Nuclear export signal within CALM is necessary for CALM-AF10-induced leukemia

Abstract

The CALM-AF10 fusion gene, which results from a t(10;11) translocation, is found in a variety of hematopoietic malignancies. Certain HOXA cluster genes and MEIS1 genes are upregulated in patients and mouse models that express CALM-AF10. Wild-type clathrin assembly lymphoid myeloid leukemia protein (CALM) primarily localizes in a diffuse pattern within the cytoplasm, whereas AF10 localizes in the nucleus; however, it is not clear where CALM-AF10 acts to induce leukemia. To investigate the influence of localization on leukemogenesis involving CALM-AF10, we determined the nuclear export signal (NES) within CALM that is necessary and sufficient for cytoplasmic localization of CALM-AF10. Mutations in the NES eliminated the capacity of CALM-AF10 to immortalize murine bone-marrow cells in vitro and to promote development of acute myeloid leukemia in mouse models. Furthermore, a fusion of AF10 with the minimal NES can immortalize bone-marrow cells and induce leukemia in mice. These results suggest that during leukemogenesis, CALM-AF10 plays its critical roles in the cytoplasm.

Keywords: AF10; chromosome translocation; histone modification; leukemia; nuclear export signal.

Figure 1

The NES region within clathrin assembly lymphoid myeloid leukemia protein (CALM) is necessary for cytoplasmic localization. (a) Schematic representations of CALM, AF10, CALM-AF10 and mutant proteins. FLAG-CALM^NES4A-AF10 and FLAG-CALM^NES4A were generated by alanine substitution of three leucine residues and one isoleucine residue in the putative CALM NES (red). FLAG-NES1-AF10 and FLAG-NES2-AF10 mutants were constructed by fusion of the NES sequences of CALM to the AF10 portion of CALM-AF10. (b) Diagrams of the plasmid constructs used in transfection experiments. (c) Subcellular distribution of CALM-AF10 and the NES point mutations FLAG-CALM-AF10, FLAG-CALM, FLAG-CALM^NES4A-AF10, FLAG-CALM^NES4A, and FLAG-NES1-AF10, FLAG-NES2-AF10 and FLAG-mAF10 in COS-7 cells. Transfected cells were stained with anti-FLAG antibody (green) and observed by fluorescence microscopy. (d) Population of cells expressing transduced genes in the nucleus and cytoplasm shown in (c). (e) Subcellular distribution of CALM-AF10 and NES mutation proteins in murine bone-marrow cells. Transduced cells were stained with anti-FLAG antibody (red). (f) Population of cells expressing transduced genes in the nucleus and the cytoplasm shown in (e). Nuclei were stained with DAPI (blue) and observed by fluorescence microscopy. The scale bar represents 20 μm in (c) and 10 μm in (e). ANTH, AP180 N-terminal homology domain binding phosphatidylinositol 4,5-bisphosphate (PIP₂); DIF and DPF, motifs interacts with AP-2; NPF, a motif interacts with the EH (Eps15 homology) domain; CBS-I and -II, putative type I and II clathrin-binding sequences; NES, nuclear export signal; PHD Type1 and 2, plant homeodomain zinc finger domains; NLS, nuclear localization signal; AT-hook, DNA-binding protein motif; OMLZ, octapeptide motif-leucine zipper domain; Q-rich, glutamine-rich region.

Figure 2

The nuclear export signal within clathrin assembly lymphoid myeloid leukemia protein (CALM) is critical for in vitro immortalization of cells by CALM-AF10. (a) Serial colony-replating assays of murine bone-marrow cells transduced with FLAG-tagged wild-type and mutant CALM-AF10. In each round of replating, 3 × 10⁴ transduced bone-marrow cells were plated. Bars represent the numbers of colonies. (b) Hoxa cluster and Meis1 expression in cells transduced with wild-type or mutant CALM-AF10. RNA transcripts were analyzed by real-time PCR of murine bone-marrow cells transduced with wild-type and mutant CALM-AF10 in vitro. Expression levels of Hoxa5, Hoxa7, Hoxa9, Hoxa10 and Meis1 were normalized against Actb expression and compared with the levels in vector-transfected whole bone-marrow cells. Data are shown as means ± SEM from three independent samples. *P < 0.05; **P < 0.01. (vs normal bone-marrow cells). CA WT, wild-type CALM-AF10.

Figure 3

The nuclear export signal within clathrin assembly lymphoid myeloid leukemia protein (CALM) is sufficient for leukemic transformation by CALM-AF10. (a) Survival of mice injected with murine bone-marrow cells transduced with FLAG-CALM-AF10 or FLAG-NES2-AF10. The leukemia-free survivals of the mice were analyzed. CALM-AF10 primary transplantation, n = 8; CALM-AF10 secondary transplantation, n = 4; NES2-AF10 primary transplantation, n = 4; NES2-AF10 secondary transplantation, n = 9. (b) Peripheral blood smears and bone-marrow cytospins were stained with May-Giemsa from CALM-AF10-transduced or NES2-AF10-transduced bone-marrow cells. Original magnification is 400×. (c) Population of blasts and segmented neutrophils in bone-marrow cells shown in (b). The scale bars represent 20 μm.

Figure 4

Characterization of leukemic cells in vivo. (a) Flow cytometric analysis of leukemic cells. Murine bone-marrow cells were prepared from mice that developed leukemia after receiving transplantation of tumor cells transduced with CALM-AF10 or NES2-AF10, and were co-stained for Gr-1, Mac-1, colony stimulating factor 1 receptor (CSF1R) and c-kit; data are representative of CALM-AF10 primary transplantation (n = 3) and NES2-AF10 primary transplantation (n = 3). (b) Hoxa cluster and Meis1 expression in mice receiving cells transduced with wild-type and mutant CALM-AF10. RNA transcripts were analyzed by real-time PCR of bone-marrow cells in mice that developed leukemia after CALM-AF10 and NES2-AF10 bone-marrow transplantation. Expression levels of Hoxa5, Hoxa7, Hoxa9, Hoxa10 and Meis1 were normalized against Actb and compared with wild-type whole bone marrow. Data are shown as means ± SEM from three independent leukemic mice. *P < 0.05; **P < 0.01 (vs normal bone-marrow cells).

Figure 5

Dot1L mainly localize in the nucleus in CALM-AF10-induced or NES2-AF10-induced leukemic cells. (a) Subcellular distribution of endogenous Dot1L in CALM-AF10-induced or NES2-AF10-induced leukemic cells. Cytospins of the cells were stained with anti-FLAG antibody (red), anti-DOT1L antibody (green) and DAPI (blue) and observed by confocal laser scanning microscopy. Note that GFP expression was not detected in the condition. (b) Subcellular distribution of endogenous Dot1L in the control vector-infected murine using fluorescence microscopy. (c) Population of leukemia cells expressing DOT1L and CALM-AF10 or FLAG-NES2-AF10 in the nucleus and the cytoplasm shown in (a) and (b). The scale bar represents 5 μm in (a) and 10 μm in (b).