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 1

FANC core complex loss of function is associated with MiTF overexpression.

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FANC core complex loss of function is associated with MiTF overexpressio...
(A) Representative Western blots illustrating MiTF expression in lymphoblasts from WT healthy (HSC93, SNW646, GM0131) or FA donors HSC99 (FANCA–/–, FA-A), HSC536 (FANCC–/–, FA-C), HSC72 (FANCA–/–, FA-A), GM16756 (FANCD2–/–, FA-D2), or FA cells complemented with the corresponding WT FANC gene (HSC536CORR and HSC72CORR). We performed at least 5 individual experiments for each cell line, with similar results. (B) qRT-PCR of MiTF mRNA expression in the indicated cell lines. In each experiment, MiTF expression was first normalized to that of Actin (internal control) and then normalized to the MiTF/Actin ratio in control HSC93 cells, which was set as 1 in each experiment. Data are shown as mean ± SEM of n = 3 (FANCM–/–, GM16756), n = 6 (HSC72, HSC72CORR, HSC536, HSC536CORR), and n = 18 (HSC93, HSC99) experiments. (C) RT-PCR analysis of MiTF-A, MiTF-C, MiTF-E, and MiTF-M isoforms in cells from WT (HSC93, SNW646) or FA (HSC72, HSC99, HSC536) donors. (D) Representative Western blots (n = 3) showing Mitf expression in WT, Fanca–/–, or Fancc–/– MEFs collected at the indicated time points after seeding. (E) qRT-PCR of Mitf expression in the same cells and conditions described in D. qRT-PCR data were normalized against Oaz1 RNA and normalized to the Mitf/Oaz1 ratio in WT cells at 24 hours, which was set as 1. Data are shown as mean ± SEM of n = 5 experiments. (F) Representative Western blot (n = 2, left) and qRT-PCR data (right) showing Mitf expression in WT, Fanca–/–, or Fancd2–/– MEFs 72 hours after seeding. RNA samples were analyzed as described in E. Data are shown as mean ± SEM of n = 3 experiments. β-Actin was used as a loading control for A, D, and F. Statistical significance was assessed using an unpaired 2-tailed t test with Welch’s correction (B and F) or 1-way ANOVA with Bonferroni’s correction (E). *P < 0.05; **P < 0.01; ***P < 0.001.