Letters to the Editor 229 Figure 1 (a–d) The BCR–ABL variants show similar tyrosine phosphorylation profiles in vitro.(a) Domain organization of the three BCR–ABL variants tested in this study. The coiled-coil (CC), DBL and pleckstrin homology (PH) domains pertain to BCR; the SH3, SH2 and TK domains pertain to ABL. The p200BCR–ABL variant lacks the PH domain, while p190BCR–ABL lacks both the PH and DBL-like domains. Each BCR–ABL variant was stably expressed in 32D cells and whole-cell lysates were subjected to immunoblot analysis with antibodies specific for (b) ABL or (c) global tyrosine phosphorylation (4G10). (d) Immunoblot analysis of STAT5 and STAT6 phosphorylation in whole cell lysates from Ba/F3 cells expressing each of the three BCR–ABL variants. (e–h) of the PH domain is sufficient to increase the lymphoid transformation potency of BCR–ABL. Donor bone marrow cells from (e) 5-FU-treated Balb/c mice or (f) non-5-FU-treated Balb/c mice were infected with equivalent titer retrovirus expressing p210BCR–ABL, p200BCR–ABL or p190BCR–ABL and transplanted into lethally irradiated recipient mice. Kaplan–Meier survival curves are shown. Mortality events due to CML-like disease, B-ALL-like disease and unrelated causes are marked with square, circle and triangle symbols, respectively. (g, h) Representative fluorescence-activated cell sorting analyzes for CML-like and B-ALL-like disease, respectively. Peripheral blood cells were measured for GFP expression on the y axis. The x axis represents staining with antibodies specific for myeloid cells (Ly6G), B cells (B220), T cells (Thy1.2), early myeloid cells (CD11b) or erythroid cells (Ter119). CML-like and B-ALL-like disease was analyzed in different mouse tissues using GFP (y axis) and either Ly6G or B220 (x axis), respectively. a slight further increase is observed with the additional deletion 2Howard Hughes Medical Institute, Portland, OR, USA of BCR exons 2–8. This is consistent with the data presented by E-mail: [email protected] Li et al., which showed that p190BCR–ABL but not p210BCR–ABL induced B-ALL in this model. Given that the associations References between the type of BCR–ABL and the disease 7 phenotype are not stringent, additional mechanisms must be 1 Deininger MW, Goldman JM, Melo JV. The molecular biology of operational. For example, pre-B cells may have a higher chronic myeloid . Blood 2000; 96: 3343–3356. likelihood of acquiring 190BCR–ABL compared with hemato- 2 Li S, Ilaria Jr RL, Million RP, Daley GQ, Van Etten RA. The P190, poietic stem cells that are thought to be the origin of CML. P210, and P230 forms of the BCR/ABL induce a similar chronic myeloid leukemia-like syndrome in mice but have Although it remains to be established that PH domain- different lymphoid leukemogenic activity. J Exp Med 1999; 189: dependent signals are critical to phenotype selection, our results 1399–1412. show a role for this domain in BCR–ABL signal transduction and 3 Lugo TG, Pendergast AM, Muller AJ, Witte ON. transforming capacity. activity and transformation potency of - oncogene products. Science 1990; 247: 1079–1082. 4 Kin Y, Li G, Shibuya M, Maru Y. The Dbl homology domain of Conflict of interest BCR is not a simple spacer in P210BCR-ABL of the Philadelphia . J Biol Chem 2001; 276: 39462–39468. 5 Ilaria Jr RL, Van Etten RA. P210 and P190(BCR/ABL) induce The authors declare no conflict of interest. the tyrosine phosphorylation and DNA binding activity of multi- ple specific STAT family members. J Biol Chem 1996; 271: 31704–31710. Acknowledgements 6 Benekli M, Baer MR, Baumann H, Wetzler M. Signal transducer and activator of transcription in . Blood 2003; This study was supported in part by the Leukemia and Lymphoma 101: 2940–2954. Society (BJD, MWD), the TJ Martell Foundation (BJD) and NHLBI 7 Melo JV. The diversity of BCR-ABL fusion proteins and their grant HL082978-01 (MWD). JWT is supported by the William relationship to leukemia phenotype. Blood 1996; 88: 2375–2384. 8 Russo C, Gao Y, Mancini P, Vanni C, Porotto M, Falasca M et al. Lawrence Foundation and the Oregon Clinical and Translational Modulation of oncogenic DBL activity by phosphoinositol Research Institute. MWD is a Scholar in Clinical Research of the phosphate binding to pleckstrin homology domain. J Biol Chem Leukemia and Lymphoma Society. BJD is an investigator of the 2001; 276: 19524–19531. Howard Hughes Medical Institute. 9 Han J, Luby-Phelps K, Das B, Shu X, Xia Y, Mosteller RD et al. Role of substrates and products of PI 3-kinase in regulating activation of Rac-related guanosine triphosphatases by Vav. Science 1998; 279: S Demehri1, T O’Hare1,2, CA Eide1,2, CA Smith1, JW Tyner1, 1,2 1 558–560. BJ Druker and MWN Deininger 10 Rossman KL, Worthylake DK, Snyder JT, Siderovski DP, Campbell 1 Division of Hematology and Medical Oncology, Oregon SL, Sondek J. A crystallographic view of interactions between Dbs Health and Science University Knight Institute, and Cdc42: PH domain-assisted guanine nucleotide exchange. Portland, OR, USA and Embo J 2002; 21: 1315–1326.

Supplementary Information accompanies the paper on the Leukemia website (http://www.nature.com/leu)

Inhibition of Syk protein tyrosine kinase induces apoptosis and blocks proliferation in T-cell non-Hodgkin’s lymphoma cell lines

Leukemia (2010) 24, 229–232; doi:10.1038/leu.2009.198; prognosis compared with aggressive B-cell lymphomas. There- published online 24 September 2009 fore, novel targeted therapies are needed. Syk is a protein tyrosine kinase involved in B-cell receptor signaling in both Peripheral T-cell lymphomas (PTCLs) are among the most normal and malignant B cells.1 Syk activation in B-cell aggressive of all lymphomas and are associated with a poor lymphomas is associated with cell growth and survival, and an

Leukemia Letters to the Editor 230 orally available Syk inhibitor is currently being tested for B-cell To determine whether Syk contributes to the growth and lymphomas in a phase I/II clinical trial, with encouraging survival of T-cell lymphomas, a small interfering RNA (siRNA) results.2 Normal T lymphocytes lack Syk expression and use a approach was used to inhibit Syk expression. Syk siRNA Syk homolog, Zap-70, in receptor signaling. However, we effectively silenced Syk expression in both SU-DHL-1 and recently showed that Syk protein is aberrantly expressed in the SR-786 cells (Figure 2a). As Syk inhibition blocks proliferation majority of PTCLs.3 The functional role of Syk in PTCL is and induces apoptosis in B-cell lymphomas,1 we determined the currently unknown. Herein, we show that silencing Syk induces effects of Syk siRNA on PTCL cells. In both SU-DHL-1 and SR- apoptosis and blocks proliferation in PTCL cells. These findings 786, Syk siRNA significantly reduced both proliferation (3H-TdR suggest that Syk represents a novel therapeutic target for patients incorporation, Figure 2b) and cell viability (annexin V assay, with PTCL. Figure 2c). Syk siRNA also silenced Syk expression in SeAx cells, To establish an in vitro model for examining the effects of Syk in which Syk is not phosphorylated (data not shown). Prolifera- inhibition, we analyzed Syk expression in multiple PTCL cell tion of SeAx cells was determined after treatment with either a lines (SeAx, HuT 78, SR-786, Karpas 299 and SU-DHL-1) by control or Syk siRNA in a manner identical to that described in both western blot (Figure 1a) and flow cytometry (Figure 1b). Figure 2b. No significant difference in proliferation (67 691 þ /À Consistent with our previously reported data showing Syk 1974 cpm versus 73 884 þ /À 3467 cpm, respectively; n ¼ 3) expression in PTCL, three of five cell lines tested (SU-DHL-1, was observed between the two treatment groups. SR-786 and SeAx) showed strong Syk expression.3 Next, we As siRNA-based inhibition of therapeutic targets is difficult to determined the phosphorylation status of Syk in these cell lines. achieve clinically, we determined whether a Syk inhibitor currently SU-DHL-1 and SR-786 cells, but not SeAx cells, showed in clinical trial, R406,2 would recapitulate the functional effects of Syk phosphorylation at Y348 (Figure 1c and d), a site autopho- Syk siRNA in PTCL cells. As expected, R406 led to depho- sphorylated on B-cell receptor engagement in B cells.4 Syk was sphorylation of Syk in both SU-DHL-1 and SR-786 (Figure 2d). also phosphorylated at Y525/526 in both SU-DHL-1 and SR-786 Furthermore, R406 significantly inhibited cell proliferation in a (data not shown). dose-dependent manner (Figure 2e), and induced cell death in

Figure 1 Syk is expressed and phosphorylated in T-cell lymphomas. Cell lines (anaplastic large cell lymphoma cell lines SU-DHL-1, SR-786 and Karpas 299, and the cutaneous T-cell lymphoma cell lines SeAx and HuT 78 were used, as indicated) were analyzed for total Syk expression by western blot (a) and flow cytometry (b). (a) Protein lysates prepared from cell lines were separated by SDS-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad, Hercules, CA, USA) and incubated with primary antibodies to Syk or b-actin (BD, Franklin Lakes, NJ, USA), followed by anti-mouse secondary antibody (Pierce, Rockford, IL, USA). (b) Fluorochrome-conjugated antibodies (BD) were used to determine Syk expression using intracellular staining and flow cytometry on a FACSCalibur instrument (BD) as per the manufacturer’s recommendations. CellQuest or FACSDiva Software (BD) were used for analysis. (c) Similarly, intracellular phospho-specific flow cytometry was used to assess Syk (Y348) phosphorylation. Cells stained with an isotype control are shown in gray (filled). (d) Western blot was carried out on protein lysates from untreated cells using antibodies to Syk, phospho-Syk(Y348) or b-actin.

Leukemia Letters to the Editor 231

Figure 2 Syk promotes cell growth and survival in T-cell lymphomas. (a–c) Syk-specific (AAGAACTGGGCTCTGGTAATT) and negative-control small interfering RNAs (siRNAs) were obtained from Qiagen (Valencia, CA, USA). SU-DHL-1 and SR-786 cells (2 Â 105 per well of a 24-well plate) were transfected with siRNA (100 nM final concentration) using HiPerFect (Qiagen) as per the manufacturer’s recommendations. (a) After transfection, Syk expression was evaluated by western blot. (b) Tritiated thymidine incorporation was determined for the last 12 h of a 48-h culture. Data shown are representative of at least three similarly performed experiments. (c) Cell viability was assessed 72 h after transfection using annexin V/propidium iodide staining and flow cytometry (pooled data from three independent experiments). (d–f) SU-DHL-1 (top row) and SR-786 (bottom row) cells were subjected to R406 and Syk phosphorylation (d), and cell proliferation (e) and viability (f) determined. R406 (a generous gift from Rigel, San Francisco, CA, USA) was dissolved in dimethylsulphoxide (DMSO) at a concentration of 40 mM and stored at À80 1C. Stock solution was diluted immediately after thawing in the appropriate volume of DMSO. Final DMSO concentration in any given assay did not exceed 0.5%. A dose titration experiment using both SU-DHL-1 and SR-786 cells revealed that Syk phosphorylation was almost completely inhibited at a concentration of 8 mM; unless otherwise indicated, studies were carried out using this concentration. (d) Cells were treated with R406 or vehicle alone (DMSO), and Syk phosphorylation was determined 24 h later by intracellular phospho-specific flow cytometry. Cells stained with an isotype control are shown in gray (filled). (e) Tritiated thymidine incorporation was determined for the last 12–15 h of culture, 72 h after treatment with the R406 concentrations shown. (f) Similarly, cell viability was assessed by annexin V (Caltag, Carlsbad, CA, USA) and propidium iodide (Sigma, St Louis, MO, USA) staining after treatment with R406 or vehicle. The data shown are representative of at least three similarly performed experiments. both lines (Figure 2f). The approximately 30% reduction in cell of ALK on Syk expression and activation are unknown, but viability observed was comparable to that observed (range 20– warrant further study as SYK upregulation has been reported in 70%) in the diffuse large B-cell lymphoma lines tested (for ALK-positive ALCLs.5 Conversely, the extent to which Syk example, DHL6, Ly3). These effects were not because of inhibition may contribute to lymphomagenesis in ALK-negative PTCLs, of the Syk homolog, Zap-70, as SU-DHL-1 lacks Zap-70 including PTCL unspecified, will require further study, ideally expression (data not shown). R406 treatment of NPM-ALK þ (and using primary tumor samples. Although ‘tonic’ B-cell receptor SykÀ) Karpas 299 cells failed to inhibit phosphorylation of AKT and signaling in diffuse large B-cell lymphomas may be Syk- ERK 1/2 or inhibit the expression of CD274, all of which are dependent,1 an analogous role for T-cell antigen receptor downstream of NPM-ALK (data not shown). In addition, R406 signaling in PTCLs has not been described. In fact, PTCLs often failed to downregulate CD274 in SR-786 or SU-DHL-1 cells. lack an intact T-cell antigen receptor-signaling apparatus, as we Therefore, the effects observed with R406 treatment are unlikely observed in both SU-DHL-1 and SR-786 cells (data not shown). because of the inhibition of NPM-ALK. In B-cell lymphomas, Syk phosphorylation also is regulated The events leading to Syk activity in PTCLs are not under- by protein tyrosine phosphatases (PTPs),6 and diminished stood. Syk can be phosphorylated through various receptors, PTP expression may contribute to Syk activity. As diminished including the b- and g-chains of the interleukin-2 receptor and PTP expression has been identified in malignant T cells, effects T-cell antigen receptor.4 that signal through receptors of PTPs on Syk regulation in PTCLs merit further study. sharing the g-chain (for example, interleukin-2) may contribute Syk can function through a variety of downstream pathways, to PTCL growth, but the extent to which such signaling is Syk- including MAPK, PI3K and PLC-g activation.4 The pathways dependent is unclear. Both SU-DHL-1 and SR-786 are cell lines responsible for the functional effects of Syk in PTCLs are derived from anaplastic lymphoma kinase (ALK)-positive unknown. Furthermore, in addition to promoting cell growth anaplastic large cell lymphomas (ALCLs). The specific effects and survival, Syk may have other functional effects in PTCLs;

Leukemia Letters to the Editor 232 for example, Syk regulates tumor cell migration and 2Department of Laboratory Medicine and Pathology, Mayo production in various solid tumors.7 Finally, though normal Clinic, Rochester, MN, USA T cells lack Syk expression, recent data indicate that Syk is E-mail: [email protected] overexpressed and functionally active in T cells from patients with systemic lupus erythematosis.8 Syk signaling in these cells occurs through preferential binding to Fc receptor gamma chain References (FcR-g), resulting in altered calcium flux and actin polymeriza- tion. These findings may offer additional clues to the function of 1 Chen L, Monti S, Juszczynski P, Daley J, Chen W, Witzig TE et al. SYK-dependent tonic B-cell receptor signaling is a rational Syk in PTCLs. treatment target in diffuse large B-cell lymphoma. Blood 2008; In summary, our data provide the first evidence of a functional 111: 2230–2237. role for Syk in survival and growth of malignant T cells, and 2 Friedberg JW, Sharman J, Schaefer-Cutillo J, Johnston PB, validate Syk as a rational therapeutic target in PTCLs. As PTCL De Vos S, LaCasce A et al. Fostamatinib disodium (FosD), an oral patients currently lack good treatment options, our findings inhibitor of Syk, is well-tolerated and has significant clinical activity support the novel therapeutic approach of Syk inhibition for in diffuse large B cell lymphoma (DLBCL) and chronic lymphocytic leukemia (SLL/CLL). Blood (ASH Annu Meet Abstr) 2008; 112:3. these malignancies. 3 Feldman AL, Sun DX, Law ME, Novak AJ, Attygalle AD, Thorland EC et al. Overexpression of Syk tyrosine kinase in peripheral T-cell lymphomas. Leukemia 2008; 22: 1139–1143. Conflict of interest 4 Sada K, Takano T, Yanagi S, Yamamura H. Structure and function of Syk protein-tyrosine kinase. J Biochem 2001; 130: 177–186. The authors declare no conflict of interest. 5 Thompson MA, Stumph J, Henrickson SE, Rosenwald A, Wang Q, Olson S et al. Differential in anaplastic lymphoma kinase-positive and anaplastic lymphoma kinase-negative ana- Acknowledgements plastic large cell lymphomas. Hum Pathol 2005; 36: 494–504. 6 Chen L, Juszczynski P, Takeyama K, Aguiar RC, Shipp MA. Protein tyrosine phosphatase receptor-type O truncated (PTPROt) regulates This research was supported in part by the Public Health Service SYK phosphorylation, proximal B-cell-receptor signaling, and Grant number P50 CA097274 from the University of Iowa/Mayo cellular proliferation. Blood 2006; 108: 3428–3433. Clinic Lymphoma Specialized Program of Research Excellence 7 Luangdilok S, Box C, Patterson L, Court W, Harrington K, Pitkin L (UI/MC Lymphoma SPORE) and the National Cancer Institute. et al. Syk tyrosine kinase is linked to cell motility and progression in squamous cell carcinomas of the head and neck. Cancer Res 2007; 1 2 1 2 67: 7907–7916. RA Wilcox , DX Sun , A Novak , A Dogan , 8 Krishnan S, Juang YT, Chowdhury B, Magilavy A, Fisher CU, 1 2 SM Ansell and AL Feldman Nguyen H et al. Differential expression and molecular associations 1 Division of Hematology, Mayo Clinic, of Syk in systemic lupus erythematosus T cells. J Immunol 2008; Rochester, MN, USA and 181: 8145–8152.

PTPN11 in childhood acute lymphoblastic leukemia occur as a secondary event associated with high hyperdiploidy

Leukemia (2010) 24, 232–235; doi:10.1038/leu.2009.200; were largely mutually exclusive with NRAS and KRAS mutations, published online 24 September 2009 suggesting a perturbing role of PTPN11 mutations on signal flow through RAS in ALL as well. In childhood ALL, high hyper- The protein tyrosine phosphatase, non-receptor type 11 (PTPN11) diploidy (450 ) is the most frequent cytogenetic gene encodes SHP-2, a phosphatase involved in many signaling aberration, accounting for 30% of the cases.5 Although several pathways, with a key role in development and hematopoiesis. lines of evidence support the view that hyperdiploidy is an early Somatic PTPN11 mutations were found with variable prevalences leukemogenic event, frequently occurring prenatally,6 the long in pediatric leukemia, most frequently associated with juvenile latency before overt leukemia strongly indicates that, also for myelomonocytic leukemia (JMML).1,2 In vitro and in vivo studies, hyperdiploidy, additional alterations in the leukemic clone are mainly in a myeloid context, demonstrated a crucial involve- required.6 Although a higher prevalence of mutations in ment of PTPN11 mutations in the hyperactivation of the RAS/ERK coding for transducers with a role in RAS signaling in high pathway.3 The oligoclonality of transgene integration sites, and hyperdiploid childhood ALL cases has been reported,7,8 the the long latency before overt leukemia in mice transduced with order of the events before overt disease in PTPN11-mutated ALL mutant PTPN11,3 suggested that additional molecular lesions are has not been addressed. needed to cooperate with PTPN11 mutations in leukemogenesis. With this aim, we genotyped the available DNA from relapsed This observation is in line with the ‘two hit model’ proposed by ALL cases with a PTPN11 at disease presentation, and Greaves4 for B-cell precursor childhood acute lymphoblastic performed single-nucleotide polymorphism (SNP) mapping analysis leukemia (ALL), in which an initiating alteration, often occurring in PTPN11-mutated cases at diagnosis to identify the presence of prenatally, gives rise to a preleukemic clone, which eventually additional genetic alterations. becomes fully malignant by subsequent acquisition of other In our previous screening, we identified 23 PTPN11 mutation- molecular alterations. positive ALL patients.2 Among these cases, four patients relapsed, We previously documented that somatically acquired PTPN11 and biological samples of three patients were available to be mutations occur in approximately 10% of B-cell precursor child- genotipically characterized (Figure 1). All patients studied were hood ALL.2 Molecular profiling indicated that PTPN11 mutations enrolled in the AIEOP-BFM ALL 2000 clinical trial, approved

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