IntroductionFanconi anemia (FA) is a rare recessive syndrome featuring progressive bone marrow (BM) failure, multiple developmental abnormalities and cancer predisposition. 1-3 BM failure and its related consequences, such as pancytopenia or acute myeloid leukemia (AML), are the major cause of morbidity and mortality of FA patients. 4 The cellular phenotype is characterized by chromosomal instability and hypersensitivity to DNA interstrand crosslink (ICL)-inducing agents such as mitomycin C (MMC), diepoxybutane, and cisplatin. [5][6][7][8] At least 13 complementation groups have been identified (FA-A, B, C, D1, D2, E, F, G, I, J, L, M, and N) and the genes for all of these groups have been cloned. 9 One of the major functions of FANC proteins is to deal with DNA damage, thus participating in an as-yet-undefined manner to the repair of DNA lesions induced by cross-linking agents. [10][11][12] However, the spectrum of clinical and cellular abnormalities of the syndrome suggests that FANC proteins could have other functions or participate in pathways other than DNA repair. 13,14 Whether the hematologic problems of the FA patients are a consequence of a defect in DNA repair or in other potential functions of the FANC proteins remains to be determined.Tumor necrosis factor-␣ (TNF-␣) is a major cytokine involved in hematopoiesis, inflammation, and apoptosis. 15,16 TNF-␣ is synthesized as a membrane-bound precursor of 26 kDa that can be processed to generate a secreted 17-kDa mature TNF-␣. 17,18 Soluble mature TNF-␣ is released from the cells by cleavage of the precursor at the Ala76-Val77 bond by the TNF-␣ converting enzyme (TACE or ADAM17) 19,20 or, less efficiently, by the matrix metalloproteinase 7 (MMP-7 or matrilysin). 21,22 TNF-␣ signals through 2 distinct cell-surface receptors, TNFR-1 and TNFR-2. 16 The binding of TNF-␣ to its receptors results, among other events, in the activation of both the mitogen-activated protein kinases (MAPKs) stress signaling cascade 16 and the NF-B transcription factor. 23 Activation of the MAPKs and NF-B plays an important role in the induction of many cytokines including the TNF-␣ itself. 24,25 TNF-␣ negatively regulates the expansion and selfrenewal of pluripotent hematopoietic stem cells (HSCs) 26,27 and has inhibitory effects on normal human hematopoietic progenitor cells as well as leukemia progenitor cells. [28][29][30] Consistently, TNF-␣ overproduction has been associated with different hematopoietic disorders such as myelodysplastic syndrome (MDS), AML, and aplastic anemia. [31][32][33] FA syndrome recapitulates all these abnormalities, that is, impaired HSC expansion and development of the myeloid lineages, MDS, aplastic anemia, and AML. 2-4 FA is also characterized by TNF-␣ overproduction, both in vivo and in vitro. Indeed, it has been reported that TNF-␣ is (1) overexpressed in BM of FA patients, 34,35 (2) increased in the serum of patients, 35,36 and (3) overproduced by Epstein-Barr virus (EBV)-transformed FA lymphoblasts. 36 Moreover, hematopoietic progenitors from ...

Fanconi anemia (FA) is a cancer-prone hereditary disease resulting from mutations in one of the 13 genes defining the FANC/BRCA pathway. This pathway is involved in the cellular resistance to DNA-cross-linking agents. How the FANC/BRCA pathway is activated and why its deficiency leads to the accumulation of FA cells with a 4N DNA content are still poorly answered questions. We investigated the involvement of ATR pathway members in these processes. We show here that RAD9 and RAD17 are required for DNA interstrand cross-link (ICL) resistance and for the optimal activation of FANCD2. Moreover, we demonstrate that CHK1 and its interacting partner CLASPIN that act downstream in the ATR pathway are required for both FANCD2 monoubiquitination and assembling in subnuclear foci in response to DNA damage. Paradoxically, in the absence of any genotoxic stress, CHK1 or CLASPIN depletion results in an increased basal level of FANCD2 monoubiquitination and focalization. We also demonstrate that the ICL-induced accumulation of FA cells in late S/G2 phase is dependent on ATR and CHK1. In agreement with this, CHK1 phosphorylation is enhanced in FA cells, and chemical inhibition of the ATR/CHK1 axis in FA lymphoblasts decreases their sensitivity to mitomycin C. In conclusion, this work describes a complex crosstalk between CHK1 and the FANC/BRCA pathway: CHK1 activates this pathway through FANCD2 monoubiquitination, whereas FA deficiency leads to a CHK1-dependent G2 accumulation, raising the possibility that the FANC/BRCA pathway downregulates CHK1 activation.