EBV latent membrane protein 2A orchestrates p27kip1 degradation via Cks1 to accelerate MYC-driven lymphoma in mice

Abstract

Epstein-Barr virus (EBV) establishes lifelong infection in B lymphocytes of most human hosts and is associated with several B lymphomas. During latent infection, EBV encodes latent membrane protein 2A (LMP2A) to promote the survival of B cells by mimicking host B-cell receptor signaling. By studying the roles of LMP2A during lymphoma development in vivo, we found that LMP2A mediates rapid MYC-driven lymphoma onset by allowing B cells to bypass MYC-induced apoptosis mediated by the p53 pathway in our transgenic mouse model. However, the mechanisms used by LMP2A to facilitate transformation remain elusive. In this study, we demonstrate a key role of LMP2A in promoting hyperproliferation of B cells by enhancing MYC expression and MYC-dependent degradation of the p27^kip1 tumor suppressor. Loss of the adaptor protein cyclin-dependent kinase regulatory subunit 1 (Cks1), a cofactor of the SCF^Skp2 ubiquitin ligase complex and a downstream target of MYC, increases p27^kip1 expression during a premalignant stage. In mice that express LMP2A, Cks1 deficiency reduces spleen weights, restores B-cell follicle formation, impedes cell cycle progression of pretumor B cells, and eventually prolongs MYC-driven tumor onset. This study demonstrates that LMP2A uses the role of MYC in the cell cycle, particularly in the p27^kip1 degradation process, to accelerate lymphomagenesis in vivo. Thus, our results reveal a novel mechanism of EBV in diverting the functions of MYC in malignant transformation and provide a rationale for targeting EBV's roles in cell cycle modulation.

Graphical abstract

Figure 1.

Cks1 knockout decreases the pretumor B-cell population in LMP2A/λ-MYC mice. (A) Spleens were isolated from 4-week-old mice at the pretumor stage, and spleen weights as a percentage of whole body weight are indicated. (B) Lymphocytes were isolated from the bone marrow (BM) and spleens (SP) of pretumor mice, and their B- and T-cell numbers and percentages were calculated by flow cytometry stained with B220-allophycocyanin and CD3ε- phycoerythrin antibodies. Data represent the mean ± standard deviation. (C) The percentage of IgM-positive cells to total splenic B cells were calculated by staining with IgM-FITC antibody. (D) Representative immunohistochemical analysis of 4-week-old mouse spleens from 3 mice of each genotype stained with B220 (original magnification ×4). The statistic differences of Cks1 knockouts against their controls are indicated with thick lines. *P < .05, **P < .01, ***P < .001, ****P < .0001, calculated by 1-way ANOVA with post hoc multiple comparison tests. MYC, λ-MYC mice; 2A MYC, LMP2A/λ-MYC mice.

Figure 2.

Loss of Cks1 restores p27^kipexpression in pretumor spleens of LMP2A/λ-MYC and λ-MYC mice. (A) Representative immunohistochemical analysis of spleens from 4-week-old mice stained with p27^kip1 (original magnification ×40). Representative B- and T-cell areas in white pulps are shown from a total of 3 mice of each genotypes. (B) Representative immunoblots of p27^kip1 and MYC expression in purified splenic B cells from 4-week-old mice. (C) P27^kip1 and (D) MYC expression were normalized to Gapdh (a loading control). The statistical differences of Cks1 knockouts against their controls are indicated with thick lines. Data represent the mean ± standard deviation. **P < .01, ****P < .0001, calculated by 1-way ANOVA with post hoc multiple comparison tests.

Figure 3.

Cks1 deficiency inhibits pretumor B-cell proliferation in LMP2A/λ-MYC mice. The effects of Cks1 status on the cell cycle phases of purified splenic pretumor B cells from 4-week-old mice are indicated in (A) pie charts and (B) column graphs. (C) The percentages of Ki-67–positive cells in purified splenic pretumor B cells. Data were calculated by 1-way ANOVA with post hoc multiple comparison tests. The statistical differences of Cks1 knockouts against their controls are indicated with thick lines. Data represent the mean ± standard deviation. *P < .05, **P < .01, ***P < .001, and ****P < .0001.

Figure 4.

Loss of Cks1 stabilizes p27^kip1in a proteasome-dependent manner. (A) Representative immunoblot analyses of p27^kip1 expression in purified splenic pretumor B cells from 4-week-old mice after treatment with 25 μg/mL CHX with or without 40 μM MG-132 (MG) at indicated time points at 37°C. (B) The fold reduction of p27^kip1 after CHX treatment was normalized to calnexin (a loading control), and the fold reduction of p27^kip1 was calculated using cells at 0 hour of each genotype as the control. Combined data from 3 to 4 different mice represent the mean ± standard deviation. **P < .01, ****P < .0001 were calculated by 2-way ANOVA with multiple comparison tests.

Figure 5.

Cks1 knockout results in the delay of tumor onset in both LMP2A/λ-MYC and λ-MYC mice. Kaplan-Meier curves indicating the percentage of tumor-free survival of indicated genotypes. P values were calculated by comparing 2 genotypes by log-rank (Mantel-Cox) test.

Figure 6.

P27^kip1levels remain high as observed in pretumor cells of tumors from Cks1 knockout LMP2A/λ-MYC and λ-MYC mice. (A) Representative immunohistochemical analysis of lymph node tumors from 3 to 6 mice of each genotype stained with hematoxylin and eosin, B220, p27^kip1, and Thr187-phosphorylated p27^kip1, using an EVOS XL core microscope with ×40 magnification. Insets are tumors imaged by a Nikon Eclipse E600 microscope with ×100 magnification and oil immersion. (B) Percentage of lymphoma cells with intense nuclear staining of Thr187-phosphorylated p27^kip1 in total lymphoma cells per microscopic field (×40 magnification) analyzed by ImageJ software (National Institutes of Health). (C) Immunoblots of p27^kip1 and MYC expression in tumor cells. (D) P27^kip1 and (E) MYC expression were normalized to Gapdh (a loading control). Data represent the mean ± standard deviation. ***P < .001, ****P < .0001, calculated by 1-way ANOVA with post hoc multiple comparison tests.

Figure 7.

Cks1 knockout does not change the occurrence of p53 and/or p19^ARFabnormalities in LMP2A/λ-MYC and λ-MYC tumors. The status of p53 and p19^ARF expression in lymph node tumors from the indicated mouse genotype. (A) Representative immunoblots of p53 and p19^ARF. (B) Combined data from several independent experiments. Statistical analyses were performed with Fisher’s exact test by comparing the frequency of p53 and p19^ARF abnormalities in Cks1^+/+ vs Cks1^−/− within the LMP2A/λ-MYC or λ-MYC background. p53⁺ and p19⁻, baseline p53 and p19^ARF levels as indicated by immunoblots; p53⁻, undetected baseline level of p53; p53⁺⁺, aberrant p53 accumulation at ∼53 kDa; p19⁺, abnormal accumulation of p19^ARF. (C) The number of tumor-free days in each indicated genotype. Medians with 95% confident intervals of ratios are indicated. Survival analyses were performed with both the log-rank (Mantel-Cox) test and Gehan-Breslow-Wilcoxon test by comparing the tumor-free days of the subgroups with (+) vs without (–) p53 and p19^ARF abnormalities for each genotype. NS, not significant.