Fanconi anemia is a genetic disorder characterized by bone marrow failure. Significant evidence supports enhanced apoptosis of hematopoietic stem/progenitor cells as a critical factor in the pathogenesis of bone marrow failure in Fanconi anemia. However, the molecular mechanism(s) responsible for the apoptotic phenotype are incompletely understood. Here, we tested whether alterations in the activation of a redox-dependent pathway may participate in the pro-apoptotic phenotype of primary Fancc ؊/؊ cells in response to oxidative stress. Our data indicate that Fancc ؊/؊ cells are highly sensitive to oxidant stimuli and undergo enhanced oxidant-mediated apoptosis compared with wild type controls. In addition, antioxidants preferentially enhanced the survival of Fancc ؊/؊ cells. Because oxidative stress activates the redox-dependent ASK1 pathway, we assessed whether Fancc ؊/؊ cells exhibited increased oxidant-induced ASK1 activation. Our results revealed ASK1 hyperactivation in H 2 O 2 -treated Fancc ؊/؊ cells. Furthermore, using small interfering RNAs to decrease ASK1 expression and a dominant negative ASK1 mutant to inhibit ASK1 kinase activity, we determined that H 2 O 2 -induced apoptosis was ASK1-dependent. Collectively, these data argue that the predisposition of Fancc ؊/؊ hematopoietic stem/progenitor cells to apoptosis is mediated in part through altered redox regulation and ASK1 hyperactivation.

Fanconi anemia (FA) is a chromosomal instability disorder characterized by a progressive bone marrow (BM) failure and an increased incidence of myeloid leukemias. Children with FA are currently being enrolled in clinical trials to evaluate the safety of retroviral-mediated gene transfer. Previously, we used Fancc ؊/؊ mice to show that Fancc ؊/؊ hematopoietic stem cells (HSCs) have a profound defect in repopulating ability. Here, we examined whether retroviral-mediated gene transfer of recombinant Fancc (rFancc) would restore the repopulating ability of Fancc ؊/؊ HSC to wild-type levels. Fancc ؊/؊ HSCs transduced with a retrovirus encoding rFancc exhibited a repopulating ability that approached wild-type levels. Interestingly, ϳ30% of primary recipients (7 of 22) transplanted with uncorrected Fancc ؊/؊ cells developed a range of hematopoietic abnormalities including pancytopenia and BM hypoplasia similar to individuals with FA. Hematopoietic abnormalities were detected in only 1 of 22 mice transplanted with Fancc ؊/؊ cells transduced with a retrovirus encoding rFancc. Moreover, several mice with hematopoietic defects had progenitors that displayed a marked resistance to IFN-␥, TNF-␣, and MIP-1␣ compared to both Fancc ؊/؊ progenitors, which are uniquely hypersensitive to these cytokines, and wild-type progenitors. These data are analogous to studies using progenitors from patients with myelodysplasia and provide functional support for clonal evolution in these mice. Collectively, these data show that gene transfer can enhance HSC repopulating ability and suppresses the tendency for clonal evolution. These studies also reveal potential detrimental effects of ex vivo manipulation for untransduced Fancc ؊/؊

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Fanconi anemia (FA) is characterized by bone marrow (BM) failure and cancer susceptibility. Identification of the cDNAs of many FA complementation types allows the potential of using gene transfer technology to introduce functional cD-NAs as transgenes into autologous stem cells and provide a cure for the BM failure in FA patients. Previous studies in FA murine models and in a phase 1 clinical trial suggest that myelopreparation is required for significant engraftment of exog-enous, genetically corrected stem cells. Since myeloid progenitors from Fancc / mice and human Fanconi anemia group C protein (FANCC) patients have increased apoptosis in response to interferon (IFN-) in vitro, we hypothesized that IFN-may be useful as a nongenotoxic, myelo-preparative conditioning agent. To test this hypothesis, IFN-was administered as a continuous infusion to Fancc / and wild-type (WT) mice for 1 week. Primitive and mature myeloid lineages were preferentially reduced in IFN-treated Fancc / mice. Further, IFN-conditioning of Fancc / recipients was sufficient as a myelopreparative regimen to allow consistent engraftment of isogenic WT repopu-lating stem cells. Collectively, these data demonstrate that Fancc / hematopoietic cell populations have increased hypersen-sitivity to IFN-in vivo and that IFN-conditioning may be useful as a nongeno-toxic strategy for myelopreparation in this

The pathogenesis of bone marrow failure in Fanconi anemia is poorly understood. Suggested mechanisms include enhanced apoptosis secondary to DNA damage and altered inhibitory cytokine signaling. Recent data determined that disrupted cell cycle control of hematopoietic stem and/or progenitor cells disrupts normal hematopoiesis with increased hematopoietic stem cell cycling resulting in diminished function and increased sensitivity to cell cycle–specific apoptotic stimuli. Here, we used Fanconi anemia complementation type C–deficient (Fancc–/–) mice to demonstrate that Fancc–/– phenotypically defined cell populations enriched for hematopoietic stem and progenitor cells exhibit increased cycling. In addition, we established that the defect in cell cycle regulation is not a compensatory mechanism from enhanced apoptosis occurring in vivo. Collectively, these data provide a previously unrecognized phenotype in Fancc–/– hematopoietic stem/progenitor cells, which may contribute to the progressive bone marrow failure in Fanconi anemia.

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