Spontaneous abrogation of the G₂DNA 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.

Figure 1. Attenuation of G₂ arrest — a new phenotype in FA.

(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).

Figure 2. Clonality of attenuated FA cells.

(A) Array-CGH showed clonal chromosomal abnormalities in PHA-stimulated PBLs from an attenuated patient. MGG stains confirmed the activated lymphoblast morphology (original magnification, ×400). Arrows indicate copy number abnormalities. (B) X-linked–based analysis of clonality; female FA patients who were heterozygous for 1 or more of the 3 SNPs in the X chromosome were identified by direct sequencing of genomic DNA. Then, allelic X-linked inactivation was evaluated in a semiquantitative manner by sequencing PBL cDNA from these patients. The left panel shows balanced expression of the heterozygous MMP1 SNP in a healthy control and a classical FA patient, whereas skewed expression was detected in the attenuated and revertant patients (asterisks). The right panel summarizes the X-linked clonality data in heterozygous PBLs from healthy female controls (n = 11), attenuated patients (n = 5), revertants (n = 1), and classical FA subjects (n = 6). The percentage of skewing expression is quantified for each case, and median values are indicated by horizontal bars. The threshold for skewed unbalanced expression was arbitrarily fixed (dashed horizontal line). Each symbol represents a patient.

Figure 3. Low expression of CHK1 and p53, but not CHK2, in attenuated FA patients.

(A) Immunoblot analysis of PHA-stimulated PBL extracts from an attenuated FA patient, 2 classical FA subjects, and a healthy donor, with increasing concentrations of MMC. (B) CHK1 and CHK2 levels were quantified by RQ-PCR in PHA-stimulated PBLs of classical FA patients (n = 11), attenuated FA patients (n = 8), and healthy donors (n = 3). RQ-PCR copy number values are expressed using the ΔCT method relative to a housekeeping gene (reverse log₂ scale). Mean values are indicated by horizontal bars. Statistical analyses were performed using the non-parametric Wilcoxon test (*P < 0.05). Each symbol represents a patient. (C) As shown in the left panel, proteasome inhibition partially restored CHK1 levels in the attenuated cells. Attenuated and control PHA-stimulated PBLs were incubated with the proteasome inhibitor MG132 or control DMSO for 4 hours. Then the level of CHK1 protein was analyzed by immunoblotting. As shown in the right panel, translational inhibition by CHX treatment revealed an increase of CHK1 decay in the attenuated PHA-stimulated PBLs compared with that in control PHA-stimulated PBLs. The CHK2 level was analyzed in parallel as a more stable protein.

Figure 4. Inhibition of CHK1 and ATR, but not CHK2, ATM, or BRCA1, mimics the attenuated phenotype in FA cells.

(A) Cell cycle analysis of MMC-induced G₂ arrest in HeLa cells transfected with combinations of siRNAs (si) against luciferase (control), FANCD2, and the checkpoint genes CHK1, CHK2, ATR, ATM, and BRCA1, as indicated. FANCD2-silenced HeLa cells displayed a strong MMC-induced G₂ arrest and were used as FA-like cells for convenience in cotransfection experiments. The immunoblot shows the knockdown of different proteins for each siRNA. Arrows and asterisks indicate typical FA G₂ arrest and its abrogation, respectively. All data were obtained in at least 3 independent experiments for each targeted gene with consistent results. (B) Abrogation of MMC-induced G₂ arrest by the CHK1 inhibitor SB-218078 (CHK1i) but not by the CHK2 inhibitor C3742 (CHK2i) in FA and non-FA EBV cells. DMSO was used as control, and all data were obtained in 3 independent experiments with consistent results. The arrows indicate the MMC-induced G₂ arrest, and the asterisk designates the G₂ checkpoint abrogation. Horizontal bars in A and B indicate the G₁ and G₂ cell cycle phases (M1 and M2, respectively).

Figure 5. CHK1 inhibitor protects FA cells from DNA damage-induced cell death in short-term cultures.

(A) Representative flow cytometry–based analysis of cell death of FA primary fibroblasts, with or without CHK1i. The fraction of cellular fragments (sub-G₁, bars) was measured in each condition. Consistent data were obtained in at least 2 experiments for each of 4 unrelated FA patients. (B) Graphic representation of the percentage of cellular fragments measured in A. Annexin V staining is shown in Supplemental Figure 5. (C) MGG staining of FA primary fibroblasts with or without CHK1i treatment with 25 ng/ml MMC (original magnification, ×160).

Figure 6. Low expression of CHK1, but not CHK2, in the leukemic cells from MDS/AML FA patients (n = 4).

Leukemic cells were purified by CD34⁺ selection; the clonality and high purity of the blast cell fraction was demonstrated in all of the 4 MDS/AML FA patients by array-CGH profiling, which detected chromosomal alterations (data not shown). Data from classical FA patient PHA-stimulated PBLs are indicated as reference (several samples were reanalyzed with consistent results). Leukemic cells from a series of non-FA patients (n = 15) with AML were analyzed as malignant reference. RQ-PCR copy number values are expressed using the ΔCT method relative to a housekeeping gene (reverse log₂ scale). Mean values are indicated by horizontal bars. Statistical analyses were performed using the non-parametric test Wilcoxon test (**P < 0.001). As expected from published data in MDS and AML in non-FA patients (32), very low levels of CHK1 transcripts were also found in the non-FA AML cells. Each symbol represents a patient.

Figure 7. Genomic instability, DNA damage response, and stepwise oncogenesis — a model of stepwise progression to MDS/AML in patients with or without constitutive genetic instability.

In FA patients, the first step is constitutive and leads to excess cell death related to the G₂ checkpoint response; the attenuation phenomenon described here rescues cell survival and allows for the accumulation of additional oncogenic events that are favored by the genetic instability. In patients that are not genetically predisposed (non-FA patients), activation of oncogenes is associated with acquired genetic instability and induction of anticancer DNA damage response (–8); further inactivation of this response, often by TP53 inactivation, allows cancer progression to late stages (–8, 32, 44, 47). Notably, no TP53 mutation or deletion was observed in the 5 MDS/AML leukemia cases that developed in attenuated FA patients in our series (data not shown).