Germ cell defects and hematopoietic hypersensitivity to gamma- interferon in mice with a targeted disruption of the Fanconi anemia C gene

Fanconi anemia (FA) is an autosomal recessive chromosome instability syndrome characterized by progressive bone marrow (BM) failure, skeletal defects, and increased susceptibility to malignancy. FA cells are hypersensitive to DNA cross-linking agents, oxygen and have cell cycle abnormalities. To develop an animal model of the disease we generated mice homozygous for a targeted deletion of exon 9 of the murine FA complementation group C gene (fac). Mutant mice had normal neonatal viability and gross morphology, but their cells had the expected chromosome breakage and DNA cross-linker sensitivity. Surprisingly, male and female mutant mice had reduced numbers of germ cells and females had markedly impaired fertility. No anemia was detectable in the peripheral blood during the first year of life, but the colony forming capacity of marrow progenitor cells was abnormal in vitro in mutant mice. Progenitor cells from fac knock-out mice were hypersensitive to interferon gamma. This previously unrecognized phenotype may form the basis for BM failure in human FA.     

Down-regulated expression of hsa-miR-181c in Fanconi anemia patients: implications in TNFα regulation and proliferation of hematopoietic progenitor cells

Fanconi anemia (FA) is an inherited genetic disorder associated with BM failure and cancer predisposition. In the present study, we sought to elucidate the role of microRNAs (miRNAs) in the hematopoietic defects observed in FA patients. Initial studies showed that 3 miRNAs, hsa-miR-133a, hsa-miR-135b, and hsa-miR-181c, were significantly down-regulated in lymphoblastoid cell lines and fresh peripheral blood cells from FA patients. In vitro studies with cells expressing the luciferase reporter fused to the TNFα 3'-untranslated region confirmed in silico predictions suggesting an interaction between hsa-miR-181c and TNFα mRNA. These observations were consistent with the down-regulated expression of TNFα mediated by hsa-miR-181c in cells from healthy donors and cells from FA patients. Because of the relevance of TNFα in the hematopoietic defects of FA patients, in the present study, we transfected BM cells from FA patients with hsa-miR-181c to evaluate the impact of this miRNA on their clonogenic potential. hsa-miR-181c markedly increased the number and size of the myeloid and erythroid colonies generated by BM cells from FA patients. Our results offer new clues toward understanding the biologic basis of BM failure in FA patients and open new possibilities for the treatment of the hematologic dysfunction in FA patients based on miRNA regulation.     

TNFα facilitates clonal expansion of JAK2V617F positive cells in myeloproliferative neoplasms

Proinflammatory cytokines such as TNFα are elevated in patients with myeloproliferative neoplasms (MPN), but their contribution to disease pathogenesis is unknown. Here we reveal a central role for TNFα in promoting clonal dominance of JAK2(V617F) expressing cells in MPN. We show that JAK2(V617F) kinase regulates TNFα expression in cell lines and primary MPN cells and TNFα expression is correlated with JAK2(V617F) allele burden. In clonogenic assays, normal controls show reduced colony formation in the presence of TNFα while colony formation by JAK2(V617F)-positive progenitor cells is resistant or stimulated by exposure to TNFα. Ectopic JAK2(V617F) expression confers TNFα resistance to normal murine progenitor cells and overcomes inherent TNFα hypersensitivity of Fanconi anemia complementation group C deficient progenitors. Lastly, absence of TNFα limits clonal expansion and attenuates disease in a murine model of JAK2(V617F)-positive MPN. Altogether our data are consistent with a model where JAK2(V617F) promotes clonal selection by conferring TNFα resistance to a preneoplastic TNFα sensitive cell, while simultaneously generating a TNFα-rich environment. Mutations that confer resistance to environmental stem cell stressors are a recognized mechanism of clonal selection and leukemogenesis in bone marrow failure syndromes and our data suggest that this mechanism is also critical to clonal selection in MPN.     

Interferon-gamma-induced apoptotic responses of Fanconi anemia group C hematopoietic progenitor cells involve caspase 8-dependent activation of caspase 3 family members

Hematopoietic progenitor cells (HPC) from mice nullizygous at the Fanconi anemia (FA) group C locus and children with Fanconi anemia group C (FA-C) are hypersensitive to interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha. This hypersensitivity results, in part, from the capacity of these cytokines to prime the fas pathway. Because fas-mediated programmed cell death in many cells involves sequential activation of specific caspases, we tested the hypothesis that programmed cell death in FA HPC involves the ordered activation of specific caspase molecules. Lysates from lymphoblasts treated with both agonistic anti-fas antibody and IFN-gamma contained activated caspase 3 family members (caspases 3, 6, and 7), as well as caspase 8, whereas activation of caspases 1, 2, 4, 9, and 10 was not detected. The apoptotic effects of fas agonists in IFN-gamma-treated human and murine FA-C cells were blocked when pretreated with inhibitors (ac-DEVD-cho, CP-DEVD-cho, Z-DEVD-FMK) of the caspase 3 protease. Inhibitors (ac-YVAD-cho, CP-YVAD-cho, Z-YVAD-FMK) of caspase 1 did not block apoptosis or caspase 3 activation. Treatment of FA cells with the fluoromethyl ketone tetrapeptide caspase 8 inhibitor (ac-IETD-FMK) did suppress caspase 3 activation. A 4-fold greater fraction of IFN-induced FA-C cells expressed caspase 3 than FA-C cells complemented by retroviral-mediated transfer of FANCC. Therefore fas-induced apoptosis in Fanconi anemia cells of the C type involves the activation of caspase 8, which controls activation of caspase 3 family members and one direct or indirect function of the FANCC protein is to suppress apoptotic responses to IFN-gamma upstream of caspase 3 activation. (Blood. 2000;96:4204-4211)     

p38γ and p38δ kinases regulate the Toll-like receptor 4 (TLR4)-induced cytokine production by controlling ERK1/2 protein kinase pathway activation

On the basis mainly of pharmacological experiments, the p38α MAP kinase isoform has been established as an important regulator of immune and inflammatory responses. However, the role of the related p38γ and p38δ kinases has remained unclear. Here, we show that deletion of p38γ and p38δ impaired the innate immune response to lipopolysaccharide (LPS), a Toll-like receptor 4 (TLR4) ligand, by blocking the extracellular signal-regulated kinase 1/2 (ERK1/2) activation in macrophages and dendritic cells. p38γ and p38δ were necessary to maintain steady-state levels of tumor progression locus 2 (TPL2), the MKK kinase that mediates ERK1/2 activation after TLR4 stimulation. TNFα, IL-1β, and IL-10 production were reduced in LPS-stimulated macrophages from p38γ/δ-null mice, whereas IL-12 and IFNβ production increased, in accordance with the known effects of TPL2/ERK1/2 signaling on the induction of these cytokines. Furthermore, p38γ/δ-deficient mice were less sensitive than controls to LPS-induced septic shock, showing lower TNFα and IL-1β levels after challenge. Together, our results establish p38γ and p38δ as key components in innate immune responses.     

Increased sensitivity of Fancc-deficient hematopoietic cells to nitric oxide and evidence that this species mediates growth inhibition by cytokines

Fanconi anemia complementation group C (Fancc)-deficient murine bone marrow progenitors demonstrate increased sensitivity to growth inhibition by interferon gamma (IFNgamma), tumor necrosis factor alpha (TNFalpha), and macrophage inflammatory protein 1alpha (MIP-1alpha). This property has been proposed as a possible pathogenic factor in the marrow failure seen in Fanconi anemia. Supporting our hypothesis that nitric oxide (NO) production might be a common effector in this sensitivity, we found that cytokine-mediated growth inhibition of Fancc(-/-) bone marrow cells was prevented by inhibiting NO synthase activity. Interestingly, Fancc(-/-) hematopoietic cells also exhibited increased growth inhibition on exposure to 2 distinct NO-generating agents, S-nitroso-N-acetyl-D, L-penicillamine (SNAP) and diethylenetriamine nitric oxide adduct (DETA/NO). In keeping with the sensitivity of Fancc(-/-) cells to IFNgamma, inducible nitric oxide synthase (iNOS) levels and nitrite release were both increased following stimulation of Fancc(-/-) macrophages with this cytokine, either alone or in combination with bacterial lipopolysaccharide. Suggesting a plausible mechanism for the increased expression of iNOS, IFNgamma-stimulated Fancc(-/-) macrophages generated higher levels of phospho-Stat1, a positive regulator of inos (nos2) gene expression. These observations, while confined to C57BL/6 Fancc(-/-) hematopoietic cells, raise the possibility that nitric oxide has a role in the pathogenesis of Fanconi anemia.     

A functionally active retrovirus vector for gene therapy in Fanconi anemia group C

Fanconi anemia (FA) is a rare genetic disorder characterized by progressive pancytopenia, congenital abnormalities, and a predisposition to malignancy. Recently, mutation in a novel gene named FACC (Fanconi anemia C complementing) has been identified as causing one type of FA. Here, we report successful functional complementation of four FA(C) cell lines using a retroviral vector to transfer a copy of the normal FACC gene. The hallmark of the FA cell phenotype is extreme sensitivity to cross-linking agents such as mitomycin C (MMC). Cell lines transduced by FACC viral vectors were distinguished by their ability to grow at concentrations of MMC several orders of magnitude higher than those concentrations inhibitory of parental controls. The genetically corrected cell lines were analyzed for susceptibility to MMC-induced chromosomal breakage and were found to have been normalized. These two different assays confirmed that our retroviral vectors were capable of transferring a functional FACC gene to lymphoid cell lines established from FA(C) patients. We next analyzed the ability of our viral vectors to functionally correct hematopoietic progenitor cells from a patient bearing a splice donor mutation. Progenitor cells were purified by an immunoaffinity column to enrich for cells with high CD34 expression. Similar to FA lymphoid cell lines, this patient's CD34-enriched cells were extremely sensitive to MMC. After infection of these progenitor cells with viral vectors bearing normal FACC, increased numbers of colonies formed both in the absence and presence of < or = 5 nmol/L MMC, but no colonies formed from uninfected cells, even in the absence of MMC. Polymerase chain amplification was used to confirm proviral DNA integration. Thus, retroviral vectors can be engineered to transfer a normal FACC gene to lymphoid cell lines and primary hematopoietic cells bearing four different FACC mutations. FA stem cells rescued by gene transduction should have a selective growth advantage within the hypoplastic FA marrow environment in vivo. These experiments suggest that gene therapy may be an effective treatment strategy for FA.     

Abnormal lymphokine production: a novel feature of the genetic disease Fanconi anemia. II. In vitro and in vivo spontaneous overproduction of tumor necrosis factor alpha

We have previously shown an unbalanced cytokine production in Fanconi anemia (FA) cells, ie, an underproduction of interleukin 6 (IL-6) during growth. Among a number of cytokines analyzed, the only other anomalies detected concern tumor necrosis factor alpha (TNF alpha). In comparison to normal cells, this cytokine is overproduced by FA lymphoblasts from the four genetic complementation groups. Indeed, up to an eight-fold increase in TNF alpha is observed in the growth medium of FA cells. Moreover, addition of anti-TNF alpha antibodies partially corrects the FA hypersensitivity to treatment by mitomycin C (MMC). Treatment of FA cells with IL-6, which partially restored an almost normal sensitivity to MMC of FA cells also reduces the TNF alpha overproduction in FA lymphoblasts. No anomalies at the molecular level (Southern and Northern blot analyses) are detected for the TNF alpha gene and its mRNA. We have investigated the in vivo situation by assaying TNF alpha levels in the serum from FA homozygotes and obligate heterozygotes. In contrast to normal healthy donors or to aplastic anemia patients in whom serum TNF alpha is present only in trace amounts, all 36 FA patients and 21 FA parents monitored show a significantly (P < .001) higher level of serum TNF alpha activity. Consequently, abnormal TNF alpha production seems to be associated with the FA genetic background.     

Dynamic regulation of pro- and anti-inflammatory cytokines by MAPK phosphatase 1 (MKP-1) in innate immune responses

Engagement of Toll-like receptors (TLRs) on macrophages leads to activation of the mitogen-activated protein kinases (MAPKs), which contribute to innate immune responses. MAPK activity is regulated negatively by MAPK phosphatases (MKPs). MKP-1, the founding member of this family of dual-specificity phosphatases, has been implicated in regulating lipopolysaccharide (LPS) responses, but its role in TLR-mediated immune responses in vivo has not been defined. Here, we show that mice deficient in MKP-1 were highly susceptible to endotoxic shock in vivo, associated with enhanced production of proinflammatory cytokines TNF-alpha and IL-6 and an anti-inflammatory cytokine, IL-10. We further examined the regulation and function of MKP-1 in macrophages, a major cell type involved in endotoxic shock. MKP-1 was transiently induced by TLR stimulation through pathways mediated by both myeloid differentiation factor 88 (MyD88) and TIR domain-containing adaptor inducing IFN-beta (TRIF). MKP-1 deficiency led to sustained activation of p38 MAPK and c-Jun N-terminal kinase (JNK) in LPS-treated macrophages. In response to TLR signals, MKP-1-deficient macrophages produced 5- to 10-fold higher IL-10, which could be blocked by a p38 MAPK inhibitor. Thus, p38 MAPK plays a critical role in mediating IL-10 synthesis in TLR signaling. TNF-alpha was found to be more abundant in MKP-1-deficient macrophages within 2 hours of TLR stimulation, but its production was rapidly down-regulated by IL-10. Our studies demonstrate that MKP-1 attenuates the activities of p38 MAPK and JNK to regulate both pro- and anti-inflammatory cytokines in TLR signaling. These results highlight the complex mechanisms by which the MAPKs regulate innate immunity.     

TNF-α signaling in Fanconi anemia

Tumor necrosis factor-alpha (TNF-α) is a major pro-inflammatory cytokine involved in systemic inflammation and the acute phase reaction. Dysregulation of TNF production has been implicated in a variety of human diseases including Fanconi anemia (FA). FA is a genomic instability syndrome characterized by progressive bone marrow failure and cancer susceptibility. The patients with FA are often found overproducing TNF-α, which may directly affect hematopoietic stem cell (HSC) function by impairing HSC survival, homing and proliferation, or indirectly change the bone marrow microenvironment critical for HSC homeostasis and function, therefore contributing to disease progression in FA. In this brief review, we discuss the link between TNF-α signaling and FA pathway with emphasis on the implication of inflammation in the pathophysiology and abnormal hematopoiesis in FA.     

Fanconi anemia genes act to suppress a cross-linker-inducible p53- independent apoptosis pathway in lymphoblastoid cell lines

Hypersensitivity to cross-linking agents such as mitomycin C (MMC) is characteristic of cells from patients suffering from the inherited bone marrow failure syndrome. Fanconi anemia (FA). Here, we link MMC hypersensitivity of Epstein-Barr virus (EBV)-immortalized FA lymphoblasts to a high susceptibility for apoptosis and p53 activation. In MMC-treated FA cells belonging to complementation group C (FA-C), apoptosis followed cell cycle arrest in the G2 phase. In stably transfected FA-C cells, plasmid-driven expression of the wild-type cytoplasmic FAC protein relieved MMC-dependent G2 arrest and suppressed p53 activation. However, in both FA and non-FA lymphoblasts, p53 seemed not to be instrumental in the induction of MMC-dependent apoptosis, since overexpression of a dominant-negative p53 mutant failed to affect cell survival. In addition, no differences in the level of Bcl-2 expression, an inhibitor of apoptosis, were detected between FA and non- FA cells either in the absence or presence of MMC. Our findings suggest that FAC and the other putative FA gene products may function in a yet to be identified p53-independent apoptosis pathway.     

Hematopoietic changes during progression from Fanconi anemia into acute myeloid leukemia: Case report and brief review of the literature

Fanconi anemia (FA) is a rare autosomal recessive disorder characterized by bone marrow (BM) failure and a wide array of physical abnormalities. Around 9% of FA patients develop acute myeloid leukemia (AML), which makes FA a good genetic model to study leukemogenesis. To date, however, no information exists on the functional integrity of the hematopoietic system of FA patients during the period in which they develop AML. Herein, we report on the characterization of hematopoietic progenitor cells from a pediatric FA patient that developed AML. Our results show that significant changes occurred in the hematopoietic system of the patient from the time he presented with FA to the time he developed AML. Such changes included marrow cellularity, frequency of CD34⁺ cells and CFC, as well as proliferation potential of progenitor cells in liquid cultures supplemented with recombinant cytokines. Interestingly, no significant changes in the karyotype of marrow cells were observed, indicating that progression from FA into AML may proceed without major chromosomal alterations (i.e. translocations and/or deletions). This study represents one of the first steps towards the cellular characterization of the hematopoietic system in FA patients that develop AML.     

Hematopoietic changes during progression from Fanconi anemia into acute myeloid leukemia: case report and brief review of the literature

Fanconi anemia (FA) is a rare autosomal recessive disorder characterized by bone marrow (BM) failure and a wide array of physical abnormalities. Around 9% of FA patients develop acute myeloid leukemia (AML), which makes FA a good genetic model to study leukemogenesis. To date, however, no information exists on the functional integrity of the hematopoietic system of FA patients during the period in which they develop AML. Herein, we report on the characterization of hematopoietic progenitor cells from a pediatric FA patient that developed AML. Our results show that significant changes occurred in the hematopoietic system of the patient from the time he presented with FA to the time he developed AML. Such changes included marrow cellularity, frequency of CD34(+) cells and CFC, as well as proliferation potential of progenitor cells in liquid cultures supplemented with recombinant cytokines. Interestingly, no significant changes in the karyotype of marrow cells were observed, indicating that progression from FA into AML may proceed without major chromosomal alterations (i.e. translocations and/or deletions). This study represents one of the first steps towards the cellular characterization of the hematopoietic system in FA patients that develop AML.     

Suppression of apoptosis in hematopoietic factor-dependent progenitor cell lines by expression of the FAC gene

Fanconi anemia (FA) is a genetically heterogeneous, inherited blood disorder characterized by bone marrow failure, congenital malformations, and a predisposition to leukemias. Because FA cells are hypersensitive to DNA cross-linking agents and have chromosomal instability, FA has been viewed as a disorder of DNA repair. However, the exact cellular defect in FA cells has not been identified. Sequence analysis of the gene defective in group C patients (FAC) has shown no significant homologies to other known genes. The FAC protein has been localized to the cytoplasm, indicating that FAC may either play an indirect role in DNA repair or is involved in a different cellular pathway. Recent evidence has indicated that FA cells may be predisposed to apoptosis, especially after treatment with DNA cross-linking agents. The demonstration that genes can suppress apoptosis has been accomplished by overexpression of such genes in growth factor-dependent cell lines that die by apoptosis after factor withdrawal. Using retroviral-mediated gene transfer, we present evidence that expression of FAC in the hematopoietic factor-dependent progenitor cell lines 32D and MO7e can suppress apoptosis induced by growth factor withdrawal. Flow cytometry and morphologic analysis of propidium iodide stained cells showed significantly lower levels of apoptosis in FAC-retroviral transduced cells after growth factor deprivation. Expression of FAC in both cell lines promoted increased viability rather than proliferation, which is consistent with other apoptosis-inhibiting genes such as Bcl- 2. These findings imply that FAC may act as a mediator of an apoptotic pathway initiated by growth factor withdrawal. Furthermore, the congenital malformations and hematologic abnormalities characterizing FA may be related to an increased predisposition of FA progenitor cells to undergo apoptosis, particularly in the absence of extracellular signals.     

Fanconi anemia type C-deficient hematopoietic stem/progenitor cells exhibit aberrant cell cycle control

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.     

Continuous in vivo infusion of interferon-gamma (IFN-gamma) enhances engraftment of syngeneic wild-type cells in Fanca-/- and Fancg-/- mice

Fanconi anemia (FA) is a heterogeneous genetic disorder characterized by bone marrow (BM) failure and cancer susceptibility. Identification of the cDNAs of FA complementation types allows the potential of using gene transfer technology to introduce functional cDNAs as transgenes into autologous stem cells and provide a cure for the BM failure in FA patients. However, strategies to enhance the mobilization, transduction, and engraftment of exogenous stem cells are required to optimize efficacy prior to widespread clinical use. Hypersensitivity of Fancc-/- cells to interferon-gamma (IFN-gamma), a nongenotoxic immune-regulatory cytokine, enhances engraftment of syngeneic wild-type (WT) cells in Fancc-/- mice. However, whether this phenotype is of broad relevance in other FA complementation groups is unresolved. Here we show that primitive and mature myeloid progenitors in Fanca-/- and Fancg-/- mice are hypersensitive to IFN-gamma and that in vivo infusion of IFN-gamma at clinically relevant concentrations was sufficient to allow consistent long-term engraftment of isogenic WT repopulating stem cells. Given that FANCA, FANCC, and FANCG complementation groups account for more than 90% of all FA patients, these data provide evidence that IFN-gamma conditioning may be a useful nongenotoxic strategy for myelopreparation in FA patients.     

Regulatory role of Toll-like receptor 2 during infection with Trypanosoma cruzi

Previous studies have emphasized the role of Toll-like receptors (TLRs) and myeloid differentiation factor 88 (MyD88) during infection with protozoan parasites. TLR2 was shown to be important for induction of cytokine synthesis by macrophages exposed to the purified glycosylphosphatidylinositol (GPI)-anchored mucin-like glycoproteins of Trypanosoma cruzi trypomastigotes (tGPIm). On the other hand, MyD88(-/-) mice, but not TLR2(-/-) mice, showed impaired cytokine production and resistance to infection with T. cruzi parasites. Here we evaluate the importance of MyD88 and TLR2 in MAPK activation and cytokine synthesis by macrophages exposed to live T. cruzi parasites and compared to tGPIm. The absence of MAPK phosphorylation in TLR2- and MyD88-deficient macrophages exposed to tGPIm correlated with the incapacity to induce cytokine release in these cells. In contrast, activation of MAPK and synthesis of pro-inflammatory cytokines were not abrogated in TLR2-deficient macrophages exposed to live T. cruzi parasites. We also showed that pretreatment with tGPIm significantly reduces cytokine release by macrophages in response to T. cruzi in a TLR2-dependent manner. Consistently, TLR2(-/-) mice were shown to produce enhanced levels of cytokines upon in vivo challenge with T. cruzi parasites. Together, these results suggest the involvement of additional TLR(s) in the pro-inflammatory response of macrophages to whole parasites, and that, in vivo, TLR2 may have a predominant immunoregulatory role during acute infection with T. cruzi parasites.     

Enhanced TNF-alpha-induced apoptosis in Fanconi anemia type C-deficient cells is dependent on apoptosis signal-regulating kinase 1

Fanconi anemia (FA) is a chromosomal instability disorder characterized by progressive bone marrow failure. Experimental evidence suggests that enhanced oxidant and myelosuppressive cytokine-mediated apoptosis of hematopoietic stem and progenitor cells contributes to the pathogenesis of marrow failure in FA. However, the molecular mechanisms responsible for the apoptotic phenotype in hematopoietic cells are incompletely understood. Recent data in Fancc-/- murine embryonic fibroblasts (MEFs) implicate increased oxidant-induced apoptotic signaling through the redox-dependent protein, apoptosis signal-regulating kinase 1 (Ask1). Here, we examined whether altered Ask1 signaling participated in the proapoptotic phenotype of primary Fancc-/- MEFs and hematopoietic progenitors treated with the myelosuppressive cytokine tumor necrosis factor-alpha (TNF-alpha). Our data indicate that TNF-alpha induces hyperactivation of Ask1 and the downstream effector p38 in Fancc-/- MEFs. In addition,Ask1 inactivation in Fancc-/- MEFs and hematopoietic progenitors restored survival to wild-type (WT) levels in the presence of TNF-alpha. Furthermore, targeting the Ask1 pathway by using either antioxidants or a p38 inhibitor protected Fancc-/- MEFs and c-kit+ cells from TNF-alpha-induced apoptosis. Collectively, these data argue that the predisposition of Fancc-/- hematopoietic progenitors to apoptosis is mediated in part through altered redox regulation and Ask1 hyperactivation.     

Overexpression of IL-3Rα on CD34+CD38- stem cells defines leukemia-initiating cells in Fanconi anemia AML

Patients with Fanconi anemia (FA) have a high risk of developing acute myeloid leukemia (AML). In this study, we attempted to identify cell-surface markers for leukemia-initiating cells in FA-AML patients. We found that the IL-3 receptor-α (IL-3Rα) is a promising candidate as an leukemia-initiating cell-specific antigen for FA-AML. Whereas IL-3Rα expression is undetectable on normal CD34(+)CD38(-) HSCs, it is overexpressed on CD34(+)CD38(-) cells from FA patients with AML. We examined the leukemia-initiating cell activity of IL-3Rα-positive FA-AML cells in a "humanized" FA xenotransplant model in which we separated AML cells into IL-3Rα-positive and IL-3Rα-negative CD34 fractions and transplanted them into irradiated recipient mice. In all 3 FA-AML samples, only IL-3Rα-positive cells showed significant levels of engraftment and developed leukemia in the recipient mice. The FA CD34(+)IL-3Rα(+) blasts isolated from leukemic mice exhibited hypersensitivity to IL-3 deprivation and JAK2-STAT5 overactivation after IL-3 treatment. Finally, treatment of FA CD34(+)IL-3Rα(+) blasts with an IL-3Rα-neutralizing antibody inhibited IL-3-mediated proliferation and STAT5 activation. These results demonstrate that IL-3Rα is a cell-surface marker present on FA-AML leukemia-initiating cells and may be a valuable therapeutic target.     

Long-term bone marrow culture in persons with Fanconi anemia and bone marrow failure

Fanconi anemia is an autosomal recessive disease characterized by a high risk of developing bone marrow (BM) failure and acute myelogenous leukemia. We studied growth of hematopoietic progenitor cells in long- term BM culture (LTBMC) in 8 persons with Fanconi anemia and BM failure. Although LTBMC were initiated with very few BM cells, an adherent layer formed in cultures from 7 persons. In these cultures, the number of nonadherent cells increased for 10 to 15 days. Cell growth continued until cultures were terminated at day 35 to 40. During the first 2 weeks of culture, most nonadherent cells were differentiated myeloid cells. By days 35 to 40, the adherent layer contained cells able to initiate secondary LTBMCs. These data indicate that hematopoietic precursors cells able to proliferate and differentiate in vitro are present in the BM of persons with Fanconi anemia and BM failure. They suggest that mechanisms other than absent precursor cells are responsible for BM failure in Fanconi anemia.