Janus Kinase 2: a Critical Target in Chronic Myelogenous Leukemia

Priority Report

Janus Kinase 2: A Critical Target in Chronic Myelogenous Leukemia

Ajoy K. Samanta,1 Hui Lin,1 Tong Sun,1 Hagop Kantarjian,2 and Ralph B. Arlinghaus1

Departments of 1Molecular Pathology and 2Leukemia, The University of Texas M.D. Anderson Cancer Center, Houston, Texas

Abstract is the increase in c-Myc expression (5), which is required for The Bcr-Abl tyrosine kinase is the causative factor in most leukemia induction (9). chronic myelogenous leukemia (CML) patients. We have shown that Bcr-Abl is associated with a cluster of signaling Materials and Methods proteins, including Janus kinase (Jak) 2, growth factor Cell culture. The mouse cell line, 32Dp210, and the human leukemic cell receptor binding protein 2–associated binder (Gab) 2, Akt, lines, K562, KBM7, and BV173 cells, were cultured in RPMI 1640 medium and glycogen synthase kinase (GSK)-3B. Treatment of CML cell supplemented with 10% fetal bovine serum (FBS). Mouse 32Dcl3 cells were lines and mouse Bcr-Abl+ 32D cells with either Jak2 short supplemented with 10% WEHI-cultured conditioned medium as a source of interfering RNA or Jak2 kinase inhibitor AG490 inhibited pTyr interleukin-3 (IL-3) in addition to 10% FBS. Gab2 and pSer Akt formation, inhibited the activation of Antibodies and reagents. pTyr antibody (clone 4G10) was purchased 473 nuclear factor-KB, and caused the activation of GSK-3B, from Upstate (Charlottesville, VA). Akt, pSer Akt (pAkt), glycogen h 9 h leading to the reduction of c-Myc. Importantly, BaF3 cells synthase kinase (GSK)-3 , pSer GSK-3 , growth factor receptor binding expressing T315I and E255K imatinib-resistant mutants of protein 2 (Grb2)–associated binder (Gab) 2, c-Myc antibodies, Jak2 antibody (conjugated with protein A-agarose), and secondary antibodies Bcr-Abl underwent apoptosis on exposure to AG490 yet were against mouse, rabbit, and goat antibodies were purchased from Santa resistant to imatinib. Similar to wild-type Bcr-Abl+ cells, Cruz Biotechnology (Santa Cruz, CA). Antibodies against pTyr452Gab2 inhibition of Jak2 by Ag490 treatment resulted in decrease of (YxxM) were obtained from Cell Signaling Technology (Beverly, MA). Anti- pSer Akt and c-Myc in imatinib-resistant cells. These results h-actin antibody, HEPES, NP40, DTT, 4-(2-aminoethyl)-benzenesulfonyl identify Jak2 as a potentially important therapeutic target for fluoride, leupeptin, and aprotinin were purchased from Sigma (St. Louis, CML. (Cancer Res 2006; 66(13): 6468-72) MO). Poly(deoxyinosinic-deoxycytidylic acid), T4 polynucleotide kinase, protein G-agarose, and enhanced chemiluminescence reagents were Introduction purchased from Roche Diagnostics (Mannheim, Germany). The apoptosis measurement kit (Annexin V) was purchased from BD PharMingen (San Chronic myelogenous leukemia (CML) is caused by hemato- Diego, CA). poietic progenitor cells that possess the Philadelphia chromo- Immunoprecipitation, Western blotting, and electrophoretic mo- some, which encodes the Bcr-Abl oncoprotein (1). The driving bility shift assay. The methods are described (10). force for the leukemia is the activated tyrosine kinase of Bcr- Apoptosis assay. Apoptosis was measured by the Annexin V/propidium Abl, which stimulates several signal transduction pathways (1), iodide method as described (2, 5). including the Janus kinase (Jak) 2 pathway (2) and, separately, Preparation of short interfering RNA of Jak2. Short interfering RNA the signal transducer and activator of transcription 5 (STAT5) (siRNA) duplexes targeting human and mouse Jak2 were purchased from Dharmacon, Inc., Lafayette, CO (i.e., siGenome SMARTpool targeting human pathway (3). Imatinib mesylate is a selective inhibitor of the Bcr- Jak2, siGenome SMARTpool targeting mouse Jak2, and siCONTROL as Abl tyrosine kinase, and this drug is the frontline treatment for nontargeted control). The human siRNAs were specific to Jak2 and did not CML patients (4). have sequence overlap with Jak1, Jak3, and Tyk2. Bcr-Abl activates the Jak2 tyrosine kinase (2, 5), a member of the Transfection of siRNAs. For transfection of siRNAby electroporation Jak family. The activation step involves phosphorylation of critical method, we followed the nucleofection protocol of the manufacturer Tyr1007 (2, 5). Activation of Jak2 is also very important for Bcr-Abl- (Amaxa, Inc., Gaithersburg, MD). mediated oncogenicity (2). AG490, a member of the tyrphostin family of protein kinase inhibitors, is a potent and specific inhibitor of Jak2 kinase (6). In a human B precursor leukemic cell line, Results treatment with AG490 markedly down-regulates tyrosine phos- Jak2 kinase inhibitor AG490 inhibited Akt phosphorylation, phorylation of Jak2 (7). Although Ag490 also inhibits Jak3, Jak3 has activated GSK-3B, and decreased the expression of c-Myc. We not been reported to be involved in Bcr-Abl+ leukemia. examined the effects of AG490 on the growth and survival of In Bcr-Abl+ 32D cells, the activation of Jak2 does not lead to 32Dp210 cells. To determine the effects on the signaling molecules STAT5 activation (2, 5), which is activated by Bcr-Abl (3, 8). One downstream of Jak2, we examined 32Dp210 cells and the CML cell major effect of the activation of Jak2 by the Bcr-Abl oncoprotein line K562 after treating the cells with AG490. We incubated 32Dp210 cells with 100 Amol/L AG490 for 1 to 5 hours and measured the effects on the levels of pSer473 Akt, pSer9 GSK-3h, and c-Myc. The pAkt and pGSK-3h (but not the unphosphorylated Note: Supplementary data for this article are available at Cancer Research Online proteins) and c-Myc protein levels were sharply reduced after 2 to (http://cancerres.aacrjournals.org/). A Requests for reprints: Ralph B. Arlinghaus, Department of Molecular Pathology, 5 hours of treatment of 32Dp210 cells with AG490 (Fig. 1 ). The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Treatment of the CML cell line K562 with 100 Amol/L AG490 also Houston, TX 77030. Phone: 712-792-8995; Fax: 713-794-1395; E-mail: rarlingh@ decreased pGSK-3h and c-Myc (Fig. 1B). The amount of reduced mdanderson.org. I2006 American Association for Cancer Research. expression was estimated by dividing the intensity of the bands doi:10.1158/0008-5472.CAN-06-0025 normalized by h-actin loading controls by the normalized intensity

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of the untreated control (listed as percentage in Fig. 1 and other figures). The reduction of both pAkt and pGSK-3h by AG490 was dose dependent (Fig. 1C). Importantly, the level of nonphosphory- lated forms of Akt and GSK-3h (Fig. 1A-C) was not significantly reduced. AG490 inhibited activation of nuclear factor-KB. It is well known that nuclear factor-nB (NF-nB) activation increases levels of c-Myc transcripts, and Akt kinase activity is known to regulate NF- nB. The cells were incubated with different concentrations of AG490 for 16 hours. Compared with the untreated control, the activation of NF-nB was strongly reduced in the presence of AG490 (IC50,12Amol/L; Fig. 1D; Supplementary Fig. S1). Therefore, the reduction of NF-nB activation by AG490 in 32Dp210 cells indicates that the Bcr-Abl/Jak2 pathway also regulates NF-nB activation. Treatment of 32Dp210 and CML cell lines with Jak2 siRNA inhibited the phosphorylation of Akt but induced the activation of GSK-3B causing reduction of c-Myc. To establish the critical role of Jak2 in this Bcr-Abl pathway, we transfected Jak2-specific siRNAs in 32Dp210 cells, K562, and other human CML cell lines. At 48 hours after transfection with Jak2 siRNAs, the transfected cells were collected, and Western blotting was carried out with the various specific antibodies. After Jak2 siRNA treatment of 32Dp210 cells, Jak2 expression was reduced >75% compared with the untreated control (Fig. 2A). Analysis of the same lysates for pAkt and pGSK-3h showed significantly lower levels of these phosphoproteins and a lower level of c-Myc protein in 32Dp210 (Fig. 2A) and K562 cells (Fig. 2B). Like AG490 treatment, Jak2 siRNA–transfected cells also showed strongly reduced activation of NF-nB compared with the control (Fig. 2C). The effect of Jak2 siRNAon K562 cells was dose dependent (Supplementary Fig. S2A). We also tested several other CML cell lines besides K562, including BV-173 and KBM7, for the effects of Jak2 knockdown (Supplementary Fig. S2B-C). Transfection with Jak2 siRNAs reduced the level of Jak2 protein, the expression of the c-Myc protein, and the phosphoproteins pAkt and pGSK-3h. Because Gab2 is involved in the activation of phosphatidylinositol 3-kinase (PI3K; refs. 1, 13), we also measured levels of pTyr Gab2 using a sequence-specific antibody that detects one of the several pYxxM sequence motifs in Gab2 (14). Importantly, pTyr Gab2 was strongly reduced in these CML cell lines following Jak2 knockdown (Supplementary Fig. S2A-C). Nontargeted siRNA had little effect on Jak2 levels and also did not affect the levels of the downstream target, pTyr Gab2 (Fig. 2D). These Jak2 knockdown results were similar to the inhibitory effects obtained with AG490 treatment of 32Dp210 and K562 cells, which also strongly reduced pTyr Gab2 (Supplementary Fig. S3). Inhibition of the Jak2 tyrosine kinase in Bcr-Abl+ cells inhibited survival. Trypan blue dye exclusion showed that cell viability was significantly reduced in 32Dp210 cells and partially reduced in K562 cells [it has other oncogenic events besides Bcr- Figure 1. AG490 down-regulates activities of Akt and NF-nB and expression of Abl (e.g., lack of p53)] in a dose-dependent manner at 72 hours c-Myc and induces activation of the GSK-3h kinase. To examine the effects of AG490 on the levels of pAkt, c-Myc, and pGSK-3h in 32Dp210 and K562 cells, of treatment with AG490 treatment (Supplementary Fig. S4A-B). cell-free detergent extracts (60 Ag/lane) were used for SDS-PAGE followed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide Western blotting. A, AG490 treatment (100 Amol/L) of 32Dp210 cells for 1 to assays showed similar results (data not shown). AG490 also 5 hours. B, AG490 (100 Amol/L) treatment of CML cell line K562 for 2 to 5 hours. C, AG490 treatment of K562 cells for 16 hours with various concentrations induced apoptosis in 32p210 cells in a dose-dependent manner (0-100 Amol/L) of AG490. D, to examine the activation of NF-nB in 32Dp210 cells using the Annexin V and DNA fragmentation assays (Supplemen- and determine the effects of AG490 on the activation of NF-nB, electrophoretic mobility shift assays (EMSA) were carried out. 32Dp210 cells were incubated with tary Fig. S4C-D). However, the K562 cells were relatively resistant different concentrations of AG490. Nuclear extracts were prepared from the to AG490 treatment (data not shown). We note that cells treated untreated and AG490-treated cells. Nuclear extracts (10 Ag each tube) were with AG490 for 16 hours had no effect on viability (Supplementary incubated with 32P-labeled duplex oligonucleotide for 15 minutes at 37jC and separated in a native 6.6% polyacrylamide gel. The NF-nB-DNA complex was Fig. S5A-B); therefore, the effects of AG490 on signal transduction detected using PhosphorImager (GE Healthcare Life Sciences, Piscataway, NJ). in Fig. 1 were not caused by nonspecific toxic effects of AG490. www.aacrjournals.org 6469 Cancer Res 2006;66: (13). July 1, 2006

Figure 2. Knockdown of Jak2 by Jak2 siRNA reduced levels of pAkt, pGab2, pGSK-3h, and c-Myc in 32Dp210 and a Bcr-Abl+ CML cell line. A to D, to examine the role of Jak2 on the expression of downstream target molecules, a pool of Jak2 siRNA was transfected into the 32Dp210 and the K562 cell line using an electroporation apparatus following the manufacturer’s protocol. After 48 hours, the expression of Jak2 and other signaling molecules were examined by Western blotting (A, B, D). The activation of NF-nB and the effect of Jak2 siRNA on NF-nB activation were determined by EMSA (C).

AG490 treatment of imatinib-resistant Bcr-Abl+ BaF3 cells complex, which contains Bcr-Abl, Jak2, Akt, and GSK-3h. Other induced apoptosis. Several mutations in the ATP-binding domain proteins, such as the PI3K and Gab2, are likely to be in the Bcr-Abl- of Bcr-Abl have been shown to render cells from CML patients Jak2 network (12, 13). resistant to imatinib (15). To determine whether the Jak2 pathway would still be required in imatinib-resistant Bcr-Abl+ cells, we treated imatinib-sensitive and imatinib-resistant Bcr-Abl+ BaF3 cells Discussion with 50 Amol/L AG490 and measured apoptosis induction after 72 We have presented evidence that Jak2 plays a critical role in the hours (Fig. 3A). The results showed that T315I-mutant Bcr-Abl+ cells, signal transduction through the Bcr-Abl/Jak2/Gab2/PI3K/Akt/ although resistant to imatinib, were sensitive to AG490 treatment. In GSK-3h network, which leads to the activation of many fact apoptosis induction by AG490 was higher in resistant than sensitive cells (Fig. 3A). Similarly, the E255K mutant was also more responsive to AG490 than the imatinib-sensitive cells. The imatinib- resistant mutant Bcr-Abl proteins used the Bcr-Abl/Jak2/Akt network for maintaining growth and survival of imatinib-resistant cells (Fig. 3B), suggesting that drugs that inhibit Jak2 may be useful for treatment of imatinib-resistant CML patients. GSK-3B is associated with Bcr-Abl/Jak2/Akt signaling network. To identify the Bcr-Abl-associated proteins (termed the network), detergent lysates of the mouse 32D and 32Dp210 cell lines were analyzed by immunoprecipitation with either Jak2 antibody-protein Aconjugate (for Jak2-associated proteins) or by the addition of other specific antibodies (e.g., GSK-3h). These same antibodies were used to identify Bcr-Abl network–associated proteins by Western blotting of the immunoprecipitates (Fig. 4). Immunoprecipitation and Western blotting of the detergent- soluble lysates from either 32D cells maintained in IL-3 or 32Dp210 cells with the anti-Jak2 antibody detected Akt and GSK- 3h (Fig. 4A) and Bcr-Abl in the 32Dp210 lysate (data not shown). If the same lysates were used for immunoprecipitation with anti-Akt and probed with the respective antibodies, Bcr-Abl (only in the 32Dp210 lysate) and GSK-3h were detected (Fig. 4B). Immunopre- h cipitation with anti-GSK-3 of these lysates followed by Western Figure 3. AG490 induces apoptosis in imatinib-resistant Bcr-Abl+ BaF3 cells. blotting with the appropriate antibodies identified cyclin D1, a A, imatinib-sensitive and imatinib-resistant BaF3 cells were treated with 5 Amol/L h imatinib and 50 Amol/L AG490 for 72 hours, and the cells were assayed for target of GSK-3 kinase, and Bcr-Abl but only in the 32Dp210 lysate + C apoptosis by Annexin V/propidium iodide staining. B, BCR-ABL BaF3-sensitive (Fig. 4 ). Controls for specificity are shown in Supplementary Fig. and BaF3-resistant cells treated with 25 Amol/L and 50 Amol/L AG490 for S6A-B. Thus, Bcr-Abl protein network constitutes a signaling 16 hours. The cell lysates were analyzed for pAkt and c-Myc.

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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2006 American Association for Cancer Research. Jak2 Mediates Bcr-Abl Activation of Akt downstream targets, including c-Myc (Fig. 4D; refs. 2, 5, 11–13). (PIP3) from phosphatidylinositol 3,4-biphosphate and then This pathway regulates c-Myc expression by (a) transcriptional attracts components of the Bcr-Abl/Jak2 network to the inner activation of c-Myc transcription through NF-nB and (b) enhanced face of the cell surface membrane through pleckstrin homology h e stability of c-Myc protein by inactivation of GSK-3 kinase by domains; ( ) the Akt activation pathway (PIP3, PDK1, and Akt); phosphorylation at Ser9 of GSK-3h by pAkt (Fig. 4D). We propose and (f ) GSK-3h. Imatinib effectively reduced the effects of Bcr- that Jak2 may be a target for treatment of CML patients, especially Abl on all members of the Bcr-Abl/Jak2 network that were those who are resistant to imatinib because AG490 treatment of examined (data not shown). Signal transduction by this Bcr-Abl/ hematopoietic cells transformed by imatinib-resistant mutants of Jak2 protein network results in phosphorylation and activation of Bcr-Abl (15) was induced to undergo apoptosis (Fig. 3A). Akt, which down-regulates the kinase activity of GSK-3h and The presence of a multimeric protein complex composed of thus leads to the stabilization of c-Myc and activates NF-nBto Bcr-Abl, Jak2, Gab2, PI3K, Akt, and GSK-3h is supported by cause elevation of c-Myc transcripts (Fig. 4D). Although the various immunoprecipitation experiments using anti-Akt, anti- possible involvement of other signaling networks cannot be Jak2, and anti-GSK-3 (Fig. 4A-C). In CML cells, Bcr-Abl activates excluded, our findings suggest that this Bcr-Abl network plays a Jak2 (2, 5). Our studies (Figs. 1 and 2) indicate that, in Bcr-Abl+ central role in the cell proliferation and survival of Bcr-Abl+ cells, a dedicated Jak2 pathway is involved in activating the leukemia cells. Gab2/PI3K/Akt pathway but not STAT5 activation (2, 5). Like Our findings with knockdown of Jak2 in CML cell lines (K562, others, we term this multimeric complex the Bcr-Abl network BV173, and KBM7) further strengthen the role of Jak2 in the Bcr- (Fig. 4D; refs. 12, 13). This Bcr-Abl network includes (a) the Bcr- Abl/Jak2/Gab2/Akt/GSK-3h network (Fig. 2; Supplementary Fig. S2). Abl/Jak2 complex (2, 5), which leads to phosphorylation of Gab2 These results were very similar to the effects of AG490 treatment at the Tyr within the YxxM motif that is significantly reduced in of K562 and 32Dp210 cells and thus confirm that the Jak2 kinase lysates of Bcr-Abl+ cells following Jak2 knockdown (Fig. 2); (b) is a major component of the Bcr-Abl network that leads to the the Bcr-Abl/Grb2/Gab2 complex, which is formed by autophos- enhancement of c-Myc expression and possibly other important phorylation of Tyr177 of Bcr-Abl and is involved in activation of targets that contribute to the oncogenic phenotype induced by the PI3K (11); (c) the p85 regulatory subunit and the p110 Bcr-Abl. catalytic subunit of the PI3K, in which the Src homology (SH) 3 The PI3K pathway is well known to be a transducer of the domain of p85 interacts with the Bcr-Abl network (13) and the Bcr-Abl tyrosine kinase signal, which is essential for cell proli- SH2 domain of p85 binds to the pYxxM in Gab2 (12, 13); (d) the feration and survival (12–14). As expected, we observed that active PI3K, which forms phosphatidylinositol 3,4,5-triphosphate treatment of 32Dp210 cells with the PI3K inhibitors wortmannin

Figure 4. Bcr-Abl and Jak2 proteins are part of a protein network. A to C, 32Dp210 cells were immunoprecipitated with several antibodies, and the immunoprecipitates were detected by Western blotting. D, model for the role of Jak2 in the Bcr-Abl/Jak2/ Gab2/PI3K/Akt/NF-nB/GSK-3h network that enhances c-Myc protein levels in Bcr-Abl+ hematopoietic cells.

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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2006 American Association for Cancer Research. Cancer Research and LY294002 diminished pAkt and c-Myc levels and, at the patients were shown to have a mutation in Jak2 that activates the same time, activated the kinase activity of GSK-3h (data not kinase (19). shown). Thus, the Bcr-Abl effects on c-Myc are likely to be Adaptor proteins, such as Gab1, Gab2, insulin receptor substrate mediated through the Bcr-Abl/Jak2/Gab2/PI3K/Akt/GSK-3h (IRS)-1, and IRS-2, have been shown to have a critical role in pathway. It has been further shown that Grb2 binding to coupling tyrosine kinase activation to PI3K recruitment in protein pTyr177 of Bcr-Abl leads to Gab2 binding and activation of PI3K networks (20). Our studies have focused on Gab2, but they do not (11). Our findings indicate that inhibition of Jak2, either by eliminate the involvement of other Gab isoforms. treatment of CML cells with AG490 or knockdown of Jak2 by use In summary, we have presented evidence to support the concept of Jak2 siRNAs, reduced the level of tyrosine phosphorylation of that the Bcr-Abl network contains Jak2/Gab2/PI3K/Akt/GSK-3h. the YxxM sequence in Gab2. The Gab2 pYxxM sequence binds Importantly, our findings have shown that mouse hematopoietic the regulatory subunit of the PI3K, which leads to the activation cells transformed by the T315I mutant of Bcr-Abl were strongly of the PI3K (Fig. 4D; ref. 14). induced to undergo apoptosis by AG490 treatment (Fig. 3A). Thus, Another important issue is whether Jak2 or another tyrosine specific inhibitors of Jak2 may be useful for the treatment of kinase downstream of Jak2 is the actual kinase that phosphor- imatinib-resistant forms of CML. ylates Gab2 at the YxxM sequence (Fig. 4D; ref. 14). The kinase specificity of Jak2 is not well known, which raises the possibility Acknowledgments that Jak2 may phosphorylate the YxxM site. Alternatively, Received 1/6/2006; revised 3/28/2006; accepted 4/25/2006. another tyrosine kinase downstream of Jak2 and activated by Grant support: NIH grant CA93792 and Hendrick Marrow Foundation. Jak2 might catalyze the phosphorylation of Gab2 at the YxxM The costs of publication of this article were defrayed in part by the payment of page sequence. charges. This article must therefore be hereby marked advertisement in accordance Jak2 with 18 U.S.C. Section 1734 solely to indicate this fact. Several fusions involving the gene have been reported to We thank Dr. Charles Sawyers (University of California at Los Angeles, Los Angeles, induce myeloid leukemias (16–18). In addition, polycythemia vera CA) for supplying BaF3 cells with imatinib-resistant Bcr-Abl mutations.

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Ajoy K. Samanta, Hui Lin, Tong Sun, et al.

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