Oncogene (2009) 28, 3261–3273 & 2009 Macmillan Publishers Limited All rights reserved 0950-9232/09 $32.00 www.nature.com/onc ORIGINAL ARTICLE The Syk regulates the survival of chronic lymphocytic leukemia B cells through PKCd and proteasome-dependent regulation of Mcl-1 expression

AD Baudot1,2, PY Jeandel2,3, X Mouska1, U Maurer4, S Tartare-Deckert2,5, SD Raynaud6, JP Cassuto7, M Ticchioni1,8 and M Deckert1,2

1INSERM UMR576, Nice, France; 2University of Nice Sophia-Antipolis, Nice, France; 3Department of Internal Medicine, CHU Nice, Nice, France; 4Institute for Molecular Medicine and Cell Research, University of Freiburg, Germany; 5INSERM UMR895, Nice, France; 6Genetics Laboratory, CHU Nice, Nice, France; 7Department of Clinical Hematology, CHU Nice, Nice, France and 8Immunology Laboratory, CHU Nice, Nice, France

B-cell chronic lymphocytic leukemia (B-CLL) is char- characterized by the accumulation in primary and acterized by accumulation of mature monoclonal CD5 þ secondary lymphoid tissues and bone marrow of slowly B cells. The disease results mainly from a failure of cells dividing mature monoclonal CD5 þ B cells, expressing to undergo , a process largely influenced by the low levels of surface immunoglobulin (sIg). Clinically, existence of constitutively activated components of B-cell CLL is heterogeneous with a stable form characterized receptor signaling and the deregulated expression of anti- by cells harboring IgM with mutated immunoglobulin apoptotic molecules. Recent evidence pointing to a critical variable heavy-chain (IgVH) region genes and an role of spleen tyrosine kinase (Syk) in ligand-independent aggressive form with cells expressing unmutated IgVH BCR signaling prompted us to examine its role in primary region genes and frequently associated with the expres- B-CLL cell survival. We demonstrate that pharmacologi- sion of the protein tyrosine kinase zeta-associated cal inhibition of constitutive Syk activity and silencing by protein at 70 kDa (ZAP-70) (Caligaris-Cappio and siRNA led to a dramatic decrease of cell viability in CLL Hamblin, 1999; Kipps, 2003). The disease results mainly samples (n ¼ 44), regardless of clinical and biological from a failure of cells to undergo cell death rather than status and induced typical apoptotic cell death with by excessive cellular proliferation. In addition to genetic mitochondrial failure followed by caspase 3-dependent or epigenetic modifications changing their intrinsic cell death. We also provide functional and biochemical survival capacities, interactions of malignant B cells evidence that Syk regulated B-CLL cell survival through a with their environment may enhance their resistance to novel pathway involving PKCd and a proteasome- apoptosis (Kipps, 2003). Studies showing that antigenic dependent regulation of the anti-apoptotic protein Mcl- stimulation through the B-cell receptor (BCR) may 1. Together, our observations are consistent with a model promote the selection and expansion of the neoplastic wherein PKCd downstream of Syk stabilizes Mcl-1 B-cell clone, supports the notion that BCR signals have through inhibitory phosphorylation of GSK3 by Akt. We a central role in the physiopathology of the disease conclude that Syk constitutes a key regulator of B-CLL (Stevenson and Caligaris-Cappio, 2004). cell survival, emphasizing the clinical utility of Syk BCR engagement regulates signaling pathways in- inhibition in hematopoietic malignancies. volved in B-cell development, proliferation, survival and Oncogene (2009) 28, 3261–3273; doi:10.1038/onc.2009.179; differentiation (Niiro and Clark, 2002; Monroe, 2006). published online 6 July 2009 In normal B-, BCR stimulation triggers the recruitment and activation of members of the Tec, Src Keywords: B-CLL; Syk; survival; PKCdelta; Mcl-1; and spleen tyrosine kinase (Syk) families of tyrosine proteasome kinases, followed by the activation of effector , including C (PKC) isoforms, phosphati- dyl 3-kinase (PI3K) and phospholipase Cg2 (PLCg2). These signaling complexes then regulate key down- Introduction stream pathways, including the Ras-MAPK and Akt pathways, whose activities determine gene transcription B-cell chronic lymphocytic leukemia (B-CLL) is the and B-cell fate (Niiro and Clark, 2002). In B-CLL cells, most common adult leukemia in Western countries. It is several studies have indicated the existence of constitu- tively activated components of the BCR-signaling path- way that participates to cell death resistance, such as Correspondence: M Deckert, INSERM UMR576, Hoˆpital de l’Archet Lyn (Contri et al., 2005), PI3K and PKCd (Barragan 1, Route Saint-Antoine de Ginestie` re, F-06202, Nice Cedex 3, France. et al., 2002; Ringshausen et al., 2002; Plate, 2004; E-mail: [email protected] Received 21 January 2009; revised 17 May 2009; accepted 17 May 2009; Ticchioni et al., 2007) and Akt (Plate, 2004; Longo et al., published online 6 July 2009 2008). These constitutively activated-signaling pathways Syk and CLL survival AD Baudot et al 3262 were proposed to participate to cell survival through BAY61-3606 results in the specific inhibition of Syk regulation of anti-apoptotic molecules including Bcl-2 activity. family members, XIAP and Mcl-1 (Cuni et al., 2004; Longo et al., 2008). Thus, several pieces of evidence Pharmacologic inhibition of Syk induces cell death in support the existence of cell-intrinsic signaling pathways B-CLL cells promoting CLL cell survival with or without BCR We next addressed the effect of Syk inhibition on CLL engagement. cell viability using flow cytometry combined to Annexin- The SYK family of protein tyrosine kinase is composed V/7-AAD staining. In all patient cells tested (n ¼ 7), the of Syk and ZAP-70, two kinases initially identified in Syk inhibitor-induced cell death at concentrations leukocyte signaling and in immunologic and allergic ranging from 0.5 to 10 mM, with a maximal cytotoxicity disorders (Latour and Veillette, 2001; Niiro and Clark, obtained for a concentration of 5 mM on six samples out 2002; Wong et al., 2004). However, studies now indicate of seven (Figure 2a). A time course of cell death that abnormal expression of Syk and ZAP-70 may also induction with 5 mM of BAY61-3606 showed a maximal contribute to hematologic malignancies (Goodman et al., cytotoxicity reached between 6 and 24 h (Figure 2b). The 2001; Crespo et al., 2006; Wossning et al., 2006; Feldman treatment for 24 h with 5 mM of BAY61-3606 or vehicle et al., 2008). Other studies indicate that constitutively alone was therefore chosen for testing the effect of Syk activated Syk may be a central determinant of B-CLL inhibition on cell survival from the 44 patient samples of (Gobessi et al., 2009) and B-cell lymphoma survival this study (Figure 2c). Under these conditions, the (Leseux et al., 2006; Chen et al., 2008; Refaeli et al., treatment with BAY61-3606 significantly reduced CLL 2008). Altogether those studies suggest that Syk is a cells survival (P 0.001, n ¼ 44). In contrast, Figure 2d major component of neoplastic B-cell survival. o shows that incubation for 24 h with 5 mM of BAY61-3606 In this study, we examined the mechanisms by which was minimally toxic to normal peripheral B-lympho- Syk regulates ligand-independent survival of patient cytes (5 to 35% loss of cell viability), whereas it caused a B-CLL cells. We demonstrate that pharmacological dramatic loss of CLL cell viability (60 to 90% loss of cell inhibition of Syk activity and depletion of its expression viability). Next, we wished to determine whether by siRNA-induced caspase 3-dependent apoptosis in differences in cytotoxicity following Syk inhibition CLL cells, regardless of biological and clinical status. could be observed in some CLL subsets. The sensitivity We also provide evidence that Syk-regulated B-CLL cell to BAY61-3606 treatment was examined on cells from survival through a novel pathway involving PKCd and a patients whose clinical and biological features are proteasome-dependent regulation of the antiapoptotic defined in Supplementary Table S1. This analysis protein Mcl-1. Our study shows that Syk constitutes a showed that Syk inhibition potently reduced B-CLL key regulator of B-CLL cell viability. cell survival, independently of IgVH gene mutation, ZAP-70 and CD38 expression (Supplementary Figure S1). The presence of chromosomal alterations disabling Results the p53 DNA damage cell checkpoint pathway has a major impact on disease progression and response to Constitutive phosphorylation of Syk in B-CLL cells therapy (Kipps, 2003). We thus examined the interaction The kinase activity of Syk is regulated through the between Syk and p53 pathways in CLL B cells. Syk autophosphorylation of two tyrosine residues (Tyr525/ inhibition triggered cell death in B-CLL cells refractory Tyr526) within its catalytic domain. To investigate to the drug fludarabine and independently of p53 the contribution of Syk activity in B-CLL cell survival, activation (Supplementary Figure S2). Together, our we first evaluated the level of Syk phosphorylation data indicate that Syk regulates cell survival in CLL, in resting primary leukemic cells. Immunoblot analysis regardless of clinical and biological status. showed a basal level of Syk phosphorylation on autophosphorylated Tyr525/Tyr526 regardless of ZAP-70 expression (Figure 1a). This event was inhibited Inhibition of Syk activity induces apoptosis of B-CLL by the incubation of leukemic cells with the specific cells Syk inhibitor BAY61-3606, a compound acting as an We next assessed the pro-apoptotic effect of BAY61- ATP competitor blocking the catalytic activity of 3606 in B-CLL cells. First, electron microscopy analysis Syk (Yamamoto et al., 2003). In contrast, the phos- showed that cells treated with BAY61-3606 exhibited phorylation of Syk on the non-autophosphorylated typical apoptotic morphology with highly condensed tyrosine residue Tyr352, and of Lyn on Tyr416, was chromatin in treated cells as compared with control cells unaffected by the Syk inhibitor (Figure 1a). Using the (Figure 3a). Apoptosis was further evidenced by flow technique of phospho-flow cytometry, we further found cytometric analysis of CLL cell viability. QVD-OPH, a that BAY61-3606 inhibited basal phosphorylation of pan-caspase inhibitor, completely reversed the decrease Syk Tyr525/Tyr526, and the increased Syk phosphor- of cell viability induced by BAY61-3606 in cells from all ylation induced on these residues following BCR patients examined (n ¼ 7) (Figure 3b). A significant stimulation (Figure 1b). In contrast, BAY61-3606 had decrease of cell viability was observed after 48 h and 72 h no effect on Syk Tyr352 phosphorylation detected on of BAY61-3606 treatment in the presence of QVD- either resting or BCR-stimulated cells (Figure 1b). These OPH, suggesting that a caspase-independent cell death data indicate that treatment of CLL B cells with process may also take place after long-term inhibition of

Oncogene Syk and CLL survival AD Baudot et al 3263 BAY61-3606 -+ -+ -+-+

P-SykY525/526

IP Syk P-SykY352

Syk

P-Src Y416 IP Lyn Lyn ZAP-70 : neg pos neg pos

#28 #14 #32 #17

P-Syk Y352 P-Syk Y525/526

15 85 - IgM/IgD 12 55

Ig Control - BAY61-3606 + BAY61-3606 24 + IgM/IgD 140 20

68 Counts Phospho-Syk (log) Figure 1 Analysis of Syk phosphorylation and inhibition in CLL cells. (a) Lysates from four CLL cell samples treated or not for 12 h with 5 mM of BAY61-3606 were immunoprecipitated (IP) with antibodies against Syk or Lyn, and immunoblotted using antibodies against phospho-Syk Tyr525/526, phospho-Syk Tyr352, pan phospho-Src Tyr416, Syk and Lyn. Status of ZAP-70 on CLL samples determined by flow cytometry (see Supplementary Table S1) is indicated (pos, positive; neg, negative). (b) CLL cells were incubating for 12 h at 37 1C with 5 mM of BAY61-3606 and stimulated or not with 10 mg/ml of anti-IgM/IgD antibodies. Intracellular phosphoflow analysis of Syk phosphorylation on Tyr352 and Tyr525/526 was then performed as described in the Materials and methods section. Mean fluorescence intensity for each determination is indicated. The experiment is representative of three patient cell samples.

Syk (Supplementary Figure S3). To confirm apoptosis cytometric detection of the mitochondrial probe induction in CLL cells exposed to BAY61-3606, we mitotracker. Incubation with BAY61-3606 caused a examined caspase-3 activation by immunobloting. Upon dramatic loss of the mitochondrial potential as shown BAY61-3606 treatment, processing of the zymogen form by the decreased percentage of mitotracker-positive cells of caspase-3 to its active 19-kDa subunit was observed (80% positive cells in untreated cells versus 23% in after 16 h, together with a marked cleavage of PARP, a BAY61-3606-treated cells). Incubation with QVD-OPH known caspase-3 substrate (Figure 3c). QVD-OPH in the presence of BAY61-3606 abolished the loss of the abolished BAY61-3606-induced caspase-3 and PARP mitochondrial potential induced by BAY61-3606 alone processing (Figure 3c). The activation of caspase-3 (Figure 3d). These results show that Syk inhibition in following Syk inhibition was confirmed by flow cyto- B-CLL cells results in early caspase-dependent apoptotic metry using an antibody against active caspase-3. cell death accompanied by mitochondrial failure. Treatment with BAY61-3606 induced cleavage of caspase-3 in 61% of cells as compared with untreated cells (23% positive). Treatment with QVD-OPH in Syk silencing induces apoptosis in B-CLL cells the presence of BAY61-3606 completely blocked the To sustain our data obtained with pharmacological apparition of cleaved caspase-3 (9% positive cells) inhibition of Syk activity, we next asked whether B-CLL (Figure 3d). Finally, we assessed whether Syk inhibition cell survival is affected by specific depletion of Syk. For results in mitochondrial permeabilization by using flow this purpose, we used siRNAs to knockdown Syk in

Oncogene Syk and CLL survival AD Baudot et al 3264 100 90 control BAY 0.5uM 90 80 BAY 2uM 80 70 BAY 5uM BAY 10uM 70 60 60 50 50 40 40 30 % viable cells % viable cells 30 control 20 #3 20 BAY 10 control 10 #13 0 BAY #14 #16 #17 #18 #20 #23 #31 0 2h 4h 6h 24h

100 90 100 80 70 80 60 50 60 40 40

% viable cells 30 20 20 10 % viable cells (of control) 0 Control BAY61-3606 Healthy CLL donors Figure 2 Pharmacologic inhibition of Syk decreases B-CLL viability. (a) CLL cells from six patients were treated for 24 h with increasing dose of BAY61-3606. Cell viability was assessed using Annexin-V-PE/7AAD staining. (b) CLL cells from two patients were treated with 5 mM of BAY61-3606, then cell viability was assessed at indicated times as described in (a). (c) CLL cells from 44 different patients were treated for 24 h with 5 mM of BAY61-3606, then cell death was assessed as described in (a) (Wilcoxon’s rank test, Po0.001). (d) Malignant B cells from nine patients and peripheral B cells from six healthy volunteers were treated for 24 h with 5 mM of BAY61-3606, then cell death was assessed as described in (a). Each square indicates the mean percentage of viable cells following treatment with BAY61-3606; the box indicates the mean±s.d., and error bars the mean±95% confidence intervals (Mann–Whitney U-test, Po0.001).

CLL cells following nucleofection. Under these condi- The prosurvival activity of Syk is associated with PKCd tions, a significant reduction of Syk was achieved in activity different patient cells, ranging from 30 to 50% of the We next investigated the nature of the biochemical levels of Syk detected in CLL cells transfected with pathways controlled by Syk in the regulation of B-CLL control siRNA (Figure 4a). These levels of Syk down- cell survival. We observed that treatment with the regulation are consistent with the nucleofection effi- phorbol diester PMA, an activator of classical and novel ciency in patient CLL cells, which routinely reached 40 PKCs, rescued the cytotoxic effect of BAY61-3606 on to 60% of transfected cells (data not shown and leukemic cells (Supplementary Figure S4a) and a (Ticchioni et al., 2007)). Downregulation of Syk resulted reduced activity of the serine/threonine kinase PKCd in a significant decrease in CLL cell viability as shown has been implicated in CLL cell death (Barragan et al., by flow cytometric analysis of Annexin-V/7-AAD 2002; Ringshausen et al., 2002). As shown in Figure 5a, staining (Figure 4a). On examination of further samples, Syk inhibition decreased the phosphorylation of PKCd siRNA-mediated downregulation of Syk expression was on Thr505 in all the tested samples (n ¼ 8/8). In contrast, shown to decrease cell viability in CLL cells from all BAY61-3606 had no significant effect on basal ERK1/2 patients tested (n ¼ 21, Po0.001) (Figure 4b). Apoptosis and PKCa/b phosphorylation. Furthermore, downre- of CLL cells transfected with Syk siRNA was further gulation of Syk expression by siRNA almost completely evidenced by analysis of caspase-3 and PARP cleavage. abolished the phosphorylation of PKCd on Thr505 in Using flow cytometry analysis, knockdown of Syk was unstimulated CLL cells as compared with control cells shown to significantly increase cleavage of caspase-3 (Figure 5b). In addition, we found that siRNA-mediated (52% of positive cells versus 37% of positive cells in downregulation of PKCd expression in B-CLL cells controls) (Figure 4c). This event was consistent with the markedly reduced leukemic cell viability (Figures 5c and increased cleavage of PARP observed in CLL cells d). These results indicate that PKCd activity is a crucial transfected with Syk siRNA (Figure 4d). Thus, Syk event downstream of Syk in promoting B-CLL cell expression is required for B-CLL cell survival. survival.

Oncogene Syk and CLL survival AD Baudot et al 3265 70 Control QVD-OPH Patient #38 60 BAY61-3606 BAY61-3606+QVD-OPH 50

40

30

% viable cells 20

10 control BAY61-3606 0 #13#22 #25 #33#34 #38 #41

#41 #38 - + - + - + - + QVD-OPH - -++ - - + + BAY61-3606

PARP

Uncleaved caspase-3 Cleaved caspase-3

Erk2

BAY61-3606+ Control QVD-OPH BAY61-3606 QVD-OPH

23% 6% 61% 9%

0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 Counts 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 Active Caspase-3

80% 84% 23% 83%

0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4

Counts 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 Mitotracker Figure 3 Pharmacological inhibition of Syk results in apoptotic cell death. (a) CLL cells from patient no. 38 were incubated for 16 h with 5 mM of BAY61-3606. CLL morphology was analysed by electron microscopy. (b) CLL cells from seven patients were incubated for 1 h with 20 mM of the pan-caspase inhibitor QVD-OPH before treatment for 24 h with 5 mM of BAY61-3606. Apoptosis was assessed using Annexin-V/7-AAD staining. Values represent the mean±s.d. of three independent measurements. (c) CLL cells from two patients were treated as described in (b). Cell lysates were separated by SDS–PAGE, transferred to nitrocellulose membrane, and immunoblotted with PARP and caspase-3 antibodies. (d) CLL cells were treated as described in (b). Upper panels: caspase-3 activity was assessed using a specific active caspase-3-FITC-labeled antibody as described in Materials and methods. Lower panels: measurement of mitochondrial potential (DCm) was assessed using MitoTracker Red 580 staining and analysed by flow cytometry. One experiment representative of three patient cell samples is shown.

Syk and PKCd regulate the expression of the showed that treatment with BAY61-3606 for 2 h led to antiapoptotic protein Mcl-1 in B-CLL cells a marked decrease of Mcl-1, but not XIAP, expression The altered balance between Bcl-2 family proteins has in all cases (n ¼ 9/9) (Figure 6a). Our pharmacological been involved in CLL cell survival, and inhibition of data were further supported by siRNA experiments, Mcl-1 expression-induced CLL cell apoptosis (Longo which showed that downregulation of Syk (Figure 6b) et al., 2008). These observations prompted us to and PKCd (Figure 6c) resulted in a substantial examine whether Syk and PKCd control the expression decrease of Mcl-1 expression in CLL cells. Mcl-1 of Mcl-1 in leukemic cells. Immunoblot analysis can be subjected to proteolytic degradation as the

Oncogene Syk and CLL survival AD Baudot et al 3266 100 Patient: #25 #26 #13 90 80 70 siCo siSyk siCo siSyk siCo siSyk 60 Syk 50 40 10.47 1 0.48 1 0.71

% viable cells 30 20 Hsp60 10 % viability: 44 28 24 14 32 22 0 siCo siSyk

Patient: #25 #13 siCo siSyk siCo siSyk

23% 38% 37% 52% Counts

Active Caspase3

#25 #13 siCosiSyk siCo siSyk Syk

PARP

Hsp60

% viability: 61 43 40 22 Figure 4 Knock down of Syk in B-CLL induces apoptosis. (a) CLL B cells were nucleofected with control siRNA (siCo) or Syk siRNA (siSyk). After 72 h, cell lysates were separated by SDS–PAGE, transferred to nitrocellulose membrane, and immunoblotted with Syk and Hsp60 antibodies. Three representative patient samples are shown. Syk downregulation relative to Hsp60 signal was quantified using the Image J software. Band intensities were normalized to the respective Hsp60 signal and expressed as fold change relative to control nucleofection. The percentage of viable cells was determined for each patient sample using Annexin-V/7-AAD staining and is indicated at the bottom of the panel. (b) CLL cells from 21 patients were nucleofected with Syk or control siRNA. Cell viability was evaluated after 72 h as above (Wilcoxon’s rank test, Po0.001). (c) Caspase-3 activity in CLL transfected with Syk siRNA was evaluated by specific staining using active caspase-3-FITC-labeled antibody. (d) PARP cleavage following Syk knockdown in B-CLL cells was assessed by immunoblotting using PARP antibody.

consequence of caspase activity during apoptosis Syk and PKCd inhibition for regulating B-CLL cell (Herrant et al., 2004). Because Syk inhibition triggered apoptosis. caspase-3 activation in leukemic cells, we therefore evaluated the impact of caspase inhibition on Mcl-1 expression. For this purpose, cells were incubated or not Inhibition of Syk leads to a proteasome-dependent with QVD-OPH before being subjected to BAY61-3606 degradation of Mcl-1 in B-CLL treatment for 2 h and immunoblot analysis. In the A recent study has shown that phosphorylation absence of QVD-OPH, inhibition of Syk led to Mcl-1 by GSK-3 targets Mcl-1 for proteasome-dependent downregulation, paralleled by caspase-3 processing. degradation (Maurer et al., 2006). Mcl-1 degradation However, in the presence of QVD-OPH, whereas the could be regulated by the proteasome (Maurer et al., levels of active caspase-3 were completely abolished, 2006). To assess whether such a mechanism could take BAY61-3606 treatment still induced the downregulation place downstream Syk and PKCd inhibition, B-CLL of Mcl-1 (Figure 6d). These results indicate that Mcl-1 cells were incubated for 1 h with QVD-OPH or with the downregulation is not a consequence of caspase activa- ubiquitin–proteasome inhibitor MG132 before treat- tion, but rather represents a primary event downstream ment with BAY61-3606 and immunoblot analysis.

Oncogene Syk and CLL survival AD Baudot et al 3267 #28 #14 #1 #2 #35 #10 #33 #25 BAY61-3606 -+-+-+ -+-+ -+-+-+ P-PKCδ

PKCδ

P-PKCα/β

PKCα

P-ERK1/2

ERK2

siCo siSyk siCo siSyk Syk

P-PKCδ

PKCδ

#13 #25

** ** ** 60 δ

siCo siPKC 40 siCo PKCδ siPKCδ

20 ERK2 % viable cells

#15 0 #10 #15 #28

Figure 5 Syk regulates PKCd-dependent CLL cell survival. (a) CLL cells from eight patients were treated for 2 h with 5 mM of BAY61- 3606. Cell lysates were separated by SDS–PAGE and immunoblotted with anti-phospho-PKCd Thr 505, anti-phospho-PKCa/b Thr 638/641, and anti-phospho-ERK1/2 antibodies. Protein loading was controlled using anti-PKCd, PKCa and anti-ERK2 antibodies. (b) B-CLL cells were transfected with control or Syk siRNAs. After 72 h, cell lysates were immunoblotted with anti-Syk and anti- phospho-PKCd Thr 505 antibodies. Protein loading was controlled using anti-PKCd antibody. (c) B-CLL cells from three patients were transfected with control or PKCd siRNAs. After 24 h, cell viability was determined using Annexin-V/ 7-AAD staining. Each histogram represents the mean percentage of viable cells following siRNA transfection. Results are the mean±s.d. of three independent determinations (**Po0.001). (d) PKCd expression following control or PKCd siRNA transfection assessed on one representative CLL sample by immunoblotting with anti-PKCd and ERK2 antibodies.

Whereas treatment with QVD-OPH had no effect, protection induced by Mcl-1 S159A was significantly blocking the proteasome with MG132 prevented stronger than that observed with wild-type Mcl-1 the downregulation of Mcl-1 induced following Syk (Po0.05). In addition, immunoblot analyses showed inhibition (Figure 7a). As a control, XIAP expression that Mcl-1 S159A was more resistant to degradation was not affected by BAY61-3606. We next examined induced by BAY61-3606, compared with wild-type whether Mcl-1 or a degradation-resistant mutant, Mcl-1 Mcl-1 (Figure 7d). Finally, we found that siRNA- S159A (Maurer et al., 2006) could rescue cell death mediated suppression of PKCd decreased both Akt and induced by the Syk inhibitor. To this end, B-CLL cells GSK-3 phosphorylation (Figure 7e). Together with our were nucleofected with V5-tagged Mcl-1 or Mcl-1 observation that inhibition of Syk also reduced S159A (Figure 7b), treated with BAY61-3606 and Akt phosphorylation in patient cell samples displaying cell viability examined using Annexin-V/7-AAD constitutive Akt activity (Supplementary Figure S5), staining. As shown in Figure 7c, ectopic expression of these results strongly support the notion that Syk either Mcl-1 or Mcl-1 S159A in CLL cells from three acts upstream of PKCd in B-CLL to regulate patients rescued the cytotoxicity induced by BAY61- Mcl-1 expression through a proteasome-dependent 3606 in CLL cells from three patients. However, the process.

Oncogene Syk and CLL survival AD Baudot et al 3268 #28 #14 #1 #4 #2 #35 #10 #33 #25 BAY61-3606 -+ -+ -+ -+-+ -+ -+ -+ -+ Mcl-1

XIAP

ERK2

δ δ

siCo siSyk siCo siSyk siCo siPKC siCo siPKC Syk PKCδ

Mcl-1 Mcl-1 Hsp60 Hsp60 #13 #25 #10 #15

- + - + QVD-OPH -- ++BAY61-3606 Mcl-1

XIAP

Uncleaved caspase 3

Cleaved caspase 3

Hsp60

#28 Figure 6 Targeting Syk and PKCd in CLL results in Mcl-1 downregulation. (a) CLL cells from nine patients were treated for 2 h with 5 mM of BAY61-3606. Cell lysates were then immunoblotted with specific anti-Mcl-1 and anti-XIAP antibodies. Protein loading was controlled using anti-ERK2 antibody. Cells were transfected with control or Syk siRNAs (b), or with control or PKCd siRNAs (c). Cell lysates were separated by SDS–PAGE and immunoblotted with antibodies against Syk (b) or PKCd (c) and Mcl-1. Protein loading was controlled using anti Hsp60 antibodies. (d) CLL cells were incubated for 1 h with 20 mM of the pan-caspase inhibitor QVD-OPH before treatment for 2 h with 5 mM of BAY61-3606. Cell lysates were immunoblotted with anti-Mcl-1, XIAP and caspase-3 antibodies. Protein loading was controlled using anti-Hsp60 antibody.

Discussion phosphorylation along with activated-signaling path- ways (Gauld et al., 2005). Accordingly, constitutive Human CLL B cells display high activities of protein activation of ERK1/2 in the absence of Akt activation kinases and effector enzymes involved in BCR signaling has been linked to anergy in some CLL subsets (Muzio and leukemic cell survival (Ringshausen et al., 2002; et al., 2008). However, our observations suggest that the Plate, 2004; Contri et al., 2005; Ticchioni et al., 2007; anergic status of CLL cells has no significant implication Longo et al., 2008). The molecular origin of activated- in Syk-dependent CLL cell survival. Indeed, inhibition signaling pathways found in mature B-cell neoplasms, of Syk had no effect on basal ERK1/2 phosphorylation, including B-CLL, is still unclear. and all cases in our cohort of B-CLL were sensitive to Constitutive phosphorylation of Syk has been re- Syk inhibition. Recent studies have associated the ported in B-CLL cells (Semichon et al., 1997; Lanham presence of ZAP-70 in unmutated CLL cases with et al., 2003; Deglesne et al., 2006), and in B-cell increased signaling following BCR ligation, including lymphoma (Leseux et al., 2006; Chen et al., 2008). A enhanced Syk phosphorylation (Chen et al., 2002, 2005). ligand-independent BCR signaling, also called tonic However, we found that neither sIg mutation status nor signaling (Monroe, 2006), has been recently involved in ZAP-70 expression had a statistically significant impact the constitutive activation of Syk found in B-cell on apoptosis induced by Syk inhibition. Thus, it is lymphoma (Chen et al., 2008). CLL cells express low possible that the activity of Syk found in resting levels of sIg, a property shared by murine anergic B cells CLL cell stems from the stereotyped BCR repertoire (Chiorazzi and Ferrarini, 2003; Stevenson and Caligaris- identified in CLL cells (Messmer et al., 2004), which may Cappio, 2004), which exhibit high levels of tyrosine affect BCR tonic signaling. Alternatively, constitutive

Oncogene Syk and CLL survival AD Baudot et al 3269 BAY BAY + QVD-OPH BAY + MG132 0 0.5 1 2 0 0.5 1 2 0 0.5 1 2 Hours Mcl-1

XIAP Hsp60

100 * **

80 vector Mcl-1 Mcl-1S159A V5 60 vector Mcl-1 Mcl-1 40 Mcl-1S159A Hsp60 20 #15 % viable cells (of control) 0 #15#10 #28

δ δ Vector Mcl-1 Mcl-1S159A siCo siPKCsiCo siPKC - + - + - + BAY61-3606 PKCδ V5 P-Akt Akt Mcl-1 P-GSK-3 GSK-3β Hsp60 ERK2 #15 #10 #15 Figure 7 Proteasome-dependent degradation of Mcl-1 following Syk inhibition in CLL cells. (a) CLL cells from one representative patient were incubated for 1 h with 20 mM of QVD-OPH or with 10 mM of the proteasome inhibitor MG132 before treatment with 5 mM of BAY61-3606 for indicated times. Cell lysates were immunoblotted with anti-Mcl-1 and XIAP antibodies. Protein loading was controlled using anti-Hsp60 antibody. (b) CLL cells from three patients were nucleofected with control plasmids, or plasmids encoding V5-tagged Mcl-1 and Mcl-1S159A. After 24 h, CLL cells were treated with 5 mM of BAY61-3606 for 24 h. Cell viability was assessed using Annexin-V-PE/7AAD staining. Each histogram represents the mean percentage of viable cells following treatment with BAY61-3606 relative to untreated cells. Results are the mean±s.d. of three independent experiments (*Po0.01). (c) The expression of V5-tagged Mcl-1 and Mcl-1S159A proteins following nucleofection of one representative CLL sample was assessed by immunoblotting using anti- V5 and Mcl-1 antibodies. (d) B-CLL cells of one representative patient was nucleofected as above and treated with 5 mM of BAY61- 3606 for 2 h. Cell lysates were then immunoblotted with anti-V5, anti-Mcl-1 and anti-Hsp60 antibodies. (e) Cells were transfected with control or PKCd siRNAs. After 72 h, cell lysates were immunoblotted with antibodies against PKCd, phospho-Akt and phospho- GSK-3 antibodies. Protein loading was controlled using anti-ERK2, anti-Akt and anti-GSK-3b antibodies.

Syk activity may arise from deregulated protein tyrosine patients. Again, the reduction of Syk expression by phosphatases, such as protein tyrosine phosphatase siRNA decreased cell viability and induced apoptosis. receptor type O (PTPROT), a phosphatase involved in We also showed that Syk inhibition triggered apoptosis tonic BCR signaling (Chen et al., 2006) and the independently of p53 activation, and in B-CLL cells promoter of which was found epigenetically inactivated resistant to the chemotherapeutic drug fludarabine. This in CLL (Motiwala et al., 2007). Thus, the constitutive indicates that Syk may regulate cell survival in activity of Syk found in CLL cells may have different aggressive CLL cases wherein chromosomal alterations molecular origins, and further investigations aimed at have disabled the p53 DNA damage checkpoint path- their identifications are warranted. way. Whether Syk survival pathway cooperates with Using BAY61-3606, a specific ATP-competitive Syk other treatments in CLL cells is unknown. inhibitor used in inflammatory and allergic diseases How Syk regulates CLL cell survival? In normal (Yamamoto et al., 2003), we showed that targeting Syk B-lymphocytes, Syk triggers multiple signaling pathways activity in B-CLL induced a typical mitochondrial downstream BCR and functional interactions between apoptotic cell death. Interestingly, a recent report Syk and Src family kinases such as Lyn are well showed that another Syk pharmacologic inhibitor described (Niiro and Clark, 2002; Monroe, 2006). (R406) also induced apoptosis in CLL cells (Gobessi Inhibition of Lyn by dasatinib triggers cell death in et al., 2009). To consolidate our pharmacological CLL cells (Veldurthy et al., 2008). BAY61-3606 had no approach, we knocked down Syk in CLL cells from 21 effect on the phosphorylation of Syk Tyr352 (a target

Oncogene Syk and CLL survival AD Baudot et al 3270 residue for Src kinases), Lyn and Erk1/2 (a primary dent mechanism, as shown by the reversal of BAY61- target of Src kinases in lymphoid cells including CLL 3606-induced Mcl-1 degradation by MG132. Further- (Veldurthy et al., 2008)), indicating that Syk inhibitor more, an ectopically expressed form of Mcl-1 mutated did not proceed through non-specific Src kinase inhibi- on the GSK-3 site (Mcl-1 S159A) (Maurer et al., 2006) tion. Several other signaling molecules may participate was more resistant to proteolysis induced by BAY61- to cell death resistance downstream of Syk, including 3606 treatment compared with Mcl-1, and more PI3K, Akt and PKC (Ringshausen et al., 2002; Plate, significantly rescued the cytotoxic effect of Syk inhibi- 2004; Holler et al., 2009). The pro-survival role of Akt in tion. Although other mechanisms cannot be excluded, CLL has been recently demonstrated (Longo et al., our observations suggest that Mcl-1 degradation is a 2008), and another pharmacologic inhibitor of Syk primary and critical event in cell death inducted by inhibited the Akt activity (Gobessi et al., 2009). In targeting Syk in CLL. Importantly, expression of PKCd agreement with this study, we also observed that was shown to be required for Akt and GSK-3 BAY61-3606 similarly affected Akt activity. However, phosphorylation, and Mcl-1 stabilization. However, a fraction of CLL cells from our cohort displayed no Syk inhibition also leads to Mcl-1 degradation in CLL detectable phosphorylation of Akt, consistent with cases in which no significant phosphorylation of Akt previous studies (Muzio et al., 2008). Despite absence was detected, indicating the existence of redundant of activated Akt, B-CLL cells from those particular pathways. In addition to Akt, several kinases were patients were totally sensitive to Syk inhibition, suggest- shown to phosphorylate the inhibitory N-terminal site ing the existence of alternative or redundant pathways of GSK-3, including PKCs (Fang et al., 2002). Thus, we downstream of Syk in CLL survival. Consistent with would like to propose that, in the absence of active Akt this notion, while having no effect on PKCa/b activity, in CLL, Syk might regulate GSK-3 through direct Syk inhibition and knock down decreased phosphoryla- phosphorylation by PKCd. tion of PKCd, a protein kinase involved in B-CLL, but The phosphorylation of Mcl-1 by GSK-3 was recently not in normal B-cell survival (Ringshausen et al., 2002). shown to target Mcl-1 for degradation by the protea- This stands in apparent contradiction with other cell some (Maurer et al., 2006). Together, these observations types, wherein PKCd is regarded as a pro-apoptotic are consistent with a model wherein PKCd downstream molecule (Reyland, 2007). However, further supporting of Syk stabilizes Mcl-1 through inhibitory phosphoryla- the important role of PKCd in Syk-mediated CLL cell tion of GSK-3. In the majority of CLL samples survival, siRNA-mediated PKCd silencing markedly displaying active Akt, our model is further supported reduced cell viability, as well as Akt phosphorylation. by our observations that inhibition of Syk also reduced How Syk regulates PKCd activity in CLL B cells is Akt phosphorylation (Supplementary Figure S5, and currently unknown. It is well known that Syk phos- data not shown), and those from Gobessi et al. (Gobessi phorylates and activates PLCg2 in B cells (Monroe, et al., 2009). How Syk/PKCd regulates the Akt/GSK-3 2006), generating lipid second messengers that activate pathway to the ubiquitin proteasome system is currently PKCs, including PKCd (Steinberg, 2004). However, under investigation. In addition, identifying which E3 neither BAY61-3606 nor Syk siRNA inhibited PLCg2 ubiquitin (s) targets Mcl-1 for degradation down- phosphorylation in CLL B cells (data not shown). Another stream Syk/PKCd signaling would be of special interest potential mechanism could implicate a direct link between to our understanding of the pathogenesis of CLL. Syk and the p85 subunit of PI3K (Moon et al., 2005), and In conclusion, our data show that Syk has a key role PI3,4,5-P3-dependent activation of PKCd (Toker et al., in B-CLL cell survival through PKCd and proteasome- 1994). Further investigations are clearly required to dependent regulation of Mcl-1 expression, indicating determine the mechanisms by which Syk regulates PKCd that Syk is an attractive target in CLL. Whether similar activity in CLL. mechanisms also regulate cell survival in other B-cell The increased survival of B-CLL cells is believed to be neoplasms needs additional investigations. a consequence of the altered balance between Bcl-2 family proteins. Indeed, high levels of Bcl-2, Mcl-1, Bcl- xL, Bax and Bim have been found in the majority of Materials and methods CLL cells (Kitada et al., 1998; Kipps, 2003; Saxena et al., 2004). Elevated levels of Mcl-1 in CLL have also Patients, cell isolation and culture been associated with increased survival, resistance to Leukemic cells were obtained at the Centre Hospitalier chemotherapy and poor prognosis (Moshynska et al., Universitaire of Nice from peripheral blood samples of 44 2004; Petlickovski et al., 2005; Longo et al., 2008; CLL patients after informed consent. The diagnosis of B-CLL Pepper et al., 2008). Although the expression of another was based on clinical and immunophenotypic criteria. All cell survival regulator, XIAP, was not affected, Syk patients included in this study were either untreated or had not inhibition or depletion regulated Mcl-1 expression at the received chemotherapy or steroids for at least 6 months before protein level. Consistent with our findings, the Syk the investigation. Peripheral mononuclear cells were isolated from peripheral blood by Ficoll–Hypaque density gradient inhibitor R406 was shown to decrease Mcl-1 expression centrifugation. Leukemic B cells were isolated by magnetic in CLL cells (Gobessi et al., 2009). However, we provide selection as previously described (Ticchioni et al., 2007). Cells additional evidence that Mcl-1 downregulation was not were cultured at a concentration of 1x106 per ml in RPMI-1640 a consequence of caspase activation and apoptosis, but medium (Gibco BRL, Paisley, UK) containing 10% FCS rather resulted from an ubiquitin-proteasome-depen- (Hyclone, Logan, UT, USA), 1 mML-glutamine and 1 mM

Oncogene Syk and CLL survival AD Baudot et al 3271 pyruvate. Note that the differences in viability of frozen versus Immunoblot and Immunoprecipitation analysis freshly isolated leukemic cells did not modify the relative effect Cell lysis, immunoprecipitation and immunoblot analyses of the different treatments on in vitro cell survival. were performed as previously described (Ticchioni et al., 2007). Reagents, antibodies, plasmids and siRNA BAY 61-3606 and phorbol myristate acetate (PMA) were Electron microscopy purchased from Sigma Aldrich (St Louis, MI, USA). QVD- Cells were fixed in situ with 1.6% glutaraldehyde in 0.1 M OPH was from MP-Biomedicals (Aurora, OH, USA). Anti- phosphate buffer pH 7.5 at room temperature, washed with bodies against caspase 3, PARP, XIAP, Akt and phosphory- same buffer and then postfixed with 1% osmium tetroxyde and lated forms of Syk, PKCd, PKCa/b, Akt, GSK-3 and HRP- 1% potassium ferricyanid. Finally, cells were rinsed with conjugated secondary antibodies were from Cell Signaling distilled water, dehydrated with ethanol and embedded in Technology (Beverly, MA, USA). Antibodies against Syk, Epon. Thin sections were contrasted with uranyl acetate and PKCd, PKCa/b, ERK2, Hsp60, Mcl-1 and siRNA against lead nitrate and observed with a Philips transmission electron PKCd were from Santa Cruz Biotechnology (Santa Cruz, CA, microscope. USA). Anti-CD5, CD19 and CD38 antibodies were from Becton Dickinson (San Jose, CA, USA). Anti-V5 antibody and siRNAs against Syk were from Invitrogen (Paisley, UK). V5- Statistical analysis S159A tagged Mcl-1 and Mcl-1 constructs were generated as The nonparametric Wilcoxon’s signed rank test for paired described (Maurer et al., 2006). measures was used to evaluate variations in leukemic cell viability following pharmacological or siRNA treatment. A Intracellular phosphoflow dynamics generalized linear model with a binomial probability distribu- Cells (5 Â 106) were incubated or not with BAY61-3606 at tion was used to compare the effect of clinical status on B-CLL 37 1C, then cells were left untreated or stimulated with goat cells viability after BAY61-3606 treatment. The Mann- anti-human-IgM/IgD for 10 min at 37 1C. Cells were washed Whitney U-test was used to evaluate differences in cell viability with cold PBS, permeabilized with Cytofix/Cytoperm solution between B-CLL and healthy volunteers after BAY61-3606 (BD Biosciences, Mountain View, CA, USA) for 30 min at 4 1C, treatment. Statistical analyses were performed using the SAS washed with Perm/Wash solution (BD Biosciences), and software. incubated for 30 min with anti-phospho-Syk (Y352), anti- phospho-Syk (Y525/526) antibodies, or isotype control anti- bodies. Cells were washed two times and stained with secondary Abbreviations antibody conjugated with Alexafluor 488. Finally, cells were washed and resuspended in Perm/Wash solution. Phospho-Syk staining was monitored by flow cytometry (BD Biosciences). B-CLL, B-chronic lymphocytic leukemia; BCR, B-cell recep- tor; ERK, extracellular signal-regulated kinase; GSK-3, Glycogen synthase kinase-3; MAPK, mitogen-activated pro- Analysis of apoptosis, caspase 3 activity and mitochondrial tein kinase; Mcl-1, myeloid cell leukemia sequence 1; PI3K, membrane potential by flow cytometry phosphatidyl 3-kinase; PKC, protein kinase C; PLCg2, Apoptosis was evaluated by flow cytometry following double phospholipase Cg2; siRNA, small-interfering RNA; XIAP, staining with annexin-V-PE and 7-AAD as previously X-linked inhibitor of apoptosis protein; ZAP-70, zeta-asso- described (Ticchioni et al., 2007). ciated protein at 70 kDa. To assess caspase-3 activity, cells were fixed and permeabi- lized with Cytofix/Cytoperm as above, then incubated for 30 min with anti-active caspase-3-FITC antibody. Analysis was performed by flow cytometry. Conflict of interest The mitochondrial membrane potential was analysed by measuring the accumulation of the mitochondrial probe The authors declare no conflict of interest. MitoTracker Red580 (Molecular probes, Eugene, OR, USA). One million of cells were incubated at 37 1C for 30 min in HBSS (Gibco BRL) with 100 nM MitoTracker Red580, washed Acknowledgements and analysed by flow cytometry. This work was supported by INSERM, ARC (Grant No Cell transfection 3111), and INCa (Grant PL-06-026). ADB is a recipient of an Transient transfections into human B-CLL cells were per- INSERM-Re´gion Provence Alpes Coˆte d’Azur PhD fellowship formed using the AMAXA nucleofection system (AMAXA in partnership with TxCell (Valbonne, France). M Deckert is a Gmbh, Koln, Germany) as described previously (Ticchioni recipient of a Contrat d’Interface Clinique with the Depart- et al., 2007). Briefly, cells were resuspended in 100 ml of Buffer ment of Clinical Hematology, CHU de Nice. We thank V, mixed with 5 mg of plasmid DNA or 2 mM of siRNA duplex, P Gounon (CCMA, Faculte´des Sciences, Nice) for electron electroporated using program U-015 and incubated at 37 1C, microscopy analysis, and M. Ciosi (INRA-CNRS, Sophia- 5% CO2 before treatment. Antipolis) for statistical analysis.

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