Improving approaches for hematopoietic stem cell (HSC) and hematopoietic progenitor cell (HPC) mobilization is clinically important because increased numbers of these cells are needed for enhanced transplantation. Chemokine stromal cell derived factor-1 (also known as CXCL12) is believed to be involved in retention of HSCs and HPCs in bone marrow. AMD3100, a selective antagonist of CXCL12 that binds to its receptor, CXCR4, was evaluated in murine and human systems for mobilizing capacity, alone and in combination with granulocyte colony-stimulating factor (G-CSF). AMD3100 induced rapid mobilization of mouse and human HPCs and synergistically augmented G-CSF–induced mobilization of HPCs. AMD3100 also mobilized murine long-term repopulating (LTR) cells that engrafted primary and secondary lethally-irradiated mice, and human CD34+ cells that can repopulate nonobese diabetic-severe combined immunodeficiency (SCID) mice. AMD3100 synergized with G-CSF to mobilize murine LTR cells and human SCID repopulating cells (SRCs). Human CD34+ cells isolated after treatment with G-CSF plus AMD3100 expressed a phenotype that was characteristic of highly engrafting mouse HSCs. Synergy of AMD3100 and G-CSF in mobilization was due to enhanced numbers and perhaps other characteristics of the mobilized cells. These results support the hypothesis that the CXCL12-CXCR4 axis is involved in marrow retention of HSCs and HPCs, and demonstrate the clinical potential of AMD3100 for HSC mobilization.

Stromal cell-derived factor-1 (SDF-1/CXCL12) enhances survival of myeloid progenitor cells. The two main questions addressed by us were whether these effects on the progenitors were direct-acting and if SDF-1/CXCL12 enhanced engrafting capability of competitive, repopulating mouse stem cells subjected to short-term ex vivo culture with other growth factors. SDF-1/CXCL12 had survival-enhancing/antiapoptosis effects on human bone marrow (BM) and cord blood (CB) and mouse BM colony-forming units (CFU)-granulocyte macrophage, burst-forming units-erythroid, and CFU-granulocyte-erythroid-macrophage-megakaryocyte with similar dose responses. The survival effects were direct-acting, as assessed on colony formation by single isolated human BM and CB CD34(+++) cells. Effects were mediated through CXCR4 and G(alpha)i proteins. Moreover, SDF-1/CXCL12 greatly enhanced the engrafting capability of mouse long-term, marrow-competitive, repopulating stem cells cultured ex vivo with interleukin-6 and steel factor for 48 h. These results extend information on the survival effects mediated through the SDF-1/CXCL12-CXCR4 axis and may be of relevance for ex vivo expansion and gene-transduction procedures.

To assess whether Fanconi anemia (FA) patients might be at risk for acute graftversus-host disease (AGvHD) despite using low-intensity conditionings, we retrospectively analyzed the incidence of AGvHD and its impact on outcome in 37 FA patients and 73 patients with acquired aplastic anemia (

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

IntroductionFanconi anemia (FA) is an autosomal recessive disorder characterized by a progressive bone marrow failure, developmental defects, and cancer. Individuals with FA have a 500-to 1000-fold increased risk of developing myeloid leukemia and a range of solid tumors that preferentially affect the head and neck, skin, and gastrointestinal and genitourinary systems. [1][2][3][4] Recent studies have identified the existence of 7 FA gene products (denoted FANCA-FANCG) that appear to function, at least in part, in a complex of signaling proteins that modulate genomic stability (for a review, see Joenje and Patel 5 ). In addition, one gene product (FANCC) appears to have a role in modulating apoptosis in addition to its role in the FANCA-FANCG complex. [6][7][8] The ways the different FA proteins interact with each other and with other biochemical pathways to maintain genomic stability and prevent tumor formation is an area of active investigation.Although the mechanisms underlying tumor formation in patients with FA are incompletely understood, epidemiologic and experimental observations raise a number of possibilities. First, the development of malignant cells from primary cells frequently requires alterations in apoptosis or cell cycle control. Because myeloid malignancies predominantly occur in the context of progressive apoptotic depletion of hematopoietic progenitors in patients with FA 9 and in Fancc Ϫ/Ϫ mice, 10 it has been hypothesized that leukemogenesis results from an accumulation of mutations that precipitates the emergence of apoptotic-resistant precursors. Second, p53 is a pivotal sensor of genotoxic and nongenotoxic stress and activates signaling pathways that result in cell cycle arrest or apoptosis. The apoptosis in FANCC-deficient lymphoblasts has previously been shown to be due to activation of protein kinase R (PKR). 11 Because p53 is a downstream effector of PKR, 12-14 inactivation or polymorphisms of p53 or proteins in the p53 pathway may be an important target for cellular transformation in FA-associated myeloid malignancies. Third, patients with FA are predisposed to solid tumors, including epithelial and squamous cell carcinomas, which have a high rate of p53 alteration in sporadic malignancies. 15,16 The risk of these solid tumors in patients with FA goes up markedly after 20 years of age and increases to a cumulative risk of 90% by 40 years of age. 2 Finally, recent studies have positioned the FA proteins in a biochemical pathway that includes BRCA1 and BRCA2. 17,18 In fact, truncating, hypomorphic mutations of BRCA2 were observed in the FA-D1 complementation group. 18 Further, studies using Brca1 and Brca2 conditional knockout mice support a role for p53 in tumor progression. [19][20][21] Although epidemiologic and biochemical data suggest a role for p53 in mediating apoptosis and cancer in FA, a previous study found that a dominant-negative p53 did not affect apoptosis in FANCC-deficient, immortalized lymphoblasts exposed to genotoxic stress. 22 However, the use of immortalized cell ...

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