Jane Yates

TLR8-dependent TNF-α overexpression in Fanconi anemia group C cells

Tumor necrosis factor alpha (TNF-alpha) production is abnormally high in Fanconi anemia (FA) cells and contributes to the hematopoietic defects seen in FA complementation group C-deficient (Fancc(-/-)) mice. Applying gene expression microarray and proteomic methods to studies on FANCC-deficient cells we found that genes encoding proteins directly involved in ubiquitinylation are overrepresented in the signature of FA bone marrow cells and that ubiquitinylation profiles of FA-C and complemented cells were substantially different. Finding that Toll-like receptor 8 (TLR8) was one of the proteins ubiquitinylated only in mutant cells, we confirmed that TLR8 (or a TLR8-associated protein) is ubiquitinylated in mutant FA-C cells and that TNF-alpha production in mutant cells depended upon TLR8 and the canonical downstream signaling intermediates interleukin 1 receptor-associated kinase (IRAK) and IkappaB kinase-alpha/beta. FANCC-deficient THP-1 cells and macrophages from Fancc(-/-) mice overexpressed TNF-alpha in response to TLR8 agonists but not other TLR agonists. Ectopically expressed FANCC point mutants were capable of fully complementing the mitomycin-C hypersensitivity phenotype of FA-C cells but did not suppress TNF-alpha overproduction. In conclusion, FANCC suppresses TNF-alpha production in mononuclear phagocytes by suppressing TLR8 activity and this particular function of FANCC is independent of its function in protecting the genome from cross-linking agents.

Cytogenetic Instability in Ovarian Epithelial Cells from Women at Risk of Ovarian Cancer

Fanconi anemia is an inherited cancer predisposition disease characterized by cytogenetic and cellular hypersensitivity to cross-linking agents. Seeking evidence of Fanconi anemia protein dysfunction in women at risk of ovarian cancer, we screened ovarian surface epithelial cells from 25 primary cultures established from 22 patients using cross-linker hypersensitivity assays. Samples were obtained from (a) women at high risk for ovarian cancer with histologically normal ovaries, (b) ovarian cancer patients, and (c) a control group with no family history of breast or ovarian cancer. In chromosomal breakage assays, all control cells were mitomycin C (MMC) resistant, but eight samples (five of the six high-risk and three of the eight ovarian cancer) were hypersensitive. Lymphocytes from all eight patients were MMC resistant. Only one of the eight patients had a BRCA1 germ-line mutation and none had BRCA2 mutations, but FANCD2 was reduced in five of the eight. Ectopic expression of normal FANCD2 cDNA increased FANCD2 protein and induced MMC resistance in both hypersensitive lines tested. No FANCD2 coding region or promoter mutations were found, and there was no genomic loss or promoter methylation in any Fanconi anemia genes. Therefore, in high-risk women with no BRCA1 or BRCA2 mutations, tissue-restricted hypersensitivity to cross-linking agents is a frequent finding, and chromosomal breakage responses to MMC may be a sensitive screening strategy because cytogenetic instability identified in this way antedates the onset of carcinoma. Inherited mutations that result in tissue-specific FANCD2 gene suppression may represent a cause of familial ovarian cancer.

p38 MAPK inhibition suppresses the TLR-hypersensitive phenotype in FANCC- and FANCA-deficient mononuclear phagocytes

Fanconi anemia, complementation group C (FANCC)-deficient hematopoietic stem and progenitor cells are hypersensitive to a variety of inhibitory cytokines, one of which, TNFα, can induce BM failure and clonal evolution in Fancc-deficient mice. FANCC-deficient macrophages are also hypersensitive to TLR activation and produce TNFα in an unrestrained fashion. Reasoning that suppression of inhibitory cytokine production might enhance hematopoiesis, we screened small molecules using TLR agonist-stimulated FANCC- and Fanconi anemia, complementation group A (FANCA)-deficient macrophages containing an NF-κB/AP-1-responsive reporter gene (SEAP). Of the 75 small molecules screened, the p38 MAPK inhibitor BIRB 796 and dasatinib potently suppressed TLR8-dependent expression of the reporter gene. Fanconi anemia (FA) macrophages were hypersensitive to the TLR7/8 activator R848, overproducing SEAP and TNFα in response to all doses of the agonist. Low doses (50nM) of both agents inhibited p38 MAPK-dependent activation of MAPKAPK2 (MK2) and suppressed MK2-dependent TNFα production without substantially influencing TNFα gene transcription. Overproduction of TNFα by primary FA cells was likewise suppressed by these agents and involved inhibition of MK2 activation. Because MK2 is also known to influence production and/or sensitivity to 2 other suppressive factors (MIP-1α and IFNγ) to which FA hematopoietic progenitor cells are uniquely vulnerable, targeting of p38 MAPK in FA hematopoietic cells is a rational objective for preclinical evaluation.

FANCL ubiquitinates β-catenin and enhances its nuclear function

Bone marrow failure is a nearly universal complication of Fanconi anemia. The proteins encoded by FANC genes are involved in DNA damage responses through the formation of a multisubunit nuclear complex that facilitates the E3 ubiquitin ligase activity of FANCL. However, it is not known whether loss of E3 ubiquitin ligase activity accounts for the hematopoietic stem cell defects characteristic of Fanconi anemia. Here we provide evidence that FANCL increases the activity and expression of β-catenin, a key pluripotency factor in hematopoietic stem cells. We show that FANCL ubiquitinates β-catenin with atypical ubiquitin chain extension known to have nonproteolytic functions. Specifically, β-catenin modified with lysine-11 ubiquitin chain extension efficiently activates a lymphocyte enhancer-binding factor-T cell factor reporter. We also show that FANCL-deficient cells display diminished capacity to activate β-catenin leading to reduced transcription of Wnt-responsive targets c-Myc and Cyclin D1. Suppression of FANCL expression in normal human CD34(+) stem and progenitor cells results in fewer β-catenin active cells and inhibits expansion of multilineage progenitors. Together, these results suggest that diminished Wnt/β-catenin signaling may be an underlying molecular defect in FANCL-deficient hematopoietic stem cells leading to their accelerated loss.

FANCA and FANCC modulate TLR and p38 MAPK dependent expression of IL-1β in macrophages

Hematopoietic stem and progenitor cells with inactivated Fanconi anemia (FA) genes, FANCA and FANCC, are hypersensitive to inflammatory cytokines. One of these, tumor necrosis factor α (TNF-α), is also overproduced by FA mononuclear phagocytes in response to certain Toll-like receptor (TLR) agonists, creating an autoinhibitory loop that may contribute to the pathogenesis of progressive bone marrow (BM) failure and selection of TNF-α-resistant leukemic stem cell clones. In macrophages, the TNF-α overproduction phenotype depends on p38 mitogen-activated protein kinase (MAPK), an enzyme also known to induce expression of other inflammatory cytokines, including interleukin 1β (IL-1β). Reasoning that IL-1β might be involved in a like autoinhibitory loop, we determined that (1) TLR activation of FANCA- and FANCC-deficient macrophages induced overproduction of both TNF-α and IL-1β in a p38-dependent manner; (2) exposure of Fancc-deficient BM progenitors to IL-1β potently suppressed the expansion of multipotent progenitor cells in vitro; and (3) although TNF-α overexpression in FA cells is controlled posttranscriptionally by the p38 substrate MAPKAPK-2, p38-dependent overproduction of IL-1β is controlled transcriptionally. We suggest that multiple inflammatory cytokines overproduced by FANCA- and FANCC-deficient mononuclear phagocytes may contribute to the progressive BM failure that characterizes FA, and that to achieve suppression of this proinflammatory state, p38 is a more promising molecular therapeutic target than either IL-1β or TNF-α alone.

Cigarette smoke induces genetic instability in airway epithelial cells by suppressing FANCD2 expression

Chromosomal abnormalities are commonly found in bronchogenic carcinoma cells, but the molecular causes of chromosomal instability (CIN) and their relationship to cigarette smoke has not been defined. Because the Fanconi anaemia (FA)/BRCA pathway is essential for maintenance of chromosomal stability, we tested the hypothesis that cigarette smoke suppresses that activity of this pathway. Here, we show that cigarette smoke condensate (CSC) inhibited translation of FANCD2 mRNA (but not FANCC or FANCG) in normal airway epithelial cells and that this suppression of FANCD2 expression was sufficient to induce both genetic instability and programmed cell death in the exposed cell population. Cigarette smoke condensate also suppressed FANCD2 function and induced CIN in bronchogenic carcinoma cells, but these cells were resistant to CSC-induced apoptosis relative to normal airway epithelial cells. We, therefore, suggest that CSC exerts pressure on airway epithelial cells that results in selection and emergence of genetically unstable somatic mutant clones that may have lost the capacity to effectively execute an apoptotic programme. Carcinogen-mediated suppression of FANCD2 gene expression provides a plausible molecular mechanism for CIN in bronchogenic carcinogenesis.

Human FANCC is hypomorphic in murine Fancc-deficient cells

Fancc suppresses cross-linker-induced genotoxicity, modulates growth-inhibitory cytokine responses, and modulates endotoxin responses. Although loss of the latter function is known to account for endotoxin-induced marrow failure in murine Fancc (mFancc)-deficient mice, some argue that cytokine and endotoxin hypersensitivities devolve simply from genomic instability. Seeking to resolve this question, we planned to ectopically express instructive human FANCC (hFANCC) mutants in murine Fancc-deficient hematopoietic stem cells. To first assure that hFANCC cDNA was competent in murine cells, we compared hFANCC and mFancc in complementation assays for cross-linking agent hypersensitivity and endotoxin hypersensitivity. We found that mFancc complemented murine Fancc-deficient cells in both assays, but that hFANCC fully suppressed only endotoxin hypersensitivity, not cross-linking agent hypersensitivity. These results support the notions that Fancc is multifunctional and that structural prerequisites for its genoprotective functions differ from those required to constrain endotoxin responses known to lead to marrow failure in Fancc-deficient mice.

PlGF enhances TLR-dependent inflammatory responses in human mononuclear phagocytes

Levels of placental growth factor (PlGF) peak during third trimester of pregnancy, a time when women are at increased risk of virus‐induced morbidity. We hypothesized PlGF might contribute to an exaggerated inflammatory response to Toll‐like receptor (TLR) activation.

The PI3K/Akt1 pathway enhances steady-state levels of FANCL

The Fanconi anemia pathway supports hematopoietic stem cell survival in response to inflammatory and metabolic stress. We show that polyubiquitination and proteasome degradation of FANCL is inhibited by Akt1 activation, revealing a potentially important mechanism for the maintenance of stem cell function.