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. 2012 Jul;95(1):49-56.
doi: 10.1016/j.antiviral.2012.05.003. Epub 2012 May 16.

Dasatinib therapy results in decreased B cell proliferation, splenomegaly, and tumor growth in a murine model of lymphoma expressing Myc and Epstein-Barr virus LMP2A

Jamie L Dargart  1 , Kamonwan Fish, Leo I Gordon, Richard Longnecker, Osman Cen
Affiliations

Dasatinib therapy results in decreased B cell proliferation, splenomegaly, and tumor growth in a murine model of lymphoma expressing Myc and Epstein-Barr virus LMP2A

Jamie L Dargart et al. Antiviral Res. 2012 Jul.

Abstract

Epstein-Barr virus (EBV) infection and latency has been associated with malignant diseases including nasopharyngeal carcinoma, Hodgkin lymphoma, Burkitt lymphoma, and immune deficiency associated lymphoproliferative diseases. EBV-encoded latent membrane protein 2A (LMP2A) recruits Lyn and Syk kinases via its SH2-domain binding motifs, and modifies their signaling pathways. LMP2A transgenic mice develop hyperproliferative bone marrow B cells and immature peripheral B cells through modulation of Lyn kinase signaling. LMP2A/λ-MYC double transgenic mice develop splenomegaly and cervical lymphomas starting at 8 weeks of age. We reasoned that targeting Lyn in LMP2A-expressing B cells with dasatinib would provide a therapeutic option for EBV-associated malignancies. Here, we show that dasatinib inhibits B cell colony formation by LMP2A transgenic bone marrow cells, and reverses splenomegaly and tumor growth in both a pre-tumor and a syngeneic tumor transfer model of EBV-associated Burkitt lymphoma. Our data support the idea that dasatinib may prove to be an effective therapeutic molecule for the treatment of EBV-associated malignancies.

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Conflict of interest statement

Disclosure statement

The authors have no potential conflicts of interest to disclose.

Figures

Figure 1
Figure 1
Dasatinib inhibits colony formation by bone marrow cells from LMP2A transgenic (TgE) mice. (A) Representative micrographs of colonies formed in the methylcellulose culture by bone marrow cells from wild-type (wt) or LMP2A transgenic (TgE line) mice in the presence of dasatinib or DMSO. The micrograph is a representative of 4 different experiments. (B) Macroscopic colonies were manually counted and calculated as explained in Materials and Methods. The normalized percentage of colonies for each dasatinib concentration for wt or TgE bone marrow cells is graphed versus the concentration of dasatinib (n=2–4). Data were analyzed using Student’s t-test. A p value of 0.05 or less was considered statistically significant.
Figure 2
Figure 2
Dasatinib decreases spleen size in LMP2A transgenic mice. LMP2A transgenic (TgE) and wild-type (wt) mice were treated intraperitoneally with dasatinib or vehicle alone daily for 14 days. The spleen mass and cell composition were analyzed on day 15. (A) The spleen mass of wt or TgE mice treated with DMSO or dasatinib (das) is shown. The percent of B220-positive (B) and CD3-positive (C) lymphocytes in the spleen of respective groups was analyzed with flow cytometry as explained in Materials and Methods. Each data point is from 2–6 mice. See Material and Methods for the details of data presentation on the graphs.
Figure 3
Figure 3
Lyn is hyperphosphorylated in Tg6/λ-MYC tumors. (A) Representative western blots of cervical tumors for phosphorylated Lyn (pLyn), total Lyn (tLyn), LMP2A, and GAPDH from 4 Tg6/λ-MYC (6M) and 4 λ-MYC (myc) transgenic mice (a non-specific band consistently detected with anti-LMP2A antibody is also seen just above the LMP2A). (B) Densitometric analysis of pLyn normalized to tLyn for each tumor sample. The raw densitometric value for each pLyn band from each sample is divided to that of tLyn from the same sample and multiplied by 100 to find the percent of pLyn for the respective mouse. Data are from 4 independent experiments, totaling 14 Tg6/λ-MYC and 15 λ-MYC transgenic mice. See Material and Methods for the details of data presentation on the graphs.
Figure 4
Figure 4
Dasatinib inhibits splenomegaly and lymphadenopathy in Tg6/λ-MYC mice. Tg6/λ-MYC (6M), λ-MYC (myc), and wild-type (wt) mice were intraperitoneally treated with dasatinib (das) or vehicle (DMSO) alone daily for 14 days; the mass of spleens (A) and lymph nodes (B) were analyzed on day 15. The percentage of B220-positive (C) and CD3-positive (D) lymphocytes in the spleens (left panels) and lymph nodes (LN - right panels) of respective groups were analyzed by flow cytometry as indicated in the Materials and Methods section. Each data point is from 2 to 6 mice. See Material and Methods for the details of data presentation on the graphs.
Figure 4
Figure 4
Dasatinib inhibits splenomegaly and lymphadenopathy in Tg6/λ-MYC mice. Tg6/λ-MYC (6M), λ-MYC (myc), and wild-type (wt) mice were intraperitoneally treated with dasatinib (das) or vehicle (DMSO) alone daily for 14 days; the mass of spleens (A) and lymph nodes (B) were analyzed on day 15. The percentage of B220-positive (C) and CD3-positive (D) lymphocytes in the spleens (left panels) and lymph nodes (LN - right panels) of respective groups were analyzed by flow cytometry as indicated in the Materials and Methods section. Each data point is from 2 to 6 mice. See Material and Methods for the details of data presentation on the graphs.
Figure 5
Figure 5
Dasatinib inhibits splenomegaly and secondary tumor formation in Rag1KO mice implanted with primary tumor from Tg6/λ-MYC mice. Lymph node tumor cells from Tg6/λ-MYC (6M) and λ-MYC (myc) transgenic mice were injected subcutaneously into the flank of Rag-1KO mice. When tumors were palpable, mice were intraperitoneally treated with dasatinib (das) or vehicle (DMSO) alone daily for 10–14 days, depending on the health and anticipated survival of the mice. The masses of spleens (A) and tumors (B) were analyzed on the day following the last day of treatment. Data of one representative experiment of three separate experiments are shown. The spleen masses from untreated wild-type (wt) and Rag-1 knock-out (rag1KO) mice are shown as reference in A. Each data point represents 3 to 6 mice. (C) Representative protein lysates of tumors from two mice in each group from panel B above were analyzed by western blotting for the phosphorylated Lyn (pLyn), total Lyn (tLyn), LMP2A, and GAPDH. Each lane indicates a single tumor of the specified genotype; the data is representative of three different immunoblot analyses. See Material and Methods for the details of data presentation on the graphs.
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