Progressive bone marrow failure is a major cause of morbidity and mortality in human Fanconi Anemia patients. In an effort to develop a Fanconi Anemia murine model to study bone marrow failure, we found that Fancd2 ؊/؊ mice have readily measurable hematopoietic defects. Fancd2 deficiency was associated with a significant decline in the size of the c-Kit ؉ Sca-1 ؉ Lineage ؊ (KSL) pool and reduced stem cell repopulation and spleen colony-forming capacity. Fancd2 ؊/؊ KSL cells showed an abnormal cell cycle status and loss of quiescence. In addition, the supportive function of the marrow microenvironment was compromised in Fancd2 ؊/؊ mice. Treatment with Sirt1-mimetic and the antioxidant drug, resveratrol, maintained Fancd2 ؊/؊ KSL cells in quiescence, improved the marrow microenvironment, partially corrected the abnormal cell cycle status, and significantly improved the spleen colony-forming capacity of Fancd2 ؊/؊ bone marrow cells. We conclude that Fancd2 ؊/؊ mice have readily quantifiable hematopoietic defects, and that this model is well suited for pharmacologic screening studies. IntroductionFanconi anemia (FA) is a rare, autosomal, recessive genetic disorder associated with severe birth defects, cancer predisposition, and bone marrow failure. Thirteen causative genes (FANCA, FANCB, FANCC, FANCD1/BRCA2, FANCD2, FANCE, FANCF, FANCG/XRCC9, FANCI, FANCL/PHF9/Pog, FANCJ/BRIP1/BACH1, FANCM/Hef, and FANCN/ PALB2) have been identified and cloned to date, and the encoded proteins are believed to work together in a common DNA damageresponse pathway to maintain genomic integrity and protect the genome from DNA damage induced by cross-linking agents. 1,2 Although deficiency in DNA cross-link repair renders all FA cells susceptible to cross-linking agents, bone marrow is the most affected organ system. Mutations in any of the different FA genes almost universally lead to bone marrow failure, which is the primary cause of mortality in FA. 3 The pathogenesis of bone marrow failure in FA remains elusive. Mutations in several genes involved in DNA damage repair, including Atr, XPD, and Ercc1, caused either hematopoietic stem cell (HSC) loss or impaired HSC function under conditions of stress. [4][5][6] These studies suggest that the maintenance of genome integrity is critical for HSC survival and function. However, the extent to which genotoxicity, resulting from impaired DNA damage repair, contributes to bone marrow failure in FA is unclear. 7 Other pathways associated with hematopoietic failure, such as altered cytokine signaling, may also contribute to FA pathogenesis. 8,9 For example, levels of proapoptotic cytokines tumor necrosis factor-␣ (TNF-␣) and interferon-␥ (IFN-␥) are elevated in FA lymphocytes, bone marrow cells, and FA patient serum samples. 10-12 FA bone marrow cells (at least of the C complementation group) are also hypersensitive to these cytokines and undergo apoptosis when exposed to even low levels of them. [13][14][15] To better understand FA, multiple murine knockout models, includingand Fancl Ϫ/Ϫ m...

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...