Qishen Pang

The Fanconi anemia protein FANCC binds to and facilitates the activation of STAT1 by gamma interferon and hematopoietic growth factors.

ABSTRACT Hematopoietic progenitor cells from Fanconi anemia (FA) group C (FA-C) patients display hypersensitivity to the apoptotic effects of gamma interferon (IFN-γ) and constitutively express a variety of IFN-dependent genes. Paradoxically, however, STAT1 activation is suppressed in IFN-stimulated FA cells, an abnormality corrected by transduction of normal FANCC cDNA. We therefore sought to define the specific role of FANCC protein in signal transduction through receptors that activate STAT1. Expression and phosphorylation of IFN-γ receptor α chain (IFN-γRα) and JAK1 and JAK2 tyrosine kinases were equivalent in both normal and FA-C cells. However, in coimmunoprecipitation experiments STAT1 did not dock at the IFN-γR of FA-C cells, an abnormality corrected by transduction of the FANCC gene. In addition, glutathione S-transferase fusion genes encoding normal FANCC but not a mutant FANCC bearing an inactivating point mutation (L554P) bound to STAT1 in lysates of IFN-γ-stimulated B cells and IFN-, granulocyte-macrophage colony-stimulating factor- and stem cell factor-stimulated MO7e cells. Kinetic studies revealed that the initial binding of FANCC was to nonphosphorylated STAT1 but that subsequently the complex moved to the receptor docking site, at which point STAT1 became phosphorylated. The STAT1 phosphorylation defect in FA-C cells was functionally significant in that IFN induction of IFN response factor 1 was suppressed and STAT1-DNA complexes were not detected in nuclear extracts of FA-C cells. We also determined that the IFN-γ hypersensitivity of FA-C hematopoietic progenitor cells does not derive from STAT1 activation defects because granulocyte-macrophage CFU and erythroid burst-forming units from STAT1−/− mice were resistant to IFN-γ. However, BFU-E responses to SCF and erythropoietin were suppressed in STAT−/− mice. Consequently, because the FANCC protein is involved in the activation of STAT1 through receptors for at least three hematopoietic growth and survival factor molecules, we reason that FA-C hematopoietic cells are excessively apoptotic because of an imbalance between survival cues (owing to a failure of STAT1 activation in FA-C cells) and apoptotic and mitogenic inhibitory cues (constitutively activated in FA-C cells in a STAT1-independent fashion).

TNF-α induces leukemic clonal evolution ex vivo in Fanconi anemia group C murine stem cells

The molecular pathogenesis of the myeloid leukemias that frequently occur in patients with Fanconi anemia (FA) is not well defined. Hematopoietic stem cells bearing inactivating mutations of FA complementation group C (FANCC) are genetically unstable and hypersensitive to apoptotic cytokine cues including IFN-γ and TNF-α, but neoplastic stem cell clones that arise frequently in vivo are resistant to these cytokines. Reasoning that the combination of genetic instability and cytokine hypersensitivity might create an environment supporting the emergence of leukemic stem cells, we tested the leukemia-promoting effects of TNF-α in murine stem cells. TNF-α exposure initially profoundly inhibited the growth of Fancc–/– stem cells. However, longer-term exposure of these cells promoted the outgrowth of cytogenetically abnormal clones that, upon transplantation into congenic WT mice, led to acute myelogenous leukemia. TNF-α induced ROS-dependent genetic instability in Fancc–/– but not in WT cells. The leukemic clones were TNF-α resistant but retained their characteristic hypersensitivity to mitomycin C and exhibited high levels of chromosomal instability. Expression of FANCC cDNA in Fancc–/– stem cells protected them from TNF-α–induced clonal evolution. We conclude that TNF-α exposure creates an environment in which somatically mutated preleukemic stem cell clones are selected and from which unaltered TNF-α–hypersensitive Fancc–/– stem cells are purged.

Oxidative Stress in Fanconi Anemia Hematopoiesis and Disease Progression

Patients with the genomic instability syndrome Fanconi anemia (FA) commonly develop progressive bone marrow failure and have a high risk of cancer. The prominent role of the FA protein family involves DNA damage response and/or repair. Oxidative stress, defined as an imbalance between the production of reactive oxygen species and antioxidant defense, is considered to be an important pathogenic factor in leukemia-prone bone marrow diseases such as FA. Cellular responses inducing resistance to oxidative stress are important for cellular survival, organism lifespan, and cancer prevention, but until recently, mammalian factors regulating resistance to oxidative stress have not been well characterized. Significant evidence supports excessive apoptosis of hematopoietic stem/progenitor cells, induced by stresses, most significantly oxidative stress, as a critical factor in the pathogenesis of bone marrow failure and leukemia progression in FA. In this brief review, we discuss the functional link between FA proteins and oxidative DNA damage response/repair, with emphasis on the implication of oxidative stress in the pathophysiology and abnormal hematopoiesis in FA.

Inflammatory ROS promote and cooperate with the Fanconi anemia mutation for hematopoietic senescence

The proinflammatory cytokine tumor necrosis factor α (TNFα) inhibits hematopoietic stem cell (HSC) expansion, interferes with HSC self-renewal and compromises the ability of HSC to reconstitute hematopoiesis. We have investigated mechanisms by which TNFα suppresses hematopoiesis using the genomic instability syndrome Fanconi anemia mouse model deficient for the complementation-group-C gene (Fancc). Examination of senescence makers, such as senescence-associated β-galactosidase, HP1-γ, p53 and p16INK4A shows that TNFα induces premature senescence in bone marrow HSCs and progenitor cells as well as other tissues of Fancc–/– mice. TNFα-induced senescence correlates with the accumulation of reactive oxygen species (ROS) and oxidative DNA damage. Neutralization of TNFα or deletion of the TNF receptor in Fancc–/– mice (Fancc–/–;Tnfr1–/–) prevents excessive ROS production and hematopoietic senescence. Pretreatment of TNFα-injected Fancc–/– mice with a ROS scavenger significantly reduces oxidative base damage, DNA strand breaks and senescence. Furthermore, HSCs and progenitor cells from TNFα-treated Fancc–/– mice show increased chromosomal aberrations and have an impaired oxidative DNA-damage repair. These results indicate an intimate link between inflammatory reactive oxygen species and DNA-damage-induced premature senescence in HSCs and progenitor cells, which may play an important role in aging and anemia.

Inflammatory Reactive Oxygen Species-Mediated Hemopoietic Suppression in Fancc-Deficient Mice

Patients with the genomic instability syndrome Fanconi anemia (FA) commonly develop progressive bone marrow (BM) failure and have a high risk of cancer. Certain manifestations of the disease suggest that the FA immune system is dysfunctional and may contribute to the pathogenesis of both BM failure and malignancies. In this study, we have investigated inflammation and innate immunity in FA hemopoietic cells using mice deficient in Fanconi complementation group C gene (Fancc). We demonstrate that Fancc-deficient mice exhibit enhanced inflammatory response and are hypersensitive to LPS-induced septic shock as a result of hemopoietic suppression. This exacerbated inflammatory phenotype is intrinsic to the hemopoietic system and can be corrected by the re-expression of a wild-type FANCC gene, suggesting a potential role of the FANCC protein in innate immunity. LPS-mediated hemopoietic suppression requires two major inflammatory agents, TNF-α and reactive oxygen species. In addition, LPS-induced excessive accumulation of reactive oxygen species in Fancc−/− BM cells overactivates the stress kinase p38 and requires prolonged activation of the JNK. Our data implicate a role of inflammation in pathogenesis of FA and BM failure diseases in general.

FAAP20: a novel ubiquitin-binding FA nuclear core-complex protein required for functional integrity of the FA-BRCA DNA repair pathway

Fanconi anemia (FA) nuclear core complex is a multiprotein complex required for the functional integrity of the FA-BRCA pathway regulating DNA repair. This pathway is inactivated in FA, a devastating genetic disease, which leads to hematologic defects and cancer in patients. Here we report the isolation and characterization of a novel 20-kDa FANCA-associated protein (FAAP20). We show that FAAP20 is an integral component of the FA nuclear core complex. We identify a region on FANCA that physically interacts with FAAP20, and show that FANCA regulates stability of this protein. FAAP20 contains a conserved ubiquitin-binding zinc-finger domain (UBZ), and binds K-63-linked ubiquitin chains in vitro. The FAAP20-UBZ domain is not required for interaction with FANCA, but is required for DNA-damage-induced chromatin loading of FANCA and the functional integrity of the FA pathway. These findings reveal critical roles for FAAP20 in the FA-BRCA pathway of DNA damage repair and genome maintenance.

The Fanconi Anemia Proteins Functionally Interact with the Protein Kinase Regulated by RNA (PKR)

Protein kinase regulated by RNA (PKR) plays critical roles in cell growth and apoptosis and is implicated as a potential pathogenic factor of Alzheimers, Parkinsons, and Huntingtons diseases. Here we report that this proapoptotic kinase is also involved in Fanconi anemia (FA), a disease characterized by bone marrow (BM) failure and leukemia. We have used a BM extract to show that three FA proteins, FANCA, FANCC, and FANCG, functionally interact with the PKR kinase, which in turn regulates translational control. By using a combined immunoprecipitation and reconstituted kinase assay, in which an active PKR kinase complex was captured from a normal cell extract, we demonstrated functional interactions between the FA proteins and the PKR kinase. In primary human BM cells, mutations in the FANCA, FANCC, and FANCG genes markedly increase the amount of PKR bound to FANCC, and this PKR accumulation is correlated with elevated PKR activation and hypersensitivity of BM progenitor cells to growth repression mediated by the inhibitory cytokines interferon-γ and tumor necrosis factor-α. Specific inhibition of PKR by 2-aminopurine in these FA BM cells attenuates PKR activation and apoptosis induction. In lymphoblasts derived from an FA-C patient, overexpression of a dominant negative mutant PKR (PKRK296R) suppressed PKR activation and apoptosis induced by interferon-γ and tumor necrosis factor-α. Furthermore, by using genetically matched wild-type and PKR-null cells, we demonstrated that forced expression of a patient-derived FA-C mutant (FANCCL554P) augmented double-stranded RNA-induced PKR activation and cell death. Thus, inappropriate activation of PKR as a consequence of certain FA mutations might play a role in bone marrow failure that frequently occurred in FA.

Nucleophosmin regulates cell cycle progression and stress response in hematopoietic stem/progenitor cells.

Nucleophosmin (NPM) is a multifunctional protein frequently overexpressed in actively proliferating cells. Strong evidence indicates that NPM is required for embryonic development and genomic stability. Here we report that NPM enhances the proliferative potential of hematopoietic stem cells (HSCs) and increases their survival upon stress challenge. Both short term liquid culture and clonogenic progenitor cell assays show a selective expansion of NPM-overexpressing HSCs. Interestingly, HSCs infected with NPM retrovirus show significantly reduced commitment to myeloid differentiation compared with vector-transduced cells, and majority of the NPM-overexpressing cells remains primitive during a 5-day culture. Bone marrow transplantation experiments demonstrate that NPM promotes the self-renewal of long term repopulating HSCs while attenuated their commitment to myeloid differentiation. NPM overexpression induces rapid entry of HSCs into the cell cycle and suppresses the expression of several negative cell cycle regulators that are associated with G1-to-S transition. NPM knockdown elevates expression of these negative regulators and exacerbates stress-induced cell cycle arrest. Finally, overexpression of NPM promotes the survival and recovery of HSCs and progenitors after exposure to DNA damage, oxidative stress, and hematopoietic injury both in vivo and in vitro. DNA repair kinetics study suggests that NPM has a role in reducing the susceptibility of chromosomal DNA to damage rather than promoting DNA damage repair. Together, these results indicate that NPM plays an important role in hematopoiesis via mechanisms involving modulation of HSC/progenitor cell cycle progression and stress response.

MTOR regulates DNA damage response through NF-κB-mediated FANCD2 pathway in hematopoietic cells

Hematopoietic stem/progenitor cells (HSPCs) function to give rise to mature blood cells. Effective DNA damage response (DDR) and maintenance of genomic stability are crucial for normal functioning of HSPCs. Mammalian target of rapamycin (mTOR) integrates signals from nutrients and growth factors to control protein synthesis, cell growth, survival and metabolism, and has been shown to regulate DDR in yeast and human cancer cells through the p53/p21 signaling cascade. Here, we show that gene targeting of mTOR in HSPCs causes a defective DDR due to a variety of DNA damage agents, mimicking that caused by deficient FANCD2, a key component of the Fanconi anemia (FA) DDR machinery. Mechanistically, mTOR−/− HSPCs express drastically reduced FANCD2. Consistent with these genetic findings, inactivation of mTOR in human lymphoblast cells by pp242 or Torin 1, mTOR kinase inhibitors, suppresses FANCD2 expression and causes a defective DDR that can be rescued by reconstitution of exogenous FANCD2. Further mechanistic studies show that mTOR deficiency or inactivation increases phosphorylation and nuclear translocation of nuclear factor (NF)-κB, which results in an enhanced NF-κB binding to FANCD2 promoter to suppress FANCD2 expression. Thus, mTOR regulates DDR and genomic stability in hematopoietic cells through a noncanonical pathway involving NF-κB-mediated FANCD2 expression.

Oxidative stress-specific interaction between FANCD2 and FOXO3a.

The molecular pathway by which Fanconi anemia (FA) proteins function in oxidative stress response has not been defined. Here we report the functional interaction of the FA protein Fanconi anemia complementation group D2 (FANCD2) and the forkhead transcription factor forkhead box O 3a (FOXO3a). FOXO3a colocalized with FANCD2 foci in response to oxidative stress. The FANCD2-FOXO3a complex was not detected in cells deficient for the FA core complex component FANCA but could be restored in corrected cells. Consistent with this, a nonmonoubiquitinated FANCD2 mutant failed to bind FOXO3a. Although both mitomycin C and ionizing radiation induced FANCD2 monoubiquitination, neither could induce the association of FANCD2 and FOXO3a. Overexpression of FOXO3a reduced abnormal accumulation of reactive oxygen species, enhanced cellular resistance to oxidative stress, and increased antioxidant gene expression in corrected but not mutant FA-D2 cells. The novel oxidative stress response pathway identified in this study, in which FANCD2 and FOXO3a converge, probably contributes to cellular antioxidant defense.