Microphthalmia transcription factor expression contributes to bone marrow failure in Fanconi anemia
Alessia Oppezzo, … , Patrycja Pawlikowska, Filippo Rosselli
Alessia Oppezzo, … , Patrycja Pawlikowska, Filippo Rosselli
Published December 26, 2019
Citation Information: J Clin Invest. 2020;130(3):1377-1391. https://doi.org/10.1172/JCI131540.
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Research Article Cell biology Hematology

Microphthalmia transcription factor expression contributes to bone marrow failure in Fanconi anemia

Abstract

Hematopoietic stem cell (HSC) attrition is considered the key event underlying progressive BM failure (BMF) in Fanconi anemia (FA), the most frequent inherited BMF disorder in humans. However, despite major advances, how the cellular, biochemical, and molecular alterations reported in FA lead to HSC exhaustion remains poorly understood. Here, we demonstrated in human and mouse cells that loss-of-function of FANCA or FANCC, products of 2 genes affecting more than 80% of FA patients worldwide, is associated with constitutive expression of the transcription factor microphthalmia (MiTF) through the cooperative, unscheduled activation of several stress-signaling pathways, including the SMAD2/3, p38 MAPK, NF-κB, and AKT cascades. We validated the unrestrained Mitf expression downstream of p38 in Fanca–/– mice, which display hallmarks of hematopoietic stress, including loss of HSC quiescence, DNA damage accumulation in HSCs, and reduced HSC repopulation capacity. Importantly, we demonstrated that shRNA-mediated downregulation of Mitf expression or inhibition of p38 signaling rescued HSC quiescence and prevented DNA damage accumulation. Our data support the hypothesis that HSC attrition in FA is the consequence of defects in the DNA-damage response combined with chronic activation of otherwise transiently activated signaling pathways, which jointly prevent the recovery of HSC quiescence.

Authors

Alessia Oppezzo, Julie Bourseguin, Emilie Renaud, Patrycja Pawlikowska, Filippo Rosselli

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Figure 5

p38 Abnormalities in HSCs from Fanca–/– mice.

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p38 Abnormalities in HSCs from Fanca–/– mice. (A) Immunohistochemical st...
(A) Immunohistochemical staining of p-p38 in BM sections of WT and Fanca–/– mice treated or not with 50 mg/kg SB203580 i.p. daily for 12 days. Scale bars: 100 μm. (B) p-p38 Protein expression level measured by FACS (right) in LSK cells from untreated WT and Fanca–/– mice or from WT mice treated i.p. with pI:pC (5 mg/kg) 24 hours before. Each point represents a single mouse. (C) Colony-forming hematopoietic progenitor frequency counted 7 days after plating of LSK cells from WT and Fanca–/– mice (n = 3) treated 24 hours before with pI:pC (5 mg/kg). LSK cells were cultured with or without SB203580 (20 μM). (D) WT and Fanca–/– colony-forming hematopoietic progenitor frequency after a dose of pI:pC and in vivo treatment with SB203580 (50 mg/kg). Each point represents a single mouse. (E) Successive seeding of WT and Fanca–/– progenitors with or without TNF-α in MethoCult 3434 medium after replating at 3 × 103 cells per dish in triplicate. Each point represents the mean value of 3 independent experiments started with 3 different mice. (F) Middle: Gating strategy to assess the percentage of cycling cells through the use of the Ki67+ intracellular marker. Bottom: percentages of cycling LT-HSCs from WT and Fanca–/– mice treated or not with a dose of SB203580 (50 mg/kg) for 24 hours. Each point represents a single mouse. (G) Bottom: Quantification of γ-H2ax foci per cell (left) or γ-H2ax-positive LSK cells (right) sorted from mice treated with a dose of pI:pC (5 mg/kg), followed or not by successive treatment with SB203580. Middle: representative image of Fanca–/– or Fanca–/– LSK cells treated with SB203580. Scale bars: 10 μm. Each point represents a single mouse. Statistical significance was assessed using an unpaired 2-tailed t test with Welch’s correction (C, D, F, and G) or 1-way ANOVA with Dunnett’s correction (B). *P < 0.05; **P < 0.01; ***P < 0.001.