enhanced chemiluminescence system (NEN Life Science Products, Waltham, MA). Densitometry was performed using the automated digitizing software, Un-Scan-It, Version 5.1 (Silk Scientific, Orem, Utah). All phosphorylation levels were normalized to total protein levels.

c-Src In Vitro Kinase Assay

Approximately 4 μL (12 units) of catalytically active recombinant pp60^c-Src (Millipore) was incubated in 50 μL of in vitro kinase reaction buffer as described (13). Reactions were terminated via the addition of SDS-containing sample buffer and subsequently Western blotted with either anti-ACTIVE c-Src or total c-Src antibody as described.

Propidium Iodide Staining of Cells

BSC-40 cells were seeded in 6-well Lab-Tek Chamber Slides (Nunc, Rochester, NY). After adherence, the cells were treated with either DMSO or Z3 at concentrations of 25, 100 or 250 μM for 16 hours. Live cells were treated with 1 μg/mL propidium iodide and then visualized using a Bio-Rad MRC-1024 confocal microscope. Same field images were captured under phase contrast and fluorescent conditions.

HEL Cell Proliferation

HEL cells were plated on 96-well dishes at 5 × 10⁴ cells per well and treated with either DMSO or 25 μM Z3 for the indicated times. The number of viable DMSO or Z3-treated cells were determined by trypan blue exclusion using a hemocytometer.

Cell Cycle Analysis

The CycleTEST^™ Plus DNA Reagent Kit (Becton Dickinson, San Jose, CA) was utilized to analyze nuclear DNA from HEL cell suspensions following the manufacturer’s instructions. Approximately 5 × 10⁵ cells were treated with either DMSO or 25 μM of Z3 for 16, 24, 48 and 72 hours. Cell suspensions were treated with the reagents stated in the protocol, filtered through a 50-μm nylon mesh and analyzed on a FACSCalibur Flow Cytometer (Becton Dickinson).

Jak2 Mutation Analysis

Residual bone marrow aspirates were obtained from patients diagnosed with ET or PV (WHO criteria) following approval by the University of Florida Institutional Review Board (approval # 428–2006). Patient de-identified mononuclear cells were isolated by separation over Ficoll-Paque (Pharmacia, Peapack, NJ). Genomic DNA was isolated from 10⁶ cells using the Easy-DNA kit (Invitrogen) following the manufacturer’s instructions. Exons 12 and 14 were amplified via PCR using previously published exon specific primers and annealing conditions (14). Each amplified exon was then subjected to direct DNA sequence analysis.

Colony Formation Assay

Marrow derived mononuclear cells were washed three times in IMDM media and plated at 4 × 10⁵ cells/mL in 1 mL methylcellulose media (0.9% methylcellulose, 30% heat inactivated FCS, 0.1 mM 2-mercaptoethanol, 0.9% BSA, 0.05% NaHCO₃, 2 mM/L glutamine, penicillin, streptomycin, 50 ng/mL SCF, and 20 ng/mL IL-3 (Stem Cell Technologies, Vancouver, BC). Z3 (25 μM) and TPO (50 ng/mL) or EPO (1 U/mL) were either added or omitted as described in the legend. The cultures were then incubated at 37°C and 5% CO₂ until assessment of colony formation at day 14. Results were expressed as the average number of colonies from duplicate cultures per 4×10⁵ cells.

RESULTS

Z3 Inhibits Jak2 Tyrosine Autophosphorylation by Interacting with a Solvent Accessible Pocket Adjacent to the ATP Binding Pocket

Taking the crystal structure for portions of the Jak3 kinase domain (pdb code 1YVJ) into consideration, a comparative structural modeling approach was employed to generate an atomic model of the kinase domain of murine Jak2 using ProModII as previously described (12).

The program SPHGEN, which identifies potential ligand binding sites based on chemical and shape characteristics, was employed to select the target pocket on Jak2 for small molecule docking. To prepare the site for docking, all water molecules were removed. Protonation of receptor residues was performed with the program Sybyl. The sphere set utilized for molecular docking was based on the position of a Jak3 inhibitor in the crystal structure of the kinase domain of Jak3 (Figure 1A). The position of a pan Jak2 inhibitor that was subsequently crystallized in the kinase domain of human Jak2 is also shown (Figure 1A). The site selected for molecular docking was adjacent to the ATP binding site of murine Jak2 and the number of spheres in the site (50–100) was in the ideal range for binding to small molecules.

FIGURE 1. 2-methyl-1-phenyl-4-pyridin-2-yl-2-(2-pyridin-2-ylethyl)butan-1-one (Z3) inhibits Jak2-V617F and Jak2-WT tyrosine autophosphorylation.

(A) The sphere set used for molecular docking was based on the position of a solvent accessible pocket adjacent to the Jak2 activation loop. For reference, the positions of the resolved structures for portions of both the Jak2 and Jak3 kinase domains are indicated. Also shown is the position of the IZA pan Jak2 inhibitor that was crystallized within the Jak2 kinase domain. (B) BSC-40 cells were transfected with empty vector control, Jak2-V617F, or Jak2-WT expression plasmids and then infected with vaccinia virus to drive high-level expression and subsequent Jak2 tyrosine autophosphorylation. The six highest scoring compounds identified by DOCK were incubated with the cells at a concentration of 100 μM each, for 16 hours. Cell lysates were immunoprecipitated with anti-Jak2 antibody and immunoblotted with anti-phosphotyrosine antibody to detect Jak2 tyrosine phosphorylation levels (top panel). The membrane was stripped and reprobed with anti-Jak2 antibody to demonstrate equal Jak2 expression among all samples (bottom panel). Shown is one of two independent experiments. (C) Shown is the molecular structure of the Z3 compound. (D) Based on contact points and energy scores, Z3 docks into a structural pocket on the Jak2 kinase domain that is adjacent to the ATP binding site.

Utilizing the DOCK program, we tested 20,000 compounds of known chemical structure, in silico, for their ability to interact with a structural pocket adjacent to the ATP binding site. The top six scoring compounds (Supplementary Figure 1) were obtained from the NCI/DTP and tested for their ability to inhibit Jak2-WT and Jak2-V617F autophosphorylation.

A Jak2 over expression system was used to first determine the affect of each inhibitor on Jak2 autophosphorylation. Specifically, BSC-40 cells were transfected with an expression vector encoding either empty vector control (no Jak2), the mutant murine Jak2 cDNA (Jak2-V617F) or the wild type Jak2 cDNA (Jak2-WT) under the control of T7 RNA polymerase. This resulted in high-level Jak2 expression and subsequent tyrosine phosphorylation independent of exogenous ligand addition. The six inhibitor compounds (Z1–Z6) were each added at a concentration of 100 μM and incubated overnight. Sixteen hours following addition of the compounds, cellular lysates were immunoprecipitated with an anti-Jak2 polyclonal antibody and then immunoblotted with an anti-phosphotyrosine monoclonal antibody to detect tyrosine phosphorylated Jak2 (Figure 1B, top). The results show that compound Z3 (methyl-1-phenyl-4-pyridin-2-yl-2-(2-pyridin-2-ylethyl)butan-1-one) most effectively inhibited both Jak2-V617F and Jak2-WT tyrosine autophosphorylation. Clearly however, the ability of Z3 to inhibit Jak2-WT autophosphorylation was greater than when compared to Jak2-V617F autophosphorylation. The membrane was then stripped and reprobed with an anti-Jak2 polyclonal antibody to demonstrate total protein loading (Figure 1B, bottom).

The structure of the Z3 compound is shown in Figure 1C. Based on the data in Figure 1B, we returned to our molecular model of Jak2 and utilized the DOCK program to determine the position of Z3 on the Jak2 structure. Based on contact points and energy scores, the Z3 inhibitor was predicted to bind to a solvent accessible pocket adjacent to the ATP binding site as we had intended (Figure 1D). Collectively, the data in Figure 1 indicate that Z3 inhibits both Jak2-WT and Jak2-V617F tyrosine autophosphorylation (ie, kinase function) via an interaction with a critical solvent accessible pocket adjacent to the ATP binding site.

Z3 Inhibits Jak2 Tyrosine Autophosphorylation in a Dose-Dependent Manner that is Independent of Cellular Cytotoxicity

In order to obtain a better understanding of the inhibitory properties of Z3, we examined whether it could inhibit Jak2-WT and Jak2-V617F tyrosine autophosphorylation in a dose-dependent manner. Specifically, we sought to determine whether Z3 inhibits phosphorylation of the critically important tyrosine 1007 residue, as phosphorylation of this residue within the activation loop of Jak2 is necessary for maximal Jak2 activation (15). To meet this end, BSC-40 cells were transfected/infected with either a Jak2-WT or a Jak2-V617F plasmid and then treated for 16 hours with Z3 at the indicated concentrations. For the Jak2-WT expressing cells, cell lysates were first immunoprecipitated with a Jak2 polyclonal antibody and then Western blotted with a phospho-specific (pY1007/pY1008) Jak2 antibody to detect phosphorylated Jak2 at this residue (Figure 2A, top). The results indicate that Z3 inhibited phosphorylation of the critically important tyrosine 1007 residue on Jak2-WT, in a dose-dependent manner. The membrane was subsequently stripped and re-probed with an anti-Jak2 polyclonal antibody to determine Jak2 expression levels amongst all samples (Figure 2A, bottom).

FIGURE 2. Z3 inhibits Jak2 tyrosine autophosphorylation in a dose-dependent manner.

(A) BSC-40 cells were transfected/infected as described above. Jak2-WT expressing cells were incubated with either vehicle control (DMSO) or with Z3 at the indicated doses. Protein cell lysates were then immunoprecipitated with anti-Jak2 antibody and immunoblotted with a phospho-specific (pY1007/pY1008) Jak2 antibody to detect phosphorylated Jak2 at this specific residue (top). The membrane was stripped and reprobed with anti-Jak2 antibody to demonstrate equal Jak2 expression among all samples (bottom). (B) Densitometrical analysis was performed on four representative Western blots to quantify Z3-mediated inhibition of WT-Jak2 phosphorylation at the tyrosine 1007 residue. Data are represented as the ratio of phosphorylated Jak2 to total Jak2 and presented as the mean +/− SEM. The IC₅₀ of Z3 for Jak2-WT was approximately 15 μM. Statistical significance between the vehicle control and Z3 treated cells was determined by a one-way analysis of variance (ANOVA) (p<0.001) (C) For Jak2-V617F expression, cells were first immunoprecipitated with a Jak2 polyclonal antibody and then Western blotted with a phospho-specific (pY1007/pY1008) Jak2 antibody (top). The membrane was then stripped and reprobed with anti-Jak2 antibody (bottom). (D) Densitometry analysis of four representative Western blots showing Z3-mediated inhibition of Jak2-V617F phosphorylation at the tyrosine 1007 residue. Data are represented as the ratio of phosphorylated Jak2 to total Jak2 and presented as the mean +/− SEM. The IC₅₀ of Z3 for Jak2-V617F was approximately 28 μM. There was a statistically significant difference between the vehicle control and Z3 treated cells (p<0.001).

In order to quantitate Jak2-WT tyrosine 1007 phosphorylation levels in the presence of increasing amounts of Z3, densitometry analysis on four representative western blots was performed (Figure 2B). We found that Jak2-WT tyrosine autophosphorylation was reduced by approximately 60 percent between the doses of 10 to 30 μM. A 100 μM dose of Z3 maximally suppressed Jak2 phosphorylation by about 92 percent. The estimated IC₅₀ of Z3 for Jak2-WT was 15 μM.

For the Jak2-V617F samples, Jak2 immunoprecipitates were immunoblotted with the same pY1007/pY1008 phospho-specific antibody (Figure 2C, top). Similar to the Jak2-WT expressing cells, Z3 inhibited Jak2-V617F tyrosine 1007 phosphorylation in a dose-dependent fashion. The membrane was then stripped and reprobed with an anti-Jak2 antibody in order to determine relative Jak2 expression levels across all samples (Figure 2C, bottom). Densitometry analysis on four representative Western blots showed that Jak2-V617F tyrosine 1007 phosphorylation was inhibited by approximately 60 percent between the concentrations of 10 to 100 μM of Z3 (Figure 2D). The IC₅₀ of Z3 for Jak2-V617F was approximately 28 μM.

To rule out the possibility that the affects of Z3 were due to non-specific cellular toxicity rather than specific Jak2 inhibition, we treated these same BSC-40 cells with either DMSO or with 25, 100 or 250 μM Z3 for 16 hours. The live cells were then stained with propidium iodide. Propidium iodide selectively stains necrotic cells and fluoresces red, but is excluded from the plasma membrane of intact cells. The results show that cells treated with 25 or 100 μM of Z3, showed propidium iodide staining similar to that of DMSO-treated cells (Supplementary Figure 2, top panels). However, cells treated with 250 μM Z3 displayed a marked increase in propidium iodide staining, indicating that this dose of Z3 is cytotoxic. The same propidium iodide fields were also visualized by phase contrast microscopy to determine total cell numbers and overall cellular morphology (Supplementary Figure 2, bottom panels). Overall, the data indicate that the range of Z3 that inhibits Jak2 tyrosine kinase autophosphorylation by 50% – 100% (25–100 μM), does so in a manner that is independent of gross cellular cytotoxicity. Taken together, our results indicate that the Z3 compound blocks Jak2 autophosphorylation in a dose-dependent manner, but is not cytotoxic to cells at concentrations that inhibit Jak2 tyrosine kinase activity. Moreover, the ability of Z3 to inhibit Jak2-WT (IC₅₀ = ~15 μM) is greater when compared to Jak2-V617F (IC₅₀ = ~28 μM).

Z3 is a Specific Inhibitor of Jak2 Tyrosine Kinase

In order to determine whether Z3 is selective for suppressing Jak2 autophosphorylation when compared to other Jak family members, we employed an autophosphorylation assay to allow for robust Jak family kinase activation independent of ligand treatment (13). Here, COS-7 cells were transiently transfected with an expression vector encoding the wild type human Tyk2 cDNA. The following day, the cells were treated with either DMSO or 25 μM of Z3 for 16 hours. The cells were then lysed and Tyk2 was immunoprecipitated from the lysates via an anti-Tyk2 antibody. The immunoprecipitated protein was then immunoblotted with an anti-phosphotyrosine antibody to evaluate the level of Tyk2 tyrosine autophosphorylation (ie, catalytic activity). We found that Z3 did not inhibit Tyk2 tyrosine kinase autophosphorylation when compared to DMSO control (Figure 3A, top), bearing in mind that this same 25 μM dose reduced Jak2-WT tyrosine kinase autophosphorylation by approximately 70% (Figure 2B). The membrane was subsequently stripped and reprobed with anti-Tyk2 antibody to demonstrate equal Tyk2 expression for both samples (Figure 3A, bottom).

FIGURE 3. Z3 selectively inhibits Jak2 tyrosine kinase activity.

(A) COS-7 cells were transiently transfected with an expression vector encoding wild type human Tyk2 cDNA. Cells were subsequently treated with either DMSO or 25 μM of Z3 for 16 hours. Protein lysates were immunoprecipitated with anti-Tyk2 antibody and then Western blotted with anti-phosphotyrosine antibodies to detect Tyk2 autophosphorylation (top). The membrane was stripped and reprobed with anti-Tyk2 antibody to show equal Tyk2 expression among all samples (bottom). (B) Catalytically active c-Src protein was incubated in kinase reaction buffer containing either DMSO, 25 μM of Z3, or 25 μM of PP2. The samples were subsequently immunoblotted with anti-phospho c-Src (pY418) antibody to determine relative c-Src tyrosine kinase activity (top). The membrane was then stripped and reprobed with c-Src antibody to assess c-Src protein levels (bottom). Shown is one of three representative experiments for each.

We next investigated the effect of Z3 on another non-receptor tyrosine kinase, c-Src. Similar to Jak2, c-Src is ubiquitously expressed and has been shown to activate a number of effectors that are also involved in aberrant cell growth. Here, catalytically active recombinant c-Src protein was incubated either in the presence of DMSO, 25 μM of Z3, or 25 μM of the Src kinase inhibitor, PP2. The reactions were incubated for 20 minutes at room temperature and then terminated by addition of SDS-containing sample buffer. The samples were separated by SDS-PAGE and subsequently Western blotted with an anti-ACTIVE c-Src (pY418) antibody to determine c-Src catalytic activity (Figure 3B, top). We found that Z3 had no effect on c-Src tyrosine kinase activity when compared to DMSO control. However, in the presence of the known c-Src inhibitor, PP2, c-Src tyrosine kinase activity was completely abolished. To demonstrate equal c-Src protein content amongst all conditions, the membrane was stripped and reprobed with anti-c-Src antibody (Figure 3B, bottom). Collectively, the results demonstrate that while Z3 suppresses Jak2 tyrosine kinase activity, it does not inhibit Tyk2 or c-Src.

Z3 Selectively Inhibits Jak2-V617F Dependent Cell Proliferation and this Correlates with Suppression of Jak2 and STAT3 Tyrosine Phosphorylation

It is known that the human erythroleukemia cell line, termed HEL, is homozygous for the Jak2-V617F mutation (16). The single point mutation leading to a V617F substitution in the JH2 domain of Jak2 has been associated with its proliferative phenotype (16). In addition, it has been shown that expression of the constitutively activated Jak2-V617F mutation is required for transformation of HEL cells. Mechanistically, the Jak2-V617F mutation promotes G1/S phase transition in HEL cells and subsequent increases cellular proliferation (17).

Due to the presence of the constitutively activated Jak2-V617F mutation in HEL cells, we wanted to determine whether Z3 could suppress Jak2-V617F dependent cell proliferation in this cell line. Here, 5 × 10⁴ HEL cells were treated with either DMSO or 25 μM of Z3 for 0, 16, 24, 48 and 72 hours. At the end of each time point, the number of viable DMSO- or Z3-treated cells was determined by trypan blue exclusion using a hemocytometer (Figure 4A). The results show that in the presence of DMSO, HEL cells were in rapid growth. However, treatment with Z3 reduced cell numbers when compared to DMSO. Analysis of variance indicated that the Z3 growth curve was significantly different from that of DMSO (p= 1.06 × 10⁻¹⁴).

FIGURE 4. Z3 selectively suppresses Jak2-dependent cell proliferation, which correlates with inhibition of Jak2 and STAT3 phosphorylation.

(A) HEL cells were treated with either DMSO or 25 μM of Z3 for 0, 16, 24, 48, or 72 hours. The numbers of viable DMSO or Z3 treated cells were assessed by trypan blue exclusion. All data points were measured in triplicate. Statistical significance between each group was analyzed using a two-way analysis of variance (ANOVA). The two conditions were found to be significantly different (p=1.06 × 10⁻¹⁴). (B) HEL and Raji cells were treated with either DMSO or with 0.1, 0.3, 1, 3, 10, or 30 μM of Z. After 72 hours, the numbers of viable cells were assessed as described above. The graph shown is the compilation of three independent experiments. Statistical significance between each group was analyzed using a two-way analysis of variance (ANOVA). The two conditions were found to be significantly different (p=5.28 × 10⁻¹¹). (C) HEL cells were treated with either DMSO or 25 μM of Z3 for 16, 24, 48, or 72 hours. Protein lysates were immunoprecipitated with anti-Jak2 antibody and then immunblotted with anti-phosphotyrosine antibody to determine total Jak2 phosphorylation levels (top). The membrane was stripped and reprobed with Jak2 antibody to demonstrate Jak2 expression amongst all samples (bottom). (D) HEL cells were treated as indicated. STAT3 phosphorylation levels were determined by first immunoprecipitating with STAT3 antibody and then immunobloting with a phospho-specific (pY705) STAT3 antibody (top). The membrane was stripped and reprobed with STAT3 to demonstrate total STAT3 protein content among all samples (bottom). Shown is one of three (A, B) or two (C and D) representative experiments.

In order to determine whether Z3 specifically suppresses Jak2-V617F dependent cell growth, as opposed to non-specific inhibition of cell proliferation, we examined the inhibitory effect of Z3 on Raji cells. The molecular mechanism responsible for the aberrant growth of Raji cells is a translocation event between the c-Myc gene on chromosome 8 and the heavy chain locus on chromosome 14 (18). Here, 5 × 10⁴ HEL or Raji cells were treated for 72 hours with either DMSO or Z3 at the indicated concentrations. The number of viable cells was then determined (Figure 4B). The results indicate that HEL cells were more sensitive to the inhibitory effects of Z3, over a wide range of doses, when compared to Raji cells (p= 5.28 × 10⁻¹¹).

We next examined whether Z3-dependent inhibition of HEL cell proliferation correlated with suppression of Jak2 tyrosine autophosphorylation. Specifically, HEL cells were treated with either DMSO or 25 μM of Z3 for the indicated times. Protein lysates were first immunoprecipitated with anti-Jak2 antibody and then Western blotted with an anti-phosphotyrosine antibody to detect total tyrosine phosphorylated Jak2 (Figure 4C, top). We found that Z3 dramatically reduced total Jak2 tyrosine autophosphorylation by 48 hours when compared to DMSO control and this correlated with inhibition of HEL cell proliferation at this time point. The membrane was then stripped and reprobed with anti-Jak2 antibody to demonstrate total Jak2 expression amongst all samples (Figure 4C, bottom).

We next sought to determine whether Z3 could inhibit Jak2 phosphorylation at Tyr 1007 in these same cells. After treatment of the cells with either DMSO or 25 μM of Z3 for the indicated times, whole cell protein lysates were Western blotted with the phospho-specific (pY1007/pY1008) Jak2 antibody to detect phosphorylated Jak2 at this residue (Supplementary Figure 3, top). The results show that Z3 inhibited Jak2 phosphorylation at tyrosine 1007 in a time-dependent manner when compared to DMSO control. The membrane was subsequently stripped and reprobed with anti-Jak2 antibody to demonstrate total Jak2 protein expression amongst all samples (Supplementary Figure 3, bottom).

STAT3 is a known substrate of Jak2 and previous studies have shown that STAT3 is constitutively phosphorylated in HEL cells (19, 20). We therefore examined whether Z3-mediated inhibition of Jak2-dependent cell proliferation in HEL cells also correlates with reduced STAT3 phosphorylation. Specifically, protein cell lysates were immunoprecipitated with a STAT3 antibody and then immunoblotted with a phospho-specific (pY705) STAT3 antibody to assess ACTIVE STAT3 levels (Figure 4D, top). We found that Z3 inhibited STAT3 tyrosine phosphorylation in HEL cells when compared to DMSO control, thus demonstrating that reduced phospho-STAT3 levels also correlate with reduced cell numbers. The membrane was stripped and reprobed with a STAT3 antibody to demonstrate equal STAT3 content amongst all samples (Figure 4D, bottom).

Overall, the data in Figure 4 demonstrate that Z3 selectively blocks Jak2-V617F pathologic cell growth and this corresponds with reduced levels of activated Jak2 and STAT3.

Z3 Exerts its effect on the Cell Cycle by Increasing the Percentage of HEL Cells in G₁ Phase while Decreasing the Number of Cells in S phase

To determine the mechanism of Z3-mediated inhibition in cell growth, we first examined whether treatment with Z3 corresponded to an increase in apoptosis in the HEL cells. Specifically, HEL cells were treated with either DMSO or 25 μM of Z3 for 16, 24, 48 or 72 hours and cells were measured for Annexin V staining via flow cytometry. Treatment of cells with Z3 did not result in a significant increase in the percentage of Annexin V-positive-stained cells when compared to DMSO-treated cells (data not shown), indicating that Z3 did not induce apoptosis in this cell type. In addition, HEL cells treated with 25 μM of Z3 stained negative for an early marker of apoptosis, APO2.7, confirming that the mechanism by which Z3 suppresses HEL cell proliferation is independent of apoptosis (data not shown).

We next examined cell cycle distribution as a function of Z3 treatment using the CycleTest^™ PLUS DNA Kit. HEL cells were treated with either DMSO or 25 μM of Z3 for 16, 24, 48 or 72 hours and cell cycle parameters were determined by FACS analysis. We found that Z3 significantly increased the percentage of cells in the G1 phase (Figure 5A) and significantly decreased the percentage of cells in S phase (Figure 5B), when compared to DMSO control treated cells. A representative experiment of the cell cycle profile for DMSO- and Z3- treated HEL cells is also shown (Figures 5C and 5D, respectively). Collectively, the results in Figure 5 reveal that the mechanism by which Z3 reduces cell numbers is by blocking G1 to S transition.

FIGURE 5. Z3 induces cell cycle arrest in Jak2-V617F-transformed human erythroleukemia cells.

HEL cells were treated with either DMSO or 25 μM of Z3 for 16, 24, 48, or 72 hours and cell-cycle effects were determined. FACSCalibur flow cytometer along with Modfit software (Verity Software) was used to analyze DNA contents. The mean and standard deviation of samples were determined. Statistical significance between each group was analyzed using a two-way analysis of variance (ANOVA). (A) Percentage of HEL cells in G₁ phase following treatment with either DMSO or Z3 for the indicated times. The two conditions were considered to be significantly different (p=0.000407). (B) Percentage of HEL cells in S phase following treatment with either DMSO or Z3 for the indicated times. The two conditions were considered to be significantly different (p=0.0256). The graphs depicted in (A) and (B) represent the data from three independent experiments. Shown is a representative cell cycle analysis profile from one of those three experiments after 72 hours treatment with either DMSO (C) or Z3 (D).

Z3 Reduces Hematopoietic Colony Formation, Ex Vivo

Somatic Jak2 mutations have been described in myeloproliferative disorders (14, 21, 22). The most common Jak2 mutation, Jak2-V617F, has been shown to be the cause of several myeloproliferative diseases including PV, ET and PMF. We have already shown that Z3 suppresses Jak2-dependent cell growth in the HEL cell line (Figure 4A). We next turned our attention to determining whether Z3 could inhibit the growth of hematopoietic progenitor cells isolated from the bone marrow of confirmed myeloproliferative disorder patients. Here, residual bone marrow aspirates were obtained from an ET patient who was Jak2-V617F positive (Figure 6A) or from a PV patient who was Jak2-F537I positive (Figure 6B). Their hematopoietic progenitor cells were cultured in a semi-solid colony assay medium in the presence of DMSO or 25 μM of Z3. In addition, since hematopoietic progenitors taken from patients with myeloproliferative disorders are known to be hypersensitive to cytokine stimulation (23), the cells taken from the ET patient were cultured in both the presence and absence of human thrombopoietin (TPO) while the cells taken from the PV patient were cultured in the presence and absence of human erythropoietin (EPO). The results show that, as expected, treatment of the ET patient’s hematopoietic progenitor cells with TPO markedly increased megakaryocyte colony formation (Figure 6A). However, this cytokine-dependent increase in cell growth was significantly blunted when the cells were cultured in the presence of Z3. Similarly, treatment of the PV patient’s progenitor cells with EPO significantly increased erythrocyte colony formation. However, treatment of the cell with Z3 significantly reduced both cytokine-independent as well as cytokine-dependent erythrocyte colony formation (Figure 6B). Collectively, the results in Figure 6 demonstrate that Z3 greatly reduces Jak2-V617F as well as Jak2-F537I mediated, human pathologic cell growth, ex vivo.

FIGURE 6. Z3 suppresses Jak2-mediated hematopoietic colony formation, ex vivo.

(A) Marrow derived mononuclear cells from an ET patient who was Jak2-V617F positive were cultured in methylcellulose media containing either DMSO (0.25%, vol/vol) or 25 μM Z3. The number of megakaryocyte colonies was assessed 14 days later. Results are expressed as the average number of colonies from duplicate cultures per 4 × 10⁵ cells. Statistical significance between each group was analyzed using Student’s t test. *, p = 0.017. (B) Marrow derived mononuclear cells from a PV patient who was Jak2-F537I positive were cultured in methylcellulose media containing either DMSO (0.25%, vol/vol) or 25 μM Z3. The number of erythrocyte colonies was assessed 14 days later. Results are expressed as the average number of colonies from duplicate cultures per 4 × 10⁵ cells. Statistical significance between each group was analyzed using Student’s t test. *, p = 0.0245; **, p = 0.0195.

DISCUSSION

Hyper-kinetic Jak2 tyrosine kinase activity has been linked to a variety of human diseases including cardiovascular disease, diabetes and cancer (24–28). In recent years, the Jak2-V617F gain-of-function mutation in myeloproliferative disorders has also been well described (5–7, 29, 30). Mutations in Exon 12 of Jak2 which cause dysregulated Jak-STAT signaling have been identified in Jak2-V617F-negative myeloproliferative disorders as well (14, 31, 32). Thus, the continued identification of novel agents that can inhibit aberrant Jak2 tyrosine kinase function will be of great value.

Here, we have used in silico homology modeling and high throughput molecular docking to identify a novel small molecule inhibitor of Jak2. Our results are significant for a number of reasons. First, Z3 inhibits Jak2-WT and Jak2-V617F autophosphorylation, but is not cytotoxic to cells at concentrations that inhibit Jak2. Second, Z3 inhibits phosphorylation of tyrosine 1007, a residue whose phosphorylation is concomitant with hyper-kinetic Jak2 function. Third, Z3 selectively inhibits Jak2 tyrosine kinase activity as it has no effect of Tyk2 and c-Src. Fourth, Z3 preferentially suppresses proliferation of HEL cells that express the Jak2-V617F mutation by inducing cell cycle arrest. This arrest in cell growth directly correlates with reduced levels of active Jak2 and STAT3 proteins. Fifth, Z3 significantly blocks the growth of hematopoietic progenitor cells isolated from the bone marrow of myeloproliferative disorder patients carrying Jak2 mutations.

The Z3 compound is yet another step in the continuing development of Jak2 inhibitors. High throughput screening of potential tyrosine kinase inhibitors identified tyrphostin B42 (AG490) as the first Jak2 inhibitor. Initially, AG490 was regarded as a specific inhibitor of Jak2 because it concomitantly inhibited Jak2 tyrosine phosphorylation levels and suppressed acute lymphoblastic leukemia by inducing cellular apoptosis (33). However, other studies suggested that it suffered from a general lack of specificity (9–11). Ensuing work by multiple groups, including our own, has identified various small molecules or in some cases, a protein mimetic, that block Jak2 kinase function (12, 34–36). Despite the fact that these agents all inhibit Jak2 kinase function, differences in both the structure as well the mechanism of action of the inhibitors is striking. For example, AG490 inhibits Jak2 and promotes cellular apoptosis (33) while our Z3 compound inhibits Jak2 and promotes cell cycle arrest. Interestingly, the TG101209 compound recently described by Pardanani et al. inhibits Jak2 and causes both increased apoptosis and cell cycle arrest (34). Finally, work by Flowers and colleagues characterized a peptide inhibitor of Jak2 that mimics the Jak2 inhibitory protein, SOCS-1 (36). The peptide mimetic was designed to recognize this autophosphorylation site of Jak2 similar to SOCS-1. They found that the peptide mimicked SOCS-1 in that it suppressed Jak2 tyrosine autophosphorylation and subsequent IFN-γ dependent signaling, independent of marked changes in apoptosis or cell cycle progression. Collectively, these works suggest that the relationship between blocking Jak2 kinase function and the eventual fate of the cell is complex and warrants further examination.

While the Jak2-V617F mutation on exon 14 is the predominant disease-associated allele in myeloproliferative disorders, several other Jak2 exon 14 mutations have been identified. For example, unique C616Y and D620E substitution mutations have been identified in V617F-negative, myeloproliferative disorder individuals (37, 38). Additionally, a number of mutations have been identified in Jak2 exon 12, including an F537 deletion in one individual and F537 duplication in another (14). Furthermore, chromosomal translocations between the Jak2 allele and other alleles, including TEL, REL, PCM1, and BCR, have all been linked to a number of hematological malignancies (2, 39–41). While each specific translocation gives rise to a unique chimeric protein, they all share one common feature; namely, they all exhibit hyper-kinetic Jak2 kinase activity and subsequent malignant hyperplasia. Thus, given the growing number of known Jak2 somatic cell mutations and chromosomal translocations, as well as the diverse human diseases that hyperkinetic Jak2 kinase activity has been associated with, identifying inhibitors that can block multiple Jak2 mutations, such as our Z3 compound, will be of great value.

In summary, our results suggest that Z3 inhibits Jak2 tyrosine kinase function. It suppresses Jak2-dependent pathologic cell growth in vitro via a Jak2/STAT3-dependent mechanism that results in cell cycle arrest. Additionally, it blocks ex vivo hematopoietic progenitor cell growth from an ET patient who harbors Jak2-V617F and a PV patient who carries a novel Jak2-F537I mutation. As such, this compound may have practical applications in Jak2 related research and perhaps serve as a lead therapeutic agent.

ABBREVIATIONS

Jak2

Janus kinase

STATs

signal transducers and activators transcription

PV

polycythemia vera

ET

essential thrombocythemia

PMF

primary myelofibrosis

HEL cells

human erythroleukemia cells

Tyr

tyrosine