Elucidating the role of altered DNA damage response in Nup98-associated leukaemia

Acute myeloid leukaemia is a heterogeneous disease characterized by uncontrolled proliferation of neoplastic haematopoietic precursor cells, which leads to the disruption of normal haematopoiesis and bone marrow failure. Impaired haematopoiesis is often associated with balanced chromosomal translocations that involve the nucleoporin Nup98 fused to around 30 different partner genes, such as the homeobox genes HOXA9 and PMX1. Nup98-associated AML is characterized by poor prognosis and poor treatment outcome for the patients. The aim of the study was to elucidate the mechanisms underlying chemotherapy-resistance. Previous experiments showed that the expression of Nup98 fusion proteins leads to changes in nuclear organization. Based on these observations, we hypothesize that the expression of Nup98 fusion proteins affect DNA double-strand break (DSB) repair. Our work shows that the expression of Nup98-HoxA9 and Nup98-HHEX in U2OS cells does not induce any DSBs. Further, we examined the repair phenotype of exogenously induced DSBs. Experiments carried out using etoposide (ETO) or neocarzinostatin (NCS) revealed that Nup98 fusion proteins affect non-homologous end joining (NHEJ). The second major DSB repair pathway, homologous recombination (HR), remains unaffected by Nup98 fusion proteins. The repair phenotype showed that at most timepoints analyzed, cells expressing Nup98 fusion proteins present less DSBs that control cells. We further performed single cell gel electrophoresis assays, also called COMET assay. This assay determines the amount of broken DNA at the single cell level. COMET assays showed that cells expressing Nup98-HoxA9 get equally damaged as control cells. Taken together, these results show that Nup98-HoxA9 induces faster DNA repair by affecting NHEJ. Additional experiments, pointed toward a role of p53 in the effect of Nup98 fusion proteins on DSB repair. Monitoring the repair phenotype in a wild-type and p53 depletion background, revealed that the effect of Nup98-HoxA9 on NHEJ is partially p53 dependent. A further search for the potentially implicated factor in the accelerated NHEJ remained inconclusive so far. In conclusion, Nup98-HoxA9 induces accelerated NHEJ in a partially p53-dependent manner.Option Biologie moléculaire du Doctorat en Sciencesinfo:eu-repo/semantics/nonPublishe

Taking a Bad Turn: Compromised DNA Damage Response in Leukemia.

Genomic integrity is of outmost importance for the survival at the cellular and the organismal level and key to human health. To ensure the integrity of their DNA, cells have evolved maintenance programs collectively known as the DNA damage response. Particularly challenging for genome integrity are DNA double-strand breaks (DSB) and defects in their repair are often associated with human disease, including leukemia. Defective DSB repair may not only be disease-causing, but further contribute to poor treatment outcome and poor prognosis in leukemia. Here, we review current insight into altered DSB repair mechanisms identified in leukemia. While DSB repair is somewhat compromised in all leukemic subtypes, certain key players of DSB repair are particularly targeted: DNA-dependent protein kinase (DNA-PK) and Ku70/80 in the non-homologous end-joining pathway, as well as Rad51 and breast cancer 1/2 (BRCA1/2), key players in homologous recombination. Defects in leukemia-related DSB repair may not only arise from dysfunctional repair components, but also indirectly from mutations in key regulators of gene expression and/or chromatin structure, such as p53, the Kirsten ras oncogene (K-RAS), and isocitrate dehydrogenase 1 and 2 (IDH1/2). A detailed understanding of the basis for defective DNA damage response (DDR) mechanisms for each leukemia subtype may allow to further develop new treatment methods to improve treatment outcome and prognosis for patients.info:eu-repo/semantics/publishe

Localisation of Nup153 and SENP1 to nuclear pore complexes is required for 53BP1 mediated DNA double-strand break repair.

The nuclear basket of nuclear pore complexes (NPCs) is composed of three nucleoporins: Nup153, Nup50 and Tpr. Nup153 has a role in DNA double-strand break (DSB) repair by promoting nuclear import of 53BP1, a mediator of DNA damage response. Here we provide evidence that loss of Nup153 compromises 53BP1 sumoylation, prerequisite for efficient accumulation of 53BP1 at DSBs. Depletion of Nup153 resulted in reduced SUMO1 modification of 53BP1 and the displacement of the SUMO protease SENP1 from NPCs. Artificial tethering of SENP1 to NPCs restored non-homologous end joining (NHEJ) in the absence of Nup153 and re-established 53BP1 sumoylation. Furthermore, Nup50 and Tpr, the two other nuclear basket nucleoporins, also contribute to proper DSB repair, in a manner distinct from Nup153. Similar to Nup153, Tpr appears implicated in NHEJ and homologous recombination (HR), whereas loss of Nup50 only affected NHEJ. Despite the requirement of all three nucleoporins for accurate NHEJ, only Nup153 is needed for proper nuclear import of 53BP1and SENP1-dependent sumoylation of 53BP1. Our data support the role of Nup153 as important regulator of 53BP1 activity and efficient NHEJ.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

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

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

Localization of Nup98 fusion proteins.

<p>HeLa cells were transiently transfected with GFP constructs and visualized after 24 hours by direct fluorescence microscopy. All fusion proteins localize to the nucleus. (<b>A</b>) GFP-Nup98 is found at the nuclear rim and in the nucleoplasm, whereas Nup98 homeodomain fusions exhibit a punctate pattern: (<b>B</b>) GFP-Nup98-HOXA9, (<b>C</b>) GFP-Nup98-HOXA10, (<b>D</b>) GFP-Nup98-HHEX, and (<b>E</b>) GFP-Nup98-PMX1. Nup98 fusions with other chromatin-binding motifs show a different punctate distribution: (<b>F</b>) GFP-Nup98-JARID1A, and (<b>G</b>) GFP-Nup98-PHF23 (<b>H</b>) GFP-Nup98-NSD1, (<b>I</b>) GFP-Nup98-NSD3 and (<b>K</b>) GFP-Nup98-RARG. Nup98 fused to partners that lack chromatin-binding domains localize more dispersed to the nucleoplasm: (<b>L</b>) GFP-Nup98-LEDGF. Disruption of the FG, the HD or the PHD domain disrupts the localization of the Nup98 chimeras: (<b>M</b>) GFP-Nup98-PMX1 N51S, (<b>N</b>) GFP-Nup98-HOXA9 ΔFG, (<b>O</b>) GFP-Nup98-HOXA9 N51S, (<b>P</b>) GFP-Nup98-HHEX ΔFG, (<b>Q</b>) GFP-Nup98-HHEX ΔHD, and (<b>R</b>) GFP-Nup98-JARID1A W1625A. (<b>S</b>) GFP-HOXA9 and (<b>T</b>) GFP-HHEX localize to the nucleoplasm. Shown are representative confocal images. Scale bars, 5 μm.</p

Mitotic localization of Nup98 chimeras.

<p>HeLa cells were transiently transfected with GFP constructs and fixed and stained after 24 hours for immunofluorescence microscopy. (<b>A</b>) CREST serum, which in particular recognizes CENP-B [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152321#pone.0152321.ref036" target="_blank">36</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152321#pone.0152321.ref055" target="_blank">55</a>], was used to detect the inner kinetochore and DAPI to visualize DNA. Nup98-HD fusion proteins (Nup98-HOXA9, Nup98-HHEX, Nup98-PMX1; green) associate with chromatin (blue), but not with the inner kinetochore (red) during prometaphase. No association with chromatin was found for Nup98 or Nup98 fused to non-HD partners (i.e. Nup98-JARID1A and Nup98-RARG). Disruption of the HD domain of Nup98-HOXA9 (Nup98-HOXA9 N51S), but not of the FG domain (Nup98-HOXA9 ΔFG) affects chromatin association of the fusion protein. Shown are single confocal sections. Scale bars, 5 μm. (<b>B</b>) Anti-Hec1 antibodies were used to detect the outer kinetochore (red), but no co-localization of Nup98-HOXA9, Nup98-HHEX, and Nup98-PMX1, respectively (green) was observed in prometaphase cells. The fusion proteins exclusively associated with chromatin (blue). Shown are single confocal sections. Scale bars, 5 μm.</p

Expression of Nup98 fusions perturbs the nuclear distribution of lamina-associated polypeptide 2α (LAP2α).

<p>HeLa cells were transiently transfected with GFP constructs and fixed and stained after 24 hours for immunofluorescence microscopy. (<b>A</b>) Lamin A/C (LA/C, red) concentrates at the nuclear envelope in HeLa control cells and in Nup98 expressing cells (green), while LAP2α (magenta) is found throughout the nucleoplasm. In HeLa cells expressing Nup98-HOXA9 (<b>A</b>) and Nup98-HHEX (<b>B</b>), LAP2α diminished from the nucleoplasm and aggregates at the nuclear periphery. Disruption of the homeodomain in HOXA9 (<b>A</b>) and HHEX (<b>B</b>) and the FG domain of Nup98 (<b>A</b> and <b>B</b>) prevent the relocation of the lamina proteins. DAPI was used to visualize DNA (merge). (<b>B</b>) Fluorescence intensity of LAP2α staining was determined along the axis shown as line in the fluorescence images and plotted as a graph. (<b>D</b>) Quantification of cells with altered LA/C and LAP2α distribution. About 400 cells were analyzed for each sample. (<b>E</b>) Western blot analysis of the expression levels of LA/C, LAP2α, and LB1.</p

Cell cycle analysis of transiently transfected HeLa cells after double thymidine block.

<p>Cell cycle analysis of transiently transfected HeLa cells after double thymidine block.</p

Electron micrographs of HeLa cells expressing Nup98 chimeras.

<p>(<b>A</b>) and (<b>B</b>) Nup98-HOXA9, (<b>C</b>) and (<b>D</b>) Nup98-HHEX, control cells expressing (<b>E</b>) GFP and (<b>F</b>) Nup98. HeLa TRex cells expressing (<b>G</b>) Nup98 and (<b>H</b>) Nup98-HOXA9. Scale bars, 2 μm (A, C, E, F, G, H); 1 μm (B and D).</p

Nup98-HOXA9 affects lamin A/C and lamin B1 distribution.

<p>HeLa cells were transiently transfected with GFP constructs and fixed and stained after 24 hours for immunofluorescence microscopy. (<b>A</b>) Lamin A/C (LA/C, red) and lamin B1 (LB1, magenta) concentrate at the nuclear envelope (NE) in HeLa cells expressing Nup98 (green), but relocate to the nucleoplasm in cells expressing Nup98-HOXA9. White arrowheads point to some lobules decorating the NE. Disruption of the homeodomain of HOXA9 (Nup98-HOXA9 N51S) and the FG domain of Nup98 (Nup98-HOXA9 ΔFG) prevent the relocation of the lamina proteins. Scale bars, 5 μm. (<b>B</b>) Fluorescence intensity of LA/C (left) and LB1 (right) staining was determined along the axis shown as line in the fluorescence images and plotted as a graph.</p