B-Cell Expansion and Lymphomagenesis Induced by Chronic CD40 Signaling Is Strictly Dependent on CD19

Published OnlineFirst June 17, 2014; DOI: 10.1158/0008-5472.CAN-13-3274

Cancer Molecular and Cellular Pathobiology Research

B-cell Expansion and Lymphomagenesis Induced by Chronic CD40 Signaling Is Strictly Dependent on CD19

Caroline Hojer1, Samantha Frankenberger1, Lothar J. Strobl1, Samantha Feicht1, Kristina Djermanovic1, Franziska Jagdhuber1, Cornelia Homig-H€ olzel€ 1, Uta Ferch2,Jurgen€ Ruland2, Klaus Rajewsky3, and Ursula Zimber-Strobl1

Abstract CD40, a member of the TNF receptor family, is expressed on all mature B cells and on most B-cell lymphomas. Recently, we have shown that constitutive activation of CD40 signaling in B cells induced by a fusion protein consisting of the transmembrane part of the Epstein–Barr viral latent membrane protein 1 (LMP1) and the cytoplasmic part of CD40 (LMP1/CD40) drives B-cell lymphoma development in transgenic mice. Because LMP1/ CD40-expressing B cells showed an upregulation of CD19, we investigated CD19's function in CD40-driven B-cell expansion and lymphomagenesis. Here, we demonstrate that ablation of CD19 in LMP1/CD40 transgenic mice resulted in a severe loss and reduced lifespan of mature B cells and completely abrogated development of B-cell lymphoma. CD19 is localized to lipid rafts and constitutively activated by the LMP1/CD40 fusion protein in B cells. We provide evidence that the improved survival and malignant transformation of LMP1/CD40-expressing B cells are dependent on activation of the MAPK Erk that is mediated through CD19 in a PI3K-dependent manner. Our data suggest that constitutively active CD40 is dependent on CD19 to transmit survival and proliferation signals. Moreover, we detected a similarly functioning prosurvival pathway involving phosphorylated CD19 and PI3K- dependent Erk phosphorylation in human diffuse large B-cell lymphoma cell lines. Our data provide evidence that CD19 plays an important role in transmitting survival and proliferation signals downstream of CD40 and therefore might be an interesting therapeutic target for the treatment of lymphoma undergoing chronic CD40 signaling. Cancer Res; 74(16); 1–11. 2014 AACR.

Introduction (6). These data suggest an important function of CD40 in B-cell CD40, a member of the TNF -receptor (TNF-R) family, is lymphomagenesis. critically involved in germinal center formation and class The B-cell coreceptor CD19 is expressed from the early fi switch recombination during T-cell dependent (TD)-immune B-cell stages onwards. CD19-de cient mice display a severe responses (1). CD40 stimulation of B cells in vitro promotes B- reduction of marginal zone and B1 and a minor reduction of – cell activation, proliferation, and survival (2). CD40 is widely follicular B-cells (7 9). In the absence of CD19, germinal fi expressed on B-cell lymphomas, frequently along with the centers cannot be formed and antigen-speci c IgG titers are CD40 ligand (CD40L), resulting in constitutive engagement of strongly reduced underlining the essential role of CD19 in – CD40 (3, 4). In addition, mutations leading to constitutive T-cell dependent-immune responses (7 9). Upon B-cell recep- activation of CD40 have been observed in multiple myelomas tor (BCR) signaling, CD19 is known to directly interact with PI3K, Lyn, Grb2, Vav, and phospholipase C via phosphorylated (5). We have shown recently that constitutively active CD40 2þ signaling leads to B-cell activation and expansion and finally to tyrosine residues mediating signaling to Ca release and PI3K – fi B-cell lymphoma development in mice with high penetrance (10 16). Pten de ciency was shown to fully restore marginal zone B and B1 cell development as well as germinal center formation in CD19 knockout mice, corroborating a prominent 1Department of Gene Vectors, Helmholtz Zentrum Munchen,€ German role for PI3K in CD19 signaling (17). Research Center for Environment and Health (GmbH), Munich, Germany. 2Institute for Clinical Chemistry and Pathobiochemistry, Technical Univer- Because physiologically both CD19 and CD40 play a crucial sity of Munich, Munich, Germany. 3Immune Regulation and Cancer, Max- role during B-cell activation and T-cell dependent-immune Delbruck-Center€ for Molecular Medicine, Berlin-Buch, Germany. responses (1, 8, 18), it is particularly interesting whether CD19 Note: Supplementary data for this article are available at Cancer Research is functionally involved in the pathogenesis of human lym- Online (http://cancerres.aacrjournals.org/). phomas originating from aberrant chronic CD40 signaling (3). C. Hojer and S. Frankenberger contributed equally to this work. In the current study, we investigated the interplay of con- Corresponding Author: Ursula Zimber-Strobl, Helmholtz Zentrum Munchen,€ stitutive active CD40 signaling and CD19 in transmitting Marchioninistrasse 25, D-81377 Munich, Germany. Phone: 49-89-3187-1523; survival and proliferation signals. We show that deletion of Fax: 49-89-3187-4225; E-mail: [email protected] CD19 in LMP1/CD40 mice leads to a reduction of mature B-cell doi: 10.1158/0008-5472.CAN-13-3274 numbers and to the complete abrogation of B-cell lymphoma 2014 American Association for Cancer Research. development. We found that CD40 requires CD19 to activate

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PI3K and Erk signaling and that CD19 is phosphorylated in HCl, pH 7.5, 2 mmol/L EDTA, freshly added protease, and diffuse large B-cell lymphoma (DLBCL) cell lines, resulting in phosphatase inhibitors) and lysed by douncing. Lipid rafts PI3K-dependent Erk activation. Our data suggest that CD19 is were isolated by ultracentrifugation (SW41 rotor in a Beckman acting as a coreceptor not only for the BCR, but also for CD40 Ultracentrifuge, 41,000 rpm, 4C, 16 hours) in a sucrose gra- and mediates critical survival and proliferation signals. dient (42.5%, 35%, 5%). Fractions 1–11 were saved and tested for GM-1 by probing with horseradish peroxidase (HRP)-con- Materials and Methods jugated choleratoxin subunit B (CALBIOCHEM). Proteins were Mice then precipitated from the fractions by trichloracid, resus- Gt(ROSA)26Sortm2(Lmp1/CD40)Uzs (designated in the text as pended in 2 Laemmli buffer, denatured at 95 C for 5 minutes, fl LMP1/CD40stop ) is described in ref. 6 and Cd19tm1(cre)Cgn and subjected to Western blot analysis. (designated in the text as CD19-Cre) mice in ref. 19. Mice were analyzed at 8 to 16 weeks of age unless stated otherwise. Mice In vivo BrdUrd assay were bred and maintained in specific pathogen-free conditions Mice were fed with 0.8 mg/mL bromodeoxyuridine (BrdUrd; and experiments were performed in compliance with the Sigma) in the drinking water for 14 days. The water was German animal welfare law and have been approved by the exchanged every 2 to 3 days. Lymphocytes were isolated from institutional committee on animal experimentation. DLBCL the blood by Pancoll gradient centrifugation according to the cell lines used in this study are described in the figure legend of manufacturer's protocol (PAN). BrdUrd incorporation was Supplementary Fig. S5B. analyzed by the APC BrdU Flow Kit (BD Biosciences).

In vitro culture Statistical analysis þ Splenic B cells were purified by CD43 depletion by "MACS" Significance was calculated by the two-tailed student t test purification according to the manufacturer's protocol (Milte- (, P < 0.05; , P < 0.01; , P < 0.001). Each experiment was nyi) as described (6). CD40 stimulation was performed with an performed at least three times if not otherwise stated. agonistic CD40 antibody clone HM40–3 in a concentration of 1.25 to 2.5 mg/mL purchased from eBioscience. For Western blot analysis, MACS purified B cells or DLBCL- cell lines were Results left untreated or treated with inhibitors 1 hour before further CD19 is required for premalignant LMP1/CD40- procedures. Inhibitors: Ly294002, wortmannin, UO126, and mediated B-cell expansion in vivo dasatinib were purchased from Cell Signaling Technology, and Analysis of premalignant B cells from young LMP1/ fl sorafenib from Bayer HealthCare. The inhibitors were used in CD40stop //CD19-cre (LMP1/CD40) transgenic mice showed the following concentrations if not otherwise stated: Ly294002 that CD19 expression levels are elevated in comparison with 20 mmol/L, wortmannin 0.1 mmol/L, UO126 10 mmol/L, sor- resting control B cells. (Fig. 1A). To analyze whether CD19 afenib 10 mmol/L, and dasatinib 0.1 mmol/L. confers an advantage to LMP1/CD40-expressing B cells in vivo,LMP1/CD40micewerecrossedontoaCD19-deficient Flow cytometry background by generating homozygous CD19-Cre mice (19). Analyses were made with a FACSCalibur (BD Biosciences) as þ Splenomegaly, a hallmark of LMP1/CD40//CD19 / mice previously described (6). Results were analyzed using CELL- was abrogated in age-matched LMP1/CD40//CD19 / mice Quest or FlowJo software. Antibodies used to stain cells for (Fig. 1B). Calculation of total B-cell numbers in the spleen FACS analysis were all purchased from BD Biosciences (murine and inguinal lymph nodes (iLN) revealed significantly lower cells) or BioLegend (human cells). B-cell numbers in LMP1/CD40//CD19 / than LMP1/ þ/ Protein isolation and immunostaining CD40//CD19 mice (Fig. 1C), whereas developing B cells Western blotting with whole-cell extracts from purified B inthebonemarrowweresimilarlyrepresentedinboth / cells and IHC were performed as described recently (6). Anti- genotypes (Supplementary Fig. S1). LMP1/CD40//CD19 bodies for Western blot analyses: anti-p105/p50, anti-IkBa (sc- B cells still had a "blast-like" appearance and showed upregulation of B-cell activation markers such as CD95 and 371), anti-CD40 (C-20) antibodies were purchased from Santa þ Cruz Biotechnology, anti-pCD19 (Y513), anti-pIkB-a (S32/36), ICAM-1 (Fig. 1D). FACS analysis revealed that IgM and þ / anti-pErk1/2 (T202/Y204), anti-Erk1/2, anti-pJnk1/2 (T183/ IgD B cells in the spleen and iLN of LMP1/CD40//CD19 Y185), anti-Jnk1/2, anti-Akt, anti-pAkt (S473), anti-GSK3b, mice were strongly reduced in comparison with LMP1/ þ/ anti-pGSK3b (S21/9), anti-mTOR, anti-pmTOR (S2448), anti- CD40//CD19 mice (Fig. 2A). The strong shift to the hi low p70 anti-pp70 (T389), anti-Lyn, anti-pLyn (Y507), anti-Pten, marginal zone B-cell phenotype (CD21 and CD23 ), which anti-tubulin and anti-c-Myc from Cell Signaling Technology, is characteristic for LMP1/CD40-expressing mice, was lost in anti-GAPDH antibody from CALBIOCHEM. the absence of CD19 (Fig. 2B). Only some residual marginal fl Cryosections were processed, stained and analyzed as pre- zone B cells could be detected by ow-cytometric analysis, viously described (6, 20). but these cells did not localize to the marginal zone (Fig. 2C). CD19 deficiency in LMP1/CD40 mice strongly affects the Lipid raft extraction expansion of both follicular B and marginal zone B cells Purified B cells were incubated in Brij lysis buffer for 30 (Fig. 2D). We concluded from these experiments that CD19 minutes on ice (1% Brij, 150 mmol/L NaCl, 20 mmol/L Tris/ is strictly required for premalignant B-cell expansion in

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A B % of max Figure 1. LMP1/CD40-mediated 0 50 100 B-cell expansion in vivo is strictly 100 101 102 103 104 dependent on CD19. A, the CD19 CD19+/– CD19–/– LMP1/CD40// LMP1/CD40// histogram shows an overlay of CD19+/– CD19–/– CD19 surface expression of the indicated genotypes. The plot is gated on viable lymphocytes. The C 20 *** 100 indicated color code at the bottom ** ) )

– 7 5 80 is valid for A D. B, size of the spleen 15 12 52 of mice with indicated genotypes. 60 C, the graphs show total B-cell 10 numbers in the spleen (Sp) and 40 14 inguinal lymph nodes (iLN) of the 5 2.3 1.3 9.7 8.6

Cells (× 10 1.1 Cells (× 10 indicated genotypes. Mean values 20 and SDs are depicted that were 0 0 calculated from at least 12 Sp iLN independent experiments. D, histograms show an overlay of cell size (forward scatter) and surface D expression of ICAM-1 and CD95 from the indicated genotypes. The plots are gated on TO-PRO-3 , þ B220 cells. % of max 0 50 100 0 50 100 0 50 100 0 500 1,000 100 101 102 103 104 100 101 102 103 104 Forward scatter CD95 ICAM1 (CD54)

CD19+/– CD19–/– LMP1/CD40//CD19+/– LMP1/CD40//CD19–/–

the periphery in young LMP1/CD40 mice, but not for CD40- S2A). At days 1 and 2 of culture, cell numbers declined similarly induced expression of activation markers. after seeding of LMP1/CD40//CD19 / , CD19 / , and þ CD19 / B cells and were significantly lower than those of þ CD19 is indispensable for LMP1/CD40-induced B-cell LMP1/CD40//CD19 / B cells (Fig. 3B). Similarly the fraction of proliferation and survival TO-PRO-3 LMP1/CD40//CD19 / B cells declined rapidly in To determine the survival of LMP1/CD40//CD19 / Bcells the first days of culture, whereas after 3 to 5 days the percen- þ in vivo in comparison with LMP1/CD40//CD19 / cells, we tages of living cells increased again (Supplementary Fig. S2A), performed BrdUrd pulse-chase assays (21). At days 7 and 14 of suggesting that a small percentage of B cells showed enhanced þ the pulse period, CD19 / ,CD19/ , and LMP1/CD40// survival while the majority died within 3 days. þ þ CD19 / mice showed similar percentages of BrdUrd Bcells, Proliferation was measured by carboxyfluorescein diacetate whereas LMP1/CD40//CD19 / mice showed an increase of succinimidyl ester (CFSE) labeling. At day 4 of splenic B-cell about 3- and 2-fold in blood and spleen, respectively (Fig. 3A). culture, the percentage of proliferating LMP1/CD40//CD19 / þ This is indicative of a higher turnover of LMP1/CD40//CD19 / B cells averaged out at 3%, whereas LMP1/CD40//CD19 / B B cells compared with the other genotypes. The increased cells contained around 10% proliferating cells (Fig. 3C), sug- decline of BrdUrd-positive B cells in LMP1/CD40//CD19 / gesting that LMP1/CD40//CD19 / B cells show impaired mice during the chase from days 14 to 70 in blood and spleen proliferation and survival in vitro compared with LMP1/ þ (Fig. 3A) indicated a reduced lifespan of LMP1/CD40//CD19 / CD40//CD19 / cells. To test whether CD40 stimulation has þ B cells. In contrast, LMP1/CD40//CD19 / B cells showed a a comparable effect as LMP1/CD40 expression, we isolated þ survival advantage in comparison with control B cells as splenic B cells of wt, CD19 / , and CD19 / mice and stim- indicated by the higher number of BrdUrd-positive cells in the ulated them with aCD40 for 7 days. Without stimulation, cell blood and spleen at day 70. Thus, CD19 expression is required numbers declined comparably in the presence and absence of for LMP1/CD40-mediated B-cell survival and proliferation. CD19; however, with CD40 stimulation cell numbers were þ LMP1/CD40//CD19 / and control B cells were isolated from clearly lower in cultures of CD19 / than CD19 / and wt the spleen and cultured in vitro for 5 days without stimulation to B cells at days 3 to 7 (Fig. 3D). The lower cell numbers in analyze their survival in vitro (Fig. 3B and Supplementary Fig. cultures of CD19 / B cells resulted from reduced proliferation

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A IgM

49±5 40±11 64±6 28±12

13±3 15±5 35±20 6±5 iLN

Figure 2. CD19 loss in LMP1/CD40- expressing B cells leads to a IgD reduction of marginal zone B-cells +/– –/– CD19 CD19 LMP1/CD40// LMP1/CD40// and follicular B-cells. A, IgM and +/– –/– CD19 CD19 IgD expression on B cells isolated CD21 from the spleen and iLN of the B indicated genotypes. The plots are 5±2 2±1 30±4 9±4 gated on living lymphocytes. B, þ TO-PRO-3 , B220 lymphocytes 72±6 74±7 48±7 59±8 from the spleen were analyzed for marginal zone (CD21highCD23low) þ and follicular (CD21intCD23 )B CD23 cells (A and B). Numbers are mean C CD19+/– CD19–/– values and SDs of percentages of gated populations. C, IHC staining of splenic cryosections for MOMA- þ þ 1 macrophages (blue) and IgM Control B-cells (red). The marginal zone B cells are indicated by arrowheads. Red: α-IgM The scale is indicated by black bars (250 mm). D, total cell numbers of Blue: α-MOMA-1 follicular and marginal zone B cells. Numbers above the bars indicate LMP1/CD40 mean values that were calculated from at least ﬁve independent experiments.

D *** *** 100 53 100 37 CD19+/– ) )

6 16 6 12 10 *** 8.5 CD19–/– 10 1.2 1.3 1 0.4 LMP1/CD40//CD19+/– Cells (× 10 Cells (× 10 LMP1/CD40//CD19–/– 1 0.1 Igb wt Igb KO Igb wt // Igb KO // Igb wt Igb KO Igb wt // Igb KO FoBLC LC MZBLC // LC

(Fig. 3E) and reduced rescue from apoptosis in response to CD19 is required for LMP1/CD40-induced B-cell lymphoma CD40 stimulation (Supplementary Fig. S2B). These data point development. to an important role of CD19 in mediating survival and proliferation signals downstream of CD40. Next, we investi- Activation of MAPK Erk by constitutive CD40 signaling is gated whether deletion of CD19 in LMP1/CD40-expressing B dependent on CD19 expression cells abolishes B-cell lymphoma development. Twenty-two Constitutive CD40 signaling in B cells in vivo leads to þ LMP1/CD40//CD19 / and 22 LMP1/CD40//CD19 / mice selective activation of the noncanonical NF-kB pathway as were monitored for lymphoma development. Mice with well as Jnk and Erk (6). LMP1/CD40//CD19 / B-cells showed þ suspected disease were sacriﬁced and lymphoid organs were increased p52 levels as seen in LMP1/CD40//CD19 / but not þ analyzed for the presence of mono- or oligoclonal B-cell in CD19 / or CD19 / B cells (Fig. 4A). Strikingly, both LMP1/ lymphomas (Supplementary Fig. S3). A high percentage of CD40//CD19 / and CD19 / B cells showed a noticeable þ LMP1/CD40//CD19 / mice showed lymphoma develop- reduction of p100 levels, independent of their p52 levels. LMP1/ ment from the age of 11 months on, whereas none of the CD40-expressing B cells further exhibited a reduction of LMP1/CD40//CD19 / mice did (Fig. 3F). This suggests that pIkBa irrespective of their CD19 status. Erk phosphorylation

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60 A 60 * Blood *** Spleen * 40 40

20 20 * Figure 3. CD19 mediates improved * survival and enhanced proliferation downstream of CD40. A, pulse/ BrdUrd + B cells (%) chase BrdUrd experiment to 0 BrdUrd + B cells (%) 0 determine the survival of B cells 0 10203040506070Days 0 10203040506070Days þ in vivo. Percentages of BrdUrd þ Pulse Chase Pulse Chase B220 B cells in the blood and the spleen were determined by FACS at the indicated time points. The B C graphs show the mean values of 50 three independent experiments. B, LMP1/CD40//CD19+/– splenic B cells were cultured for 5 15 ) 10.1% days. Each day, cell numbers were 4 * * * LMP1/CD40//CD19–/– determined. A and B, the symbols 10 ** ** Counts corresponding to the four different 3.1% +/– genotypes are valid for A and B. CD19 0 1 2 3 4 Asterisks indicate significant 5 10 10 10 10 10 Cells (× 10 –/– differences between the CD19- CD19 CFSE proficient and -deficient LMP1/ 0 +/– CD40-expressing B cells. C, LMP1/CD40//CD19 012345 LMP1/CD40//CD19–/– splenic B cells were labeled with Days CFSE before culture. At day 4, the proliferation profile of TO-PRO-3 cells was determined by FACS. The D E numbers indicate the percentage 150 +/+ α of proliferating B cells. D, splenic B CD19 CD40 Day 4 36% cells were cultured for 7 days in the CD19+/– αCD40 absence and presence of an 100 agonistic CD40 antibody. E, each CD19–/– αCD40 Counts day, cell numbers were 26% CD19+/+ determined. Histograms showing 50 0 1 2 3 4 the proliferation profile of TO-PRO- (day 0 = 100) +/– 10 10 10 10 10 þ CD19 3 CFSE-labeled splenic CD19 / CFSE / –/–

Relative cell numbers CD19 and CD19 B cells after 4 or 7 0 days of culture in the presence of 012347 44% Day 7 an agonistic CD40 antibody. F, loss Days of CD19 abrogates lymphoma development in LMP1/CD40 mice. The graph shows the Counts 80 24% lymphoma incidence in LMP1/ F þ/ +/– CD40//CD19 and LMP1/CD40// 70 LMP1/CD40//CD19 (n = 22) 0 1 2 3 4 / 10 10 10 10 10 CD19 mice aged up to 80 60 LMP1/CD40//CD19–/– (n = 22) CFSE weeks. 50 CD19+/– αCD40 40 CD19–/– αCD40 30 20 10

Lymphoma incidence (%) 0 01020304050 60 70 80 Weeks

was impaired in LMP1/CD40//CD19 / compared with CD19 signaling is constitutively activated in LMP1/CD40- þ LMP1/CD40//CD19 / B cells, whereas the levels of pJnk expressing B cells were comparable (Fig. 4B). c-Myc levels were increased in Because we found that CD19 is required for LMP1/CD40- þ LMP1/CD40//CD19 / but not in LMP1/CD40//CD19 / B mediated activation of Erk, we explored whether CD19 is cells (Fig. 4C), which might be either due to the impaired constitutively activated in premalignant LMP1/CD40-expres- proliferation of LMP1/CD40 CD19-deﬁcient B cells or to the sing B cells. Upon antigenic triggering of the BCR, CD19 is decreased stability of c-Myc in the absence of CD19 as rep- recruited to lipid rafts (24) and phosphorylated on tyrosine orted previously (22, 23). residues Y513 and Y482 by Lyn. Upon binding to CD19, Lyn is

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Because CD19 is known to activate Akt, we also studied the –/+ activity of Akt and its downstream kinases. Akt phosphoryla-

–/–

9 91D tion at S473 was similar or slightly higher in LMP1/CD40 B cells 1DC//04DC þ than in control CD19 / B cells (Fig. 5C). CD19 deﬁciency led

/04DC/1PML to an impairment of Akt S473 phosphorylation in both LMP1/ CD40 and control B cells. Besides, we detected lower phos- b –/–

/ +/– phorylation of the downstream kinases GSK3 and pp70 after

1PML 9

1 deletion of CD19, whereas mTOR expression and phosphory-

D fi C CD19 lation were not affected by CD19 de ciency but was higher in 100 kDa p100 LMP1/CD40-expressing than in control B cells. Pten, a negative A regulator of Akt signaling, was strongly downregulated in 52 kDa p52 CD19-deficient B cells. κ α 34 kDa pI B To explore whether not only the LMP1/CD40 fusion pro- 34 kDa IκBα tein, but also exogenous CD40 signaling leads to the phos- ex vivo þ/ 53/56 kDa Lyn phorylation of CD19, we stimulated isolated CD19 BcellswithaCD40.Long-termCD40stimulationfor48 hours resulted in highly phosphorylated CD19 and simulta- 44 kDa B pErk1/2 neously in strong increases in Lyn and Erk phosphorylation 42 kDa suggesting that the activation of Erk is mediated through 44 kDa CD19 in the course of CD40 stimulation (Fig. 5D). To proof 42 kDa Erk1/2 that Lyn mediates CD19 phosphorylation, we treated the cells with dasatinib, an inhibitor of Lyn and other src family 54 kDa pJnk1/2 kinases (26). Interestingly, dasatinib did not only inhibit BCR 46 kDa signaling in murine B cells (Supplementary Fig. S4A), but also CD40-mediated CD19-, Erk-, and Lyn phosphorylation 54 kDa Jnk1/2 (Fig. 5E). These data suggest that CD40 signaling activates 46 kDa Lyn, which in turn phosphorylates CD19, resulting finally in the phosphorylation of Akt and Erk. C Because pCD19 recruits downstream effectors such as PI3K, 65 kDa c-Myc we investigated whether LMP1/CD40 mediates Akt and Erk phosphorylation through CD19 in a PI3K-dependent manner. 54 kDa Tubulin Titration of the two chemical inhibitors LY294002 and wortmannin revealed comparable inhibition of Akt and Erk phos- Figure 4. CD19 is required for CD40-mediated Erk but not NF-kB and Jnk phorylation already at very low concentrations in LMP1/CD40- activation in vivo. A, B, and C, Western blot analyses after staining with expressing B cells (Fig. 6A and B), whereas Jnk activation was the indicated antibodies. Equal protein loading was controlled by anti-Lyn not affected (Fig. 6B). To analyze whether Akt acts upstream of (A) and anti-tubulin (C) staining. Erk, we treated the cells with the Akt inhibitor AKTVIII. Similar to PI3K inhibition, Erk but not Jnk phosphorylation was þ thought to phosphorylate itself and thereby amplify the impaired upon treatment with AKTVIII in LMP1/CD40 B response (25). Indeed, Lyn was more highly phosphorylated cells (Fig. 6C). in LMP1/CD40-expressing B cells in comparison with con- To analyze whether PI3K inhibition impairs the survival of trol B cells, but the increased phosphorylation was abrogat- LMP1/CD40-expressing B cells, splenic B cells were cultured ed in CD19-deficient LMP1/CD40-expressing B cells (Fig. in vitro in the presence of the PI3K inhibitor LY294002 (Fig. 5A), suggesting a positive autoregulatory loop between 6D and Supplementary Fig. S4B). PI3K inhibition led to a constitutively active CD19 and pLyn. This is in accordance strong decline of cell survival of LMP1/CD40 B cells already with previous observations showing that Lyn phosphoryla- at day 1 of the B-cell culture. These data suggest that tion is significantly impaired in CD19-deficient B cells (12). sustained CD40 signaling in B cells uses CD19 to mediate Moreover, the phosphorylated form of CD19 was detected in survival signals in a PI3K-dependent manner. Summarizing lipid rafts from LMP1/CD40-expressing B cells but not from our results, we postulate that CD40 activates Lyn that control B cells and pLyn levels were higher in lipid rafts of subsequently phosphorylates CD19. Activated CD19 recruits LMP1/CD40 in comparison with control B cells (Fig. 5B). PI3K, resulting in Akt-Mek-Erk activation (Fig. 6E). After staining with a CD40 antibody, a band of 56 kDa was detected in the lipid raft fraction #2 of LMP1/CD40 but not CD19 is phosphorylated in DLBCL cell lines in control B cells, whereas the endogenous CD40 receptor Kloo and colleagues have shown that constitutive acti- (43 kDa) localized to the nonlipid raft fraction (Fig. 5B). vation of the PI3K signaling pathway is a common feature of These data suggest that pCD19 is constitutively activated ABC DLBCL cell lines (27). Therefore, we investigated the and recruited together with LMP1/CD40 and pLyn to lipid status of CD19 phosphorylation in DLBCL cell lines of the rafts. ABC (U2932, HBL1, TMD8 RIVA, Oci-Ly3, and Oci-Ly10) and

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–/– Lipid raft fractions +/– Non-raft

91DC/ A 91DC//04 B Ctrl LMP1/CD40 1 2 3 4 5 1 2 3 4 5 Ctrl L/C

/04DC/1P

56 kDa Δ

DC/1PML –/+

–/– 43 kDa CD40 Figure 5. The CD19 pathway is 91DC constitutively activated in LMP1/ ML 95 kDa pCD19 CD40 B cells. A, Western blot with CD19 53/56 kDa pLyn protein extracts of splenic B cells 53/56 kDa pLyn from the indicated genotypes 53/56 kDa Lyn stained with the indicated GM-1 antibodies. B, lipid rafts were isolated from LMP1/CD40// –/+ þ/ –/– αCD40 CD19 (LMP1/CD40) and C 91DC/ 91D þ D CD19 / (ctrl) B cells in ﬁve fractions (1–5) and analyzed by C//0 24 h 48 h 0.5 h 5 h / 0 h 1 h

Western blot analysis, which was 04DC/1PML

4D stained with the indicated –/+ 95 kDa pCD19

/1P

antibodies. Purity of the fractions –/– 91DC was controlled by GM-1 staining 44 kDa with choleratoxin-HRP on a dot ML pErk1/2 CD19 42 kDa blot. Nonlipid raft fractions (nonraft) pmTOR of control (ctrl) and LMP1/CD40- 289 kDa 44 kDa mTOR 42 kDa Erk1/2 expressing B cells (L/C) are shown. pAkt (S473) 60 kDa The LMP1/CD40 fusion protein Akt 53/56 kDa pLyn usually detected at 34 kDa was pp70 70 kDa detected in lipid raft preparations p70 56 kDa Lyn as a 56 kDa band. This might be pGSK3β 51 kDa due to a dimeric or oligomeric form GSK3β of LMP1/CD40, which is also 55 kDa PTEN 36 kDa GAPDH detected after overexpression of 54 kDa Tubulin LMP1/CD40 in cell lines (data not shown). C, Western blot analyses αCD40 with protein extracts of the E indicated genotypes were stained with antibodies against Akt and Akt þ target genes. D, CD19 / splenic B cells were stimulated with an agonistic CD40 antibody (aCD40) 0 h 48 h for the indicated time points. 48 h DMSO 48 h Dasatinib Western blot analyses with protein extracts of the stimulated B cells 95 kDa pCD19 were stained with the indicated antibodies. GAPDH was used as loading control. E, Western blot 44 kDa pErk1/2 analyses with cell extracts of 42 kDa þ splenic CD19 / B cells cultured in the absence or presence of aCD40 44 kDa Erk1/2 without (DMSO) or with dasatinib. 42 kDa 53/56 kDa pLyn 56 kDa Lyn

60 kDa pAkt

36 kDa GAPDH

GCB subtypes (SU-DHL-6, SU-DHL-4, and BJAB). As con- chose HBL1 and U2932, to analyze whether similar to LMP1/ trols, we used two Hodgkin lymphoma cell lines (KM-H2 and CD40-expressing B cells, Erk is activated through CD19 in a L428), which are CD19 negative. We detected phosphory- PI3K-dependent manner in DLBCL cell lines. The PI3K lated CD19 in several DLBCL cell lines of the ABC and GCB inhibitor LY294002 impaired Erk and Akt but not Jnk subtype (Fig. 7A). Total CD19 levels varied between different phosphorylation in both cell lines (Fig. 7B). The Raf inhib- cell lines being in tendency lower in ABC than in GCB- itor, sorafenib, was not able to reduce Erk phosphorylation, DLBCL cell lines (Fig. 7A and Supplementary Fig. S5B). We similar as in LMP1/CD40-expressing B cells (Supplementary

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LMP1/CD40

A Ctrl B Wortmannin LY294002 Figure 6. Erk phosphorylation is μ μ mol/L mol/L LMP1/CD40______mediated through PI3K signaling in LMP1/CD40-expressing B cells. A, Ctrl LY______Wt Western blot analysis with protein

5210.

OSMD 520.0 0 0 20 0.1 μmol/L extracts derived from LMP1/

*1.0

*0 þ

o o/w /

2.0 5.

.0 01 44 kDa CD40//CD19 B cells treated for

2 5 0 pErk1/2 42 kDa one hour without (w/o) or with 44 kDa pErk1/2 44 kDa PI3K-inhibitors LY294002 or 42 kDa Erk1/2 42 kDa wortmannin in different 44 kDa Erk1/2 54 kDa concentrations as indicated. 42 kDa pJnk1/2 þ/ 46 kDa Untreated CD19 B cells (ctrl) 60 kDa pAkt 54 kDa and DMSO-treated LMP1/CD40 Jnk1/2 cells (DMSO) served as controls. 60 kDa Akt 46 kDa Erk and Akt served as loading controls. B, protein extracts Ctrl LMP1/CD40 derived from LMP1/CD40// þ C D CD19 / (LMP1/CD40) B cells that

AKTVIII AKTVIII 70 LMP1/CD40 + DMSO were treated for 1 hour without (0)

o o/w / μ LMP1/CD40 + LY or with the PI3K inhibitors w 1 2.5 1 2.5 mol/L 60 LY294002 (LY) and wortmannin 44 kDa +/– pErk1/2 CD19 + DMSO 42 kDa 50 (Wt) were investigated by immunoblot analysis. Untreated (0) 44 kDa þ/ Erk1/2 40 * CD19 B cells served as control 42 kDa * (ctrl). Jnk and Erk served as loading 30 control. C, protein extracts derived 54 kDa þ pJnk1/2 from control CD19 / (ctrl) and þ 46 kDa 20 LMP1/CD40//CD19 / (LMP1/ CD40) B cells that were treated for 54 kDa Jnk1/2 10 1 hour without (w/o) or with the Akt 46 kDa % of TO-PRO-3–negative cells inhibitor AKTVIII were investigated 0 by Western blot analysis and 54 kDa 012345 Tubulin Days probed with the indicated antibodies. Tubulin was used as loading control. D, splenic B cells LMP1/CD40 CD19 from LMP1/CD40 mice were E cultured for up to 5 days with the PI3K inhibitor LY294002 (LY). As

JAK3 Lyn Lyn Vav control, B cells from LMP1/CD40// þ/ TRAF6 LY294002 CD19 (LMP1/CD40) and control PI3K þ/ Wortmannin mice (CD19 ) were cultured in the presence of DMSO. Mean Dasatinib percentages of TO-PRO-3 cells TRAF1 and SD are shown at the indicated Akt AKT VIII TRAF3 time points. E, schematic depiction TRAF2 of the signaling pathway activated TRAF5 in LMP1/CD40-expressing B cells. Mek1 UO126 Our data suggest that LMP1/CD40 activates Lyn that in turn phosphorylates CD19. Erk1/2 Phosphorylated CD19 recruits PI3K, leading to the activation of Akt, Mek, and Erk. Survival Jnk1/2 RelB p52/p50 proliferation

Fig. S5A). CD19 phosphorylation as well as Erk and Lyn dasatinib almost completely removes CD19 phosphoryla- phosphorylation was abrogated in the presence of dasatinib tion, we used this drug to determine the effect of CD19 in all cell lines tested, whereas sorafenib increased Erk phosphorylation on the survival of DLBCL cell lines. Except activation (Fig. 7C). In contrast PI3K and Mek inhibition of two cell lines (SU-DHL-4 and Oci-Ly3), all cell lines were resulted in decreased Erk but not CD19 and Lyn phosphor- sensitive to the treatment with dasatinib and displayed a ylation, indicating that PI3K and Lyn are acting upstream of reduced survival in comparison to untreated control DLBCL þ Mek. These data suggest that like in LMP1/CD40 B cells, cell lines (Fig. 7D). Of note, in the group of dasatinib- active Lyn phosphorylates CD19, leading to Erk activation in sensitive cell lines, those cell lines displaying lower levels a PI3K-dependent manner. After having established that of constitutive Erk phosphorylation were more sensitive for

OF8 Cancer Res; 74(16) August 15, 2014 Cancer Research

CD19 in Chronically CD40-Activated B Cells

A GCB- ABC- B DLBCL DLBCL HL HBL-1 U2932

200492YL 200

binefaroS

621OU 621OU

492YL SU-DHL-6 TMD8 Oci-Ly3 SU-DHL-4 BJAB HBL1 RIVA Oci-Ly10 U2932 KM-H2 L428

o/w

100 kDa pCD19 w pErk1/2 36 kDa GAPDH Erk1/2

100 kDa CD19 pAkt

Figure 7. In DLBCL cell lines, CD19 Akt is phosphorylated by Lyn and mediates Erk activation in a PI3K- 42/44 kDa pErk1/2 pJnk1/2 dependent manner. A, Western blot analysis of DLBCL cell lines as Jnk1/2 well as two Hodgkin lymphoma cell 42/44 kDa Erk1/2 lines (HL) were stained with the Tubulin indicated antibodies. GAPDH was used as loading control. B and C, 36 kDa GAPDH protein extracts were prepared from the indicated DLBCL cell lines. Before protein extraction, SU-DHL-6 TMD8 BJAB RIVA cells were incubated for 1 hour C without inhibitor (w/o), with DMSO, or with the PI3K inhibitor LY294002, the Mek1/2-inhibitor Sorafenib Sorafenib DMSO LY294002 UO126 DMSO LY294002 UO126 Dasatinib Dasatinib Sorafenib Sorafenib DMSO LY294002 UO126 DMSO LY294002 UO126 Dasatinib UO126, Raf inhibitor sorafenib, or Dasatinib Lyn inhibitor dasatinib. Western 100 kDa pCD19 blot analyses were performed and stained with the indicated 53/56 kDa pLyn antibodies. Tubulin (B) or GAPDH (C) served as loading control. D, 53/56 kDa Lyn DLBCL cell lines were treated with the indicated concentrations of pErk1/2 dasatinib. The graph shows the 42/44 kDa relative percentage of TO-PRO-3 42/44 kDa Erk1/2 cells of DLBCL cell lines treated with dasatinib for 3 days in 36 kDa GAPDH comparison with the untreated cell lines. 125 D SU-DHL-4

100 Oci-Ly3 BJAB 75 U2932 HBL1 50 RIVA % Living cells TMD8 (untreated = 100%) 25 Oci-Ly10 SU-DHL-6 0 0 25 50 100 200 400 nmol/L Dasatinib

dasatinib treatment. These data suggest that CD19/PI3K- stream of the BCR (28). Because CD19 was highly expressed on dependent Erk activation is a prosurvival signaling pathway the surface of LMP1/CD40-expressing B cells, we investigated in DLBCL cell lines. whether their improved survival depends on CD19. We found that ablation of CD19 has a severe effect on the survival and proliferation as well as lymphomagenesis of LMP1/CD40- Discussion expressing B cells. The reduced proliferation and increased CD19 has been shown to directly interact with, and to apoptosis of CD19 deﬁcient in comparison with control B cells activate PI3K (11, 13, 14), the critical survival signal down- in the presence of CD40 stimulation underline our assumption

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Hojer et al.

that CD19 transmits proliferation and survival signals down- localizedinlipidraftsofNHL-B.Therefore,weanalyzed stream of CD40. CD40 and CD40-L expression in the DLBCL cell lines. All The failure of LMP1/CD40 to activate Erk in the absence DLBCL cell lines expressed CD40 (Supplementary Fig S6A), of CD19 might be the reason for this phenotype, since we but expression of CD40-L was either extremely low or showed recently that Erk is crucial for the survival advan- undetectable (data not shown). Thus, it is unlikely that tage of LMP1/CD40-expressing B cells (6). We show that CD19 phosphorylation is mediated by CD40-CD40L auto- Y513-phosphorylated CD19 is recruited together with stimulation in DLBCL cell lines, but CD40 auto-aggregation LMP1/CD40 and pLyn into lipid rafts, suggesting its con- might be responsible for CD19 phosphorylation in DLBCL stitutive activation (24). Our data suggest that CD40- cell lines. Probably, CD19 phosphorylation in DLBCL cell induced CD19 phosphorylation is mediated by the kinase lines does not completely reflect CD19 phosphorylation in Lyn because (i) Lyn is hyperphosphorylated in LMP1/CD40- situ. Most DLBCL cell lines express high levels of CD40 expressing B cells; (ii) CD40 stimulation of B cells results (Supplementary Fig. S6A). It is therefore tempting to spec- in the activation of Lyn and CD19; and (iii) CD19 phoshory- ulate that in vivo lymphoma cells constantly receive CD40 lation is inhibited by the chemical Lyn inhibitor dasatinib. signals by lymphoma-infiltrating CD40-L–expressing cells We observed a strong downregulation of Pten in CD19- such as activated T cells or myeloid cells, which might deficient B cells, which might reflect a rescue mechanism of increase CD19 phosphorylation. Increased CD19 phosphor- CD19-deficient B cells, to maintain some basal PI3K activity. ylation after CD40 stimulation of BJAB cells is underlining Our data suggest that CD19 is acting not only as a cor- this hypothesis (Supplementary Fig. S6B). Collectively, our eceptor for the BCR, but also for CD40. We postulate that data suggest an important role of CD19 in enhancing and phosphorylated CD19 activates PI3K and subsequently Akt, sustaining chronic B-cell activation, survival, and prolifera- Mek, and Erk. We assume that BCR signaling is not involved tion. Because aberrant chronic activation of both BCR and in this activation process, because genetic truncation of the CD40 signaling can contribute to B-cell lymphomagenesis signaling tail of Igb does not reduce the constitutive Erk (3, 6, 30, 31), CD19 may emerge as a critical player in this activation in LMP1/CD40-expressing B cells (own unpub- process. The important role of CD19 in lymphoma progres- lished results). Akt might not directly activate Mek but sion is underlined by the observations that in the absence of rather uses additional signaling mediators for the activation CD19, lymphoma development is completely abrogated in of Mek and Erk. Because sorafenib treatment rather resulted LMP1/CD40 mice and delayed in c-mycTg mice (23). Our in increased Erk activation, it is unlikely that Raf kinases are data provide evidence that molecules interfering with CD19 involved in activation of this signaling pathway. It might be signaling might be valuable new tools in the treatment of B- that PI3K/Akt is acting through the IKK complex to activate cell lymphomas undergoing chronic BCR or CD40 signaling. Mek and Erk because links between PI3K and NF-kBaswell as NF-kB and MAPK-signaling have already been described Disclosure of Potential Conflicts of Interest (27, 29). No potential conflicts of interest were disclosed. CD19 is phosphorylated in several DLBCL cell lines of the ABC as well as GCB subtype. Like in LMP1/CD40-expressing Authors' Contributions B cells, the phosphorylation of Erk is dependent on Lyn, Conception and design: C. Hojer, S. Frankenberger, S. Feicht, J. Ruland, U. Zimber-Strobl PI3K, and Mek activity in DLBCL cell lines. Abrogation of Development of methodology: S. Frankenberger, S. Feicht, U. Ferch Lyn and downstream CD19 phosphorylation by dasatinib Acquisition of data (provided animals, acquired and managed patients, resulted in impaired survival of most DLBCL cell lines, provided facilities, etc.): C. Hojer, K. Djermanovic, F. Jagdhuber, C. Homig-€ Holzel,€ U. Zimber-Strobl suggesting that Lyn phosphorylation mediates through Analysis and interpretation of data (e.g., statistical analysis, biostatistics, CD19 an important survival signal also in DLBCL cell lines. computational analysis): C. Hojer, S. Frankenberger, L.J. Strobl, S. Feicht, Only two cell lines, Oci-Ly3 and SU-DHL-4, were resistant to K. Djermanovic, U. Ferch, J. Ruland, U. Zimber-Strobl Writing, review, and/or revision of the manuscript: C. Hojer, S. Franken- dasatinib treatment. One of these cell lines, Oci-Ly3 carries a berger, L.J. Strobl, J. Ruland, K. Rajewsky, U. Zimber-Strobl constitutive CARD11 mutation, the other cell line, SU-DHL- Study supervision: U. Zimber-Strobl 4, an IgH-bcl2 translocation leading to the overexpression of Bcl2 (30). These mutations might circumvent the dependen- Acknowledgments cy on CD19 phosphorylation. We could not provide the final The authors thank Gabriele Marschall-Schroter€ for excellent technical assis- tance and Daniel Krappmann and Gerhard Laux for providing the DLBCL cell proof that similar like in LMP1/CD40-expressing cells, CD19 lines. phosphorylation is mediated through CD40 signaling in DLBCL cell lines. Both chronic BCR and CD40 signaling Grant Support might lead to CD19 phosphorylation in DLBCL cell lines. This work was supported by grants from the Deutsche Krebshilfe (109131) Chronic BCR signaling has been described in the ABC and Sander Stiftung (2011.076.1; U. Zimber-Strobl) and from Deutsche For- DLBCL cell lines HBL1, TMD8, and Oci-Ly10 (30). Evidence schungsgemeinschaft (SFB684, TRR54; J. Ruland and U. Zimber-Strobl). The costs of publication of this article were defrayed in part by the for chronic CD40 signaling in some aggressive B-cell lineage payment of page charges. This article must therefore be hereby marked non-Hodgkin's lymphomas (NHL-B) has been provided by advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this Pham and colleagues (3). They described a "CD40 signalo- fact. some," consisting of CD40, CD40L, TRAF2, and TRAF6 as Received November 19, 2013; revised March 24, 2014; accepted April 15, 2014; well as components of the NF-kB signaling pathway that are published OnlineFirst June 17, 2014.

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www.aacrjournals.org Cancer Res; 74(16) August 15, 2014 OF11

B-cell Expansion and Lymphomagenesis Induced by Chronic CD40 Signaling Is Strictly Dependent on CD19

Caroline Hojer, Samantha Frankenberger, Lothar J. Strobl, et al.

Cancer Res Published OnlineFirst June 17, 2014.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-13-3274

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