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 4

NIPA interacts with FANCD2.

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NIPA interacts with FANCD2. (A) FLAG coimmunoprecipitation of Phoenix E ...
(A) FLAG coimmunoprecipitation of Phoenix E cells transiently transfected with FLAG-hNIPA. (B) FANCD2 coimmunoprecipitation of Phoenix E cells transiently transfected with FLAG-hNIPA. (C) NIPA coimmunoprecipitation of HeLa cells with endogenous levels of NIPA. (D) FANCD2 coimmunoprecipitation of HeLa cells with endogenous levels of NIPA. (E) Western blot analysis of HeLa cells retrovirally transfected with pLMP miRCtrl or miRNipa for FANCD2, normalized to GAPDH. (F) Western blot analysis of Nipa+/+ and Nipa–/– primary MEFs for FANCD2, NIPA, and GAPDH. Quantification of relative FANCD2 protein levels normalized to GAPDH of Nipa+/+ and Nipa–/– primary MEFs. n = 13 Nipa+/+; n = 13 Nipa–/–. (G) Quantification of relative nonubiquitinated (Non-UB) and monoubiquitinated (Mono-UB) FANCD2 protein levels of Nipa+/+ and Nipa–/– primary MEFs analyzed by Western blot. n = 13 Nipa+/+; n = 13 Nipa–/–. (H) Western blot analysis of nuclear and cytosolic extracts of Nipa+/+ and Nipa–/– primary MEFs for FANCD2, lamin A/C, and tubulin. (I) Quantification of relative (rel.) FANCD2 protein levels normalized to lamin A/C (nuclear) or tubulin (cytosolic) of Nipa+/+ and Nipa–/– primary MEFs. n = 8 Nipa+/+; n = 8 Nipa–/–. (J) Western blot analysis of non- and mono-UB FANCD2 levels of Nipa+/+ and Nipa–/– primary MEFs (steady state) treated with cycloheximide for the indicated times. n =4 Nipa+/+; n = 4 Nipa–/–. (K) Western blot analysis of non- and mono-UB FANCD2 levels of Nipa+/+ and Nipa–/– primary MEFs (6 hours, 0.5 μM MMC) treated with cycloheximide for the indicated times. n = 5 Nipa+/+; n = 5 Nipa–/–. (L) Immunofluorescence for FANCD2, NIPA, and DAPI in untreated and 4-hour MG132–treated (5 μM) in HeLa cells retrovirally transfected with pLMP miRCtrl or miRNipa. Representative confocal microscopy images are shown. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. A paired 2-tailed Student’s t test Original magnification, ×63. (F, G, and I) or Dunnett’s test (J and K) was used for statistical analyses. Data are presented as mean ± SD. See also Supplemental Figures 6–9.