**2. FA hematopoiesis**

42 Cancer Prevention – From Mechanisms to Translational Benefits

Table 1. Complementation groups and interaction proteins of Fanconi Anemia.

Fig. 1. Function of the FA pathway. Eight FA proteins form a nuclear core complex, which acts as ubiquitin ligase. FANCM interacts with FAAP24, FAAP100 as well as MHF1 and MFH2, resulting in complex chromatin loading and controlling the processing and

stabilization of stalled forks, respectively. In response to DNA damage or replication stress, nuclear core complex monoubiquitinates two other FA proteins, FANCD2 and FANCI, which then recruit other downstream FA proteins FANCD1, FANCJ, and FANCN to damaged DNA and involved in DNA repair, cell-cycle control to repair ICL (interstrand

Many studies indicate that FA proteins might play specific roles in hematopoiesis by governing the responses of hematopoietic cells to both genotoxic and cytotoxic stresses. Loss of FA functions causes excessive apoptosis of HSC and progenitor cells (HSC/P) cells leading to BMF in the early stage of FA. As the disease progresses, apoptosis as well as genomic instability impose a selective pressure on FA HSC/P cells and promote the

crosslink) lesions and to maintain genome stability.

Hematological abnormalities are among the most important clinical features of FA. Children with FA often develop pancytopenia during the first few years of life. Complications of BM failure (BMF) are the major causes of morbidity and mortality of FA, and 80% of FA patients die from BMF (Bagby *et al.,* 2003; Buchwald *et al.,* 1998; Fagerlie *et al.,* 2001; Kutler *et al.,* 2003; Lensch *et al.,* 1999; Liu *et al.,* 2000). In addition, patients with FA have high risk of developing myelodysplasia (MDS) or acute myeloblastic leukemia (AML) (Bagby *et al.,* 2003; Buchwald *et al.,* 1998; D'Andrea *et al.,* 2003; Fagerlie *et al.,* 2001; Kennedy *et al.,* 2005; Tischkowitz *et al.,* 2003). During the BMF-MDS-AML progression, FA patients frequently develop clonal chromosomal abnormalities in the BM HSC/P cells. In fact, secondary occurred clonal cytogenetic abnormalities, such as 3q addition, 5q deletion and monosomy 7, are common in in children with FA who have evolved to MDS and AML and non-FA patients with MDS and AML after alkylating agents treatment (Freie *et al.,* 2004; Fridman *et al.,* 2003; Futaki *et al.,* 2002; Giaccia *et al.,* 1998; Lina-Fineman *et al.,* 1995; Rubin *et al.,* West *et al.,* 2000).

Excessive apoptosis and subsequent failure of the HSC compartment led to progressive BMF in FA patients have been documented from *in vitro* and *in vivo* studies. However, the molecular etiology of BMF and leukemia in FA remains to be elucidated. Compelling evidence suggest that altered expression of certain growth factors and cytokines, such as reduced expression of interleukin-6 (IL-6) and granulocyte-macrophage colony stimulating factor (GM-CSF) but increased secretion of mitotic inhibitor TNF- in patient BM cells, may in part be responsible for hematopoietic disease progression in FA (de Cremoux *et al.,* 1996; Dufour *et al.,* 2003; Rosselli *et al.,* 1992; 1994; Schultz *et al.,* 1993; Stark *et al.,* 1993). It is conceivable that these alterations may change the BM microenvironment (for instance, leading to factor deprivation or constant exposure to mitogenic inhibitors) and cause deregulation of cellular homeostasis. It has also been shown that FA BM cells are hypersensitive to a variety of extracellular cytokines, including interferon- (IFN-) and tumor necrosis factor (TNF- (Dufour *et al.,* 2003; Fagerlie *et al.,* 2001; Haneline *et al.,* 1998; Koh *et al.,* 1999; Li X *et al.,* 2004; Li Y *et al.,* 2004; Nakata *et al.,* 2004; Pang *et al.,* 2001a, 2001b, 2002; Rathbun *et al.,* 1997, 2000; Reid *et al.,* 2006; Rosselli *et al.,* 1992; Schultz *et al.,* 1993; Si *et al.,* 2006; Wang *et al.,* 1998; Whitney *et al.,* 1996), which may subsequently lead to cell apoptosis. Indeed, studies of FA patients have demonstrated that BM from FA patients has decreased number of colony-forming progenitors, as well as a reduction in colony size (Doneshbod–Skibba *et al.,* 1980; Gluckman *et al.,* 1989). These data demonstrate defective hematopoiesis in FA (Bagby *et al.,* 2003; Fagerlie *et al.,* 2001; Tischkowitz *et al.,* 2003).

In contrast to FA patients, mouse models deficient for several FA genes, including *Fanca*, *Fancc*, *Fancd2* and *Fancg*, do not show no spontaneous hematological defects or leukemia development (Cheng *et al.,* 2000; Whitney *et al.,* 1996; Wong & Buchwald, 2002; Yang *et al.,* 2001). Studies in the Fanca and Fancc mouse models show that while blood count and the

Inflammatory ROS in Fanconi Anemia Hematopoiesis and Leukemogenesis 45

by complementation of functional FANCA into FA-A lymphocytes. In addition, the constitutive activation of the PI3K-Akt pathway in FA cells upregulates the expression of IL-1*β* through an NF-*κ*B independent mechanism and this overproduction activates the proliferation of tumour cells (Ibanez *et al.,* 2009). IL-6 is the chief stimulator of the production of most acute phase proteins (Scheller *et al.,* 2011), whereas the other implicated cytokines influence subgroups of acute phase proteins. Recent studies demonstratethe presence of a defect in IL-6 production in FA patients (Coussens *et al.,* 2002; Cumming *et al.,* 1996), suggesting that this cytokine may partly be responsible for pancytopenia associated with BMF, the major clinical feature of FA, in FA patients. In addition, it has been reported that *Fancc-/-* HSC/P cells had altered growth and apoptosis responses to combinations of stimulatory cytokines, most dramatically in response to a combination of factors that

Even in steady state, hematopoietic cells are exposed to various ROS, which are routinely generated during metabolic or inflammatory process. ROS induce a variety of responses in hematopoietic cells, including cellular proliferation and growth inhibition (Howlett *et al.,* 2002; Ichijo *et al.,* 1997). Like cells from other tissues, hematopoietic cells have developed several mechanisms to prevent the damage induced by oxidative stress. First, antioxidant enzymes, including superoxide dismutases (SODs), catalase, glutathione peroxidases and peroxiredoxins, can directly eliminate ROS. Secondly, other cellular enzymes can function to repair DNA damage induced by ROS in hematopoietic tissues. While FA murine models do not recapitulate some of the major FA clinical manifestations such as BM failure and leukemia, hematopoietic cells from FA knockout mice exhibit extreme oxidant sensitivity. Extensive studies have demonstrated FA oxidant hypersensitivity by using primary and immortalized cell cultures as well as *ex vivo* materials from patients (Bogliolo *et al.,* 2002; Cohen–Haguenauer *et al.,* 2006; Cumming *et al.,* 1996; Futaki *et al.,* 2002; Hadjur *et al.,* 2001; Kruyt *et al.,* 1998; Pagano *et al.,* 2005; Park *et al.,* 2004; Saadatzadeh *et al.,* 2004). It has also been shown that reoxygenation-generated oxidative stress, which is associated with significant DNA damage and inhibition of colony formation capacity (Ames *et al.,* 1993; Hammond *et al.,* 2003; Chen *et al.,* 2000), induced senescence of bone marrow progenitor cells from *Fancc-/-* mice compared to their counterparts. While these studies suggest a correlation between oxidative stress and FA disease progression, the mechanism by which oxidative stress influences the function of FA HSC/P cells has not been systematically studied. A number of hypotheses regarding the effect of oxidative stress in FA have been suggested, including the proposal that ROS could damage DNA and inability of FA HSC/P cells to repair such damage would result in exacerbated genomic instability leading to

Three major FA core complex components, FANCA, FANCC, and FANCG (Bagby *et al.,* 2003; Kennedy *et al.,* 2000; Green *et al.,* 2009), were found to interact with a variety of cellular factors that primarily function in redox-related processes (Table 2), such as FANCC protein interacts with NADPH cytochrome P450 reductase and glutathione S-transferase P1-1 (Cumming *et al.,* 1996; Kruyt *et al.,* 1998), which are involved in either triggering or detoxifying reactive intermediates including ROS. It has also been demonstrated that *Fancc- /-* mice with deficiency in the anti-oxidative enzyme Cu/Zn superoxide dismutase

included interleukin-3 (IL-3) and IL-6 (Aubé *et al.,* 2002).

**4. FA oxidant hypersensitivity** 

apoptosis and malignant transformation.

number of committed BM progenitors are normal in FA mice as compared to WT mice; however, when subjected to sublethal dose of DNA cross-linking agent mitomycin C (MMC), which does not affect WT mouse cells, to the mutant mice experienced progressive decrease of all peripheral blood parameters, as well as early and committed progenitors, and eventually died within 8 weeks (Chen *et al.,* 1996; Whitney *et al.,* 1996). These results suggest that loss of FA genes in mouse models results in compromised defects in response to environmental insults (Chen *et al.,* 1996; Whitney *et al.,* 1996; Pang *et al.,* 2000; Rathbun *et al.,* 1997; Haneline *et al.,* 1998; Wong & Buchwald, 2002).

Similar to FA-C patients, BM cells from *Fancc-/-* mice show compromised colony growth capacity following IFN-, TNF- and MIP-1 treatment (Haneline *et al.,* 1998). Literatures suggest that IFN- and TNF- suppress colony growth forming ability of FA mouse BM cells by upregulating other cellular receptors, such as the fas receptor (CD95) (Young *et al.,* 1997). Increase in CD95 expression has been found in CD34+ cells from children with FA as well as the CD34+ fraction of hematopoietic progenitors in *Fancc-/-* mice, which is associated with increased apoptosis (Cumming *et al.,* 1996; Otsuki *et al.,* 1999). The hypersensitivity of *Fancc- /-* hematopoietic cells to IFN- and TNF- is also mediated through activation of the RNAdependent protein kinase (PKR) pathway, which is reported to initiate apoptosis in some instances, as an elevated level of activated PKR was found in *Fancc-/-* mouse embryonic fibroblasts (Pang *et al.,* 2001, 2002; Zhang *et al.,* 2004). Several groups independently showed compromised hematopoietic engraftment and reconstitution after BM transplantation of FA HSCs (Haneline *et al.,* 2003; Zhang *et al.,* 2007). Deregulation of apoptotic responses in hematopoietic cells may account at least in part for the nearly universal development of BM failure in children with inactivating FA mutations.
