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. 2008 Sep 3;100(17):1247-59.
doi: 10.1093/jnci/djn253. Epub 2008 Aug 26.

Identification of chromatin remodeling genes Arid4a and Arid4b as leukemia suppressor genes

Mei-Yi Wu  1 , Karen W Eldin, Arthur L Beaudet
Affiliations

Identification of chromatin remodeling genes Arid4a and Arid4b as leukemia suppressor genes

Mei-Yi Wu et al. J Natl Cancer Inst. .

Abstract

Background: Leukemia evolves through a multistep process from premalignancy to malignancy. Epigenetic alterations, including histone modifications, have been proposed to play an important role in tumorigenesis. The involvement of two chromatin remodeling genes, retinoblastoma-binding protein 1 (Rbbp1/Arid4a) and Rbbp1-like 1 (Rbbp1l1/Arid4b), in leukemogenesis was not characterized.

Methods: The leukemic phenotype of mice deficient for Arid4a with or without haploinsufficiency for Arid4b was investigated by serially monitoring complete blood counts together with microscopic histologic analysis and flow cytometric analysis of bone marrow and spleen from the Arid4a(-/-) mice or Arid4a(-/-)Arid4b(+/-) mice. Regulation in bone marrow cells of downstream genes important for normal hematopoiesis was analyzed by reverse transcription-polymerase chain reaction. Genotypic effects on histone modifications were examined by western blotting and immunofluorescence analysis. All statistical tests were two-sided.

Results: Young (2-5 months old) Arid4a-deficient mice had ineffective blood cell production in all hematopoietic lineages. Beyond 5 months of age, the Arid4a(-/-) mice manifested monocytosis, accompanied by severe anemia and thrombocytopenia. These sick Arid4a(-/-) mice showed bone marrow failure with myelofibrosis associated with splenomegaly and hepatomegaly. Five of 42 Arid4a(-/-) mice and 10 of 12 Arid4a(-/-)Arid4b(+/-) mice progressed to acute myeloid leukemia (AML) and had rapid further increases of leukocyte counts. Expression of Hox genes (Hoxb3, Hoxb5, Hoxb6, and Hoxb8) was decreased in Arid4a-deficient bone marrow cells with or without Arid4b haploinsufficiency, and FoxP3 expression was reduced in Arid4a(-/-)Arid4b(+/-) bone marrow. Increases of histone trimethylation of H3K4, H3K9, and H4K20 (fold increases in trimethylation = 32, 95% confidence interval [CI] = 27 to 32; 45, 95% CI = 41 to 49; and 2.2, 95% CI = 1.7 to 2.7, respectively) were observed in the bone marrow of Arid4a-deficient mice.

Conclusions: Arid4a-deficient mice initially display ineffective hematopoiesis, followed by transition to chronic myelomonocytic leukemia (CMML)-like myelodysplastic/myeloproliferative disorder, and then transformation to AML. The disease processes in the Arid4a-deficient mice are very similar to the course of events in humans with CMML and AML. This mouse model has the potential to furnish additional insights into the role of epigenetic alterations in leukemogenesis, and it may be useful in developing novel pharmacological approaches to treatment of preleukemic and leukemic states.

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Figures

Figure 1
Figure 1
A chronic myelomonocytic leukemia (CMML)–like myelodysplastic/myeloproliferative disorder in Arid4a-deficient mice. A) Complete blood counts (white blood cell, lymphocyte, neutrophil, monocyte, red blood cell [RBC], hemoglobin, platelet) in wild-type mice at 2–5 months of age (n = 35), in Arid4a−/− mice at 2–5 months of age (n = 30), in wild-type mice more than 5 months old (n = 25), and in Arid4a−/− mice more than 5 months old with symptoms of CMML (n = 25). Means (and 95% confidence intervals) for cell concentrations are shown, and P values were calculated using Student t test. B) Wright–Giemsa staining of peripheral blood from an Arid4a−/− mouse with symptoms of CMML, showing teardrop poikilocytes (a, black arrowheads), red cells with Howell–Jolly bodies (b, black arrowhead), and nucleated red cells (c, black arrowhead). Immature (d, white arrowhead) and maturing (d, black arrowhead) mononuclear cells were also observed, together with phagocytosis of RBC by a monocyte (e, arrowhead). Ten separate analyses were performed. Scale bars = 5 μm. C) Survival of Arid4a−/− (n = 25) mice and wild-type (n = 25) mice. D) Reticulin staining of paraffin sections of bone marrow from a wild-type mouse and a sick Arid4a−/− mouse. The Arid4a−/− sample shows fibrous tissue stained with black color. Scale bars = 20 μm. E) Flow cytometric analysis of apoptotic cells in bone marrow from a wild-type and a sick Arid4a−/− mouse. The percentages of cells positive for annexin V are indicated. Five separate cytometric analyses were performed. F) Splenomegaly and G) hepatomegaly in a sick Arid4a−/− mouse. Hematoxylin and eosin–stained sections of H) spleen and I) liver from a wild-type mouse and a sick Arid4a−/− mouse. Extramedullary hematopoiesis was found in the Arid4a−/− spleen and Arid4a−/− liver, which were infiltrated with nucleated elements of blood cells. Ten separate analyses were performed. Scale bars = 20 μm. J) Flow cytometric analysis of cells from spleen in a wild-type mouse and an Arid4a−/− mouse stained with Ter119 surface antigen. The percentages of cells positive for the antigen are indicated. Twenty separate analyses were performed.
Figure 2
Figure 2
Growth and survival of the Arid4a−/−Arid4b+/− mice. A) Growth retardation in an Arid4a−/−Arid4b+/− mouse compared with a wild-type littermate at 12 days of age. B) Growth curve of the Arid4a−/−Arid4b+/− (n = 7) and wild-type (n = 10) littermates by mean body weight plotted against age with 95% confidence intervals. C) Survival of Arid4a−/−Arid4b+/− (n = 31) and wild-type (n = 25) mice.
Figure 3
Figure 3
Acute myeloid leukemia (AML)–like phenotype in Arid4a−/−Arid4b+/− mice. A) Bone marrow smears from a wild-type mouse and an Arid4a−/−Arid4b+/− mouse with AML were stained with Wright–Giemsa. Black arrowheads indicate blasts. White arrowheads indicate red blood cell precursors in both wild-type and AML bone marrow (for both wild-type and the Arid4a−/−Arid4b+/− bone marrow smears, original magnifications are the same). Five separate analyses were performed. Scale bars = 20 μm. B) Wright–Giemsa staining of peripheral blood smears from the AML Arid4a−/−Arid4b+/− mice showing blasts (a, black arrowhead), lymphocytes (a, white arrowhead), increased numbers of immature cells (b–d, arrowheads), and phagocytosis of cells by a macrophage (e). Eight separate analyses were performed. Scale bars = 5 μm.
Figure 4
Figure 4
Hematopoietic lineage analysis of the Arid4a−/− mice and Arid4a−/−Arid4b+/− leukemic mice. A) Comparison of cell populations in bone marrow and spleen (HSC, CMP, granulocyte, monocyte, erythroid cell, T cell, and B cell) and cell counts in peripheral blood (neutrophil, monocyte, hemoglobin, RBC, lymphocyte, and platelet) between wild-type mice (n = 5), the Arid4a−/− mice with CMML-like phenotype (n = 5), and the Arid4a−/−Arid4b+/− mice with acute myeloid leukemia (AML) (n = 5). Means (and 95% confidence intervals) for all cell populations and cell counts are shown, and P values were calculated using Student t test. B) Hematopoietic lineage tree displaying the combined impact of the Arid4a mutation with or without the Arid4b mutations in bone marrow and peripheral blood of mice with CMML-like or AML phenotype. Increased and decreased cell populations are indicated by red and green, respectively. HSC, hematopoietc stem cell; CMP, common myeloid progenitor; CLP, common lymphoid progenitor; GMP, granulocyte and monocyte progenitor; MkP, megakaryocyte progenitor; EP, erythroid progenitor.
Figure 5
Figure 5
Development of myeloid sarcoma in Arid4a−/−Arid4b+/− mice. A) Splenomegaly and B) hepatomegaly in the acute myeloid leukemia (AML) Arid4a−/−Arid4b+/− mice relative to spleen and liver from wild-type littermates. C) Histologic analysis of spleen and liver from a wild-type mouse and an AML Arid4a−/−Arid4b+/− mouse. Paraffin sections were stained with hematoxylin and eosin. Black arrowheads indicate mitotic leukemic cells in spleen and liver from the Arid4a−/−Arid4b+/− mouse. White arrowheads indicate hepatocytes. D) Histologic analysis of lungs from a wild-type and an Arid4a−/− mouse. Paraffin sections were stained with hematoxylin and eosin. Blood vessels in the Arid4a−/− lungs showed a marked increase of leukemic cells.
Figure 6
Figure 6
Histone modifications in bone marrow cells of mice lacking Arid4a. A) Western blot analysis of acid-extracted proteins from bone marrow of a wild-type and an Arid4a−/− mouse. Total proteins were transferred to a nitrocellulose membrane and stained with Ponceau S (top), followed by staining with antibodies against histones H3, H4, H2AX, H3K4me3, H3K9me3, and H4K20me3 (bottom). Ratios of histones were quantified by densitometry. Five separate experiments were performed. B) Immunofluorescence analysis of bone marrow cells or primary mouse embryo fibroblasts from wild-type and the Arid4a−/− mice using antibodies against H3K4me3, H3K9me3, and H4K20me3. DNA was counterstained with DAPI. Images were analyzed by deconvolution microscopy. Three separate experiments were performed, all with similar results.
Figure 7
Figure 7
Gene expression analysis of bone marrow cells from wild-type, Arid4a−/−, and Arid4a−/−Arid4b+/− mice. A) Reverse transcriptase–polymerase chain reaction was performed to analyze the genes indicated, with Hprt serving as the control gene. Three separate experiments were performed. B) Pathways by which Arid4a and Arid4b might regulate hematopoiesis through control of the Hox and Fox genes. In the scenario shown, Arid4a controls erythropoiesis, possibly by positively regulating Pitx2 and Hoxb6 genes. Arid4a also controls the expression of Hoxb8, whose product blocks differentiation of hematopoietic stem cells and common myeloid progenitors. Control of B lymphopoiesis by Arid4a may be achieved by increasing expression of Hoxb3. Arid4a, together with Arid4b, increases expression of Foxp3, which acts on regulatory T (TR) cells to suppress conventional T (Tc) cells. Foxp3 also functions as a tumor suppressor gene. However, it is unclear whether Foxp3 suppresses leukemia malignancies.
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