Published OnlineFirst on August 31, 2010 as 10.1158/0008-5472.CAN-10-0607

Tumor and Stem Cell Biology Cancer Research A Specific Need for CRKL in p210BCR-ABL–Induced Transformation of Mouse Hematopoietic Progenitors

Ji-Heui Seo1, Lisa J. Wood2,3, Anupriya Agarwal4, Thomas O'Hare3,4, Collin R. Elsea4, Ian J. Griswold4, Michael W.N. Deininger4, Akira Imamoto1, and Brian J. Druker3,4

Abstract CRKL (CRK-like) is an adapter predominantly phosphorylated in cells that express the tyrosine ki- nase p210BCR-ABL, the fusion product of a (9;22) chromosomal translocation causative for chronic myeloid leukemia. It has been unclear, however, whether CRKL plays a functional role in p210BCR-ABL transformation. Here, we show that CRKL is required for p210BCR-ABL to support interleukin-3–independent growth of myeloid progenitor cells and long-term outgrowth of B-lymphoid cells from fetal liver–derived hematopoietic progen- itor cells. Furthermore, a synthetic phosphotyrosyl peptide that binds to the CRKL SH2 domain with high affinity blocks association of endogenous CRKL with the p210BCR-ABL complex and reduces c-MYC levels in K562 human leukemic cells as well as in mouse hematopoietic cells transformed by p210BCR-ABL or the imatinib-resistant mutant T315I. These results indicate that the function of CRKL as an adapter protein is essential for p210BCR-ABL–induced transformation. Cancer Res; 70(18); OF1–11. ©2010 AACR.

Introduction 22q11.21 (16). Overexpression of CRKL enhances p190BCR-ABL– induced transformation and leukemogenesis in fibroblasts and Patients with chronic myeloid leukemia (CML) harbor a in a transgenic mouse model (17, 18). CRKL links tyrosine kinase Philadelphia (Ph) due to a translocation between substrates to downstream effectors containing SH3-binding 9 and 22 (1). The resulting BCR-ABL fusion gene motifs. CRKL is constitutively phosphorylated by BCR-ABL in gives rise to a p210BCR-ABL oncoprotein (2, 3). Forced expression neutrophils of CML patients, and the degree of CRKL phosphor- of the p210BCR-ABL protein renders hematopoietic cells growth ylation is a marker of BCR-ABL kinase activity (19, 20). factor independent (4, 5) and induces proliferation of myeloid Direct binding of CRKL to p210BCR-ABL is mediated by as- progenitors and bone marrow–derived B-lymphoid cells (6–9). sociation of the CRKL SH3n domain with a proline-rich motif BCR-ABL Transplantation of p210 –transduced murine bone mar- in ABL (21). However, a mutant BCR-ABL lacking the SH3- row cells into lethally irradiated syngeneic mice induces a CML- binding motif still transforms myeloid cells (21), suggesting like myeloproliferative disorder, showing the leukemogenic that CRKL remains associated with the p210BCR-ABL complex BCR-ABL potential of p210 in vivo (10–13). The tyrosine kinase by interactions with other . Cell-penetrating peptides BCR-ABL activity of p210 is essential for its oncogenic potential designed to bind with high affinity to the CRKL SH3n domain both in vitro and in vivo (13, 14); hence, the ABL inhibitor block the proliferation of primary CML blast cells (22). These imatinib has been a successful treatment for CML (15). SH3-binding peptides also bind to the CRK gene products with CRKL (CRK-like), a member of the CRK family of adapter high affinity (23), identifying a commonly encountered difficulty proteins, consists of an NH2-terminal SH2 domain followed in studies designed to provide definitive evidence for a role by two SH3 domains: SH3n and SH3c (16). Although related that CRKL may play in p210BCR-ABL–induced transformation. to CRK, CRKL is a distinct gene located on chromosome We and others generated mouse mutants with targeted disruptions at the Crkl locus (24, 25). Crkl deficiency in 129/Sv and C57BL/6 backgrounds results in embryonic le- Authors' Affiliations: 1Ben May Department for Cancer Research, Committees on Developmental Biology, Genetics, Cancer Biology, and thality that mimics 22q11 deletion syndrome, also known Cell Physiology, University of Chicago, Chicago, Illinois and 2Oregon as DiGeorge syndrome (24). The phenotype seems to be sen- Health & Science University School of Nursing; 3Howard Hughes Medical Institute, Oregon Health & Science University; 4Division of sitive to genetic background and disappears in a mixed ge- Hematology and Medical Oncology, Oregon Health & Science netic background involving 129/Sv and Black Swiss (25). University Cancer Institute, Portland, Oregon Using this Crkl-“insensitive” background, it was shown that Note: J-H. Seo and L.J. Wood are co-first authors. Crkl deficiency did not inhibit leukemogenesis caused by Corresponding Author: Akira Imamoto, Ben May Department for Cancer p190BCR-ABL in a mouse model for acute lymphoblastic leuke- Research, Committees on Developmental Biology, Genetics, Cancer Biology, and Cell Physiology, University of Chicago, 929 East 57th Street, mia (ALL; ref. 25). Disappearance of the embryonic phenotype, GCIS-W332, Chicago, IL 60637. Phone: 773-834-1258; Fax: 773-702- however, indicates that this genetic background provides an 4476; E-mail: [email protected]. alternative mechanism that compensates for loss of Crkl. doi: 10.1158/0008-5472.CAN-10-0607 Biological differences between p210BCR-ABL and p190BCR-ABL ©2010 American Association for Cancer Research. may also explain the results of the study mentioned above.

www.aacrjournals.org OF1 Seo et al.

− p190BCR-ABL causes Ph+ ALL, which is relatively refractory to poietic progenitor cells. For colony-forming assays, Wt, Crkl+/ , − − imatinib therapy (26). A recent study shows that a significant or Crkl / cells harvested from fetal livers of embryonic day 13.5 subset of leukemia cells from p190BCR-ABL transgenic mice is (E13.5) murine embryos were incubated overnight in prestimu- insensitive to imatinib and quickly takes over the culture par- lation medium [Iscove's modified Dulbecco's medium, 15% FBS, ticularly when stromal cells are present (27). Because imatinib 5% WEHI-conditioned medium, 1 μg/mL ciprofloxacin, murine is still effective at blocking the kinase activity of p190BCR-ABL interleukin (IL)-3 (6 ng/mL), IL-6 (10 ng/mL), and stem cell BCR-ABL in these resistant cells, this phenomenon suggests a kinase- factor (50 ng/mL)]. Cells were transduced with p210 – independent mechanism in p190BCR-ABL–induced leukemia. expressing retrovirus in the presence of 2 μg/mL polybrene p190BCR-ABL can sustain survival and proliferation of leukemia (Sigma-Aldrich) by two rounds of spinoculation (29), and 8 × 4 cells in the presence of imatinib (thus without substantial ABL 10 cells per 35-mm dish were plated in triplicate in Methocult tyrosine kinase activity), which may explain the ability of M3234 medium (StemCell Technologies) with or without IL-3 p190BCR-ABL to induce leukemia in the transgenic model with- (100 pg/mL). Colonies were scored at day 12. out Crkl. To directly address the requirement for CRKL for Whitlock-Witte cultures were performed as described (8). +/− BCR-ABL Briefly, cells harvested from the fetal liver of E13.5 Wt, Crkl , p210 transformation, we have therefore used hematopoi- −/− BCR-ABL etic progenitor cells from the fetal liver of Crkl-deficient mouse or Crkl mouse embryos were infected with p210 or embryos in a Crkl-sensitive genetic background. Our results are control retrovirus in lymphoid medium (RPMI 1640 plus 10% μ further supported by studies with a synthetic peptide that blocks FBS and 0.05 mmol/L 2-mercaptoethanol) containing 8 g/mL association of CRKL with the p210BCR-ABL protein complex in polybrene by two rounds of spinoculation. Approximately 5×106 cells were plated per 60-mm dish in triplicate. Cul- mouse hematopoietic progenitor cells and in human K562 leuke- tureswerefedevery3daysbyadding2mLoflymphoidme- mia cells. The current study shows an essential role for CRKL dium. Viable nonadherent cells were counted at day 14 after in p210BCR-ABL–induced transformation of hematopoietic transduction, and viability was determined by trypan blue progenitor cells. dye exclusion.

Materials and Methods Analysis of cell surface markers Immunophenotyping of p210BCR-ABL–transformed lym- Mice phoid cells expanded from Whitlock-Witte cultures as well − − − The Institutional Animal Use and Care Committees of the as fresh fetal liver cells from E13.5 Wt, Crkl+/ ,andCrkl / University of Chicago and the Oregon Health & Science Uni- embryos was performed by fluorescence-activated cell sorting +/− versity approved animal studies. Crkl mice were described (FACS) using a FACSCalibur instrument (BD Biosciences). previously (24). All mice and embryos were examined in a Whitlock-Witte culture-derived lymphoid cells transformed mixed 129S4/SvJaeSor × C57BL/6J background. Embryos with p210BCR-ABL werestainedwithphycoerythrin(PE)- +/− were derived by timed matings between Crkl parents. conjugated B220, Sca1, or c-Kit. For analysis of hematopoietic progenitor populations in fetal liver, lineage-committed (Lin+) Cell lines hematopoietic cells were identified with the biotinylated mouse Certified BaF3 and K562 cells were obtained from the lineage antibody cocktail (BD Biosciences) and streptavidin- − American Type Culture Collection and grown in the recom- conjugated PE-Cy7 (Caltag Laboratories, Inc.). Lin cells were mended culture medium. The BaF3 transfectant (expressing then stained with the following antibodies: PE-conjugated the p210BCR-ABL mutant T315I mutation) has been previously c-Kit, allophycocyanin (APC)-conjugated Thy1.1, and FITC- described (28). Transformed p210BCR-ABL B-lymphoid cells conjugated Sca1. The percentage of stem cells in each fetal derived from wild-type (Wt) and Crkl-deficient fetal liver cells liver cell population was calculated by determining the num- − were generated as described below. None of the cell lines ber of Lin ,c-Kit+,Sca1+,andThy1.1+ cells. To evaluate used in this study were cultured for longer than 6 months the composition of B-cell progenitors in the fetal liver of − − − from initial purchase or characterization. No further authen- Wt, Crkl+/ ,andCrkl / mice,cellswerestainedwithB220- tication of cell line characteristics was done. APC, CD43-PE, IgM-APC, and IgD-APC. Based on immunophe- − notype, cells were characterized as pro-B (B220low,CD43+,IgM , − − − − Retrovirus preparation IgD ), pre-B (B220low, CD43 ,IgM ,IgD ), immature B (B220moderate, − − − Bosc23 cells (28) were maintained in DMEM supplemen- CD43 ,IgM+,IgD), or mature B (B220high,CD43,IgMlow,IgDhigh) ted with 10% fetal bovine serum (FBS). Hematopoietic pro- cells. Monoclonal antibodies raised against B220, Sca1, Thy1.1, genitor cells were infected with p210BCR-ABL retrovirus or c-Kit, and CD43 were purchased from BD Biosciences, and for control green fluorescent protein (GFP) retrovirus generated IgM and IgD from Southern Biotechnology Associates. by transfecting Bosc23 cells with MSCV-p210-IRES-GFP (13) or empty MSCV-IRES GFP vector, and the viral supernatant Peptides, immunoprecipitation, and immunoblotting was harvested at 48 hours after transfection. The purity of synthetic peptides was >95%. The amino acid sequences used were as follows: pYELP peptide, Myeloid and lymphoid outgrowth assays GVSEpYELPEDPRWELPR; CRKL pY207 peptide, GIPEPAHA- − − Due to the lethal phenotype of Crkl / embryos at late ges- pYAQPQTTTPLPA; and CRK pY221 peptide, GGPEPGP- tation (24), the fetal liver was harvested as a source of hemato- pYAQPSVNTPLPN. Control peptides included a tyrosine or

OF2 Cancer Res; 70(18) September 15, 2010 Cancer Research CRKL and BCR-ABL Transformation

phenylalanine residue in place of phosphotyrosine (pY). Co- kinase inhibitor imatinib, PEP-1 (a 21–amino acid peptide immunoprecipitation, SDS-PAGE, and immunoblotting were carrier) was used to deliver phosphorylated or control peptides performed using standard protocols. In addition to fetal into the cell (30). The phosphorylated or control peptide was liver–derived mouse lymphoid cells, we used the human preincubated with PEP-1 for 1 hour at a molar ratio of 1:20. CML blast crisis cell line K562 and murine cell lines BaF3- The peptide mix was then added to the cell culture and in- p210BCR-ABL or BaF3-p210BCR-ABL with T315I mutation. Cell cubated for the indicated amount of time before washout lysates were prepared in lysis buffer [15% glycerol, 1% NP40, and lysis. Imatinib, kindly provided by E. Buchdunger (No- 50 mmol/L Tris (pH 7.4), 0.2 mol/L NaCl, 2.5 mmol/L MgCl2] vartis), was prepared freshly as a 10 mmol/L stock solution including protease inhibitor cocktail (Roche), 1 mmol/L sodi- in sterile PBS. um orthovanadate, and 10 mmol/L NaF. For peptide inhibition Immunoblots were performed according to a standard experiments, phosphopeptide or control peptide was added to method and probed using specific antibodies. To probe phos- precleared cell lysates and incubated for 1 hour at room tem- phorylation at Y649 in signal transducer and activator of perature. Stable association of CRKL or CRK with p210BCR-ABL transcription 5A (STAT5A) and at Y699 in STAT5B, we used was assessed by coimmunoprecipitation with mouse anti-ABL mouse monoclonal anti–phospho-STAT5 (clone ST5P-4A9; monoclonal antibody (clone 8E9; Santa Cruz Biotechnology) Invitrogen). Total STAT5 (STAT5A and STAT5B) was deter- and anti-mouse IgG-conjugated Sepharose beads (Sigma), mined by rabbit anti-STAT5 (Santa Cruz Biotechnology). The followed by SDS-PAGE and immunoblot analysis with rabbit BCL-X and MYC gene products were probed with rabbit – anti-CRKL (C20) or anti-CRK antibody (Santa Cruz Biotech- anti BCL-XL(SantaCruzBiotechnology)andrabbitpoly- nology). Prior control experiments performed with isotype- clonal anti-Myc (Upstate). matched, irrelevant antibody confirm the specificity of bands detected in the high-stringency anti-ABL immunoprecipitates. Cell cycle analysis To assess the biological effects of the phosphopeptide on Cell cycle analysis was carried out by FACS. Cells were col- BCR-ABL function, and to compare its activity to the ABL lected by centrifugation, washed with PBS, and permeabilized

Figure 1. CRKL is required for p210BCR-ABL–induced growth factor independence in myeloid cells. Equal numbers of fetal liver cells from E13.5 Wt, Crkl+/−, and Crkl−/− littermate embryos were infected with p210BCR-ABL retrovirus and cultured (A) alone or (B) supplemented with 100 pg/mL of mouse IL-3. Myeloid colonies were counted 12 d after infection. Columns, average from six independent assays in triplicate; bars, SD. *, P = 0.01 and 0.007, for Crkl+/− and Crkl−/− fetal liver cells, respectively, compared with Wt; **, P = 0.0005, between Crkl−/− and Crkl+/− cells. C, differences in relative proportion of colonies of CFU-GM, CFU-M, or CFU-GEMM lineages formed by Wt, Crkl+/−, or Crkl−/− cells.

www.aacrjournals.org Cancer Res; 70(18) September 15, 2010 OF3 Seo et al.

in 70% ethanol at −20°C for 30 minutes before DNA staining. Results Approximately 1 × 106 permeabilized cells were incubated with 50 μg/mL propidium iodide (Roche) or 3 μmol/L 4′,6- CRKL is required for p210BCR-ABL–induced diamidino-2-phenylindole (DAPI; Invitrogen) and 0.1 mg/mL IL-3–independent myeloid outgrowth RNase A in staining buffer [100 mmol/L Tris (pH 7.4), BCR-ABL expression in hematopoietic progenitor cells re- 150 mmol/L NaCl, 1 mmol/L CaCl2, 0.5 mmol/L MgCl2,and sults in growth factor–independent myeloid colonies in 0.1% NP40] for 15 to 30 minutes. Stained cells were analyzed methylcellulose (6). To assess the requirement of CRKL in − in a FACSCalibur or BD LSR II flow cytometer (BD Bios- this process, fetal liver cells harvested from E13.5 Wt, Crkl+/ , − − ciences) using ModFit LT or FACSDiva software. and Crkl / embryos were infected with retrovirus that cotransduces p210BCR-ABL and GFP. Transduction efficiency − − − Proliferation assays of Wt, Crkl+/ ,andCrkl / cells was similar, as comparable − − p210BCR-ABL–transformed Wt and Crkl / hematopoietic numbers of cells were GFP+ by FACS analysis following cells (5 × 104 per well) that were expanded from Whitlock- transduction (data not shown). Cells were seeded into meth- Witte cultures were plated into 24-well flat-bottomed plates ylcellulose in the presence or absence of IL-3. Growth factor– in RPMI 1640 containing 5% FBS and 50 μmol/L 2-mercapto- dependent and growth factor–independent colonies were ethanol. Cells were counted daily for 3 days. Results represent counted on day 12. We found that p210BCR-ABL expression in counts obtained from triplicate wells performed with cells Wt cells leads to the formation of cytokine-independent mye- derived from three independent Whitlock-Witte cultures. loid colonies of granulocyte-macrophage colony-forming unit

Figure 2. The requirement for CRKL in generating p210BCR-ABL B-lymphoid cells. A, fetal liver cells infected with p210BCR-ABL retrovirus were cultured under conditions favoring outgrowth of transformed B lymphocytes. Following incubation at 37°C for 14 d, viable cells were counted. Columns, average from three independent assays in triplicate with Wt, Crkl+/−,orCrkl−/− matched littermates; bars, SD. *, P = 0.05; **, P = 0.0005, Crkl+/− and Crkl−/− compared with Wt. B, the p210BCR-ABL– transformed lymphoid cells from Wt cultures were B220+, whereas Crkl−/− cells were largely B220−. Crkl−/− cells expressed markers of more primitive progenitors, such as c-Kit and Sca1. C, immunoblotting of BCR-ABL and CRKL in Wt and Crkl-deficient p210BCR-ABL–transformed lymphoid cells with anti-ABL and anti-CRKL antibodies, with actin as a loading control.

OF4 Cancer Res; 70(18) September 15, 2010 Cancer Research CRKL and BCR-ABL Transformation

Figure 3. Crkl deficiency does not affect distribution of stem cell and B-cell progenitor populations in fetal liver cells isolated from E13.5 Wt, Crkl+/−, and Crkl−/− mouse embryos. FACS profiling of stem cells (Thy1.1+ and Sca1+), pre-B cells (B220low), pro-B cells (IgMlow,CD43+), immature (CD43−, IgM+), and mature (CD43−, IgMlow) B-cell progenitors revealed no significant differences in the distribution of cells from Wt, Crkl+/−,orCrkl−/− embryos.

(CFU-GM), macrophage CFU (CFU-M), and granulocyte- data suggest that (a) Crkl deficiency does not compromise erythrocyte-macrophage-megakaryocyte CFU (CFU-GEMM) the generation of hematopoietic colonies in response to physio- lineages. In contrast, there was a 3-fold reduction in the logic stimuli and (b) decreased myeloid colony formation in − − number of growth factor–independent colonies formed by Crkl / cultures is not due to decreased transduction efficiency − − − Crkl / cells (Fig. 1A). The number of Crkl+/ colonies was of early myeloid progenitors in the absence of CRKL. reduced by 2-fold, and there were significantly fewer − − − cells formed by Crkl / cells than Crkl+/ cells, suggesting a CRKL is required for p210BCR-ABL–induced dosage-sensitive effect of CRKL on p210BCR-ABL–induced lymphoid outgrowth − transformation (Fig. 1A). In the presence of IL-3, Wt, Crkl+/ , To further explore the requirement of CRKL for trans- − − − − and Crkl / cells gave similar numbers of myeloid colonies formation by p210BCR-ABL, we assessed outgrowth of Crkl / (Fig. 1B). There was no difference in the relative proportion lymphoid cells induced by p210BCR-ABL. Hematopoietic pro- of colonies of CFU-GEMM, CFU-GM, or CFU-M lineages genitor cells harvested from the fetal livers of 13.5-day-old − − − − − − formed by Wt, Crkl+/ ,orCrkl / cells (Fig. 1C). Importantly, Wt, Crkl+/ ,andCrkl / embryos were transduced with all colonies were GFP+ by fluorescence microscopy and con- p210BCR-ABL retrovirus and cultured under conditions favoring tained the provirus by PCR screening (data not shown). These outgrowth of transformed lymphoid cells (8). A similar

www.aacrjournals.org Cancer Res; 70(18) September 15, 2010 OF5 Seo et al.

− cells differed between fetal liver cells derived from Wt, Crkl+/ , − − − and Crkl / embryos. The percentage of stem cells (Lin , c-Kit+, − − − Sca1+,Thy1.1+) was the same in Wt, Crkl+/ ,andCrkl / fetal liver cells (Fig. 3). Moreover, we found no difference in the percentage − − − of pro-B (B220low, CD43+, IgM ,IgD), pre-B (B220low, CD43 , − − − − IgM ,IgD), immature B (B220moderate,CD43,IgM+,IgD), or − mature B (B220high,CD43 ,IgMlow,IgDhigh) cells in the fetal liver − − − of Wt, Crkl+/ ,orCrkl / embryos (Fig. 3). We conclude that − − − reduction in B-cell outgrowth from Crkl / and Crkl+/ fetal liver in Whitlock-Witte cultures was not due to a deficiency in B-cell progenitors. This finding is consistent with previous findings that there was no difference in the ability of hema- − − topoietic progenitor cells derived from Crkl / fetal livers to − − reconstitute the bone marrow of irradiated Rag2 / recipients compared with Wt with similar numbers of circulating B220+ cells (32).

Phosphotyrosine-mediated interactions play a major role in association of CRKL with the p210BCR-ABL complex Biological functions of adapter proteins such as CRKL rely on their ability to link partners through protein interaction domains (16). We previously showed that CRKL associates with fibroblast growth factor receptors, FGFR1 and FGFR2, Figure 4. pYELP peptide inhibits association of BCR-ABL and CRKL. through its SH2 domain (33). Based on a CRKL SH2 A, a phosphotyrosyl peptide, pYELP, dissociates CRKL from the domain-binding motif identified in FGFR1, we synthesized p210BCR-ABL complex in vitro. The 17–amino acid peptide pYELP or a phosphotyrosyl peptide (pYELP) exhibiting high affinity control peptides without phosphotyrosine (YELP and FELP) were added ∼ to cell lysates prepared from B-lymphoid cells derived from for the CRKL SH2 domain but 30-fold weaker binding to p210BCR-ABL–transduced Wt fetal liver. After 1-h incubation at room the SH2 domain of CRK (34). Although the peptide sequence temperature, stable association of CRKL or CRK with p210BCR-ABL was based on an FGFR1 autophosphorylation site, pYELP was assessed by coimmunoprecipitation (IP) with anti-ABL antibody peptide efficiently disrupted association of endogenous followed by immunoblot (IB) analysis with anti-CRKL or anti-CRK antibody. BCR-ABL B, pYELP peptide inhibits association of CRKL with p210BCR-ABL in vivo. CRKL with the p210 protein complex when added to B-lymphoid cells derived from Wt fetal liver transduced with cell lysates from Wt cells (Fig. 4A). Conversely, this peptide p210BCR-ABL retrovirus cultured in Whitlock-Witte medium were had no inhibitory effect on association of CRK with the incubated with pYELP or similar phosphopeptide corresponding to p210 protein complex (Fig. 4A). In the same experiment, con- CRKL pY207 or CRK pY221 along with PEP-1 carrier peptide for trol peptides without phosphotyrosine (YELP and FELP) did 2 h before cell lysates were prepared. Association of CRKL and BCR-ABL p210BCR-ABL was evaluated by coimmunoprecipitation. not affect association of CRKL with the p210 complex. Further, we observed that pYELP peptide also inhibited their association in vivo in a dose-dependent manner when the − − − percentage of Wt, Crkl+/ , and Crkl / cells were GFP+ following peptide is introduced into the cell along with a cell mem- retroviral transduction with p210BCR-ABL (data not shown), but brane penetrating peptide carrier PEP-1 (Fig. 4B; ref. 30). Of − − − fewer cells were observed in Crkl+/ (P = 0.05) and Crkl / (P = note, appropriate control experiments were performed with 0.0005) cultures at day 14 after transduction compared with an isotype-matched, irrelevant antibody and these experi- − − Wt cultures (Fig. 2A). We also observed fewer cells in Crkl / ments confirmed the specificity of bands detected in the − relative to Crkl+/ cultures, suggesting a dosage-sensitive effect high-stringency anti-ABL immunoprecipitates (data not of CRKL on p210BCR-ABL–induced lymphoid transformation. shown). In total, these results indicate that pYELP peptide Consistent with previous reports (31), all cells emerging from can block the adapter function of CRKL in vitro and in vivo. − − Wt fetal liver expressed the B220 antigen; however, Crkl / cells CRKL is constitutively phosphorylated at Y207 in leukemia − were largely B220 and expressed markers of more primitive cells expressing p210BCR-ABL (35). Y207 and Y221 in CRKL and progenitors, such as c-Kit and Sca1 (Fig. 2B). There was no CRK, respectively, are thought to play a role in regulating difference in p210BCR-ABL levels shown between these cells adapter functions through intramolecular associations − − (Fig. 2C), indicating that poor outgrowth of cells from Crkl / fe- (34, 36). Interestingly, however, phosphotyrosyl peptides tal liver was not due to compromised expression of p210BCR-ABL. based on the internal SH2 domain-binding motif in CRKL These results suggest that p210BCR-ABL fails to induce B220+ and CRK (pY207 or pY221 peptide, respectively) failed to disso- lymphoid cells in the absence of CRKL but could also be attrib- ciate these adapters from the p210BCR-ABL complex at concen- − − − utable to skewed progenitor populations in Crkl+/ and Crkl / trations up to 10 μmol/L (Fig. 4B; data not shown), providing cells compared with Wt cells. We therefore investigated by evidence that CRKL functions as an adapter despite phos- FACS whether the composition of hematopoietic progenitor phorylation at Y207 in leukemia cells.

OF6 Cancer Res; 70(18) September 15, 2010 Cancer Research CRKL and BCR-ABL Transformation

We used the ability of pYELP peptide to dissociate CRKL decrease in phospho-STAT5 in CRKL-deficient p210BCR-ABL– from the p210BCR-ABL complex in Wt B-lymphoid cells trans- transformed cells relative to their Wt counterparts as well as BCR-ABL formed by p210 . Treatment with either pYELP peptide a corresponding reduction in BCL-XL (Fig. 6A). Consistent − − or imatinib led to minor decreases in the number of cells in with this, p210BCR-ABL–transformed Crkl / cells proliferated – G0-G1,S,andG2, whereas the population in sub G0-G1 was significantly more slowly than transformed Wt cells (Fig. 6B). markedly increased (P < 0.01) in mouse B-lymphoid cells trans- Next, we determined the effect of the blocking peptide pYELP formed by p210BCR-ABL in Whitlock-Witte culture (Fig. 5A). Treat- on the phosphorylation of STAT5 (at Y694 or Y699 in STAT5A ment of K562 cells with either pYELP peptide or imatinib led to or STAT5B, respectively) in both mouse and human cells. On BCR-ABL a significant increase in G0-G1 population (P < 0.01) and an in- treatment of p210 –transformed lymphoid cells with crease in the sub–G0-G1 population (P < 0.01) accompanied by pYELP peptide, phosphorylation of STAT5 was reduced in decreases in the S and G2 populations (P < 0.01; Fig. 5B). These a dose-dependent manner, whereas control peptides had lit- results indicate that blockage of CRKL-mediated pathways or tle effect (Fig. 6C, left). Similar effects of pYELP peptide were ABL catalytic activity had consistently similar effects in both also observed in the human CML cell line K562 (Fig. 6C, cell types, manifested as increased apoptosis in p210BCR-ABL– right). In both cases, reduced phospho-STAT5 levels were transformed B-lymphoid cells or as G1 arrest in K562 cells. again associated with a similar decrease in BCL-XL levels (Fig. 6C). Taken together, these results indicate an important CRKL is required for p210BCR-ABL–induced high role for CRKL in the cell survival network that p210BCR-ABL BCL-XL and phospho-STAT5 levels aberrantly activates. p210BCR-ABL constitutively activates the transcription factor STAT5 (encoded by the Stat5A and Stat5B genes), CRKL is required for p210BCR-ABL–induced high which is required for transforming activity (37, 38). It has c-MYC levels been reported that p210BCR-ABL promotes expression of the The proto-oncogene c-MYC is required for BCR-ABL trans- prosurvival factor BCL-XL through STAT5 (39). Given the formation (40) and plays an essential role in G1 to S transition adapter role of CRKL in BCR-ABL–mediated activation of in cell cycle (41). We therefore determined whether c-MYC − − STAT5 signaling cascades, we investigated whether the phe- levels were dependent on CRKL. We found that Crkl / − − notypic defect observed in p210BCR-ABL–transformed Crkl / lymphoid cells expressing BCR-ABL had a striking reduction hematopoietic cells was a result of failure of BCR-ABL to ac- in c-MYC protein levels compared with Wt cells (Fig. 7A). This tivate STAT5 in the absence of CRKL. We observed a striking difference in Crkl-deficient lymphoid cells may again be

BCR-ABL Figure 5. pYELP peptide leads to G0-G1 cell cycle arrest with reduced cell survival. A, p210 –transformed mouse B-lymphoid cells isolated by Whitlock-Witte culture were treated with either pYELP, control peptide (CTR), or imatinib and cell cycle analysis was performed by DAPI staining using FACS 12 h after the treatment. B, K562 cells were treated with either pYELP or imatinib and cell cycle analysis was performed as described above. The percentage of cells in various stages of the cell cycle is shown to the right of cell cycle profiles. Columns, mean of four independent experiments; bars, SD. *, P < 0.01, significant difference from the control cells.

www.aacrjournals.org Cancer Res; 70(18) September 15, 2010 OF7 Seo et al.

Figure 6. CRKL is required for p210BCR-ABL–induced STAT5- dependent downstream molecular cascades. A, cell lysates from p210BCR-ABL–transformed Crkl- deficient and Wt lymphoid cells were immunoblotted with antibodies recognizing phospho-STAT5

(p-STAT5) and BCL-XL, with actin as a loading control. B, growth rates of BCR-ABL–transformed Wt and Crkl−/− cells. Cells were plated in triplicate, and viable cell counts were determined every 24 h by trypan blue exclusion. C, pYELP inhibits p210-induced upregulation

of BCL-XL and tyrosine phosphorylation of STAT5. p210BCR-ABL–transformed mouse B-lymphoid cells isolated by Whitlock-Witte culture and K562 cells were treated with pYELP or control peptides (YELP and FELP). Cell lysates were prepared 12 h after peptide treatment. Immunoblots were probed with

anti–BCL-XL, anti–phospho-STAT5 (Y694), or anti-STAT5 antibodies.

attributable to their primitive differentiation status (Fig. 2) signaling protein in p210BCR-ABL leukemogenesis has been the rather than CRKL-dependent properties. To address this is- focus of much attention. To address the role of CRKL in sue, we used pYELP peptide to dissociate CRKL from the transformation by p210BCR-ABL, we analyzed the ability of p210BCR-ABL protein complex in Wt B-lymphoid cells trans- p210BCR-ABL to transform hematopoietic progenitor cells de- formed by p210BCR-ABL. As expected, treatment with the rived from mice harboring a null mutation in the Crkl locus. ABL inhibitor imatinib resulted in reduced c-MYC protein In the current study, we used a Crkl-sensitive genetic back- levels in B-lymphoid cells derived from Wt fetal liver by ground in which Crkl deficiency results in a lethal phenotype p210BCR-ABL (Fig. 7B). When pYELP peptide was introduced during late embryogenesis (24). On the contrary, although an- into transformed B-lymphoid cells using the carrier peptide other Crkl knockout strain was used to assess the role of CRKL PEP-1, c-MYC protein levels were efficiently reduced in p190BCR-ABL–induced leukemogenesis, the genetic back- (Fig. 7B). In contrast, control peptides FELP or YELP without ground used was insensitive to CRKL as evidenced by disap- − − tyrosine phosphorylation had little effect. pYELP peptide pearance of an overt phenotype in Crkl / homozygous mice also had a similar effect on c-MYC levels in the CML blast (25). − − crisis K562 cells (Fig. 7C). We found that p210BCR-ABL–transduced Crkl / progenitors Emergence of imatinib-resistant CML cells leads to thera- showedamarkeddecreaseingrowthfactor–independent py failure in some patients treated with imatinib (42). There- myeloid colony formation compared with its Wt counter- fore, we tested the effect of pYELP peptide on BaF3 cells parts. Likewise, we found that compared with Wt, transfor- BCR-ABL BCR-ABL − − expressing p210 Wt or the p210 T315I mutant, mation of Crkl / progenitor cells by p210BCR-ABL resulted which is insensitive to imatinib. We found that imatinib failed in greatly reduced numbers of lymphoid cells under Whitlock- BCR-ABL to reduce c-MYC levels in cells that express the p210 Witte conditions. BCR-ABL provides both proliferative and T315I mutant, whereas treatment with pYELP peptide was survival signals (44, 45), and a reduction in myeloid colony for- able to reduce c-MYC levels in these cells (Fig. 7D). These re- mation; failed outgrowth of B-lymphoid populations in Whitlock- sults provide evidence that signaling pathways mediated by Witte cultures indicates a reduced ability of p210BCR-ABL to CRKL play an essential role in the induction or maintenance promote survival and/or proliferation in Crkl-deficient cells. BCR-ABL of elevated levels of the oncoprotein c-MYC in p210 – Our results suggest that p210BCR-ABL–transformed mouse transformed cells. B-lymphoid cells are particularly sensitive to CRKL-dependent pathways for survival (Fig. 5). Consistent with our results using − − Discussion a CRKL SH2 blocking peptide, p210BCR-ABL–transformed Crkl / fetal liver cells outgrown from Whitlock-Witte cultures showed Since the discovery that CRKL is a major substrate of BCR- an abnormal cell cycle profile as well as reduced BCL-XLlevels ABL in CML patient neutrophils (19, 20, 43), the role of this compared with p210BCR-ABL–transformed Wt counterparts.

OF8 Cancer Res; 70(18) September 15, 2010 Cancer Research CRKL and BCR-ABL Transformation

These CRKL-deficient lymphoid cultures, however, presented Anecdotal evidence for compromised survival included the − − cell markers different from B-lymphoid cells (Fig. 2), thus mak- inability of p210BCR-ABL–transduced Crkl / cells to recover from ing it difficult to directly interpret these results in light of a role storage in liquid nitrogen or ultralow-temperature freezer, that CRKL may play in p210BCR-ABL–induced transformation. whereas Wt fetal liver counterparts transformed by p210BCR-ABL were easily reestablished from low-temperature storage. This was, however, an obstacle to further investigation of − − Crkl / cells after introduction of p210BCR-ABL by additional experimental manipulations such as transfection. It has been reported that p210BCR-ABL induces expression of the antiapoptotic gene BCL-XL (39). One explanation for our observations above is that loss of CRKL as a transcrip- tional coactivator of STAT5 may reduce STAT5-dependent transcription of BCL-XL, thus rendering susceptibility to ap- optosis. Tyrosine phosphorylation at Y694 or Y699 in STAT5A and STAT5B, respectively, is essential for STAT5 dimeriza- tion required for its transcriptional activity (46). Thus, reduc- tion of tyrosine phosphorylation of STAT5 at these sites in p210BCR-ABL–transformed cells on treatment with pYELP peptide likely contributes to increased apoptotic cell death. CRKL is associated with STAT5 in p210BCR-ABL–expressing cells (47), and glutathione S-transferase fusion proteins car- rying either the SH2 or the first of the two SH3 domains of CRKL were able to pull down STAT5 in lysates of cells ex- pressing p210BCR-ABL (40, 48). As the tyrosine phosphoryla- tion site of STAT5 is essential for dimerization required for its transcriptional activity, binding of the CRKL SH2 domain to this site would be counterproductive to the function of STAT5. Although it is possible that the SH3 domain may me- diate their physical interaction in the cell, CRKL-STAT5 com- plexes are not found in cells that do not express p210BCR-ABL. Thus, interactions between CRKL and STAT5 may be stabi- lized in a tertiary protein complex in which CRKL SH2 do- main may also play a role. It remains to be resolved how the CRKL-STAT5 complex is maintained in p210BCR-ABL– expressing cells. Previous studies have indicated that c-MYC plays an es- sential role in p210BCR-ABL–mediated transformation (40, 48). The c-MYC oncoprotein is overexpressed in many human neoplasms and has been found to regulate cell cycle (41). It is noteworthy that MYC is a putative STAT5 target gene (49, 50). However, p210BCR-ABL can transform STAT5A/STAT5B compound homozygous bone marrow cells (51), thus suggesting that c-MYC may be induced by STAT5- dependent and STAT5-independent pathways downstream Figure 7. CRKL is required for p210BCR-ABL–induced high c-MYC levels. of p210BCR-ABL. Our results indicate that disruption of CRKL BCR-ABL A, c-MYC protein levels were determined in p210 –transformed Wt SH2–mediated interactions results in a reduction of c-MYC Crkl−/− or lymphoid cells derived from fetal liver. The blot was reprobed BCR-ABL– with anti-actin antibody for loading controls. B, c-MYC levels were protein levels in p210 expressing cells. Therefore, decreased by pYELP treatment in p210BCR-ABL–transformed B-lymphoid CRKL is likely involved in multiple pathways. Future studies cells from Wt fetal liver. Peptide was added to B-lymphoid cells at are warranted to elucidate more precise pathways that the different concentrations, and resulting cell lysates were probed with adapter protein CRKL mediates in p210BCR-ABL–induced c-MYC and c-ABL antibodies. C, blocking CRKL adapter function BCR-ABL– leukemogenesis. As CRKL SH2 blocking peptide can reduce with pYELP peptide decreases c-MYC levels in murine p210 BCR-ABL transformed B-lymphoid cells from Whitlock-Witte cultures and in human c-MYC levels in the imatinib-resistant p210 mutant K562 CML cells. Note that pYELP reduces c-MYC levels in both cell T315I, analysis of CRKL-mediated pathways may provide types. D, pYELP treatment decreased c-MYC levels in BaF3 cells useful therapeutic targets for treatment of CML patients. transformed with Wt p210BCR-ABL and T315I mutant p210BCR-ABL. Note that although T315I is resistant to imatinib, the cells still respond to the treatment with pYELP peptide. Cells were treated with either pYELP Disclosure of Potential Conflicts of Interest peptide mixed with the carrier peptide PEP-1 or imatinib for 2 h. Cell lysates were probed with antibodies against c-MYC or c-ABL. No potential conflicts of interest were disclosed.

www.aacrjournals.org Cancer Res; 70(18) September 15, 2010 OF9 Seo et al.

Acknowledgments kemia Research Foundation, Howard Hughes Medical Institute Research Resources Program, and National Institute of Dental and Craniofacial We thank R. Duggan and D. Leclerc (University of Chicago Flow Cytometry Research/NIH grant R01 DE015883 (A. Imamoto). Facility) for technical assistance and P. Nash and D.L. Guris for critical reading The costs of publication of this article were defrayed in part by the of the manuscript. payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate Grant Support this fact.

National Cancer Institute, Specialized Center of Research Award from the Leukemia and Lymphoma Society, Clinical Scientist Award from the Received 02/18/2010; revised 06/18/2010; accepted 07/07/2010; published Burroughs Wellcome Fund, and T.J. Martell Foundation (B.J. Druker) and Leu- OnlineFirst 08/31/2010.

References 1. Kurzrock R, Gutterman JU, Talpaz M. The molecular genetics of tyrosine phosphoprotein in chronic myelogenous leukemia cells. Philadelphia chromosome-positive leukemias. N Engl J Med 1988;319: Blood 1994;84:2912–8. 990–8. 20. ten Hoeve J, Arlinghaus RB, Guo JQ, Heisterkamp N, Groffen J. 2. Bartram CR, de Klein A, Hagemeijer A, et al. Translocation of c-ab1 Tyrosine phosphorylation of CRKL in Philadelphia+ leukemia. Blood oncogene correlates with the presence of a Philadelphia chromo- 1994;84:1731–6. some in chronic myelocytic leukaemia. Nature 1983;306:277–80. 21. Heaney C, Kolibaba K, Bhat A, et al. Direct binding of CRKL to BCR- 3. Heisterkamp N, Stephenson JR, Groffen J, et al. Localization of the ABL is not required for BCR-ABL transformation. Blood 1997;89: c-ab1 oncogene adjacent to a translocation break point in chronic 297–306. myelocytic leukaemia. Nature 1983;306:239–42. 22. Kardinal C, Konkol B, Schulz A, et al. Cell-penetrating SH3 domain 4. Daley GQ, Baltimore D. Transformation of an interleukin 3-dependent blocker peptides inhibit proliferation of primary blast cells from CML hematopoietic cell line by the chronic myelogenous leukemia-specific patients. FASEB J 2000;14:1529–38. P210bcr/ protein. Proc Natl Acad Sci U S A 1988;85:9312–6. 23. Posern G, Zheng J, Knudsen BS, et al. Development of highly selec- 5. Hariharan IK, Adams JM, Cory S. -abl oncogene renders myeloid tive SH3 binding peptides for Crk and CRKL which disrupt Crk- cell line factor independent: potential autocrine mechanism in chronic complexes with DOCK180, SoS and C3G. Oncogene 1998;16: myeloid leukemia. Oncogene Res 1988;3:387–99. 1903–12. 6. Gishizky ML, Witte ON. Initiation of deregulated growth of multipo- 24. Guris DL, Fantes J, Tara D, Druker BJ, Imamoto A. Mice lacking the tent progenitor cells by bcr-abl in vitro. Science 1992;256:836–9. homologue of the human 22q11.2 gene CRKL phenocopy neuro- 7. Gishizky ML, Witte ON. BCR/ABL enhances growth of multipotent cristopathies of DiGeorge syndrome. Nat Genet 2001;27:293–8. progenitor cells but does not block their differentiation potential 25. Hemmeryckx B, Reichert A, Watanabe M, et al. BCR/ABL P190 in vitro. Curr Top Microbiol Immunol 1992;182:65–72. transgenic mice develop leukemia in the absence of Crkl. Oncogene 8. McLaughlin J, Chianese E, Witte ON. In vitro transformation of imma- 2002;21:3225–31. ture hematopoietic cells by the P210 BCR/ABL oncogene product of 26. Ottmann OG, Druker BJ, Sawyers CL, et al. A phase 2 study of the Philadelphia chromosome. Proc Natl Acad Sci U S A 1987;84: imatinib in patients with relapsed or refractory Philadelphia 6558–62. chromosome-positive acute lymphoid leukemias. Blood 2002;100: 9. Young JC, Witte ON. Selective transformation of primitive lymphoid 1965–71. cells by the BCR/ABL oncogene expressed in long-term lymphoid or 27. Mishra S, Zhang B, Cunnick JM, Heisterkamp N, Groffen J. Resis- myeloid cultures. Mol Cell Biol 1988;8:4079–87. tance to imatinib of bcr/abl p190 lymphoblastic leukemia cells. Can- 10. Daley GQ, Van Etten RA, Baltimore D. Induction of chronic myelog- cer Res 2006;66:5387–93. enous leukemia in mice by the P210bcr/abl gene of the Philadelphia 28. Pear WS, Nolan GP, Scott ML, Baltimore D. Production of high-titer chromosome. Science 1990;247:824–30. helper-free retroviruses by transient transfection. Proc Natl Acad Sci 11. Elefanty AG, Hariharan IK, Cory S. bcr-abl, the hallmark of chronic U S A 1993;90:8392–6. myeloid leukaemia in man, induces multiple haemopoietic neo- 29. Kotani H, Newton PB, Zhang S, et al. Improved methods of retroviral plasms in mice. EMBO J 1990;9:1069–78. vector transduction and production for gene therapy. Hum Gene 12. Kelliher MA, McLaughlin J, Witte ON, Rosenberg N. Induction of a Ther 1994;5:19–28. chronic myelogenous leukemia-like syndrome in mice with v-abl 30. Morris MC, Depollier J, Mery J, Heitz F, Divita G. A peptide carrier for and BCR/ABL. Proc Natl Acad Sci U S A 1990;87:6649–53. the delivery of biologically active proteins into mammalian cells. Nat 13. Zhang X, Ren R. Bcr-Abl efficiently induces a myeloproliferative Biotechnol 2001;19:1173–6. disease and production of excess interleukin-3 and granulocyte- 31. Scherle PA, Dorshkind K, Witte ON. Clonal lymphoid progenitor cell macrophage colony-stimulating factor in mice: a novel model for lines expressing the BCR/ABL oncogene retain full differentiative chronic myelogenous leukemia. Blood 1998;92:3829–40. function. Proc Natl Acad Sci U S A 1990;87:1908–12. 14. Lugo TG, Pendergast AM, Muller AJ, Witte ON. Tyrosine kinase 32. Peterson AC, Marks RE, Fields PE, Imamoto A, Gajewski TF. T cell activity and transformation potency of bcr-abl oncogene products. development and function in CrkL-deficient mice. Eur J Immunol Science 1990;247:1079–82. 2003;33:2687–95. 15. Druker BJ, Talpaz M, Resta DJ, et al. Efficacy and safety of a specific 33. Moon AM, Guris DL, Seo JH, et al. Crkl deficiency disrupts Fgf8 sig- inhibitor of the BCR-ABL tyrosine kinase in chronic myeloid leuke- naling in a mouse model of 22q11 deletion syndromes. Dev Cell mia. N Engl J Med 2001;344:1031–7. 2006;10:71–80. 16. Feller SM. Crk family adaptors-signalling complex formation and bi- 34. Seo JH, Suenaga A, Hatakeyama M, Taiji M, Imamoto A. Structural ological roles. Oncogene 2001;20:6348–71. and functional basis of a role for CRKL in a fibroblast growth factor 17. Hemmeryckx B, van Wijk A, Reichert A, et al. Crkl enhances leuke- 8-induced feed-forward loop. Mol Cell Biol 2009;29:3076–87. mogenesis in BCR/ABL P190 transgenic mice. Cancer Res 2001;61: 35. de Jong R, ten Hoeve J, Heisterkamp N, Groffen J. Tyrosine 207 in 1398–405. CRKL is the BCR/ABL phosphorylation site. Oncogene 1997;14: 18. Senechal K, Heaney C, Druker B, Sawyers CL. Structural require- 507–13. ments for function of the Crkl adapter protein in fibroblasts and he- 36. Kobashigawa Y, Sakai M, Naito M, et al. Structural basis for the matopoietic cells. Mol Cell Biol 1998;18:5082–90. transforming activity of human cancer-related signaling adaptor 19. Nichols GL, Raines MA, Vera JC, Lacomis L, Tempst P, Golde DW. protein CRK. Nat Struct Mol Biol 2007;14:503–10. Identification of CRKL as the constitutively phosphorylated 39-kD 37. Shuai K, Halpern J, ten Hoeve J, Rao X, Sawyers CL. Constitutive

OF10 Cancer Res; 70(18) September 15, 2010 Cancer Research CRKL and BCR-ABL Transformation

activation of STAT5 by the BCR-ABL oncogene in chronic myeloge- cyclin D2 in BCR/ABL-induced proliferation of hematopoietic cells. nous leukemia. Oncogene 1996;13:247–54. Cancer Res 2002;62:535–41. 38. Sillaber C, Gesbert F, Frank DA, Sattler M, Griffin JD. STAT5 activa- 46. Stoecklin E, Wissler M, Moriggl R, Groner B. Specific DNA binding of tion contributes to growth and viability in Bcr/Abl-transformed cells. STAT5, but not of glucocorticoid receptor, is required for their func- Blood 2000;95:2118–25. tional cooperation in the regulation of gene transcription. Mol Cell 39. Gesbert F, Griffin JD. Bcr/Abl activates transcription of the Bcl-X Biol 1997;17:6708–16. gene through STAT5. Blood 2000;96:2269–76. 47. Rhodes J, York RD, Tara D, Tajinda K, Druker BJ. CrkL functions as a 40. Sawyers CL, Callahan W, Witte ON. Dominant negative MYC blocks nuclear adaptor and transcriptional activator in Bcr-Abl-expressing transformation by ABL oncogenes. Cell 1992;70:901–10. cells. Exp Hematol 2000;28:305–10. 41. Pelengaris S, Khan M, Evan G. c-MYC: more than just a matter of life 48. Afar DE, Goga A, McLaughlin J, Witte ON, Sawyers CL. Differential and death. Nat Rev Cancer 2002;2:764–76. complementation of Bcr-Abl point mutants with c-MYC. Science 42. Quintas-Cardama A, Kantarjian H, Cortes J. Flying under the radar: 1994;264:424–6. the new wave of BCR-ABL inhibitors. Nat Rev Drug Discov 2007;6: 49. Huang M, Dorsey JF, Epling-Burnette PK, et al. Inhibition of Bcr-Abl 834–48. kinase activity by PD180970 blocks constitutive activation of STAT5 43. Oda A, Miyakawa Y, Druker BJ, et al. Crkl is constitutively tyrosine and growth of CML cells. Oncogene 2002;21:8804–16. phosphorylated in platelets from chronic myelogenous leukemia 50. Lord JD, McIntosh BC, Greenberg PD, Nelson BH. The IL-2 receptor patients and inducibly phosphorylated in normal platelets stimulated promotes lymphocyte proliferation and induction of the c-MYC, bcl- by thrombopoietin. Blood 1996;88:4304–13. 2, and bcl-x genes through the trans-activation domain of STAT5. 44. Hess P, Pihan G, Sawyers CL, Flavell RA, Davis RJ. Survival signal- J Immunol 2000;164:2533–41. ing mediated by c-Jun NH(2)-terminal kinase in transformed B lym- 51. Sexl V, Piekorz R, Moriggl R, et al. STAT5a/b contribute to interleukin phoblasts. Nat Genet 2002;32:201–5. 7-induced B-cell precursor expansion, but abl- and bcr/abl-induced 45. Jena N, Deng M, Sicinska E, Sicinski P, Daley GQ. Critical role for transformation are independent of STAT5. Blood 2000;96:2277–83.

www.aacrjournals.org Cancer Res; 70(18) September 15, 2010 OF11