Tumor necrosis factor alpha (TNF-␣) production is abnormally high in IntroductionThe Fanconi anemia (FA) proteins play an important role in regulating genome stability, 1 but there is little evidence that the loss of the genoprotection per se in FA cells accounts for the molecular pathogenesis of the bone-marrow failure characteristic of this disease. In fact there is evidence that at least some of these proteins are multifunctional 2 and participate in canonical signaling pathways in hematopoietic cells. [2][3][4][5][6][7][8] Fanconi anemia, complementation group C (FANCC)-deficient cells, for example, are hypersensitive to the apoptotic effects of tumor necrosis factor-␣ (TNF-␣). [4][5][6][7][8][9] In addition, FA cells overproduce TNF-␣ for reasons that have not yet been fully explained. [10][11][12] Most importantly, there is clear evidence that overproduction of and hypersensitivity to TNF-␣ in hematopoietic cells of Fancc Ϫ/Ϫ mice results in bone marrow hypoplasia 13,14 and that long-term ex vivo exposure of murine Fancc Ϫ/Ϫ hematopoietic cells to both growth factors and TNF-␣ results in the evolution of cytogenetically marked preleukemic clones. 9 Therefore, the hematopoietic phenotype of FA may evolve from the overproduction of precisely the cytokine to which FA stem cells are hypersensitive. We designed gene expression microarray experiments by using marrow cells from both patients with FA and normal volunteers in part to seek potential clues to the mechanisms by which FA cells overproduce TNF-␣.Recognizing that transcriptomal analysis would not reveal aspects of the FA phenotype that were controlled translationally or posttranslationally, we also conducted a proteomics analysis. We based our experimental design on an accepted function of the FA "nuclear core complex," that is, its capacity to facilitate monoubiquitinylation of both Fanconi anemia, complementation group I and Fanconi anemia, complementation group D2 (FANCD2). 15,16 Although it is clear that monoubiquitinylation, at least of FANCD2, is required for the avoidance of genotoxicity, 17 it seemed to us unlikely that 8 individual FA genes encoding the "core complex proteins" should have evolved to control the monoubiquitinylation of merely 1 or 2 nuclear proteins. Therefore, reasoning that ubiquitinylation of a variety of other proteins might also be influenced by the core FA proteins, we designed a proteomics survey of ubiquitinylated proteins in FA-C cells and isogenic controls. We reasoned that this approach might lead to the identification of other proteins underubiquitinylated in mutant cells. As reported herein, the gene expression microarray analysis revealed a significant overrepresentation of overexpressed ubiquitin pathway genes in the mutant cells. We therefore took into account the alternative possibility that some ubiquitinylated proteins might be found uniquely in the mutant cells.Indeed, one such protein, Toll-like receptor 8 (TLR8), did appear in the ubiquitin-positive fractions only in FANCC-mutant cells. Given that TLR8 activ...

Fanconi anemia, complementation group C (FANCC)-deficient hematopoietic stem and progenitor cells are hypersensitive to a variety of inhibitory cytokines, one of which, TNF␣, can induce BM failure and clonal evolution in Fancc-deficient mice. FANCC-deficient macrophages are also hypersensitive to TLR activation and produce TNF␣ in an unrestrained fashion. Reasoning that suppression of inhibitory cytokine production might enhance hematopoiesis, we screened small molecules using TLR agonist-stimulated FANCCand Fanconi anemia, complementation IntroductionBM failure is a nearly universal complication of Fanconi anemia (FA), an inherited disease caused by biallelic inactivating mutations of any one of 15 genes. [1][2][3][4] FA gene products collectively facilitate responses to DNA damage, 1 and therefore it is often presumed (notwithstanding a lack of direct evidence supporting the idea) that hematopoietic defects simply reflect attrition of hematopoietic stem cells (HSCs) that have specifically suffered excessive DNA damage. An alternative explanation is that the FA proteins are multifunctional and play a direct role in stem cell maintenance, and therefore, DNA damage in FA HSCs is not necessarily required to suppress their function. [5][6][7][8][9] In normal cells, for example, Fanconi anemia, complementation group C (FANCC) modulates the hematopoietic inhibitory effects of TNF␣, IFN␥, and MIP-1␣, each of which normally function to suppress hematopoiesis. 6,10-12 FANCC influences TNF␣ responsiveness at least in part by modulating the activation state of the IFN-inducible double-stranded RNA activated protein kinase. 13 FANCC also suppresses the activation potential of certain TLR pathways in normal mononuclear phagocytes. 14 Therefore, in hematopoietic tissues, FANCC deficiency results in a TLR-dependent overproduction of TNF␣, one of the cytokines to which the stem-cell pool is uniquely intolerant. 10,[15][16][17][18][19] These abnormalities are important elements in the pathogenesis of BM failure. 6,20 There is also experimental evidence that this TNF␣-inhibitory loop is a selective pressure that enhances the ultimate emergence of TNF-resistant leukemic and preleukemic clones. [21][22][23] Therefore, interdiction of TNF␣-induced BM failure, particularly in ways that might have an additional favorable influence on IFN␥-and MIP-1␣-activated signaling pathways, may improve BM function and reduce the likelihood of clonal evolution by improving the fitness landscape and altering the coefficient of selection. 21 Seeking small molecules with these attributes, in the present study we exploited the TLR-hypersensitive phenotype as a screening tool to identify therapeutic agents that might suppress that pathway in FA cells. Using a TLR8-hypersensitive, FANCCdeficient mononuclear phagocyte cell line that we described previously, 14 we screened 75 small molecules, approximately 50 of which were kinase inhibitors. We identified 2 inhibitors, BIRB 796 and dasatinib, that functioned to suppress the TLR-dependent overproduction...

Key Points TLR-activated FANCA- and FANCC-deficient macrophages overproduce IL-1β. IL-1β suppresses in vitro expansion of Fancc-deficient multipotent hematopoietic progenitor cells.

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. (Cancer Res 2006; 66(18): 9017-25)