MicroRNA miR-125b causes leukemia

Abstract

MicroRNA miR-125b has been implicated in several kinds of leukemia. The chromosomal translocation t(2;11)(p21;q23) found in patients with myelodysplasia and acute myeloid leukemia leads to an overexpression of miR-125b of up to 90-fold normal. Moreover, miR-125b is also up-regulated in patients with B-cell acute lymphoblastic leukemia carrying the t(11;14)(q24;q32) translocation. To decipher the presumed oncogenic mechanism of miR-125b, we used transplantation experiments in mice. All mice transplanted with fetal liver cells ectopically expressing miR-125b showed an increase in white blood cell count, in particular in neutrophils and monocytes, associated with a macrocytic anemia. Among these mice, half died of B-cell acute lymphoblastic leukemia, T-cell acute lymphoblastic leukemia, or a myeloproliferative neoplasm, suggesting an important role for miR-125b in early hematopoiesis. Furthermore, coexpression of miR-125b and the BCR-ABL fusion gene in transplanted cells accelerated the development of leukemia in mice, compared with control mice expressing only BCR-ABL, suggesting that miR-125b confers a proliferative advantage to the leukemic cells. Thus, we show that overexpression of miR-125b is sufficient both to shorten the latency of BCR-ABL-induced leukemia and to independently induce leukemia in a mouse model.

Fig. 1.

MicroRNA-125b overexpression leads to an increase in WBCs associated with a macrocytic anemia in mice. C57BL/6 mice (CD45.1) were transplanted with lineage-negative hematopoietic fetal liver cells (CD45.2) that were infected with an empty retroviral vector (XZ) expressing GFP or infected with the same vector expressing GFP and miR-125b. Peripheral blood was harvested once a month for CBCs and flow cytometry analysis. (A) MicroRNA-125b expression level relative to controls in WBCs (after lysis of red cells) of mice from two different batches at 16 wk posttransplantation. (B) Percent of GFP⁺ cells in the peripheral blood of mice transplanted with miR-125b or control XZ infected cells over time. The first time point corresponds to the percent of GFP⁺ cells 3 d after the first infection in in vitro culture. t test: ***P < 0.0001. (C) Whole-blood counts of mice transplanted with control (n = 20) or miR-125b infected cells (n = 18) showing numbers of WBC, the absolute numbers of neutrophils, lymphocytes, monocytes, eosinophils and basophils, RBCs, platelets, percent hematocrit, hemoglobin concentration, and MCV (mean volume of red blood cells) at 16 wk posttransplantation. Combined data from three independent experiments are shown. Mann–Whitney test: *P < 0.05; ***P < 0.0001. (D) Representative peripheral blood flow cytometry at 16 wk posttransplantation from mice transplanted with XZ control or miR-125b–infected cells. Cd11b⁺Gr1⁺ cells correspond to granulocytes; Cd11b⁺Gr1⁻ cells correspond to monocytes.

Fig. 2.

MicroRNA-125b overexpression causes leukemia in mice. Of 20 mice transplanted with miR-125b–infected lineage-negative fetal liver cells in three different batches, 10 developed a fatal hematologic malignancy. Peripheral blood samples, bone marrow, and spleen single-cell suspensions were collected from moribund mice. Donor-derived cells (CD45.2) were stained by using antibodies against specific lineage markers to determine the proportion of the different lineages by flow cytometry analysis: myeloid cells (CD11b and Gr1), B cells (B220), and T cells (CD4 and CD8). Peripheral blood smears were stained with May Grunwald Giemsa (MGG). Five micrometer sections of paraffin-embedded bone marrow and spleen tissue were prepared on slides and stained with H&E. (A) Survival curve of mice transplanted with miR-125b or control vector-infected cells. (B) Quantitative RT-PCR on peripheral blood of leukemic mice with T-ALL (n = 3), B-ALL (n = 1), or MPN (n = 2) compared with healthy mice transplanted with miR-125b or control vector-infected cells at 16 wk posttransplantation. (C) Representative MGG staining of peripheral blood smear (100×) and H&E staining of bone marrow (100×) and spleen (100×) sections from a control mouse killed at 16 wk posttransplantation. (D) Representative flow cytometry from a mouse with a MPN (mouse 1) compared with peripheral blood staining of a control mouse. Peripheral blood, bone marrow, and spleen of mouse 1 are full of neutrophils (CD11b⁺Gr1⁺). (E) Corresponding MGG staining of peripheral blood smear (100×) and H&E staining of bone marrow (100×) and spleen (100×) sections showing an excess of myeloid cells. (F) Representative flow cytometry from a mouse with B-ALL (mouse 2) compared with bone-marrow staining of a control mouse. Note the presence of lymphoblasts (B220⁺) in the peripheral blood, bone marrow, and spleen. (G) Corresponding MGG staining of peripheral blood smear (100×) and H&E staining of bone marrow (100×) and spleen (100×) sections. (H) Representative flow cytometry from a mouse with T-ALL (mouse 4) compared with bone-marrow staining of a control mice. Presence of CD4⁺CD8⁺ lymphoblasts in the peripheral blood, bone marrow, and spleen. (I) Corresponding blood smear (100×) showing a monocyte and a lymphoblast (arrow). Bone marrow (100×) and spleen (100×) sections with excess of lymphoblasts.

Fig. 3.

MicroRNA-125b overexpression decreases the latency of BCR-ABL–induced leukemia. BALB/c mice were transplanted with 5-FU–treated bone-marrow cells that were infected with retroviral vectors expressing BCR-ABL and an empty vector XZ or infected with retroviral vectors expressing BCR-ABL and miR-125b. (A) Example of BCR-ABL RT-PCR on bone-marrow cells from a mouse transplanted with BCR-ABL–infected cells or miR-125b plus BCR-ABL–infected cells. PCR was processed with a negative control corresponding to cDNA from an untransplanted mouse and a positive control corresponding to the BCR-ABL construct. (C) Survival curve. Mice transplanted with BCR-ABL and miR-125b–infected bone-marrow cells (n = 20) have a median survival of 21 d. Mice transplanted with BCR-ABL and XZ-infected bone-marrow cells (n = 19) have a median survival of 35 d. Combined data from three independent experiments are shown. Log-rank test gives a P value <0.0001.