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Expression of Leukemia-Associated Nup98 Fusion Proteins Generates an Aberrant Nuclear Envelope Phenotype.

Fahrenkrog B, Martinelli V, Nilles N, Fruhmann G, Chatel G, Juge S, Sauder U, Di Giacomo D, Mecucci C, Schwaller J - PLoS ONE (2016)

Bottom Line: Chromosomal translocations involving the nucleoporin NUP98 have been described in several hematopoietic malignancies, in particular acute myeloid leukemia (AML).In the resulting chimeric proteins, Nup98's N-terminal region is fused to the C-terminal region of about 30 different partners, including homeodomain (HD) transcription factors.Our findings unravel Nup98 fusion-associated NE alterations that may contribute to leukemogenesis.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biology and Medicine, Université Libre de Bruxelles, Charleroi, Belgium.

ABSTRACT
Chromosomal translocations involving the nucleoporin NUP98 have been described in several hematopoietic malignancies, in particular acute myeloid leukemia (AML). In the resulting chimeric proteins, Nup98's N-terminal region is fused to the C-terminal region of about 30 different partners, including homeodomain (HD) transcription factors. While transcriptional targets of distinct Nup98 chimeras related to immortalization are relatively well described, little is known about other potential cellular effects of these fusion proteins. By comparing the sub-nuclear localization of a large number of Nup98 fusions with HD and non-HD partners throughout the cell cycle we found that while all Nup98 chimeras were nuclear during interphase, only Nup98-HD fusion proteins exhibited a characteristic speckled appearance. During mitosis, only Nup98-HD fusions were concentrated on chromosomes. Despite the difference in localization, all tested Nup98 chimera provoked morphological alterations in the nuclear envelope (NE), in particular affecting the nuclear lamina and the lamina-associated polypeptide 2α (LAP2α). Importantly, such aberrations were not only observed in transiently transfected HeLa cells but also in mouse bone marrow cells immortalized by Nup98 fusions and in cells derived from leukemia patients harboring Nup98 fusions. Our findings unravel Nup98 fusion-associated NE alterations that may contribute to leukemogenesis.

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Lamina-associated polypeptide 2α (LAP2α) is altered in Nup98 fusion expressing leukemic cells.(A) Mouse bone marrow cells were transduced with retroviral particles to express untagged Nup98-HOXA9 and Nup98-HHEX and stained for immunofluorescence microscopy. Expression of these fusion proteins induced lobulations in the NE as evident from the lamin B1 staining (LB1, green). Whereas lamin A/C is not expressed in mouse BM cells (LA/C, magenta), LAP2α (red) is evenly distributed in nuclei from total bone marrow (TBM) cells and lineage minus precursor cells (Lin-), it is diminished from the nucleoplasm and aggregates at the nuclear periphery of mouse BM cells expressing Nup98-HHEX and Nup98-HOXA9, respectively. Electron micrographs of mouse bone marrow cells transduced with (B) Nup98-HHEX and (C) Nup98-HOXA9. Scale bars, 2 μm (C); 1 μm (B). (D) Lamin B1 staining of patient-derived bone marrow cells revealed irregular NE contour. Lamin A/C is not consistently expressed in patient cells and LAP2α is diminished from the nucleoplasm and aggregates at the nuclear periphery. Patient 1 harbored a Nup98-RAP1GDS1, patient 2 a Nup98-NSD1, and patient 3 a Nup98-DDX10 fusion, respectively. Scale bars, 10 μm.
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pone.0152321.g006: Lamina-associated polypeptide 2α (LAP2α) is altered in Nup98 fusion expressing leukemic cells.(A) Mouse bone marrow cells were transduced with retroviral particles to express untagged Nup98-HOXA9 and Nup98-HHEX and stained for immunofluorescence microscopy. Expression of these fusion proteins induced lobulations in the NE as evident from the lamin B1 staining (LB1, green). Whereas lamin A/C is not expressed in mouse BM cells (LA/C, magenta), LAP2α (red) is evenly distributed in nuclei from total bone marrow (TBM) cells and lineage minus precursor cells (Lin-), it is diminished from the nucleoplasm and aggregates at the nuclear periphery of mouse BM cells expressing Nup98-HHEX and Nup98-HOXA9, respectively. Electron micrographs of mouse bone marrow cells transduced with (B) Nup98-HHEX and (C) Nup98-HOXA9. Scale bars, 2 μm (C); 1 μm (B). (D) Lamin B1 staining of patient-derived bone marrow cells revealed irregular NE contour. Lamin A/C is not consistently expressed in patient cells and LAP2α is diminished from the nucleoplasm and aggregates at the nuclear periphery. Patient 1 harbored a Nup98-RAP1GDS1, patient 2 a Nup98-NSD1, and patient 3 a Nup98-DDX10 fusion, respectively. Scale bars, 10 μm.

Mentions: Next we asked if the aberrant NE phenotype could also be observed in leukemic cells carrying Nup98 fusions. Lacking established human leukemia cell lines expressing Nup98 fusions we first immortalized mouse bone marrow (BM) cells by retroviral expression of untagged Nup98-HOXA9 and Nup98-HHEX. The cells were expanded by serial plating and immortalized cell lines were established [32]. As shown in Fig 6A and S6 Fig, Nup98-HOXA9 and Nup98-HHEX expressing mouse BM cells, similar to normal total bone marrow (TBM) cells and lineage marker-depleted (Lin-) BM progenitor cells, expressed no or only very small amounts of lamin A/C, in contrast to the BM-derived B cell line Ba/F3. Lamin B1 staining revealed that Nup98-HOXA9 and Nup98-HHEX expressing cells have strongly lobulated nuclei in contrast to TBM and Lin- cells, with a slight increase in the nucleoplasmic pool of lamin B1 (Fig 6A, first row). These NE lobulations were also readily detectable on electron microscopic level (Fig 6B and 6C). In TBM and Lin- BM cells LAP2α localized to the nucleoplasm, whereas Nup98-HOXA9 and Nup98-HHEX expressing cells showed only weak labeling and aggregation of LAP2α. The expression levels of LAP2α, lamin A/C and lamin B1 on mRNA level were similar in all cell types (S6 Fig). We also analyzed lamin B1, lamin A/C and LAP2α localization in bone marrow derived leukemic blasts of three patients with Nup98 alterations (S3 Table). As shown in Fig 6D, only one of the patients (patient 3 with NUP98-DDX10) expressed some lamin A/C, while the other two did not (patient 1 with NUP98-RAP1GDS1, patient 2 with NUP98-NSD1). Lamin B1 localized to the NE and the nucleoplasm revealing nuclear lobulation, whereas LAP2α was barely detectable and often aggregated, indicating that alterations in LAP2α localization also occur in leukemic blasts with NUP98 translocations. Collectively these data indicate that the aberrant NE phenotype observed in transfected HeLa cells is also found in murine bone marrow cells immortalized by Nup98 fusions as well as in primary tumor cells from leukemia patients carrying translocations leading to Nup98 fusions.


Expression of Leukemia-Associated Nup98 Fusion Proteins Generates an Aberrant Nuclear Envelope Phenotype.

Fahrenkrog B, Martinelli V, Nilles N, Fruhmann G, Chatel G, Juge S, Sauder U, Di Giacomo D, Mecucci C, Schwaller J - PLoS ONE (2016)

Lamina-associated polypeptide 2α (LAP2α) is altered in Nup98 fusion expressing leukemic cells.(A) Mouse bone marrow cells were transduced with retroviral particles to express untagged Nup98-HOXA9 and Nup98-HHEX and stained for immunofluorescence microscopy. Expression of these fusion proteins induced lobulations in the NE as evident from the lamin B1 staining (LB1, green). Whereas lamin A/C is not expressed in mouse BM cells (LA/C, magenta), LAP2α (red) is evenly distributed in nuclei from total bone marrow (TBM) cells and lineage minus precursor cells (Lin-), it is diminished from the nucleoplasm and aggregates at the nuclear periphery of mouse BM cells expressing Nup98-HHEX and Nup98-HOXA9, respectively. Electron micrographs of mouse bone marrow cells transduced with (B) Nup98-HHEX and (C) Nup98-HOXA9. Scale bars, 2 μm (C); 1 μm (B). (D) Lamin B1 staining of patient-derived bone marrow cells revealed irregular NE contour. Lamin A/C is not consistently expressed in patient cells and LAP2α is diminished from the nucleoplasm and aggregates at the nuclear periphery. Patient 1 harbored a Nup98-RAP1GDS1, patient 2 a Nup98-NSD1, and patient 3 a Nup98-DDX10 fusion, respectively. Scale bars, 10 μm.
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pone.0152321.g006: Lamina-associated polypeptide 2α (LAP2α) is altered in Nup98 fusion expressing leukemic cells.(A) Mouse bone marrow cells were transduced with retroviral particles to express untagged Nup98-HOXA9 and Nup98-HHEX and stained for immunofluorescence microscopy. Expression of these fusion proteins induced lobulations in the NE as evident from the lamin B1 staining (LB1, green). Whereas lamin A/C is not expressed in mouse BM cells (LA/C, magenta), LAP2α (red) is evenly distributed in nuclei from total bone marrow (TBM) cells and lineage minus precursor cells (Lin-), it is diminished from the nucleoplasm and aggregates at the nuclear periphery of mouse BM cells expressing Nup98-HHEX and Nup98-HOXA9, respectively. Electron micrographs of mouse bone marrow cells transduced with (B) Nup98-HHEX and (C) Nup98-HOXA9. Scale bars, 2 μm (C); 1 μm (B). (D) Lamin B1 staining of patient-derived bone marrow cells revealed irregular NE contour. Lamin A/C is not consistently expressed in patient cells and LAP2α is diminished from the nucleoplasm and aggregates at the nuclear periphery. Patient 1 harbored a Nup98-RAP1GDS1, patient 2 a Nup98-NSD1, and patient 3 a Nup98-DDX10 fusion, respectively. Scale bars, 10 μm.
Mentions: Next we asked if the aberrant NE phenotype could also be observed in leukemic cells carrying Nup98 fusions. Lacking established human leukemia cell lines expressing Nup98 fusions we first immortalized mouse bone marrow (BM) cells by retroviral expression of untagged Nup98-HOXA9 and Nup98-HHEX. The cells were expanded by serial plating and immortalized cell lines were established [32]. As shown in Fig 6A and S6 Fig, Nup98-HOXA9 and Nup98-HHEX expressing mouse BM cells, similar to normal total bone marrow (TBM) cells and lineage marker-depleted (Lin-) BM progenitor cells, expressed no or only very small amounts of lamin A/C, in contrast to the BM-derived B cell line Ba/F3. Lamin B1 staining revealed that Nup98-HOXA9 and Nup98-HHEX expressing cells have strongly lobulated nuclei in contrast to TBM and Lin- cells, with a slight increase in the nucleoplasmic pool of lamin B1 (Fig 6A, first row). These NE lobulations were also readily detectable on electron microscopic level (Fig 6B and 6C). In TBM and Lin- BM cells LAP2α localized to the nucleoplasm, whereas Nup98-HOXA9 and Nup98-HHEX expressing cells showed only weak labeling and aggregation of LAP2α. The expression levels of LAP2α, lamin A/C and lamin B1 on mRNA level were similar in all cell types (S6 Fig). We also analyzed lamin B1, lamin A/C and LAP2α localization in bone marrow derived leukemic blasts of three patients with Nup98 alterations (S3 Table). As shown in Fig 6D, only one of the patients (patient 3 with NUP98-DDX10) expressed some lamin A/C, while the other two did not (patient 1 with NUP98-RAP1GDS1, patient 2 with NUP98-NSD1). Lamin B1 localized to the NE and the nucleoplasm revealing nuclear lobulation, whereas LAP2α was barely detectable and often aggregated, indicating that alterations in LAP2α localization also occur in leukemic blasts with NUP98 translocations. Collectively these data indicate that the aberrant NE phenotype observed in transfected HeLa cells is also found in murine bone marrow cells immortalized by Nup98 fusions as well as in primary tumor cells from leukemia patients carrying translocations leading to Nup98 fusions.

Bottom Line: Chromosomal translocations involving the nucleoporin NUP98 have been described in several hematopoietic malignancies, in particular acute myeloid leukemia (AML).In the resulting chimeric proteins, Nup98's N-terminal region is fused to the C-terminal region of about 30 different partners, including homeodomain (HD) transcription factors.Our findings unravel Nup98 fusion-associated NE alterations that may contribute to leukemogenesis.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biology and Medicine, Université Libre de Bruxelles, Charleroi, Belgium.

ABSTRACT
Chromosomal translocations involving the nucleoporin NUP98 have been described in several hematopoietic malignancies, in particular acute myeloid leukemia (AML). In the resulting chimeric proteins, Nup98's N-terminal region is fused to the C-terminal region of about 30 different partners, including homeodomain (HD) transcription factors. While transcriptional targets of distinct Nup98 chimeras related to immortalization are relatively well described, little is known about other potential cellular effects of these fusion proteins. By comparing the sub-nuclear localization of a large number of Nup98 fusions with HD and non-HD partners throughout the cell cycle we found that while all Nup98 chimeras were nuclear during interphase, only Nup98-HD fusion proteins exhibited a characteristic speckled appearance. During mitosis, only Nup98-HD fusions were concentrated on chromosomes. Despite the difference in localization, all tested Nup98 chimera provoked morphological alterations in the nuclear envelope (NE), in particular affecting the nuclear lamina and the lamina-associated polypeptide 2α (LAP2α). Importantly, such aberrations were not only observed in transiently transfected HeLa cells but also in mouse bone marrow cells immortalized by Nup98 fusions and in cells derived from leukemia patients harboring Nup98 fusions. Our findings unravel Nup98 fusion-associated NE alterations that may contribute to leukemogenesis.

Show MeSH
Related in: MedlinePlus