Leukemia & Lymphoma, A p ril 2015; 56(4): 1 08 8-1095
informa
© 2014 In fo rm a UK, Ltd. ISSN: 1042-8194 p r in t / 1029-2403 o n lin e
healthcare
DOI: 10.3109/10428194.2014.941832
ORIGINAL ARTICLE: RESEARCH
Expression and pro-survival function of phospholipase Cy2 in diffuse large B-cell lymphoma Minh Q. Huynh1, Jennifer GoRmann1, Stefan Gattenloehner3, Wolfram Klapper2, Hans-Heinrich Wacker4, Annette Ramaswamy5, Alwina Bittner1, Ulrich Kaiser6 & Andreas Neubauer1 1Department o f Hematology, Oncology and Immunology, University Hospital, Marburg, Germany, 2Department o f Pathology, Hematopathology Section and Lymph Node Registry, University Hospital, Kiel, Germany,3Institute o f Pathology, University Hospital, Giessen, Germany,4Institute o f Hematopathology, Kiel, Germany,5Institute o f Pathology, University Hospital, Marburg, Germany and 6Department o f Internal Medicine II, St. Bernward Hospital, Hildesheim, Germany
Abstract
expressed in a s ig n ific a n t n u m b e r o f DLBCLs a n d has p ro g n o s tic
kinase (Btk), phospholipase Cy2 (PLCy2) and protein kinase Cp (PKCp), which is important for nuclear factor kB (NFkB) regulation [8-11]. NFkB is in particular up-regulated in DLBCL with activated B cell-like (ABC) subtype [12]. This above described signaling cascade also activates Ras and Akt [13-16]. There are several preclinical and clinical phase I/II studies showing promising data concerning DLBCL treat ment targeting the BCR signaling pathway, e.g. dasatinib (Src kinase inhibitor), PRT318 (Syk inhibitor), ibrutinib (Btk inhibitor) and enzastaurin (PKCP inhibitor) [17-22], These small molecule inhibitors are also currently being tested in ongoing clinical trials in patients with DLBCL (http://www. clinicaltrials.gov/). It has been shown that the activity of Syk and PLCy2 is well correlated with the sensitivity of DLBCL to dasatinib, and that inhibition of Syk with the specific inhibitor PRT060318 results in reduced lymphoma survival and cell cycle arrest
im p lic a tio n s . In h ib itio n o f PLCy2 co u ld b e a n e w ta r g e t fo r
[21,
ly m p h o m a tr e a tm e n t.
Immunohistological studies showed that Syk, B cell linker protein (BLNK) and PLCy2 are strongly expressed in primary mediastinal B-cell lymphoma, another subtype of DLBCL, and lymphocyte predominant Hodgkin lymphoma, but not in classical Hodgkin lymphoma [23-25], In conclusion, these data suggest that PLCy2 could be a treatment target for PLCy2 positive lymphomas. In this study, we intended to evaluate the expression frequency of PLCy2 in DLBCL and its role in lymphoma survival.
D iffu s e la rg e B -cell ly m p h o m a (DLBCL) can b e cu re d in a b o u t 6 0 % o f cases w ith im m u n o -c h e m o th e ra p y . H o w e v e r, a la rg e su b set o f p a tie n ts w ith DLBCL d o n o t g o in to rem issio n , o r re la p s e a fte r firs t-lin e th e ra p y . F u rth e r th e ra p y o p tio n s a re th e re fo r e n e e d e d . P h o s p h o lip a s e C y2 (PLCy2) is o n e o f th e k e y re g u la to rs o f th e B cell re c e p to r sig n a lin g p a th w a y , w h ic h ta rg e ts several p ro p ro life ra tiv e fa c to rs , such as n u c le a r fa c to r
k
B (N F k B), Ras a n d
A k t. Using im m u n o h is to c h e m is try , w e fo u n d t h a t PLCy2 w as s tro n g ly ex pressed in 6 3 % o f cases o f DLBCL. T h e PLC in h ib ito r U 73122
h ad
an
in h ib ito r y
e ffe c t on
cell
p ro life ra tio n
an d
in d u c e d a p o p to s is a n d G 0/G 1 cell cycle a rre s t. C o -tre a tm e n t w ith e n z a s ta u rin o r th e Src in h ib ito r p p 2 t o g e th e r w ith U 7 3 1 2 2 h a d an a d d itiv e e ffe c t on cell p ro life ra tio n c o m p a re d to U 7 3 1 2 2 a lo n e . U n e x p e c te d ly , stro n g PLCy2 expression w as associated w ith b e tte r o v e ra ll s u rv iv a l. In co n clu s io n , PLCy2 is stro n g ly
Keywords: PLCy2, DLBCL, overall survival, signaling pathway, PKCp, B cell receptor
In tro d u c tio n
Cell survival and development of normal B cells are dependent on B cell receptor (BCR) signaling [1-3]. There are also critical hints that BCR signaling is essential for lymphomagenesis and survival of malignant lymphoma B cells, especially in diffuse large B-cell lymphoma (DLBCL) [4-7]. Antigen binding to the BCR leads to phosphorylation of members of the Src tyrosine kinase family (e.g. Src, Lyn, Fyn, Lck), resulting in activation of the signaling cascade involving spleen tyrosine kinase (Syk), Bruton tyrosine
22],
M e th o d s Patient materials, cell line and reagents
Paraffin-embedded tissue materials of 86 primary DLBCLs were obtained from the archival tissue bank of the Institute of Pathology of Kiel. All patients were treated in a random-
Correspondence: Minh Q. Huynh, Universitatsklinikum Giessen und Marburg, Standort Marburg, Klinik fur Hamatologie, Onkologie und Immunologie, Baldinger Strafie, 35033 Marburg, Germany. Tel: + 49-6421-58-63004. Fax: + 49-6421-58-63005. E-mail: [email protected] Received 17 February 2014; revised 26 June 2014; accepted 30 June 2014 1088
PLC72 and diffuse large B cell lymphoma ized, multicenter, phase III study [26]. Inform ed consent was provided according to the Declaration of Helsinki. Tissue sam ples of Burkitt lym phom a {n= 3), follicular lym phom a grade III (n = 4), follicular lym phom a grade I [n= 6), m ande cell lym phom a (n = 3) and gastric m ucosa-associated lym phoid tissue (MALT) lym phom a (n = 2) were selected from the tissue bank of the Institute of Pathology of Marburg. Pri m ary lym phom a cells were collected from peripheral blood (total n = 6; m antle cell lym phom a n = 1, B-cell chronic lymphocytic leukem ia (CLL) n = 1, DLBCL n = 1, unclassi fied lym phom a n = 2, hairy cell leukem ia n = 1) or pleural effusion (total n = 2; DLBCL n — 1, follicular lym phom a n = 1) using TRIzol and erythrocyte lysis buffer (both from Sigma, St. Louis, MO) according to the m anufacturer’s pro tocol. W ritten consents from all patients were available. The DLBCL cell lines U2932, SuDHL-4 and SuDHL-6 were purchased from Deutsche Sam m lung von M ikroorganismen und Zellkulturen GmbH (DSMZ, Braunschweig, Germany). The specific PLC inhibitor U73122 (Biosource, Nivelles, Belgium), its inactive analog U73343 and the Src kinase inhibitor pp2 were also purchased (Calbiochem, Darmstadt, Germany). Enzastaurin was obtained from Eli Lilly and Com pany (Indianapolis, IN). Im m u n o c y to lo g y , im m u n o h is to c h e m is tr y a n d tis s u e m ic ro a r ra y
U ntreated DLBCL cells w ere w ashed with phosphatebuffered saline (PBS) an d centrifuged at 700 rpm for 7 m in on a glass slide using a Cytospin 2 Centrifuge (Shandon, Nutley, NJ). Cytospins were air-dried for 30 m in at room tem perature, fixed w ith m ethanol for 5 m in, w ashed three tim es w ith PBS and perm eabilized w ith ice-cold acetone for 45 s. Slides w ere w ashed three tim es w ith PBS. Im m unocytological staining was perform ed w ith a Vectastain ABC Kit (rabbit im m unoglobulin G [IgG]) from Vector Labora tories (Peterborough, UK). The prim ary antibody against PLCy2 (Q-20) was obtained from Santa Cruz Biotechnology (Heidelberg, Germ any). The prim ary antibody was diluted 1:50 in Antibody D iluent (Dako Cytomation, Glostrup, Denm ark). A rabbit isotype antibody served as control (Invitrogen, Carlsbad, CA). Antibody binding was stained with DAB (3,3’-diam inobenzidine) C hrom ogen (Dako, Carpinteria, CA) and counterstained with M ayer’s hem alum solution (Merck, D arm stadt, Germ any). Im m unohistochem ical staining of paraffin-em bedded prim ary DLBCL was perform ed as previously described [27], Im m unhistochem ical exam ination was done according to an intensity scoring system (0 = negative; 1 = low = < 20% positive cells, 2 = high = > 20% positive cells). Tissue m icroarray (TMA) was conducted as previously described [28], In brief, tissue cores of 0.6 m m diam eter were obtained from representa tive regions of archival paraffin-em bedded m aterial and th en re-em bedded in a new paraffin block. Five-m icrom eter sections were th en cut from TMA blocks and stained with antibodies against PLCy2 (Q-20), CD10 (Novocastra, Newcastle u p o n Tyne, UK), Bcl-6 and Mum-1 (both Dako, Glostrup, D enm ark). Classification of DLBCLs into germ i nal center B cell-like (GCB) and non-GCB/ABC subtypes was done according to the Hans algorithim [29].
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M e a s u r e m e n t o f cell v ia b ility a n d p r o life r a tio n
Cells were treated with or without small molecule inhibitors for 24 h and then incubated for 1 h at 37°C in 100 pL RPMI 1640 m edium with 20 pL CellTiter 96 AQueous One Solution Reagent (Promega, Madison, WI) in 96-well plates, and light absorbance was m easured at 492 nm with an enzyme linked im m unosorbent assay (ELISA) reader (TECAN GmbH, Crailsheim, Germany). Cell proliferation was determ ined using the trypan blue exclusion m ethod (Sigma-Aldrich, St. Louis, MO). T ra n s fe c tio n o f PLC y2 siR NA
Transfections with 3 pg PLCy2-specific short interfering RNA (siRNA) (Santa Cruz Biotechnology) or 3 pg non-silencing siRNA (Qiagen, Hilden, Germany) were done using the Amaxa transfection system (Lonza Cologne GmbH, Cologne, Germany), according to the m anufacturer’s instructions. F lo w c y to m e tr ic a n a ly s is o f a p o p to s is , cell c yc le a n d p h o s p h o -p ro te in s
Flow cytometric analysis was done on an LSRII m achine from BD Biosciences using Flowjo v7.6.5 for Windows software (Tree Star, Inc., Ashland, OR). Apoptosis was m easured by staining cells with Annexin-fluorescein iso thiocyanate (FITC) and propidium iodide (PI) according to the m anufacturer's instructions (Alexis Biochemicals, Griinberg, Germany). For cell cycle analysis, cells were fixed with ice-cold 70% ethanol and stored at —20°C for at least 2 h. RNA was degraded with RNAse A (10 pg/pL) (Qiagen) for 15 m in at room tem perature and DNA stained with PI (200 pg/mL) (Sigma). Rabbit anti-phospho-Src (Y418), rab bit anti-phospho-PLCy2 (Y759) (Cell Signaling, Danvers, MA) and rabbit anti-phospho-PKCpi/II (Thr500) antibody (Millipore, Temecula, CA) were used as prim ary antibodies. The secondary antibody goat anti-rabbit Alexa Fluor 488 was purchased from Invitrogen (Eugene, OR). Q u a n t it a t iv e PCR
Quantitative polymerase chain reaction (PCR) was per formed on an ABI Prism 7700 sequence detection system (PE Applied Biosystems, Foster City, CA) using a QuantiTect SYBR Green Kit (Qiagen) according to the m anufac turer’s instructions. The Plcg2 prim ers were: 5'-ATG GCT GGA CGC GGA CTA CC-3' (forward) and 5'-CTT GGC CAT GTG ACG CTG TT-3’ (reverse). The Gapdh prim ers were 5'-CTCCTCCACCTTTGACGCTG-3' (forward) and 5'-ACC ACCCTGTTGCTGTAGCC-3’ (reverse). All prim ers were obtained from MWG Biotech (Ebersberg, Germany). The amplification conditions were 1 X 95°C for 15 m in followed by 45 X (94°C for 15 s, 58.5°C for 30 s, 72°C for 30 s). Analysis of quantitative PCR was perform ed according to the ACT m ethod as previously described [27], W e s te rn b lo t
Cells were incubated with dimethylsulfoxide (DMSO) (control), 5 pM U73122 or 5 pM U73343. After 3 h cells were lysed with RIPA buffer, supplem ented with 1 pL protease inhibitor cocktail set III (Calbiochem, Darmstadt, Germany), 10 pL phenylmethylsulfonyl fluoride (PMSF) (200 mM)
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(Sigma-Aldrich), 10 (iL phosphatase inhibitor cocktail I and 10 uL phosphatase inhibitor cocktail II (both from SigmaAldrich). The protein concentration was m easured with the BCA™ Protein Assay Kit (Pierce, Rockford, IL), accordingto the m anufacturer's protocol. Cell lysates were electrophoresed on 10% sodium dodecyl sulfate (SDS)-polyacrylamide gels and transferred onto nitrocellulose m em branes (Bio-Rad, Richmond, CA). M em branes were blocked with 5% milk pow der in Tris-buffered saline (TBS; 20 mM Tris-HCl, pH 7.6,150 mM NaCl) and prim ary antibodies were incubated overnight at 4°C. Horseradish peroxidase (HRP) conjugated secondary antibodies were incubated for 1 h at room tem perature. Pro teins were detected with a C hem ilum inescence Detection Kit (Pierce Biotechnology, Rockford, USA). The following prim ary antibodies were used: rabbit anti-phospho-PLCG2 (Y1217), rabbit anti-phospho-p65 (S276), rabbit anti-p65, rabbit anti-phospho-AKT (Ser473), rabbit anti-AKT, rabbit anti-phospho-p44/42 (Thr202/Tyr204), rabbit anti-p44/42 (all from Cell Signaling), rabbit anti-phospho-PKCBI/II (Thr500) (Millipore), m ouse anti-PKCB (BD Biosciences Pharmingen, Heidelberg, Germany), rabbit anti-PLCG2, m ouse anti-PKC, m ouse anti-(3-actin and m ouse anti-a-tubulin (all from Santa Cruz Biotechnology). Goat m onoclonal anti-rabbit or goat m onoclonal anti-m ouse secondary antibodies were also obtained from Santa Cruz Biotechnology. Statistical analysis
All statistical analyses were perform ed with GraphPad Prism Version 5.0 for Windows (GraphPad Software, Inc., La Jolla, CA). Statistical significance was determ ined if p was < 0.05. Correlation of PLCy2 expression and DLBCL subtype (GCB
vs. non-GCB/ABC), age-adjusted international prognostic index (alPI), lactate dehydrogenase (LDH) level and treat m ent arm s was done using Fisher's exact test. Overall survival (OS) was calculated with the log-rank test. Cell proliferation of siRNA transfected lym phom a cells was calculated using an unpaired, two-tailed f-test. Results PLCy2 is s tro n g ly expressed in B cell n o n -H o d g k in ly m p h o m a a nd especially in a larg e subset o f DLBCL
We stained several entities of aggressive (DLBCL, n = 86; Burkitt lymphoma, n = 3; follicular lym phom a grade 3, n = 4) and indolent B cell non-Hodgkin lym phom a (B-NHL) (fol licular lym phom a grade 1, n = 6; gastric MALT lymphoma, n = 2; m antle cell lymphoma, n = 3) for PLCy2 expression. PLCy2 was strongly expressed in all cases of Burkitt lym phom a, follicular lym phom a grade 1 and 3, gastric MALT lym phom a and m antle cell lymphoma. Out of 86 cases of DLBCL, PLCy2 was strongly expressed in 54 (63%) cases and weak or no expression was seen in 32 cases (37%). Three DLBCL cell lines (U2932, SuDHL-4 and SuDHL-6) were also positive for PLCy2 expression [Figure 1(A)]. SuDHL-4 and SuDHL-6 are of germinal GCB subtype and U2932 of ABC subtype. Furtherm ore, we investigated w hether PLCy2 expression correlates with GCB and non-GCB/ABC subtype. To analyze this, we utilized the TMA m ethod. Fifty-five of the same 86 cases of DLBCL were available for TMA. According to the Hans algorithm, staining of CD10, Bcl-6 and Mum-1 was perform ed [29]. Twenty-four (44%) cases were of GCB subtype and 31 (56%) of non-GCB/ABC subtype. Of the 36 PLCy2
Figure 1. Expression of PLCy2 in lymphoma cells. (A) Immunohistochemistry and immunocytology of PLCy2: exemplary cases of B-NHL; all photographed at X 60 (except i: X 10). a and b: DLBCL with strong expression of PLCy2; c: case of DLBCL with weak PLCy2 expression; d-f: SuDHL-4 GCB subtype, SuDHL-6 GCB subtype, U2932 ABC subtype; g: Burkitt lymphoma; h: follicular lymphoma grade III; i: follicular lymphoma grade I; j: mantle cell lymphoma; k: gastric MALT lymphoma. (B) Correlation of PLCy2 expression with DLBCL subtype (tissue microarray; n = 55).
PLCy2 and diffuse large B cell lymphoma
positive lymphomas, 18 belonged to the GCB subtype and also 18 belonged to the non-GCB/ABC subtype. There was no significant correlation between PLCy2 expression (immunohistochemistry) and DLBCL subtype (TMA) [Fisher’s exact test p = 0.2564; Figure 1(B)]. Lymphoma proliferation is dependent on PLCy2 activation
In order to investigate the functional role of PLCy2 in lym phoma cells, we treated primary cells from patients who suffered from different B cell malignancies ( n = 8) and three DLBCL cell lines with U73122, a specific inhibitor of PLC, and its inactive analog U73343. Primary lymphoma cells of different lymphoma entities were collected from peripheral blood ( n = 6) or pleural effu sion [ n = 2). Reverse transcription (RT)-PCR and Western blot confirmed strong expression of PLCy2 in primary lym phoma cells (see Supplementary Figure 1 available online at http://informahealthcare.com/doi/abs/10.3109/10428194.201 4.941832). Compared to U73343 and untreated cells (DMSO control), inhibition of PLCy2 with U73122 resulted in reduced cell proliferation in all patient samples [Figure 2(A)], Additionally, we also observed reduced cell viability in all DLBCL cell lines (U2932, SuDHL-4 and SuDHL-6) after treatment with U73122, which occurred in a dose-dependent manner. U73343 did not reduce lymphoma proliferation
significantly [Figure 2(B)], To further exclude non-specific inhibition of cell proliferation with U73122, we treated the PLCyl positive Jurkat cell line (cells derived from a T cell leukemia), which expressed low PLCy2 with U73122 and U73343. PLCy2 mRNA in Jurkat cells was lower as compared to U2932, SuDHL-4 and SuDHL-6 [see Supplementary Figure 2(A) available online at http://informahealthcare.com/ doi/abs/10.3109/10428194.2014.941832]. Treatment of Jurkat cells with U73122 resulted in non-significant cell viability inhibition [Figure 2(B)]. In addition, no direct inhibition of PLCy2 and its targets were observed [see Supplementary Figure 2(B) available online at http://informahealthcare.com/ doi/abs/10.3109/10428194.2014.941832]. After 24 h treatment of lymphoma cell lines with U73122, apoptosis and the cell cycle were measured by flow cyto metric analysis. U73122 induced apoptosis [Figure 2(C)] and G0/G1 cell cycle arrest [Figure 2(D)] in lymphoma cells. Western blot analysis showed specific inhibition of phospho-PLCG2 and its downstream targets such as phospho-PKCB, p-p65 and p-AKT after treatment with U73122, but not with U73343. Phosphorylation of p44/42 was only inhibited in SuDHL-6 cells [Figure 3(A)], To confirm the specific effect of U73122 on lymphoma survival, DLBCL cell lines were then transfected with a specific siRNA against PLCy2. A non-specific siRNA was used as control. Cells were collected 48 h after transfection
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Figure 2. Lymphoma proliferation is dependent on PLCy2 signaling. (A) Trypan blue exclusion assay. Primary lymphoma cells were treated with or without U73122 and U73343. Cell proliferation was reduced with U73122. U73343 served as control. (B-D) DLBCL lines were treated with and without U73122 and U73343.U73122 decreased cell viability (B, MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)2H-tetrazolium] assay), induced apoposis (C, FITC-Annexin/PI assay) and G0/G1 cell cycle arrest (D, PI staining).
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Figure 3. Inhibition and knockdown of PLC72. (A) Western blot: cells were incubated with (1) DMSO, (2) 5 pM U73122 or (3) 5 pM U73343 for 3 h. Inhibition of phospho-PLCG2 and its targets phospho-PKCB, phospho-p65, phospho-AKT and phospho-p44/42 was shown with U73122, but not with U73343. (B) siRNA transfection: specific knockdown of Plcg2 with PLCy2 specific siRNA, but not with ns (non-specific) siRNA. (C) Significant decrease of cell viability with Plcg2 siRNA (MTS; p £ 0.05). Each experiment was done in triplicate and data represent mean value ± standard error (SE)(n = 3).
and cell proliferation was measured by trypan blue exclu sion assay. Western blots were performed to confirm spe cific knockdown of PLCy2 [Figure 3(B)], Transfection with a PLCy2 siRNA resulted in significant reduction of lymphoma viability; p < 0.05 [Figure 3(C)]. A d d itive e ffe c t on cell p ro liferatio n by seq u en tial in h ib itio n o f S rc-PLC y2-P K C p signaling p a th w a y
We compared treatm ent of U73122 with the Src kinase inhibitor pp2 and the PKC|3 inhibitor enzastaurin. We
Patient 5
Patient 6
could show in primary lymphoma cells that U73122 was, with respect to cell proliferation, at least as effective as pp2 and enzastaurin (Figure 4). Co-treatment of U73122 with pp2 or enzastaurin showed an additive effect on cell prolif eration in all DLBCL cell lines [Figures 5(A) and 5(B)], Flow cytometric analysis confirmed a specific effect of pp2 on Src and PLCy2 phosphorylation. U73122 and enzastaurin had an inhibitory effect on phospho-PKCB (see Supplementary Figure 3 available online at http://informahealthcare.com/ doi/abs/10.3109/10428194.2014.941832).
Patient 7
Patient 8
Figure 4. Treatment of primary lymphoma cells with U73122, pp2 and enzastaurin. Trypan blue exclusion assay: U73122 had at least similar effect on cell proliferation compared with pp2 and enzastaurin. In five out of eight patients [patient 2, 3, 4, 6 and 8) U73122 was more effective than pp2 and enzastaurin.
PLCy2 and diffuse large B cell lymphoma (A)
Co-treatment with pp2 and U73122
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Figure 5. Additive effect of U73122 and other small molecule inhibitors on lymphoma survival. (A, B) Cell proliferation and viability. Co-treatment of U73122 with pp2 or enzastaurin showed additive effect on cell proliferation and viability.
Inverse correlation of PLCy2 expression with OS
In addition, we correlated PLCy2 expression with OS. Three-year survival data were available for all patients ( n = 86) and long term survival data (range from 6 to 143 months) were available for 71 out of 86 cases. DLBCL with strong PLCy2 expression showed a trend toward better OS after a 3-year follow-up (logrank test p = 0.0975). Furthermore, strong PLCy2 expression
months
correlated with significantly better survival after long-term fol low-up (log-rank test p = 0.0385) (Figure 6). There was no cor relation between PLCy2 expression and alPI (0-1 vs. 2-3), LDH level and treatment arm (arm A = standard chemotherapy; arm B = chemotherapy + autologous transplant) (see Supplemen tary Table I available online at http://informahealthcare.com/ doi/abs/10.3109/10428194.2014.941832).
PLCgamma2 high
months
PLCgamma2 low/neg.
Figure 6. PLCy2 expression and OS. PLCy2 expression was correlated with OS after 3-year (A) and long-term (range from 6 to 143 months) (B) follow up. Strong expression of PLCy2 was associated with better OS (3-year follow-up, p = 0.0975 and long-term follow-up, p = 0.0385).
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D is c u s s io n
The outcome of patients suffering from B-cell lymphoma has been improved through the use of biological agents such as CD20-specific antibodies. The PLCy2-PKCP signaling cascade is essential for normal B cell development and, probably, B cell lymphoma survival. We found, in several lymphoma entities such as follicular lymphoma, gastric MALT lymphoma, mantle cell lymphoma, Burkitt lymphoma and DLBCL, strong expression of PLCy2 in immunohistochemical studies. These data suggest a crucial role for lymphoma survival and a potential new therapy target. Here, we have focused on the expression frequency of PLCy2 in DLBCL {n = 86 ) and its potential functional role for lymphoma survival. We could show via immunohistochemical staining that a large subset of primary DLBCL (63%) strongly expressed PLCy2, and only 37% showed weak or no expression level. Using TMA of the same cases (n = 55) we subclassified cases of DLBCL into GCB and non-GCB/ABC subtype, according to the Hans algorithm [29], However, we are aware that it has often been discussed whether the Hans classifier can reliably distinguish between GCB and non-GCB/ABC subtypes [30], In our study, 24 out of 55 cases (44%) were classified as GCB subtype and 31 cases (56%) were of non-GCB/ABC subtype. Our results were concordant with the published data of Hans etal. (42% GCB vs. 58% nonGCB/ABC) [29]. There was no significant correlation between the expression level of PLC72 (immunohistochemistry) and DLBCL subtype (TMA). We were able to show that inhibition or knockdown of PLCy2 killed primary lymphoma cells and DLBCL cell lines. We treated lymphoma cells with the specific PLC inhibitor U73122. U73122 induced apoptosis and cell cycle arrest in primary lymphoma cells, as well as lymphoma cell lines. Western blot analysis confirmed specific inhibition of PLCy2 and its downstream targets PKCfi, p65 and Akt. Desphosphorylation of p44/42 was seen only in SuDHL-6 cells, but was hardly detectable in SuDHL-4 and U2932. Supporting our data, Cheng et al. have recently shown that inactivation of PLCy2 and consequently Akt with the Syk inhibitor PRT318, but not p44/42, was associated with decreased DLBCL sur vival and increased cell cycle arrest [22]. Knockdown of PLCy2 with a specific siRNA showed the same effect on cell survival as with U73122. Furthermore, we compared the inhibitory effect of U73122 with pp2 (Src tyrosine kinase inhibitor) and enzastaurin (PKC[3) on lymphoma cells. Cell survival inhibi tion was similar between all three small molecule inhibitors in primary cells and DLBCL lines. However, co-treatment with U73122 and pp2 or U73122 and enzastaurin showed an additive effect on cell survival. PLCy2 is mainly activated by the BCR-Src-Btk signaling pathway. However, published data show high sensitivity for BCR inhibition only in ABC-DLBCL, but not in GCBDLBCL cells. We suggest that in lymphoma cells, other signaling pathways besides the BCR pathway are involved in PLCy2 activation, as it has been described that the B-cell activating factor receptor (BAFF-R) and fibroblast growth factor receptor (FGFR) are additionally involved in PLCy2 activation [2,31].
Altogether, PLCy2 is strongly expressed in a large number of cases of DLBCL, and lymphoma survival is dependent on PLCy2 signaling. Next, we investigated whether PLCy2 expression was associated with clinical treatm ent outcome. Strong expres sion of PLCy2 was associated with better OS. These data were unexpected, since the PLCy2-PKCp signaling cas cade activates pro-survival factors such as NFkB, Ras and Akt [11,14,15]. We speculate that lymphoma cells need PLCy2 for survival, whereas strong PLCy2 expression results in more cell proliferation and thus higher suscep tibility to cytotoxic drugs. In addition, PLCy2 expression was independent of the alPI or LDH level. An imbalance of treatm ent arms (arm A = standard chemotherapy; arm B = chemotherapy + autologous transplant) and PLCy2 expression could be ruled out. All patients were treated in a multicenter clinical study that was conducted in the prerituximab era [26], Therefore, a confirmatory analysis using patients treated with rituximab needs to be performed. In contrast to PLC72, it has been shown that patients with DLBCL with low PKCfill expression have better 3-year OS after immunochemotherapy [32], Targeting PLCy2 for lymphoma treatment is highly attractive, since it has been shown that the Btk inhibitor ibrutinib and the PKCP inhibi tor enzastaurin have promising anti-lymphoma activity [17,18,20]. To our knowledge, there is currently no PLCy2 inhibitor being investigated in clinical studies. In conclusion, our data show that the Src-PLCy2-PKCP signaling pathway has an important role for lymphoma sur vival. Treatment of patients with DLBCL could benefit from inhibition of PLCy2, at least in those cases with refractory or relapsed lymphoma after first-line immuno-chemotherapy. We found a significant inverse correlation of PLCy2 expression and DLBCL OS. These data have to be confirmed in further studies of patients treated with immuno-chemotherapy. A c k n o w le d g e m e n t s
The authors would like to thank the University Medi cal Center Giessen and Marburg for a research grant to M.Q.H., Deutsche Forschungsgemeinschaft (KFO 210, to A.N.), German Jose Carreras foundation (to A.N.) and the Dr. Reinfried Pohl foundation (to A.N.).
Potential conflict o f interest: Disclosure forms provided by the authors are available with the full text of this article at www.informahealthcare.com/lal.
R e fe r e n c e s
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[4] Bogusz AM, Baxter RH, Currie T, et al. Quantitative immunofluorescence reveals the signature of active B-cell receptor signaling in diffuse large B-cell lymphoma. Clin Cancer Res 2012; 18:6122-6135. [5] Young RM, Staudt LM. Targeting pathological B cell receptor signalling in lymphoid malignancies. Nat Rev Drug Discov 2013;12: 229-243. [6] Davis RE, Ngo VN, Lenz G, et al. Chronic active B-cell-receptor signalling in diffuse large B-cell lymphoma. Nature 2010;463:88-92. [7] Chen L, Monti S, Juszczynski P, et al. SYK-dependent tonic B-cell receptor signaling is a rational treatm ent target in diffuse large B-cell lymphoma. Blood 2008;111:2230-2237. [8] Takata M, Sabe H, Hata A, et al. Tyrosine kinases Lyn and Syk regulate B cell receptor-coupled Ca2 + mobilization through distinct pathways. EMBO 11994;13:1341-1349. [9] KurosakiT, MaedaA, IshiaiM, etal. Regulationofthe phospholipase C-gamma2 pathway in B cells. Immunol Rev 2000;176:19-29. [10] Marshall AJ, Niiro H, Yun TJ, et al. Regulation of B-cell activation and differentiation by the phosphaddylinositol 3-kinase and phospholipase Cgamma pathway. Immunol Rev 2000;176:30-46. [11] Tan JE, Wong SC, Gan SK, et al. The adaptor protein BLNK is required for b cell antigen receptor-induced activation of nuclear factor-kappa B and cell cycle entry and survival of B lymphocytes. J Biol Chem 2001;276:20055-20063. [12] Pavan A, Spina M, Canzonieri V, et al. Recent prognostic factors in diffuse large B-cell lymphoma indicate NF-kappaB pathway as a target for new therapeutic strategies. Leuk Lymphoma 2008;49:2048-2058. [13] Ying H, Li Z, Yang L, et al. Syk mediates BCR- and CD40-signaling integration during B cell activation. Immunobiology 2011;216:566-570. [14] Zheng Y, Liu H, Coughlin J, et al. Phosphorylation of RasGRP3 on threonine 133 provides a mechanistic link between PKC and Ras signaling systems in B cells. Blood 2005; 105:3648-3654. [15] Gold MR, Scheid MP, Santos L, et al. The B cell andgen receptor activates the Akt (protein kinase B)/glycogen synthase kinase-3 signaling pathway via phosphaddylinositol 3-kinase. I Immunol 1999;163:1894-1905. [16] Aagaard-Tillery KM, Jelinek DF. Phosphaddylinositol 3-kinase acdvadon in normal hum an B lymphocytes. J Immunol 1996; 156: 4543-4554. [17] Advani RH, Buggy JJ, Sharman JP, et al. Bruton tyrosine kinase inhibitor ibrutinib (PCI-32765) has significant activity in patients with relapsed/refractory B-cell malignancies. J Clin Oncol 2013;31:88-94. [18] Dasmahapatra G, Patel H, Dent P, et al. The Bruton tyrosine kinase (BTK) inhibitor PCI-32765 synergistically increases proteasome inhibitor activity in diffuse large-B cell lymphoma (DLBCL) and mantle cell lymphoma (MCL) cells sensitive or resistant to bortezomib. Br J Haematol 2013;161:43-56.
S u p p le m e n ta ry m a te ria l a v a ila b le o n lin e Supplementary Figures 1-3 and Table I showing further results.
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[19] Yang Y, Shaffer AL 3rd, Emre NC, et al. Exploiting synthetic lethality for the therapy of ABC diffuse large B cell lymphoma. Cancer Cell 2012;21:723-737. [20] Robertson MJ, Kahl BS, Vose JM, etal. Phase II study of enzastaurin, a protein kinase C beta inhibitor, in patients with relapsed or refractory diffuse large B-cell lymphoma. J Clin Oncol 2007;25:1741-1746. [21] Yang C, Lu P, Lee FY, et al. Tyrosine kinase inhibition in diffuse large B-cell lymphoma: molecular basis for antitum or activity and drug resistance of dasatinib. Leukemia 2008;22:1755-1766. [22] Cheng S, Coffey G, Zhang XH, et al. SYK inhibition and response prediction in diffuse large B-cell lymphoma. Blood 2011; 118:6342-6352. [23] Pozzobon M, Marafioti T, Hansmann ML, et al. Intracellular signalling molecules as immunohistochemical markers of normal and neoplastic human leucocytes in routine biopsy samples. Br J Haematol 2004;124:519-533. [24] Marafioti T, Pozzobon M, Hansmann ML, et al. Expression of intracellular signaling molecules in classical and lymphocyte predominance Hodgkin disease. Blood 2004;103:188-193. [25] Marafioti T, Pozzobon M, Hansmann ML, et al. Expression pattern of intracellular leukocyte-associated proteins in primary mediastinal B cell lymphoma. Leukemia 2005;19:856-861. [26] Kaiser U, Uebelacker I, Abel U, et al. Randomized study to evaluate the use of high-dose therapy as part of primary treatment for "aggressive" lymphoma. J Clin Oncol 2002;20:4413-4419. [27] Huynh MQ, Wacker HH, Wundisch T, et al. Expression profiling reveals specific gene expression signatures in gastric MALT lymphomas. Leuk Lymphoma 2008;49:974-983. [28] de Jong D, Rosenwald A, Chhanabhai M, et al. Im m uno histochemical prognostic markers in diffuse large B-cell lymphoma: validation of tissue microarray as a prerequisite for broad clinical applications—a study from the Lunenburg Lymphoma Biomarker Consortium. J Clin Oncol 2007;25:805-812. [29] Hans CP, Weisenburger DD, Greiner TC, et al. Confirmation of the molecular classification of diffuse large B-cell lymphoma by immunohistochemistry using a tissue microarray. Blood 2004; 103:275-282. [30] Salles G, de Jong D, Xie W, et al. Prognostic significance of immunohistochemical biomarkers in diffuse large B-cell lymphoma: a study from the Lunenburg Lymphoma Biomarker Consortium. Blood 2011;117:7070-7078. [31] Chen J, Williams IR, Lee BH, et al. Constitutively activated FGFR3 mutants signal through PLCgamma-dependent and -independent pathways for hematopoietic transformation. Blood 2005;106:328-337. [32] Riihijarvi S, Koivula S, Nyman H, et al. Prognostic impact of protein kinase C beta II expression in R-CHOP-treated diffuse large B-cell lymphoma patients. Mod Pathol 2010;23:686-693.
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© 2014 In fo rm a UK, Ltd. ISSN: 1042-8194 p r in t / 1029-2403 o n lin e
healthcare
DOI: 10.3109/10428194.2014.941832
ORIGINAL ARTICLE: RESEARCH
Expression and pro-survival function of phospholipase Cy2 in diffuse large B-cell lymphoma Minh Q. Huynh1, Jennifer GoRmann1, Stefan Gattenloehner3, Wolfram Klapper2, Hans-Heinrich Wacker4, Annette Ramaswamy5, Alwina Bittner1, Ulrich Kaiser6 & Andreas Neubauer1 1Department o f Hematology, Oncology and Immunology, University Hospital, Marburg, Germany, 2Department o f Pathology, Hematopathology Section and Lymph Node Registry, University Hospital, Kiel, Germany,3Institute o f Pathology, University Hospital, Giessen, Germany,4Institute o f Hematopathology, Kiel, Germany,5Institute o f Pathology, University Hospital, Marburg, Germany and 6Department o f Internal Medicine II, St. Bernward Hospital, Hildesheim, Germany
Abstract
expressed in a s ig n ific a n t n u m b e r o f DLBCLs a n d has p ro g n o s tic
kinase (Btk), phospholipase Cy2 (PLCy2) and protein kinase Cp (PKCp), which is important for nuclear factor kB (NFkB) regulation [8-11]. NFkB is in particular up-regulated in DLBCL with activated B cell-like (ABC) subtype [12]. This above described signaling cascade also activates Ras and Akt [13-16]. There are several preclinical and clinical phase I/II studies showing promising data concerning DLBCL treat ment targeting the BCR signaling pathway, e.g. dasatinib (Src kinase inhibitor), PRT318 (Syk inhibitor), ibrutinib (Btk inhibitor) and enzastaurin (PKCP inhibitor) [17-22], These small molecule inhibitors are also currently being tested in ongoing clinical trials in patients with DLBCL (http://www. clinicaltrials.gov/). It has been shown that the activity of Syk and PLCy2 is well correlated with the sensitivity of DLBCL to dasatinib, and that inhibition of Syk with the specific inhibitor PRT060318 results in reduced lymphoma survival and cell cycle arrest
im p lic a tio n s . In h ib itio n o f PLCy2 co u ld b e a n e w ta r g e t fo r
[21,
ly m p h o m a tr e a tm e n t.
Immunohistological studies showed that Syk, B cell linker protein (BLNK) and PLCy2 are strongly expressed in primary mediastinal B-cell lymphoma, another subtype of DLBCL, and lymphocyte predominant Hodgkin lymphoma, but not in classical Hodgkin lymphoma [23-25], In conclusion, these data suggest that PLCy2 could be a treatment target for PLCy2 positive lymphomas. In this study, we intended to evaluate the expression frequency of PLCy2 in DLBCL and its role in lymphoma survival.
D iffu s e la rg e B -cell ly m p h o m a (DLBCL) can b e cu re d in a b o u t 6 0 % o f cases w ith im m u n o -c h e m o th e ra p y . H o w e v e r, a la rg e su b set o f p a tie n ts w ith DLBCL d o n o t g o in to rem issio n , o r re la p s e a fte r firs t-lin e th e ra p y . F u rth e r th e ra p y o p tio n s a re th e re fo r e n e e d e d . P h o s p h o lip a s e C y2 (PLCy2) is o n e o f th e k e y re g u la to rs o f th e B cell re c e p to r sig n a lin g p a th w a y , w h ic h ta rg e ts several p ro p ro life ra tiv e fa c to rs , such as n u c le a r fa c to r
k
B (N F k B), Ras a n d
A k t. Using im m u n o h is to c h e m is try , w e fo u n d t h a t PLCy2 w as s tro n g ly ex pressed in 6 3 % o f cases o f DLBCL. T h e PLC in h ib ito r U 73122
h ad
an
in h ib ito r y
e ffe c t on
cell
p ro life ra tio n
an d
in d u c e d a p o p to s is a n d G 0/G 1 cell cycle a rre s t. C o -tre a tm e n t w ith e n z a s ta u rin o r th e Src in h ib ito r p p 2 t o g e th e r w ith U 7 3 1 2 2 h a d an a d d itiv e e ffe c t on cell p ro life ra tio n c o m p a re d to U 7 3 1 2 2 a lo n e . U n e x p e c te d ly , stro n g PLCy2 expression w as associated w ith b e tte r o v e ra ll s u rv iv a l. In co n clu s io n , PLCy2 is stro n g ly
Keywords: PLCy2, DLBCL, overall survival, signaling pathway, PKCp, B cell receptor
In tro d u c tio n
Cell survival and development of normal B cells are dependent on B cell receptor (BCR) signaling [1-3]. There are also critical hints that BCR signaling is essential for lymphomagenesis and survival of malignant lymphoma B cells, especially in diffuse large B-cell lymphoma (DLBCL) [4-7]. Antigen binding to the BCR leads to phosphorylation of members of the Src tyrosine kinase family (e.g. Src, Lyn, Fyn, Lck), resulting in activation of the signaling cascade involving spleen tyrosine kinase (Syk), Bruton tyrosine
22],
M e th o d s Patient materials, cell line and reagents
Paraffin-embedded tissue materials of 86 primary DLBCLs were obtained from the archival tissue bank of the Institute of Pathology of Kiel. All patients were treated in a random-
Correspondence: Minh Q. Huynh, Universitatsklinikum Giessen und Marburg, Standort Marburg, Klinik fur Hamatologie, Onkologie und Immunologie, Baldinger Strafie, 35033 Marburg, Germany. Tel: + 49-6421-58-63004. Fax: + 49-6421-58-63005. E-mail: [email protected] Received 17 February 2014; revised 26 June 2014; accepted 30 June 2014 1088
PLC72 and diffuse large B cell lymphoma ized, multicenter, phase III study [26]. Inform ed consent was provided according to the Declaration of Helsinki. Tissue sam ples of Burkitt lym phom a {n= 3), follicular lym phom a grade III (n = 4), follicular lym phom a grade I [n= 6), m ande cell lym phom a (n = 3) and gastric m ucosa-associated lym phoid tissue (MALT) lym phom a (n = 2) were selected from the tissue bank of the Institute of Pathology of Marburg. Pri m ary lym phom a cells were collected from peripheral blood (total n = 6; m antle cell lym phom a n = 1, B-cell chronic lymphocytic leukem ia (CLL) n = 1, DLBCL n = 1, unclassi fied lym phom a n = 2, hairy cell leukem ia n = 1) or pleural effusion (total n = 2; DLBCL n — 1, follicular lym phom a n = 1) using TRIzol and erythrocyte lysis buffer (both from Sigma, St. Louis, MO) according to the m anufacturer’s pro tocol. W ritten consents from all patients were available. The DLBCL cell lines U2932, SuDHL-4 and SuDHL-6 were purchased from Deutsche Sam m lung von M ikroorganismen und Zellkulturen GmbH (DSMZ, Braunschweig, Germany). The specific PLC inhibitor U73122 (Biosource, Nivelles, Belgium), its inactive analog U73343 and the Src kinase inhibitor pp2 were also purchased (Calbiochem, Darmstadt, Germany). Enzastaurin was obtained from Eli Lilly and Com pany (Indianapolis, IN). Im m u n o c y to lo g y , im m u n o h is to c h e m is tr y a n d tis s u e m ic ro a r ra y
U ntreated DLBCL cells w ere w ashed with phosphatebuffered saline (PBS) an d centrifuged at 700 rpm for 7 m in on a glass slide using a Cytospin 2 Centrifuge (Shandon, Nutley, NJ). Cytospins were air-dried for 30 m in at room tem perature, fixed w ith m ethanol for 5 m in, w ashed three tim es w ith PBS and perm eabilized w ith ice-cold acetone for 45 s. Slides w ere w ashed three tim es w ith PBS. Im m unocytological staining was perform ed w ith a Vectastain ABC Kit (rabbit im m unoglobulin G [IgG]) from Vector Labora tories (Peterborough, UK). The prim ary antibody against PLCy2 (Q-20) was obtained from Santa Cruz Biotechnology (Heidelberg, Germ any). The prim ary antibody was diluted 1:50 in Antibody D iluent (Dako Cytomation, Glostrup, Denm ark). A rabbit isotype antibody served as control (Invitrogen, Carlsbad, CA). Antibody binding was stained with DAB (3,3’-diam inobenzidine) C hrom ogen (Dako, Carpinteria, CA) and counterstained with M ayer’s hem alum solution (Merck, D arm stadt, Germ any). Im m unohistochem ical staining of paraffin-em bedded prim ary DLBCL was perform ed as previously described [27], Im m unhistochem ical exam ination was done according to an intensity scoring system (0 = negative; 1 = low = < 20% positive cells, 2 = high = > 20% positive cells). Tissue m icroarray (TMA) was conducted as previously described [28], In brief, tissue cores of 0.6 m m diam eter were obtained from representa tive regions of archival paraffin-em bedded m aterial and th en re-em bedded in a new paraffin block. Five-m icrom eter sections were th en cut from TMA blocks and stained with antibodies against PLCy2 (Q-20), CD10 (Novocastra, Newcastle u p o n Tyne, UK), Bcl-6 and Mum-1 (both Dako, Glostrup, D enm ark). Classification of DLBCLs into germ i nal center B cell-like (GCB) and non-GCB/ABC subtypes was done according to the Hans algorithim [29].
1089
M e a s u r e m e n t o f cell v ia b ility a n d p r o life r a tio n
Cells were treated with or without small molecule inhibitors for 24 h and then incubated for 1 h at 37°C in 100 pL RPMI 1640 m edium with 20 pL CellTiter 96 AQueous One Solution Reagent (Promega, Madison, WI) in 96-well plates, and light absorbance was m easured at 492 nm with an enzyme linked im m unosorbent assay (ELISA) reader (TECAN GmbH, Crailsheim, Germany). Cell proliferation was determ ined using the trypan blue exclusion m ethod (Sigma-Aldrich, St. Louis, MO). T ra n s fe c tio n o f PLC y2 siR NA
Transfections with 3 pg PLCy2-specific short interfering RNA (siRNA) (Santa Cruz Biotechnology) or 3 pg non-silencing siRNA (Qiagen, Hilden, Germany) were done using the Amaxa transfection system (Lonza Cologne GmbH, Cologne, Germany), according to the m anufacturer’s instructions. F lo w c y to m e tr ic a n a ly s is o f a p o p to s is , cell c yc le a n d p h o s p h o -p ro te in s
Flow cytometric analysis was done on an LSRII m achine from BD Biosciences using Flowjo v7.6.5 for Windows software (Tree Star, Inc., Ashland, OR). Apoptosis was m easured by staining cells with Annexin-fluorescein iso thiocyanate (FITC) and propidium iodide (PI) according to the m anufacturer's instructions (Alexis Biochemicals, Griinberg, Germany). For cell cycle analysis, cells were fixed with ice-cold 70% ethanol and stored at —20°C for at least 2 h. RNA was degraded with RNAse A (10 pg/pL) (Qiagen) for 15 m in at room tem perature and DNA stained with PI (200 pg/mL) (Sigma). Rabbit anti-phospho-Src (Y418), rab bit anti-phospho-PLCy2 (Y759) (Cell Signaling, Danvers, MA) and rabbit anti-phospho-PKCpi/II (Thr500) antibody (Millipore, Temecula, CA) were used as prim ary antibodies. The secondary antibody goat anti-rabbit Alexa Fluor 488 was purchased from Invitrogen (Eugene, OR). Q u a n t it a t iv e PCR
Quantitative polymerase chain reaction (PCR) was per formed on an ABI Prism 7700 sequence detection system (PE Applied Biosystems, Foster City, CA) using a QuantiTect SYBR Green Kit (Qiagen) according to the m anufac turer’s instructions. The Plcg2 prim ers were: 5'-ATG GCT GGA CGC GGA CTA CC-3' (forward) and 5'-CTT GGC CAT GTG ACG CTG TT-3’ (reverse). The Gapdh prim ers were 5'-CTCCTCCACCTTTGACGCTG-3' (forward) and 5'-ACC ACCCTGTTGCTGTAGCC-3’ (reverse). All prim ers were obtained from MWG Biotech (Ebersberg, Germany). The amplification conditions were 1 X 95°C for 15 m in followed by 45 X (94°C for 15 s, 58.5°C for 30 s, 72°C for 30 s). Analysis of quantitative PCR was perform ed according to the ACT m ethod as previously described [27], W e s te rn b lo t
Cells were incubated with dimethylsulfoxide (DMSO) (control), 5 pM U73122 or 5 pM U73343. After 3 h cells were lysed with RIPA buffer, supplem ented with 1 pL protease inhibitor cocktail set III (Calbiochem, Darmstadt, Germany), 10 pL phenylmethylsulfonyl fluoride (PMSF) (200 mM)
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M.Q. H uynh e ta l.
(Sigma-Aldrich), 10 (iL phosphatase inhibitor cocktail I and 10 uL phosphatase inhibitor cocktail II (both from SigmaAldrich). The protein concentration was m easured with the BCA™ Protein Assay Kit (Pierce, Rockford, IL), accordingto the m anufacturer's protocol. Cell lysates were electrophoresed on 10% sodium dodecyl sulfate (SDS)-polyacrylamide gels and transferred onto nitrocellulose m em branes (Bio-Rad, Richmond, CA). M em branes were blocked with 5% milk pow der in Tris-buffered saline (TBS; 20 mM Tris-HCl, pH 7.6,150 mM NaCl) and prim ary antibodies were incubated overnight at 4°C. Horseradish peroxidase (HRP) conjugated secondary antibodies were incubated for 1 h at room tem perature. Pro teins were detected with a C hem ilum inescence Detection Kit (Pierce Biotechnology, Rockford, USA). The following prim ary antibodies were used: rabbit anti-phospho-PLCG2 (Y1217), rabbit anti-phospho-p65 (S276), rabbit anti-p65, rabbit anti-phospho-AKT (Ser473), rabbit anti-AKT, rabbit anti-phospho-p44/42 (Thr202/Tyr204), rabbit anti-p44/42 (all from Cell Signaling), rabbit anti-phospho-PKCBI/II (Thr500) (Millipore), m ouse anti-PKCB (BD Biosciences Pharmingen, Heidelberg, Germany), rabbit anti-PLCG2, m ouse anti-PKC, m ouse anti-(3-actin and m ouse anti-a-tubulin (all from Santa Cruz Biotechnology). Goat m onoclonal anti-rabbit or goat m onoclonal anti-m ouse secondary antibodies were also obtained from Santa Cruz Biotechnology. Statistical analysis
All statistical analyses were perform ed with GraphPad Prism Version 5.0 for Windows (GraphPad Software, Inc., La Jolla, CA). Statistical significance was determ ined if p was < 0.05. Correlation of PLCy2 expression and DLBCL subtype (GCB
vs. non-GCB/ABC), age-adjusted international prognostic index (alPI), lactate dehydrogenase (LDH) level and treat m ent arm s was done using Fisher's exact test. Overall survival (OS) was calculated with the log-rank test. Cell proliferation of siRNA transfected lym phom a cells was calculated using an unpaired, two-tailed f-test. Results PLCy2 is s tro n g ly expressed in B cell n o n -H o d g k in ly m p h o m a a nd especially in a larg e subset o f DLBCL
We stained several entities of aggressive (DLBCL, n = 86; Burkitt lymphoma, n = 3; follicular lym phom a grade 3, n = 4) and indolent B cell non-Hodgkin lym phom a (B-NHL) (fol licular lym phom a grade 1, n = 6; gastric MALT lymphoma, n = 2; m antle cell lymphoma, n = 3) for PLCy2 expression. PLCy2 was strongly expressed in all cases of Burkitt lym phom a, follicular lym phom a grade 1 and 3, gastric MALT lym phom a and m antle cell lymphoma. Out of 86 cases of DLBCL, PLCy2 was strongly expressed in 54 (63%) cases and weak or no expression was seen in 32 cases (37%). Three DLBCL cell lines (U2932, SuDHL-4 and SuDHL-6) were also positive for PLCy2 expression [Figure 1(A)]. SuDHL-4 and SuDHL-6 are of germinal GCB subtype and U2932 of ABC subtype. Furtherm ore, we investigated w hether PLCy2 expression correlates with GCB and non-GCB/ABC subtype. To analyze this, we utilized the TMA m ethod. Fifty-five of the same 86 cases of DLBCL were available for TMA. According to the Hans algorithm, staining of CD10, Bcl-6 and Mum-1 was perform ed [29]. Twenty-four (44%) cases were of GCB subtype and 31 (56%) of non-GCB/ABC subtype. Of the 36 PLCy2
Figure 1. Expression of PLCy2 in lymphoma cells. (A) Immunohistochemistry and immunocytology of PLCy2: exemplary cases of B-NHL; all photographed at X 60 (except i: X 10). a and b: DLBCL with strong expression of PLCy2; c: case of DLBCL with weak PLCy2 expression; d-f: SuDHL-4 GCB subtype, SuDHL-6 GCB subtype, U2932 ABC subtype; g: Burkitt lymphoma; h: follicular lymphoma grade III; i: follicular lymphoma grade I; j: mantle cell lymphoma; k: gastric MALT lymphoma. (B) Correlation of PLCy2 expression with DLBCL subtype (tissue microarray; n = 55).
PLCy2 and diffuse large B cell lymphoma
positive lymphomas, 18 belonged to the GCB subtype and also 18 belonged to the non-GCB/ABC subtype. There was no significant correlation between PLCy2 expression (immunohistochemistry) and DLBCL subtype (TMA) [Fisher’s exact test p = 0.2564; Figure 1(B)]. Lymphoma proliferation is dependent on PLCy2 activation
In order to investigate the functional role of PLCy2 in lym phoma cells, we treated primary cells from patients who suffered from different B cell malignancies ( n = 8) and three DLBCL cell lines with U73122, a specific inhibitor of PLC, and its inactive analog U73343. Primary lymphoma cells of different lymphoma entities were collected from peripheral blood ( n = 6) or pleural effu sion [ n = 2). Reverse transcription (RT)-PCR and Western blot confirmed strong expression of PLCy2 in primary lym phoma cells (see Supplementary Figure 1 available online at http://informahealthcare.com/doi/abs/10.3109/10428194.201 4.941832). Compared to U73343 and untreated cells (DMSO control), inhibition of PLCy2 with U73122 resulted in reduced cell proliferation in all patient samples [Figure 2(A)], Additionally, we also observed reduced cell viability in all DLBCL cell lines (U2932, SuDHL-4 and SuDHL-6) after treatment with U73122, which occurred in a dose-dependent manner. U73343 did not reduce lymphoma proliferation
significantly [Figure 2(B)], To further exclude non-specific inhibition of cell proliferation with U73122, we treated the PLCyl positive Jurkat cell line (cells derived from a T cell leukemia), which expressed low PLCy2 with U73122 and U73343. PLCy2 mRNA in Jurkat cells was lower as compared to U2932, SuDHL-4 and SuDHL-6 [see Supplementary Figure 2(A) available online at http://informahealthcare.com/ doi/abs/10.3109/10428194.2014.941832]. Treatment of Jurkat cells with U73122 resulted in non-significant cell viability inhibition [Figure 2(B)]. In addition, no direct inhibition of PLCy2 and its targets were observed [see Supplementary Figure 2(B) available online at http://informahealthcare.com/ doi/abs/10.3109/10428194.2014.941832]. After 24 h treatment of lymphoma cell lines with U73122, apoptosis and the cell cycle were measured by flow cyto metric analysis. U73122 induced apoptosis [Figure 2(C)] and G0/G1 cell cycle arrest [Figure 2(D)] in lymphoma cells. Western blot analysis showed specific inhibition of phospho-PLCG2 and its downstream targets such as phospho-PKCB, p-p65 and p-AKT after treatment with U73122, but not with U73343. Phosphorylation of p44/42 was only inhibited in SuDHL-6 cells [Figure 3(A)], To confirm the specific effect of U73122 on lymphoma survival, DLBCL cell lines were then transfected with a specific siRNA against PLCy2. A non-specific siRNA was used as control. Cells were collected 48 h after transfection
(A)
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Jurkat [unclassified B-ccll lymphoma, perlph. blood|
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Figure 2. Lymphoma proliferation is dependent on PLCy2 signaling. (A) Trypan blue exclusion assay. Primary lymphoma cells were treated with or without U73122 and U73343. Cell proliferation was reduced with U73122. U73343 served as control. (B-D) DLBCL lines were treated with and without U73122 and U73343.U73122 decreased cell viability (B, MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)2H-tetrazolium] assay), induced apoposis (C, FITC-Annexin/PI assay) and G0/G1 cell cycle arrest (D, PI staining).
1092
M.Q. Huynh eta I. U2932
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Figure 3. Inhibition and knockdown of PLC72. (A) Western blot: cells were incubated with (1) DMSO, (2) 5 pM U73122 or (3) 5 pM U73343 for 3 h. Inhibition of phospho-PLCG2 and its targets phospho-PKCB, phospho-p65, phospho-AKT and phospho-p44/42 was shown with U73122, but not with U73343. (B) siRNA transfection: specific knockdown of Plcg2 with PLCy2 specific siRNA, but not with ns (non-specific) siRNA. (C) Significant decrease of cell viability with Plcg2 siRNA (MTS; p £ 0.05). Each experiment was done in triplicate and data represent mean value ± standard error (SE)(n = 3).
and cell proliferation was measured by trypan blue exclu sion assay. Western blots were performed to confirm spe cific knockdown of PLCy2 [Figure 3(B)], Transfection with a PLCy2 siRNA resulted in significant reduction of lymphoma viability; p < 0.05 [Figure 3(C)]. A d d itive e ffe c t on cell p ro liferatio n by seq u en tial in h ib itio n o f S rc-PLC y2-P K C p signaling p a th w a y
We compared treatm ent of U73122 with the Src kinase inhibitor pp2 and the PKC|3 inhibitor enzastaurin. We
Patient 5
Patient 6
could show in primary lymphoma cells that U73122 was, with respect to cell proliferation, at least as effective as pp2 and enzastaurin (Figure 4). Co-treatment of U73122 with pp2 or enzastaurin showed an additive effect on cell prolif eration in all DLBCL cell lines [Figures 5(A) and 5(B)], Flow cytometric analysis confirmed a specific effect of pp2 on Src and PLCy2 phosphorylation. U73122 and enzastaurin had an inhibitory effect on phospho-PKCB (see Supplementary Figure 3 available online at http://informahealthcare.com/ doi/abs/10.3109/10428194.2014.941832).
Patient 7
Patient 8
Figure 4. Treatment of primary lymphoma cells with U73122, pp2 and enzastaurin. Trypan blue exclusion assay: U73122 had at least similar effect on cell proliferation compared with pp2 and enzastaurin. In five out of eight patients [patient 2, 3, 4, 6 and 8) U73122 was more effective than pp2 and enzastaurin.
PLCy2 and diffuse large B cell lymphoma (A)
Co-treatment with pp2 and U73122
Trypan blue exclusion assay
(B )
Cell viability assay, MTS
Sequential inhibition with U73122 and Enzastaurin
Trypan blue exclusion assay
Cell viability assay, MTS
0 pM
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Figure 5. Additive effect of U73122 and other small molecule inhibitors on lymphoma survival. (A, B) Cell proliferation and viability. Co-treatment of U73122 with pp2 or enzastaurin showed additive effect on cell proliferation and viability.
Inverse correlation of PLCy2 expression with OS
In addition, we correlated PLCy2 expression with OS. Three-year survival data were available for all patients ( n = 86) and long term survival data (range from 6 to 143 months) were available for 71 out of 86 cases. DLBCL with strong PLCy2 expression showed a trend toward better OS after a 3-year follow-up (logrank test p = 0.0975). Furthermore, strong PLCy2 expression
months
correlated with significantly better survival after long-term fol low-up (log-rank test p = 0.0385) (Figure 6). There was no cor relation between PLCy2 expression and alPI (0-1 vs. 2-3), LDH level and treatment arm (arm A = standard chemotherapy; arm B = chemotherapy + autologous transplant) (see Supplemen tary Table I available online at http://informahealthcare.com/ doi/abs/10.3109/10428194.2014.941832).
PLCgamma2 high
months
PLCgamma2 low/neg.
Figure 6. PLCy2 expression and OS. PLCy2 expression was correlated with OS after 3-year (A) and long-term (range from 6 to 143 months) (B) follow up. Strong expression of PLCy2 was associated with better OS (3-year follow-up, p = 0.0975 and long-term follow-up, p = 0.0385).
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M .Q . H uynh e ta l.
D is c u s s io n
The outcome of patients suffering from B-cell lymphoma has been improved through the use of biological agents such as CD20-specific antibodies. The PLCy2-PKCP signaling cascade is essential for normal B cell development and, probably, B cell lymphoma survival. We found, in several lymphoma entities such as follicular lymphoma, gastric MALT lymphoma, mantle cell lymphoma, Burkitt lymphoma and DLBCL, strong expression of PLCy2 in immunohistochemical studies. These data suggest a crucial role for lymphoma survival and a potential new therapy target. Here, we have focused on the expression frequency of PLCy2 in DLBCL {n = 86 ) and its potential functional role for lymphoma survival. We could show via immunohistochemical staining that a large subset of primary DLBCL (63%) strongly expressed PLCy2, and only 37% showed weak or no expression level. Using TMA of the same cases (n = 55) we subclassified cases of DLBCL into GCB and non-GCB/ABC subtype, according to the Hans algorithm [29], However, we are aware that it has often been discussed whether the Hans classifier can reliably distinguish between GCB and non-GCB/ABC subtypes [30], In our study, 24 out of 55 cases (44%) were classified as GCB subtype and 31 cases (56%) were of non-GCB/ABC subtype. Our results were concordant with the published data of Hans etal. (42% GCB vs. 58% nonGCB/ABC) [29]. There was no significant correlation between the expression level of PLC72 (immunohistochemistry) and DLBCL subtype (TMA). We were able to show that inhibition or knockdown of PLCy2 killed primary lymphoma cells and DLBCL cell lines. We treated lymphoma cells with the specific PLC inhibitor U73122. U73122 induced apoptosis and cell cycle arrest in primary lymphoma cells, as well as lymphoma cell lines. Western blot analysis confirmed specific inhibition of PLCy2 and its downstream targets PKCfi, p65 and Akt. Desphosphorylation of p44/42 was seen only in SuDHL-6 cells, but was hardly detectable in SuDHL-4 and U2932. Supporting our data, Cheng et al. have recently shown that inactivation of PLCy2 and consequently Akt with the Syk inhibitor PRT318, but not p44/42, was associated with decreased DLBCL sur vival and increased cell cycle arrest [22]. Knockdown of PLCy2 with a specific siRNA showed the same effect on cell survival as with U73122. Furthermore, we compared the inhibitory effect of U73122 with pp2 (Src tyrosine kinase inhibitor) and enzastaurin (PKC[3) on lymphoma cells. Cell survival inhibi tion was similar between all three small molecule inhibitors in primary cells and DLBCL lines. However, co-treatment with U73122 and pp2 or U73122 and enzastaurin showed an additive effect on cell survival. PLCy2 is mainly activated by the BCR-Src-Btk signaling pathway. However, published data show high sensitivity for BCR inhibition only in ABC-DLBCL, but not in GCBDLBCL cells. We suggest that in lymphoma cells, other signaling pathways besides the BCR pathway are involved in PLCy2 activation, as it has been described that the B-cell activating factor receptor (BAFF-R) and fibroblast growth factor receptor (FGFR) are additionally involved in PLCy2 activation [2,31].
Altogether, PLCy2 is strongly expressed in a large number of cases of DLBCL, and lymphoma survival is dependent on PLCy2 signaling. Next, we investigated whether PLCy2 expression was associated with clinical treatm ent outcome. Strong expres sion of PLCy2 was associated with better OS. These data were unexpected, since the PLCy2-PKCp signaling cas cade activates pro-survival factors such as NFkB, Ras and Akt [11,14,15]. We speculate that lymphoma cells need PLCy2 for survival, whereas strong PLCy2 expression results in more cell proliferation and thus higher suscep tibility to cytotoxic drugs. In addition, PLCy2 expression was independent of the alPI or LDH level. An imbalance of treatm ent arms (arm A = standard chemotherapy; arm B = chemotherapy + autologous transplant) and PLCy2 expression could be ruled out. All patients were treated in a multicenter clinical study that was conducted in the prerituximab era [26], Therefore, a confirmatory analysis using patients treated with rituximab needs to be performed. In contrast to PLC72, it has been shown that patients with DLBCL with low PKCfill expression have better 3-year OS after immunochemotherapy [32], Targeting PLCy2 for lymphoma treatment is highly attractive, since it has been shown that the Btk inhibitor ibrutinib and the PKCP inhibi tor enzastaurin have promising anti-lymphoma activity [17,18,20]. To our knowledge, there is currently no PLCy2 inhibitor being investigated in clinical studies. In conclusion, our data show that the Src-PLCy2-PKCP signaling pathway has an important role for lymphoma sur vival. Treatment of patients with DLBCL could benefit from inhibition of PLCy2, at least in those cases with refractory or relapsed lymphoma after first-line immuno-chemotherapy. We found a significant inverse correlation of PLCy2 expression and DLBCL OS. These data have to be confirmed in further studies of patients treated with immuno-chemotherapy. A c k n o w le d g e m e n t s
The authors would like to thank the University Medi cal Center Giessen and Marburg for a research grant to M.Q.H., Deutsche Forschungsgemeinschaft (KFO 210, to A.N.), German Jose Carreras foundation (to A.N.) and the Dr. Reinfried Pohl foundation (to A.N.).
Potential conflict o f interest: Disclosure forms provided by the authors are available with the full text of this article at www.informahealthcare.com/lal.
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S u p p le m e n ta ry m a te ria l a v a ila b le o n lin e Supplementary Figures 1-3 and Table I showing further results.
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