Loss of the Fanconi anemia–associated protein NIPA causes bone marrow failure
Stefanie Kreutmair, … , Justus Duyster, Anna Lena Illert
Stefanie Kreutmair, … , Justus Duyster, Anna Lena Illert
Published April 27, 2020
Citation Information: J Clin Invest. 2020;130(6):2827-2844. https://doi.org/10.1172/JCI126215.
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Research Article Hematology

Loss of the Fanconi anemia–associated protein NIPA causes bone marrow failure

Abstract

Inherited bone marrow failure syndromes (IBMFSs) are a heterogeneous group of disorders characterized by defective hematopoiesis, impaired stem cell function, and cancer susceptibility. Diagnosis of IBMFS presents a major challenge due to the large variety of associated phenotypes, and novel, clinically relevant biomarkers are urgently needed. Our study identified nuclear interaction partner of ALK (NIPA) as an IBMFS gene, as it is significantly downregulated in a distinct subset of myelodysplastic syndrome–type (MDS-type) refractory cytopenia in children. Mechanistically, we showed that NIPA is major player in the Fanconi anemia (FA) pathway, which binds FANCD2 and regulates its nuclear abundance, making it essential for a functional DNA repair/FA/BRCA pathway. In a knockout mouse model, Nipa deficiency led to major cell-intrinsic defects, including a premature aging phenotype, with accumulation of DNA damage in hematopoietic stem cells (HSCs). Induction of replication stress triggered a reduction in and functional decline of murine HSCs, resulting in complete bone marrow failure and death of the knockout mice with 100% penetrance. Taken together, the results of our study add NIPA to the short list of FA-associated proteins, thereby highlighting its potential as a diagnostic marker and/or possible target in diseases characterized by hematopoietic failure.

Authors

Stefanie Kreutmair, Miriam Erlacher, Geoffroy Andrieux, Rouzanna Istvanffy, Alina Mueller-Rudorf, Melissa Zwick, Tamina Rückert, Milena Pantic, Teresa Poggio, Khalid Shoumariyeh, Tony A. Mueller, Hiroyuki Kawaguchi, Marie Follo, Cathrin Klingeberg, Marcin Wlodarski, Irith Baumann, Dietmar Pfeifer, Michal Kulinski, Martina Rudelius, Simone Lemeer, Bernhard Kuster, Christine Dierks, Christian Peschel, Nina Cabezas-Wallscheid, Jesus Duque-Afonso, Robert Zeiser, Michael L. Cleary, Detlev Schindler, Annette Schmitt-Graeff, Melanie Boerries, Charlotte M. Niemeyer, Robert A.J. Oostendorp, Justus Duyster, Anna Lena Illert

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Figure 6

Fancd2 restoration can overcome the defects of Nipa-deficient cells.

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Fancd2 restoration can overcome the defects of Nipa-deficient cells. (A...
(A) Immunofluorescence for FANCD2, NIPA and DAPI, in untreated and 6-hour MMC–treated (0.5 μM) Nipa+/+ and Nipa–/– primary MEFs retrovirally transfected with pBABENipaWT or pBABENipaΔF-box. Representative confocal microscopy images are shown. Original magnification, ×63. (B) Average fluorescence intensity of FANCD2 staining in the nucleus and the cytosol of the Nipa+/+ and Nipa–/– primary MEFs (+6 hour MMC) shown in A analyzed by image cytometry. Box-and-whisker plots: 10th to 90th percentile, with outliers aligned. Nuclear FANCD2: n = 4622 Nipa+/+; n = 5035 Nipa–/–; n = 722 Nipa–/– + Nipa WT; n = 315 Nipa–/– + Nipa ΔF-box. Cytosolic FANCD2: n = 3929 Nipa+/+; n = 4414 Nipa–/–; n = 593 Nipa–/– + Nipa WT; n =287 Nipa–/– + Nipa ΔF-box. Reported P values in the figure from Dunnett’s test. (C) Nipa+/+ or Nipa–/– BMCs were retrovirally infected with pMIGempty, pMIGNipaWT, or pMIGNipaΔF-box and used for in vitro CFU assay. Quantification of colonies is shown. n = 4 Nipa+/+; n = 4 Nipa–/–; n = 4 Nipa–/– + Nipa WT; n = 4 Nipa–/– + Nipa ΔF-box. Reported P values in the figure from Dunnett’s test. (D) Representative images of CFU assay described in C. Scale bars: 1000 μm. (E) Cell survival assay of Nipa+/+ and Nipa–/– primary MEFs lentivirally infected with pCRempty or pCRFancd2 measured after 5 days of culture with the indicated concentrations of MMC. Data from 2 independent tests are shown. n = 9 Nipa+/+ + pCRempty; n = 6 Nipa–/– + pCRempty; n = 6 Nipa–/– + pCRFancd2. One-way ANOVA, P = 0.004 (10 nM); P = 0.014 (20 nM). Reported P values in the figure from unpaired 2-tailed Student’s t test. (F) Nipa+/+ or Nipa–/– BMCs were lentivirally infected with pCRempty or pCRFancd2 and used for in vitro CFU assay. Representative images and quantification of colonies are shown. Scale bar: 1000 μm. n = 2 Nipa+/+ + pCRempty; n =2 Nipa–/– + pCRempty; n = 2 Nipa–/– +pCRFancd2. One-way ANOVA, P = 0.015. Reported P values in the figure from unpaired 2-tailed Student’s t test. (G) Representative Western blot of BMCs used for CFU assay shown in A. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Data are represented as mean ± SD. See also Supplemental Figures 11 and 12.