Spontaneous abrogation of the G2 DNA damage checkpoint has clinical benefits but promotes leukemogenesis in Fanconi anemia patients
Raphael Ceccaldi, Delphine Briot, Jérôme Larghero, Nadia Vasquez, Catherine Dubois d’Enghien, Delphine Chamousset, Maria-Elena Noguera, Quinten Waisfisz, Olivier Hermine, Corinne Pondarre, Thierry Leblanc, Eliane Gluckman, Hans Joenje, Dominique Stoppa-Lyonnet, Gérard Socié, Jean Soulier
Raphael Ceccaldi, Delphine Briot, Jérôme Larghero, Nadia Vasquez, Catherine Dubois d’Enghien, Delphine Chamousset, Maria-Elena Noguera, Quinten Waisfisz, Olivier Hermine, Corinne Pondarre, Thierry Leblanc, Eliane Gluckman, Hans Joenje, Dominique Stoppa-Lyonnet, Gérard Socié, Jean Soulier
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Research Article

Spontaneous abrogation of the G2 DNA damage checkpoint has clinical benefits but promotes leukemogenesis in Fanconi anemia patients

Abstract

DNA damage checkpoints in the cell cycle may be important barriers against cancer progression in human cells. Fanconi anemia (FA) is an inherited DNA instability disorder that is associated with bone marrow failure and a strong predisposition to cancer. Although FA cells experience constitutive chromosomal breaks, cell cycle arrest at the G2 DNA damage checkpoint, and an excess of cell death, some patients do become clinically stable, and the mechanisms underlying this, other than spontaneous reversion of the disease-causing mutation, are not well understood. Here we have defined a clonal phenotype, termed attenuation, in which FA patients acquire an abrogation of the G2 checkpoint arrest. Attenuated cells expressed lower levels of CHK1 (also known as CHEK1) and p53. The attenuation could be recapitulated by modulating the ATR/CHK1 pathway, and CHK1 inhibition protected FA cells from cell death. FA patients who expressed the attenuated phenotype had mild bone marrow deficiency and reached adulthood, but several of them eventually developed myelodysplasia or leukemia. Better understanding of attenuation might help predict a patient’s clinical course and guide choice of treatment. Our results also highlight the importance of evaluating the cellular DNA damage checkpoint and repair pathways in cancer therapies in general.

Authors

Raphael Ceccaldi, Delphine Briot, Jérôme Larghero, Nadia Vasquez, Catherine Dubois d’Enghien, Delphine Chamousset, Maria-Elena Noguera, Quinten Waisfisz, Olivier Hermine, Corinne Pondarre, Thierry Leblanc, Eliane Gluckman, Hans Joenje, Dominique Stoppa-Lyonnet, Gérard Socié, Jean Soulier

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Figure 1

Attenuation of G2 arrest — a new phenotype in FA.

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Attenuation of G2 arrest — a new phenotype in FA. (A) Cell cycle ana...
(A) Cell cycle analysis of PHA-stimulated PBLs from a classical FA patient (FA), an attenuated FA patient (ATT), a revertant (REV; somatic mosaic), and a healthy control (Ctrl). The arrow indicates the typical MMC-induced G2 arrest of classical FA cells, and the asterisk designates the G2 checkpoint abrogation in the attenuated cells; as expected, revertant cells demonstrated no G2 arrest. (B) Cell cycle analysis of primary fibroblasts (fibro) from the same patients, showing G2 arrest (arrow) and confirming the constitutive FA phenotype and dissociation with PBLs in attenuated and revertant patients. Horizontal bars in A and B indicate the G1 and G2 cell cycle phases (M1 and M2, respectively). (C) Immunoblot analysis showed that attenuated FA PBLs lacked the large monoubiquitinated 162-kDa isoform of FANCD2 (asterisk), like classical FA cells and unlike revertant cells; primary fibroblasts retained the FA phenotype. (D) Attenuated cells still have a high number of MMC-induced chromosomal breaks, like classical FA cells and unlike revertant cells (original magnification, ×630). Arrows indicate the chromosome breaks. Breaks scoring are shown in Supplemental Figure 1. (E) Classification of the FA patients as having classical, revertant, and attenuated PBL phenotypes and ordering by class age: fewer than 5 years (n = 9; 9.5%), 5–9 years (n = 31; 32.6%), 10–14 years (n = 15; 15.8%), 15–19 years (n = 11; 11.6%), 20–24 years (n = 10; 10.5%), 25–29 years (n = 9; 9.5%), and more than 30 years (n = 10; 10.5%). For the clarity of the figure, 2 patients whose fibroblasts were later found to be intermediate for G2 arrest are not shown (see text).