A c-Myc and Surface CD19 Signaling Amplification Loop Promotes Lymphoma Development and Progression in Mice This information is current as of September 30, 2021. Jonathan C. Poe, Veronique Minard-Colin, Evgueni I. Kountikov, Karen M. Haas and Thomas F. Tedder J Immunol published online 23 July 2012 http://www.jimmunol.org/content/early/2012/07/23/jimmun ol.1201000 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published July 23, 2012, doi:10.4049/jimmunol.1201000 The Journal of Immunology

A c-Myc and Surface CD19 Signaling Amplification Loop Promotes B Cell Lymphoma Development and Progression in Mice

Jonathan C. Poe, Veronique Minard-Colin, Evgueni I. Kountikov, Karen M. Haas, and Thomas F. Tedder

Malignant B cells responding to external stimuli are likely to gain a growth advantage in vivo. These cells may therefore maintain surface CD19 expression to amplify transmembrane signals and promote their expansion and survival. To determine whether CD19 expression influences this process, Em-Myc transgenic (c-MycTg) mice that develop aggressive and lethal B cell lymphomas were made CD19 deficient (c-MycTgCD192/2). Compared with c-MycTg and c-MycTgCD19+/2 littermates, the median life span of Tg 2/2 Tg 2/2 Tg c-Myc CD19 mice was prolonged by 81–83% (p < 0.0001). c-Myc CD19 mice also lived 42% longer than c-Myc Downloaded from littermates following lymphoma detection (p < 0.01). Tumor cells in c-MycTg and c-MycTgCD192/2 mice were B lineage derived, had a similar phenotype with a large blastlike appearance, invaded multiple lymphoid tissues, and were lethal when adoptively transferred into normal recipient mice. Importantly, reduced lymphomagenesis in c-MycTgCD192/2 mice was not due to reduc- tions in early B cell numbers prior to disease onset. In mechanistic studies, constitutive c-Myc expression enhanced CD19 expression and phosphorylation on active sites. Reciprocally, CD19 expression in c-MycTg B cells enhanced c-Myc phosphoryla-

tion at regulatory sites, sustained higher c-Myc protein levels, and maintained a balance of cyclin D2 expression over that of cyclin http://www.jimmunol.org/ D3. These findings define a new and novel c-Myc:CD19 regulatory loop that positively influences B cell transformation and lymphoma progression. The Journal of Immunology, 2012, 189: 000–000.

D19 is a B cell-specific transmembrane glycoprotein that increases in CD19 expression by 15–29% induce autoantibody by virtue of its extensive cytoplasmic domain establishes production in mice (21). Thus, strict regulation of CD19 expres- C intrinsic B cell signaling thresholds and regulates BCR- sion is important for normal B cell signal transduction, develop- dependent and BCR-independent signal transduction pathways ment, and function.

(1). CD19 is a central signaling component of cell surface com- As with normal B cells, malignant B cells responding vigorously by guest on September 30, 2021 plexes that can include CD21, CD81, and CD225 (2-5). Because to external and internal stimuli may gain survival and growth CD21 is a receptor for C3 complement fragments, CD19 also links advantages that promote lymphoma development, survival, and the innate and adaptive arms of the immune system (6, 7). CD19 is proliferation (22). CD19 is expressed on nearly all malignancies of expressed on the cell surface from the late pro-B to early pre- B cell origin, which includes 80% of acute lymphoblastic leuke- B cell stages, with increasing expression as B cells mature in mias, 88% of B cell lymphomas, and 100% of B cell leukemias (8, humans (8–11) and in mice (12–14). CD19 surface density is 11). CD19 expression is also retained at relatively high levels on similar on mature conventional B cells from different peripheral the majority of B cell tumors, including most pre-B, common, and lymphoid tissues, and B cell activation by anti-IgM Abs, LPS, or null-acute lymphoblastic leukemias, non-Hodgkin’s lymphomas, IL-4 does not significantly alter CD19 expression (12, 15). B cell chronic lymphocytic leukemias, prolymphocytic leukemias, Humans genetically deficient for CD19 expression and gene- and hairy cell leukemias (8, 9, 11, 23). However, whether CD19 2/2 targeted (CD19 ) mice have B cells that are hyporesponsive expression itself contributes to malignant B cell transformation or to transmembrane signals and generate weak -dependent growth in vivo is unknown. humoral immune responses (16–18). By contrast, transgenic Because of its ubiquitous expression in B cell malignancies and mice that overexpress CD19 have hyperresponsive B cells and importance in establishing B cell signaling thresholds, the con- develop autoantibodies with age (12, 13, 17, 19, 20). Even modest tribution of CD19 expression to the malignant transformation of B cells and severity of lymphomas was investigated in Em-Myc Tg Department of Immunology, Duke University Medical Center, Durham, NC 27710 transgenic (c-Myc ) mice (24–26). c-Myc is one of the most Received for publication April 4, 2012. Accepted for publication June 27, 2012. frequently dysregulated proteins in human malignancies. Burkitt’s This work was supported by National Institutes of Health Grants AI56363 and lymphoma and diffuse large B cell lymphomas in humans are AI057157, a grant from the Lymphoma Research Foundation (to T.F.T.), an Arm- characterized by translocations between Ig gene promoter regions strong Family Fellowship in Cancer Research from the Duke Comprehensive Cancer and the MYC proto-oncogene, leading to aberrant c-Myc over- Center (to J.C.P.), and a Special Fellow Award from the Leukemia and Lymphoma Tg Society (to K.M.H.). expression. Similarly, c-Myc mice, in which the Myc proto- Address correspondence and reprint requests to Prof. Thomas F. Tedder, Duke Uni- oncogene is under the control of the IgH enhancer, develop ag- versity, Box 3010 Medical Center, 207 Research Drive, Jones Building, Room 353, gressive B cell-derived lymphomas at an early age, and have Durham, NC 27710. E-mail address: [email protected] a ∼90% mortality rate by 20 wk of age, with a median age of Abbreviations used in this article: Cdk4, cyclin-dependent kinase 4; FSC, forward death at ∼12 wk (24, 25). c-MycTg lymphomas develop from the scatter; HSA, heat-stable Ag; WT, wild-type. B220low pre-B and immature B cell pools, and Ig gene rear- Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 rangement analyses indicate that most are monoclonal (24). Rare

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1201000 2 c-Myc AND CD19 COREGULATE B CELL LYMPHOMA DEVELOPMENT genetic events coupled with c-Myc overexpression are thus re- were assessed for the percentage of BrdU-positive B cells. Briefly, cells quired for malignant B cell transformation (25). Using c-MycTg were fixed in 0.5% paraformaldehyde, permeabilized with 3 N HCl con- mice deficient in CD19 expression, a c-Myc:CD19 regulatory loop taining 0.5% Tween 20, neutralized with 0.1 M disodium tetraborate, and stained with FITC-conjugated anti-BrdU Ab (BD Biosciences) and PE- was identified in the current study that positively influences B cell conjugated anti-mouse B220 Ab, followed by flow cytometry analysis. transformation and lymphoma progression. Analysis of protein phosphorylation and expression levels B cells were isolated from single-cell suspensions of splenocytes from disease- Materials and Methods free c-MycTg and c-MycTgCD192/2 mice, or from CD19+/+ and/or CD192/2 Mouse breeding and survival analysis littermatestoapurityof$95% by removal of T cells and NK cells with Tg Thy1.2 magnetic beads, and any residual monocytic cells by plastic adher- c-Myc [C57BL/6J-TgN(IghMyc)22Bri/J] hemizygous mice were from The 3 6 Tg ence. A total of 5 10 B cells was lysed in a buffer containing 1% Nonidet Jackson Laboratory. c-Myc mice were crossed with CD19-deficient P-40, 150 mM NaCl, 50 mM Tris-HCl (pH 8.0), 1 mM Na orthovanadate, 2 mice (17), and F1 generation mice were interbred to obtain CD19 wild-type mM EDTA, 50 mM NaF, and 1 mM PMSF, with solubilized proteins sep- (WT), heterozygous, and deficient littermates hemizygous for the c-Myc Tg Tg +/2 Tg 2/2 arated on 10% SDS-PAGE gels, followed by transfer to nitrocellulose transgene (c-Myc ,c-Myc CD19 ,andc-Myc CD19 , respectively). membranes. Immunoblotting was performed with Abs specific for CD19 c-Myc transgene presence was determined by PCR, as follows: forward phosphorylated at regulatory tyrosines on the cytoplasmic tail, or for c-Myc primer, 59-CAGCTGGCGTAATAGCGAAGAG-39; reverse primer, 59- 9 and Pax5 proteins. Phospho-CD19 blots were then stripped and reprobed CTGTGACTGGTGAGTACTCAACC-3 , according to The Jackson Labo- with Abs reactive with CD19 protein. For analysis of ERK and JNK acti- ratory genotyping protocols. c-MycTg,c-MycTgCD19+/2,andc-MycTg 2/2 vation, immunoblotting was performed with Abs specific for the kinase- CD19 littermates were monitored daily for the first appearance of active (dually phosphorylated) forms of ERK or JNK, followed by reprobing lymphadenopathy, usually characterized by swelling of the cervical and with Abs reactive with ERK2 protein. c-Myc phosphorylation was assessed brachial lymph nodes resulting in the characteristic “water wings” ap- Downloaded from Tg by immunoblotting using Abs reactive with c-Myc when phosphorylated at pearance described for c-Myc mice (25). Mice were also monitored for T58/S62. The membranes were then reprobed with Abs reactive with c-Myc the rate of tumor progression and for mortality. Notably, cachexia requiring and ERK2 proteins. D-type cyclin and Cdk4 expression was assessed by euthanasia developed in some mice without detectable lymphadenopathy. immunoblotting with Abs specific for these proteins, followed by reprobing These animals were thus grouped into a survival, but not observable lym- with Abs for ERK2. Densitometry analysis of protein bands in immunoblots phoma, category. As required by the guidelines of the Institutional Animal was performed using NIH Image software version 1.60. Care and Use Committee of Duke University, mice were euthanized if a pre- determined level of distress was reached prior to natural death. For these mice, cDNA gene arrays http://www.jimmunol.org/ the date of euthanasia was used as the date of death in survival studies. Comparative statistical analysis of survival was performed by the log-rank test. GEArray Q Series mouse gene expression arrays specific for genes in the Kaplan–Meier survival plots were generated using the GraphPad Prism soft- cell cycle pathway were from SuperArray (Bethesda, MD). Purified splenic 7 Tg +/+ ware package. For adoptive transfer experiments, B220+ cells from c-MycTg or B cells (1 3 10 ) were isolated from disease-free Myc CD19 and Tg 2/2 c-MycTgCD192/2 mice were injected i.p. in sterile DPBS into BALB/c scid c-Myc CD19 littermates, and total RNA was isolated using a RNeasy (SCID) recipient mice (The Jackson Laboratory), with the survival of re- kit (Qiagen, Valencia, CA). RNA (2.5 mg) was then subjected to cDNA cipient mice assessed, as described above. All procedures were approved synthesis and hybridization to the arrays, according to the manufacturer’s by the Duke University Institutional Animal Care and Use Committee. instructions. Reagents used for cDNA synthesis not provided in the kits included [32P]dCTP (ICN) and SuperScript II reverse transcriptase (Invi- Reagents and Abs trogen, Carlsbad, CA). cDNA probe hybridization was analyzed using

a phosphorimager. Gene densitometry analysis was performed using the by guest on September 30, 2021 FITC-, PE-, or PE-Cy5–conjugated rat anti-mouse Abs reactive with CD19, ScanAlyze program, version 2.5 (M. Eisen, Stanford University). B220, heat-stable Ag (HSA), CD43, and CD5 were from BD Pharmingen (San Diego, CA). FITC-conjugated goat anti-mouse Ab reactive with IgM was from Southern Biotechnology (Birmingham, AL). Anti-mouse Thy1.2 Results Ab-coated magnetic beads were from Dynal (Lake Success, NY). Rabbit CD19 deficiency prolongs the life span of c-MycTg mice polyclonal Abs reactive with c-Myc, Pax5, ERK2, cyclin D2, cyclin D3, and cyclin-dependent kinase 4 (Cdk4) were from Santa Cruz Biotechnol- The influence of CD19 expression on survival was evaluated for ogy (Santa Cruz, CA). Rabbit polyclonal Abs reactive with c-Myc phos- c-MycTg mice, and c-MycTg littermates heterozygous for CD19 phorylated at threonine 58 (T58)/serine 62 (S62), CD19 phosphorylated at expression (c-MycTgCD19+/2) or lacking CD19 expression (c- tyrosine 513 (Y513), and CD19 protein were from Cell Signaling Tech- Tg 2/2 Tg 2/2 nology (Beverly, MA). Rabbit polyclonal Abs reactive with the dually Myc CD19 ). The life span of c-Myc CD19 mice was Tg phosphorylated (kinase-active) forms of ERK and JNK were from Promega significantly prolonged in comparison with both c-Myc and c- (Madison, WI). MycTgCD19+/2 littermates (p , 0.0001, Fig. 1A). The median Tg 2/2 In vitro proliferation age of mortality for c-Myc CD19 mice was 24.3 wk, com- pared with 13.4 wk for c-MycTg littermates (81% increase) and Tg Tg 2/2 Primary tumor cells from c-Myc or c-Myc CD19 mice were mixed 13.3 wk for c-MycTgCD19+/2 littermates (83% increase). Never- at a 1:1 ratio and then labeled with eFluor 670 cell proliferation dye theless, a 100% penetrance of lymphomas occurred in c-MycTg (eBioscience), according to the manufacturer’s instructions. The level of 2/2 fluorescence intensity was then assessed by flow cytometry (excitation 633 CD19 mice, as all succumbed to disease by 76 wk of age. This nm/emission 670 nm) immediately after labeling of cells, or following indicates that although CD19 expression is not required for c- a period of cell culture. Myc–derived lymphomas to occur, it accelerates malignant trans- Comparison of tissue B cell numbers and cell surface protein formation and/or disease severity. expression levels During these survival studies, mice were monitored daily for the onset of lymphoma as assessed by the visual enlargement of one or Leukocytes were isolated from the blood, bone marrow (bilateral femurs), more peripheral lymph nodes until the time of death. The mean age spleens, cervical lymph nodes, and peritoneal cavities of 6- to 8-wk-old Tg 2/2 disease-free c-MycTg,c-MycTgCD192/2, or nontransgenic littermates. at which visible lymphoma onset occurred in c-Myc CD19 Tg Cells were then labeled with predetermined optimal concentrations of Abs mice was significantly delayed in comparison with c-Myc lit- reactive with cell surface molecules of interest to distinguish termates (40% increase, p , 0.01, Fig. 1B), and there was a pro- populations and to evaluate mean fluorescence intensity values by flow longed survival period between initial lymphoma detection and cyometry analysis. death (49% increase, p , 0.01). This represented a 42% greater BrdU incorporation mean life span for c-MycTgCD192/2 mice that had lymph node Tg Tg 2/2 involvement (p , 0.01). Lymphoma-free c-Myc and c-Myc CD19 mice, or WT littermates, Tg were injected i.p. with sterile-filtered BrdU (100 mg/g body weight) in Some c-Myc mice die of B cell tumors that do not induce PBS. After 3 h, tissues were harvested and single leukocyte populations lymphadenopathy, but rather invade solid organs or the CNS, The Journal of Immunology 3

FIGURE 1. CD19 expression promotes lymphomagenesis in c-MycTg mice. c-MycTg, c-MycTgCD19+/2, and c-MycTgCD192/2 littermates were monitored daily for the onset of lymphadenopathy and mortality, or a moribund state requiring euthanasia. (A) CD19 expression accelerates death of c-MycTg mice. The log-rank test was used to determine the level of significance between curves in the Kaplan–Meier survival plots. Numbers (arrows) indicate median ages of death for c-MycTg and c-MycTgCD192/2 littermates. (B) CD19 expression accelerates lymphoma development and progression in c-MycTg mice. Values represent mean (6SEM) age at disease onset, duration of disease, and age at death (or euthanasia) for mice of each genotype in the

pool of littermates described in (A). “Observable lymphoma” includes all mice with detectable lymphadenopathy prior to death, whereas “Wasting disease” Downloaded from includes all mice that succumbed to disease without detectable lymph node involvement. Statistical analysis used the Student t test. Significant differences in means between genotypes are indicated, **p # 0.01. culminating in a wasting disease characterized by a state of ca- cluding pro/pre- plus immature B cells in the bone marrow and chexia (24, 25). We also observed this phenomenon, with 21 of 60 immature B cells in all other tissues, were similar between Tg 2/2 Tg Tg Tg 2/2 (35%) c-Myc CD19 mice and 11 of 57 (19%) c-Myc mice c-Myc and c-Myc CD19 mice (Fig. 4A). Notably, mature http://www.jimmunol.org/ succumbing to disease without accompanying lymph node en- largement. When mice that presented with wasting disease were compared, c-MycTgCD192/2 mice had an 82% increased mean survival age relative to c-MycTg littermates (p = 0.01, Fig. 1B). This indicates that tumors in c-MycTgCD192/2 mice were also less aggressive when tissues other than the lymph nodes were invaded. c-MycTg and c-MycTgCD192/2 mice that were moribund from high tumor burden (gross lymph node enlargement) had associated leukemia, characterized by the presence of large B220+ B cell by guest on September 30, 2021 blasts with a high forward scatter (FSC) profile and a B220low phenotype relative to WT B cells (Fig. 2A). Likewise, the lymph nodes and spleens of these c-MycTg and c-MycTgCD192/2 mice were dominated by the presence of these B cells (Fig. 2B), as published (24, 25). Importantly, B cells from these mice were found to transfer aggressive tumors in adoptive transfer experi- ments, confirming their malignant nature. When B cells were isolated from moribund c-MycTg and c-MycTgCD192/2 mice and transferred into SCID recipient mice by i.p. injection, B cell blasts appeared in the blood by day 10 (Fig. 3A). In addition, the sur- vival of SCID recipient mice was similar between groups re- ceiving B cells from c-MycTg mice and c-MycTgCD192/2 mice (Fig. 3B). Also, in SCID recipient mice euthanized during the survival study due to humane endpoint requirements, c-MycTg and c-MycTgCD192/2 B cell blasts were found to similarly invade the lymph nodes, bone marrow, and spleen, and persisted in the peritoneal cavity injection site (Fig. 3C). Finally, primary tumor cells from c-MycTg and c-MycTgCD192/2 mice were found to survive in culture. These primary tumor cells were of similar size FIGURE 2. B cell blasts dominate the blood and lymphoid tissues of Tg Tg 2/2 based on their FSC profiles, were all B220+IgM+, and had similar c-Myc and c-Myc CD19 mice with high tumor burden. Immunoflu- rates of spontaneous proliferation (Fig. 3D). The doubling time for orescence staining followed by flow cytometry analysis was performed on blood (A) and lymph node (B) cells isolated from representative c-MycTg and each primary tumor in culture was 12 h (data not shown). Thus, Tg 2/2 2 2 c-Myc CD19 mice with lymphadenopathy that had reached humane B cells isolated from c-MycTg and c-MycTgCD19 / mice with endpoint criteria. Blood and lymph nodes from WT littermates were assessed high tumor burden were equally aggressive when transferred into for comparison. (A)Theleft panels show blood cells with the FSC and side normal mice, and proliferated at similar rates ex vivo. scatter (SSC) profiles of . Gated regions in the middle panels + B cell numbers are similar between c-MycTg and c-MycTg indicate the percentage of B220 cells of the total lymphocytes shown at left. CD192/2 mice The vertical dashed line in the panels at right is shown to delineate cells that were positive or negative for CD19 expression based on control Ab staining. Tg 2/2 Reduced tumorigenesis in c-Myc CD19 mice did not result (B) Gated regions indicate the percentage of B220+ cells out of total lymph from reduced B cell numbers. Total B220low B cell numbers, in- node cells. These results are representative of $5 mice of each genotype. 4 c-Myc AND CD19 COREGULATE B CELL LYMPHOMA DEVELOPMENT Downloaded from http://www.jimmunol.org/ by guest on September 30, 2021

FIGURE 3. Malignant B cells from c-MycTg and c-MycTgCD192/2 mice with established tumors transfer malignancy and have similar proliferation rates. (A) Malignant B cells from c-MycTg and c-MycTgCD192/2 mice transfer lymphoma after adoptive transfer. Tumor cells (2.5 3 106 spleen B220+ cells) from 2 c-MycTg and c-MycTgCD192/2 donor mice with enlarged lymph nodes approaching humane endpoint guidelines were adoptively transferred i.p. into SCID recipient mice (3 each per donor mouse for a total of 6 SCID mice receiving c-MycTg tumor B cells and 6 SCID mice receiving c-MycTgCD192/2 tumor B cells). Blood from recipient mice was assessed at day 0 and at time points thereafter by immunofluorescence staining, followed by flow cytometry analysis. Gated regions represent the FSC/side scatter (SSC) profile of lymphocytes, and indicate the appearance of transferred B220+ B cells in the blood of SCID recipients by day 10 (middle panels) that were absent at day 0 (left panels). CD19 expression levels correlatedwithdonorgenotypes(right panels). (B) Survival of SCID recipient mice receiving malignant B cells from either c-MycTg or c-MycTgCD192/2 mice was similar. Values represent mean (6SEM) days of survival for each group of recipient mice described in (A). (C) Transferred malignant B cells disseminate into all lymphoid tissues. Analysis of various tissues isolated from SCID recipient mice euthanized due to humane endpoint criteria revealed a dominant invasion by B220+ donor cells. (D) Primary tumors from c-MycTg and c-MycTgCD192/2 mice are B lineage cells with similar rates of pro- liferation. Primary tumors from a c-MycTg mouse and a c-MycTgCD192/2 mouse were identified that grew spontaneously in culture, and were mixed at a 1:1 ratio and labeled with a vital dye to assess cell divisions. CD19 staining during flow cytometry analysis was used to differentiate between cells from c- MycTg and c-MycTgCD192/2 mice (left panel). The expression levels of B220 and IgM were also assessed (middle panels). Proliferation was assessed by flow cytometry analysis of vital dye fluorescence dilution after 36 h of culture in comparison with dye fluorescence at 0 h (right panel). In the middle and right panels, c-MycTg cells are represented by the black, open histograms and c-MycTgCD192/2 cells are represented by the gray, open histograms. The filled gray histograms represent the level of background fluorescence intensity using isotype control Abs (middle panels) or cells not stained with vital dye (right panel).

(B220high) B cell numbers were reduced in the spleen and lymph reductions in early B cell numbers or to detectable changes in nodes of c-MycTgCD192/2 mice compared with c-MycTg litter- B cell proliferation prior to disease onset. mates, as occurs in CD192/2 mice (17). However, reduced B220high B cell numbers do not explain the enhanced survival of Constitutive c-Myc expression augments CD19 surface density c-MycTgCD192/2 mice, as lymphomas in c-MycTg mice generally Because genetic alterations in CD19 expression have dramatic derive from B220low B cells (24, 25). Also, the level of short-term effects on B cell function in humans and mice (12, 13, 16–20), proliferation of B cells in vivo was similar between lymphoma- whether CD19 expression levels are altered in c-MycTg mice was free c-MycTg and c-MycTgCD192/2 mice as determined by BrdU therefore assessed. B cell subset analysis in c-MycTg mice uptake (Fig. 4B). Therefore, the reduced lymphomagenesis and revealed that surface CD19 expression was detectable at higher enhanced survival of c-MycTgCD192/2 mice were not due to levels on bone marrow CD43+B220low pre-B cells, on HSAhigh The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/

FIGURE 5. Constitutive c-Myc expression enhances CD19 surface density. (A) CD19 expression by normal B cell subsets in c-MycTg mice. Bone marrow, spleen, and peritoneal cavity (Per. cavity) cells were ob-

Tg by guest on September 30, 2021 FIGURE 4. B cell development and proliferation in c-MycTg and tained from lymphoma-free c-Myc mice or their WT littermates (n =3 c-MycTgCD192/2 mice. (A) Normal B cell numbers in c-MycTg and each) and stained with CD19 and other Abs to identify the indicated B cell c-MycTgCD192/2 mice. Bone marrow (bilateral femurs), spleens, cervical subsets by flow cytometry analysis. Gated regions in dot plots indicate the lymph nodes, and blood were isolated from 6- to 8-wk-old lymphoma-free B cell subset of interest, with CD19 expression shown in four-decade c-MycTg (n = 6) and c-MycTgCD192/2 (n = 7) mice. Isolated leukocytes or single-color histograms derived from these gates. Shaded histograms rep- whole blood was then labeled with B220 Abs to determine mean (6SEM) resent negative control staining using nonspecific Abs. The bar graph high low 6 numbers of leukocytes, B cells, B220 mature B cells, and B220 pre- shows mean fluorescence intensities (MFI SEM) for immature and Tg B/immature B cells in each tissue. Blood analysis represents cells per mature B cells (WT = gray bars, c-Myc = black bars). Significant dif- Tg milliliter. Statistical analysis used the Student t test, with significant dif- ferences in mean MFI values between B cell subsets from WT and c-Myc , B ferences between means indicated, **p , 0.01. (B) B cell proliferation littermates are indicated, **p 0.01. ( ) CD19 expression by blood B Tg Tg +/2 +/2 rates were similar in c-MycTg and c-MycTgCD192/2 mice. BrdU incor- cells from c-Myc , c-Myc CD19 , or nontransgenic CD19 litter- + A poration was measured for tissue B cells isolated from lymphoma-free mates. Blood leukocytes were stained to identify CD19 B cells as in ( ) Tg Tg 2/2 $ c-Myc and c-Myc CD19 mice and their WT littermates (n = 4 per by flow cytometry analysis. Results represent those from 3 mice of each C Tg group). Mean values (6SEM) indicate percentages of BrdU-positive B genotype. ( ) CD19 expression by malignant B cells in c-Myc mice. cells of total B cells in each tissue. Immunofluorescence staining of CD19 and B220 expression on spleen B cells from a representative c-MycTg mouse with aggressive lymphade- nopathy and splenomegaly, in comparison with B cells from a WT litter- mate. The dashed line indicates the mean MFI for CD19 expression in WT. low B220 immature B cells in the bone marrow and spleen (42 and Similar results were observed in three mice for each group. 37%, respectively, p , 0.01), and on HSAlowB220high mature bone marrow and spleen B cells (14 and 17%, respectively, p , 0.01) as determined by immunofluorescence staining (Fig. 5A). A similar increase in CD19 expression on mature blood B cells was ob- c-Myc and CD19 influence the phosphorylation of one another served in c-MycTg and c-MycTgCD19+/2 mice when compared at regulatory sites with nontransgenic littermates (Fig. 5B). CD19 expression was CD19 regulates signal transduction by amplifying Src family ki- not significantly different on peritoneal CD5+B220low B1a B cells, nase activity (27, 28) and through the Ras signaling pathway (29– however (Fig. 5A), most likely because B1a cells express CD19 at 32). CD19 phosphorylation at regulatory tyrosines in its cyto- the highest level among B cell subsets (13). CD19 expression even plasmic tail, especially the signal-initiating Y513 motif, is re- remained high on B cell blasts with very low B220 expression quired for its regulatory activity (27, 28, 33–35). Therefore, the levels that were detectable in some tumor-bearing c-MycTg mice effect of c-Myc overexpression on CD19 phosphorylation was (Fig. 5C). Thus, constitutive c-Myc expression enhanced CD19 examined by immunoblot analyses. Compared with WT litter- expression, possibly leading to heightened transmembrane and mates, CD19 was hyperphosphorylated at Y513 in spleen B cells cytoplasmic signaling. from c-MycTg mice (Fig. 6A). Total CD19 protein was also in- 6 c-Myc AND CD19 COREGULATE B CELL LYMPHOMA DEVELOPMENT

FIGURE 6. Constitutive c-Myc expression drives CD19 phosphorylation, whereas CD19 expression reciprocally drives c-Myc phosphorylation. (A) Con- stitutive c-Myc expression in B cells drives CD19 hyperphosphorylation. Lysates of purified spleen B cells from lymphoma-free c-MycTg and WT mice were assessed by immunoblotting using Abs specific for CD19 phosphorylated at Y513 (pCD19) and Pax5 protein. Phospho-CD19 blots were stripped and reprobed with Abs reactive with total CD19 protein. Brackets at right indicate the multiple bands characteristic of CD19 protein migration on SDS-PAGE gels. Two experiments producing similar results are shown. (B) CD19 expression drives c-Myc hyperphosphorylation in c-MycTg mice. Lysates of purified spleen B cells from lymphoma-free c-MycTg,c-MycTgCD192/2, WT, and CD192/2 littermates were assessed by immunoblotting using Abs reactive with c-Myc phos- phorylated at T58/S62 (pc-Myc). Membranes were subsequently stripped and reprobed with Abs reactive with total c-Myc protein, as well as with total ERK2 protein to indicate equivalent protein loading. Brackets at right indicate the multiple bands characteristic of c-Myc protein migration. Two of three experiments producing similar results are shown. (C) Constitutive c-Myc and CD19 expression drives ERK activation. Lysates of purified spleen B cells from c-MycTg and c-MycTgCD192/2 littermates were assessed by immunoblotting using Abs specific for the kinase-active forms of ERK1/2 or JNK1/2 ( ERK1/2 and JNK1/2, p p Downloaded from respectively). The membranes were stripped and reprobed using Abs reactive with total ERK2 protein. Two experiments producing similar results are shown. creased (Fig. 6A), correlating with its enhanced surface expression Discussion (Fig. 5), which together may at least partially explain the observed To our knowledge, these studies demonstrate for the first time that increase in CD19 Y513 phosphorylation. Notably, although the CD19 and c-Myc expression synergize functionally to influence

transcription factor Pax5 is required for CD19 expression in http://www.jimmunol.org/ B cells (36, 37), augmented CD19 expression and phosphorylation in c-MycTg B cells did not correlate with an obvious change in Pax5 expression relative to WT B cells (Fig. 6A). c-Myc protein accumulates in the presence of active Ras (38), which depends on c-Myc phosphorylation at S62 in the N termi- nus (39). S62 phosphorylation stabilizes c-Myc protein, and is mediated by the ERK members of the MAPK family (38). c-Myc phosphorylation at this site was dramatically reduced in c-MycTg 2 2 CD19 / B cells compared with c-MycTg B cells (Fig. 6B). Re- by guest on September 30, 2021 duced c-Myc phosphorylation paralleled reduced c-Myc protein levels in c-MycTgCD192/2 B cells relative to c-MycTg B cells, markedly so in some mice assessed. Correlating with reduced c-Myc phosphorylation and protein levels, ERK activation was impaired in B cells from c-MycTgCD192/2 mice, most strikingly ERK2 (Fig. 6C). By contrast, ERK-related MAPKs that function independently of the Ras pathway in B cells, JNK1 and JNK2, were activated similarly between c-MycTg and c-MycTgCD192/2 B cells. Thus, CD19 deficiency impaired c-Myc phosphorylation and protein levels, most likely through abrogation of the Ras-ERK signaling cascade that regulates c-Myc function.

CD19 deficiency alters D-type cyclin expression in c-MycTg mice Because c-Myc is a potent regulator of cell cycle progression, the FIGURE 7. CD19 regulates D-type cyclin expression in c-MycTg B cells. influence of CD19 deficiency on c-Myc function may culminate in (A)CD19expressiondrivescyclin D2 but inhibits cyclin D3 transcription in the altered expression of cell cycle regulatory genes. To assess this, c-MycTg B cells. Cell cycle pathway gene expression arrays were used to cDNA arrays were used to quantify cell cycle gene expression by screen RNA transcripts from purified splenic B cells of lymphoma-free Tg Tg 2/2 spleen B cells from lymphoma-free c-Myc and c-Myc CD19 c-MycTg or c-MycTgCD192/2 littermates. The positions of cyclin D2 and mice. Although most cell cycle genes were expressed at nearly cyclin D3 on the arrays are directly below the underlined numbers 1 and 2, identical levels between genotypes, c-MycTgCD192/2 B cells respectively. The position of Cdk4 on the arrays is directly below the exhibited marked reductions in cyclin D2 expression and recipro- underlined number 3. The bar graph shows relative cDNA hybridization to the cyclin D3 Tg genes on the arrays. (B) CD19 expression drives cyclin D2 protein produc- cally augmented expression of relative to c-Myc Tg B cells (Fig. 7A). Expression of a key catalytic binding partner of tion, but inhibits cyclin D3 production in c-Myc B cells. Lysates of purified spleen B cells from lymphoma-free c-MycTg,c-MycTgCD192/2,WT,and D-type cyclins, cyclin-dependent kinase 4 (Cdk4), was not altered 2 2 CD19 / littermates were assessed by immunoblotting using Abs specific for in c-MycTgCD192/2 B cells. Parallel alterations in cyclin D2 and Tg cyclin D2, cyclin D3, or Cdk4, followed by reprobing of the membranes with cyclin D3 were observed at the protein level between c-Myc and Abs reactive with ERK2 as a loading control. The bar graph shows the mean Tg 2/2 c-Myc CD19 B cells (Fig. 7B). Thus, CD19 deficiency sig- (6SEM) relative density of protein bands from three independent immuno- nificantly altered the pattern of cyclin gene and protein expression blotting experiments. Statistical analysis was performed using the Student t in c-MycTg B cells. test, with significant differences between genotypes indicated, **p , 0.01. The Journal of Immunology 7

spontaneous B cell lymphoma onset and severity. CD19 deficiency (Fig. 7). These cyclins critically govern the G1-S–phase transition of significantly prolonged the life span of c-MycTg mice, with cell cycle in B cells, and cyclin D2 expression relies on ERK ac- a .80% increase in the median age of survival when compared tivity (45). That CD19 deficiency reduced ERK activity in c-MycTg with CD19-expressing littermates (Fig. 1A). CD19 expression at B cells (Fig. 6C) supports the observed reduction of cyclin D2 a threshold level regulated lymphomagenesis, because the median expression (Fig. 7). Cyclin D2 expression requires c-Myc tran- survival age was similarly short in c-MycTg mice with either WT scriptional activity in a Burkitt’s lymphoma-like cell line (46), and or heterozygous (50% reduced) levels of CD19 expression. Im- c-Myc targets the cyclin D2 promoter (47). Whereas cyclin D2 is portantly, CD19 deficiency influenced survival through a signifi- the predominant D-type cyclin expressed by normal B cells (48), cantly delayed age of lymphoma onset and a prolonged period of both cyclin D2 and cyclin D3 are expressed in Burkitt’s lymphomas survival after lymphoma detection, both of which were aspects of (49). Importantly, cyclin D3 can compensate for cyclin D2 defi- c-MycTgCD192/2 mice (Fig. 1B). Supporting these data, it was ciency in promoting B cell proliferation, but this substitution may recently demonstrated that adoptively transferred B lymphoma prolong S-phase transition (48). Thus, reciprocally enhanced cyclin cell lines were more tumorigenic when CD19 was expressed, and D3 expression despite a reduction in cyclin D2 may explain why that CD19 expression in B lymphoma cell lines positively corre- lymphomas still developed with high penetrance in c-MycTg lated with c-Myc protein expression and stability (40). Thereby, CD192/2 mice, but at a significantly delayed rate and with less the malignant outcome of c-Myc translocations in B cells is sig- severity. Because changes in cyclin expression were observed in nificantly influenced by their expression of CD19. nonmalignant B cells, even subtle changes in cell cycle progres- Mechanistically, CD19 deficiency reduced c-Myc phosphory- sion may affect the outcome of malignant conversion or provide lation at S62 in c-MycTg mice (Fig. 6B), a regulatory site that a growth advantage to premalignant clones. The impact of these Downloaded from cooperates with T58 to balance c-Myc protein stability (39). S62 differences may become less obvious once rapidly dividing ma- phosphorylation precedes that of T58, and functions to directly lignant clones evolve during progression to aggressive lymphoma. stabilize c-Myc protein. In turn, T58 phosphorylation is accom- These data define a novel, interdependent pathway between panied by dephosphorylation of S62, which enhances c-Myc c-Myc and CD19 that influences both normal and malignant B cell degradation via the ubiquitin/proteasome pathway. Supporting function. Notably, however, the similar aggressiveness of adoptively Tg Tg 2/2 the latter, tumor penetrance is enhanced in mice expressing mutant transferred B cells from c-Myc and c-Myc CD19 mice with http://www.jimmunol.org/ T58A c-Myc proteins relative to WT c-Myc (41). Reduced c-Myc end-stage disease (Fig. 3) suggests that CD19 expression may no phorphorylation in B cells from c-MycTgCD192/2 mice most longer have a significant impact on proliferation once aggressive likely reflects a lack of initial S62 phosphorylation because this is tumors are established, highlighting the importance of CD19 ex- mediated by ERK activity, and ERK activation was impaired in pression during early lymphoma development. CD19 expression is c-MycTgCD192/2 B cells (Fig. 6C). Thus, the direct effects of also likely to contribute to tumorigenesis and progression by CD19 expression on c-Myc phosphorylation and stability explain influencing tumor cell survival and dissemination, intracellular at least in part why CD19 facilitates the development and pro- activation of negative regulatory pathways, and secretion of immu- gression of malignant B cells, as also reported by Chung et al. nosuppressive cytokines or soluble factors, all of which may have (40), whereby constitutive and stabilized c-Myc protein dysregu- significant effects on disease outcome. Alternatively, CD19 defi- by guest on September 30, 2021 lates multiple signaling pathways. ciency in the B cell lineage may have secondary consequences that Supporting the existence of a c-Myc:CD19 amplification loop in affect lymphoma development and progression. For example, CD19 c-MycTg mice, constitutive c-Myc expression also augmented deficiency leads to significant developmental changes within some CD19 expression levels on normal pre-B, immature, and mature B cell subsets, with accompanying effects on T cell-dependent and B cells (Figs. 5A, 5B, 6A). Although the mechanism through T cell-independent immune responses (50, 51). As such, B cell which constitutive c-Myc expression resulted in augmented CD19 CD19 deficiency may also negatively influence antitumor immune expression is currently unknown, it has been previously shown responses by altering T cell activation or innate cell functions that that the surface expression level of CD19 on pre-B cell colonies influence lymphoma development and progression. derived from cyclin D2-deficient mice is decreased when com- The biochemical analyses carried out in this study suggest the pared with pre-B cell colonies derived from WT mice (42), and following operational model of lymphomagenesis resulting from this cyclin D2 was upregulated dramatically in c-MycTg B cells rela- c-Myc:CD19 pathway: constitutive c-Myc overexpression due to tive to WT B cells (Fig. 7). Increased CD19 expression correlated chromosomal translocations induces a signaling program that relies with enhanced CD19 Y513 phosphorylation (Fig. 6A). Of the nine significantly on CD19. In this process, c-Myc induces CD19 over- conserved tyrosines within the CD19 cytoplasmic domain, Y513 expression and hyperphosphorylation (Fig. 6A), which activates the phosphorylation precedes that of the other sites (27, 28). Phos- Ras-ERK signaling cascade (Fig. 6C) and promotes c-Myc phos- phorylated Y513 is then bound by and amplifies the activity of phorylation at the stabilizing S62 site (Fig. 6B). Stabilized c-Myc in Src-family kinases, which in turn phosphorylate CD19 at other turn governs a unique genetic program dominated by cyclin D2 sites (27, 28), including Y391, which recruits and activates Vav overexpression that enhances lymphomagenesis. Thus, retention of (29–32). Y513 also shares binding activity with PI3K, bringing it CD19 expression by most B cell malignancies may confer a growth to the cell membrane (43, 44). Thus, heightened CD19 expression advantage that further subverts tumor immunity. Thereby, drugs that and phosphorylation in c-MycTg mice most likely amplified im- inhibit CD19 amplification of Src-family kinase or Ras-ERK ac- portant B cell signaling and survival pathways that enhanced tivity may improve current lymphoma therapies. lymphomagenesis. Malignant transformation remained a c-Myc– dependent process, however, rather than enhanced CD19 expres- sion independently driving tumor development, because B cell Disclosures lymphomas have never been observed in CD19Tg mice that T.F.T. is a paid consultant and shareholder for Angelica Therapeutics. overexpress CD19 by 3-fold (12, 13, 17, 19, 20). Although the short-term rate of B cell proliferation in vivo was Tg Tg 2/2 References similar between c-Myc and c-Myc CD19 mice (Fig. 4B), 1. Fujimoto, M., J. C. Poe, M. Inaoki, and T. F. Tedder. 1998. CD19 regulates CD19 regulated the ratio of cyclin D2 and cyclin D3 expression B lymphocyte responses to transmembrane signals. Semin. 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