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. 2012 Sep 1;189(5):2318-25.
doi: 10.4049/jimmunol.1201000. Epub 2012 Jul 23.

A c-Myc and surface CD19 signaling amplification loop promotes B cell lymphoma development and progression in mice

Jonathan C Poe  1 , Veronique Minard-Colin, Evgueni I Kountikov, Karen M Haas, Thomas F Tedder
Affiliations

A c-Myc and surface CD19 signaling amplification loop promotes B cell lymphoma development and progression in mice

Jonathan C Poe et al. J Immunol. .

Abstract

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, Eμ-Myc transgenic (c-Myc(Tg)) mice that develop aggressive and lethal B cell lymphomas were made CD19 deficient (c-Myc(Tg)CD19⁻/⁻). Compared with c-Myc(Tg) and c-Myc(Tg)CD19⁺/⁻ littermates, the median life span of c-Myc(Tg)CD19⁻/⁻ mice was prolonged by 81-83% (p < 0.0001). c-Myc(Tg)CD19⁻/⁻ mice also lived 42% longer than c-Myc(Tg) littermates following lymphoma detection (p < 0.01). Tumor cells in c-Myc(Tg) and c-Myc(Tg)CD19⁻/⁻ 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-Myc(Tg)CD19⁻/⁻ mice was not due to reductions 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-Myc(Tg) B cells enhanced c-Myc phosphorylation at regulatory sites, sustained higher c-Myc protein levels, and maintained a balance of cyclin D2 expression over that of cyclin D3. These findings define a new and novel c-Myc:CD19 regulatory loop that positively influences B cell transformation and lymphoma progression.

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Figures

Figure 1
Figure 1
CD19 expression promotes lymphomagenesis in c-MycTg mice. c-MycTg, c-MycTgCD19+/− and c-MycTgCD19−/− 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-MycTgCD19−/− littermates. (B) CD19 expression accelerates lymphoma development and progression in c-MycTg mice. Values represent mean (± SEM) 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, while “Wasting disease” includes all mice that succumbed to disease without detectable lymph node involvement. Statistical analysis used the Student’s t test. Significant differences in means between genotypes are indicated (**), p ≤ 0.01.
Figure 2
Figure 2
B cell blasts dominate the blood and lymphoid tissues of c-MycTg and c-MycTgCD19−/− mice with high tumor burden. Immunofluorescence staining followed by flow cytometry analysis was performed on blood (A) and lymph node (B) cells isolated from representative c-MycTg and c-MycTgCD19−/− mice with lymphadenopathy that had reached humane endpoint criteria. Blood and lymph nodes from WT littermates were assessed for comparison. In (A), the left panels show blood cells with the FSC and side scatter (SSC) profiles of lymphocytes. Gated regions in the middle panels indicate the percentage of B220+ cells out of the total lymphocytes shown at left. The vertical dashed line in the panels at right are shown to delineate cells that were positive or negative for CD19 expression based on control Ab staining. In (B), gated regions indicate the percentage of B220+ cells out of total lymph node cells. These results are representative of ≥ 5 mice of each genotype.
Figure 3
Figure 3
Malignant B cells from c-MycTg and c-MycTgCD19−/− mice with established tumors transfer malignancy and have similar proliferation rates. (A) Malignant B cells from c-MycTg and c-MycTgCD19−/− mice transfer lymphoma after adoptive transfer. Tumor cells (2.5 × 106 spleen B220+ cells) from 2 c-MycTg and c-MycTgCD19−/− 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-MycTgCD19−/− tumor B cells). Blood from recipient mice was assessed at day 0 and at time points thereafter by immunofluorescence staining followed flow cytometry analysis. Gated regions represent the FSC/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 correlated with donor genotypes (right panels). (B) Survival of SCID recipient mice receiving malignant B cells from either c-MycTg or c-MycTgCD19−/− mice was similar. Values represent mean (± SEM) days of survival for each group of recipient mice. (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-MycTgCD19−/− mice are B lineage cells with similar rates of proliferation. Primary tumors from a c-MycTg mouse and a c-MycTgCD19−/− 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-MycTgCD19−/− 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-MycTgCD19−/− 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).
Figure 4
Figure 4
B cell development and proliferation in c-MycTg and c-MycTgCD19−/− mice. (A) Normal B cell numbers in c-MycTg and c-MycTgCD19−/− mice. Bone marrow (bilateral femurs), spleens, cervical lymph nodes, and blood were isolated from 6–8 wk-old lymphoma-free c-MycTg (n = 6) and c-MycTgCD19−/− (n = 7) mice. Isolated leukocytes or whole blood were then labeled with B220 Abs to determine mean (± SEM) numbers of leukocytes, B cells, B220high mature B cells and B220low pre-B/immature B cells in each tissue. Blood analysis represents cells per milliliter. Statistical analysis used the Student’s t test, with significant differences between means indicated (**), p < 0.01. (B) B cell proliferation rates were similar in c-MycTg and c-MycTgCD19−/− mice. BrdU incorporation was measured for tissue B cells isolated from lymphoma-free c-MycTg and c-MycTgCD19−/− mice, and their WT littermates (n = 4 per group). Mean values (± SEM) indicate percentages of BrdU-positive B cells out of total B cells in each tissue.
Figure 5
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 obtained from lymphoma-free c-MycTg mice or their WT littermates (n = 3 each) and stained with CD19 and other Abs to identify the indicated B cell subsets by flow cytometry analysis. Gated regions in dot plots indicate the B cell subset of interest, with CD19 expression shown in four-decade single-color histograms derived from these gates. Shaded histograms represent negative control staining using non-specific Abs. The bar graph shows mean fluorescence intensities (MFI ± SEM) for immature and mature B cells (WT = gray bars, c-MycTg = black bars). Significant differences in mean MFI values between B cell subsets from WT and c-MycTg littermates are indicated (**), p < 0.01. (B) CD19 expression by blood B cells from c-MycTg, c-MycTgCD19+/− or non-transgenic CD19+/− littermates. Blood leukocytes were stained to identify CD19+ B cells as in (A) by flow cytometry analysis. Results represent those from ≥ 3 mice of each genotype. (C) CD19 expression by malignant B cells in c-MycTg mice. Immunofluorescence staining of CD19 and B220 expression on spleen B cells from a representative c-MycTg mouse with aggressive lymphadenopathy and splenomegaly, in comparison with B cells from a WT littermate. The dashed line indicates the mean MFI for CD19 expression in WT. Similar results were observed in 3 mice for each group.
Figure 6
Figure 6
Constitutive c-Myc expression drives CD19 phosphorylation, while CD19 expression reciprocally drives c-Myc phosphorylation. (A) Constitutive c-Myc expression in B cells drives CD19 hyper-phosphorylation. 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 hyper-phosphorylation in c-MycTg mice. Lysates of purified spleen B cells from lymphoma-free c-MycTg, c-MycTgCD19−/−, WT and CD19−/− littermates were assessed by immunoblotting using Abs reactive with c-Myc phosphorylated 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 out 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-MycTgCD19−/− littermates were assessed by immunoblotting using Abs specific for the kinase-active forms of ERK1/2 or JNK1/2 (pERK1/2 and pJNK1/2, respectively). The membranes were stripped and reprobed using Abs reactive with total ERK2 protein. Two experiments producing similar results are shown.
Figure 7
Figure 7
CD19 regulates D-type cyclin expression in c-MycTg B cells. (A) CD19 expression drives cyclin D2 but inhibits cyclin D3 transcription in c-MycTg B cells. Cell cycle pathway gene expression arrays were used to screen RNA transcripts from purified splenic B cells of lymphoma-free c-MycTg or c-MycTgCD19−/− littermates. The positions of cyclin D2 and cyclin D3 on the arrays are directly below the underlined numbers 1 and 2, respectively. The position of Cdk4 on the arrays is directly below the underlined number 3. The bar graph shows relative cDNA hybridization to the genes on the arrays. (B) CD19 expression drives cyclin D2 protein production but inhibits cyclin D3 production in c-MycTg B cells. Lysates of purified spleen B cells from lymphoma-free c-MycTg, c-MycTgCD19−/−, WT and CD19−/− littermates were assessed by immunoblotting using Abs specific for cyclin D2, cyclin D3 or Cdk4, followed by reprobing of the membranes with Abs reactive with ERK2 as a loading control. The bar graph shows the mean (± SEM) relative density of protein bands from 3 independent immunoblotting experiments. Statistical analysis was performed using the Student’s t test, with significant differences between genotypes indicated (**), p < 0.01.
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