**Inflammatory ROS in Fanconi Anemia Hematopoiesis and Leukemogenesis**

Wei Du

*Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio USA* 

#### **1. Introduction**

40 Cancer Prevention – From Mechanisms to Translational Benefits

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Fanconi anemia (FA) is a genetic disorder characterized by bone marrow failure (BMF), clonal proliferation of hematopoietic stem cells, and transformation to leukemia and other cancers (Ames *et al.,* 1995; Boglilo *et al.,* 2002; Cohen-Haguenauer *et al.,* 2006; Cumming *et al.,* 2001; Fagerlie *et al.,* 2001; Jonkers *et al.,* 2001; Suematsu *et al.,* 2003). Somatic cell fusion studies show FA is genetically heterogeneous. So far mutations in 15 genes have been identified in FA or FA-like patients (Cohen-Haguenauer *et al.,* 2006; Joenje *et al.,* 1987; Jonkers *et al.,* 2001; Lensch *et al.,* 1999; Stoepker *et al.,* 2011; Yamamoto *et al.,* 2011). The genes encoding the groups A (FANCA), B (FANCB), C (FANCC), D1 (FANCD1/BRCA2), D2 (FANCD2), E (FANCE), F (FANCF), G (FANCG), -I (FANCI/KIAA1794), J (FANCJ/ BRIP1), L (FANCL), M (FANCM), N (FANCN/PALB2), O/RAD51C and P/SLX4 proteins have been cloned (de Winter *et al.,* 1998, 2000a, 2000b; Howlett *et al.,* 2002; Joenje *et al.,* 2000; Letitus *et al.,* 2004; Levran *et al.,* 2005; Lo Ten Foe *et al.,* 1996; Meetei *et al.,* 2003, 2004, 2005; Meindl *et al.,* 2010; Reid *et al.,* 2006; Smogorzewska *et al.,* 2007; Somyajit *et al.,* 2010; Strathdee *et al.,* 1992; Timmers *et al.,* 2001; Xia *et al.,* 2006; Yamamoto *et al.,* 2011). The latter two genes are still thought of as tentative as they do not fall within a defined category biologically and the patients carrying these gene mutations are limited. The majority of mutations are found in *FANCA*, *FANCC* and *FANCC* genes in FA patients (Table 1). Recent studies on the biological function of these FA proteins have demonstrated that eight of the FA proteins (namely, FANCA, B, C, E, F, G, L, and M) form a nuclear multiprotein complex (Collins *et al.,* 2005; D'Andrea *et al.,* 2003; de Winter *et al.,* 2000; Meetei *et al.,* 2003; Smogorzewska *et al.,* 2007; Tischkowitz *et al.,* 2003; Walsh *et al.,* 1994), which functions as a nuclear E3 ubiquitin ligase that monoubiquitinates downstream FANCD2/FANCI dimer in response to DNA damage or DNA replication stress. This FANCD2/FANCI heterodimer then recruits other downstream FA proteins including FANCD1 (which is the breast cancer protein BRCA2), and the recently identified FANCJ, FANCN, FANCO and another breast cancer protein, BRCA1 (D'Andrea *et al.,* 2010), to nuclear loci containing damaged DNA and consequently influence important cellular processes such as DNA replication, cell-cycle control, and DNA damage repair. The core complex also interacts with the FAAP100 and FAPP24 proteins, which are also crucial components in the pathway (Ciccia *et al.,* 2007; Horejsi *et al.,* 2009; Collis *et al.,* 2008, Fig 1). FANCM and its interacting proteins, such as FAAP24 and MHF1, MHF2, also play a role in controlling the processing and stabilization of stalled replication forks (Schwab *et al.,* 2010; Luke-Glaser *et al.,* 2010; Singh *et al.,* 2010).

Inflammatory ROS in Fanconi Anemia Hematopoiesis and Leukemogenesis 43

development of mutant clones, which could be transformed to leukemia (Cumming *et al.,* 1996, 2001; Fagerlie *et al.,* 2001; Haneline *et al.,* 1998, 1999, 2003; Koh *et al.,* 1999; Li X *et al.,* 2004; Li Y *et al.,* 1997; Maciejewski *et al.,* 1995; Nakata *et al.,* 2004; Pang *et al.,* 2001a, 2001b, 2002; Rathbun *et al.,* 1997, 2000; Si *et al.,* 2006; Walsh *et al.,* 1994; Wang *et al.,* 1998; Whitney

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* 

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

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

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

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

(Dufour *et al.,* 2003; Fagerlie *et al.,* 2001; Haneline *et al.,* 1998;

in patient BM cells, may

 (IFN-) and

factor (GM-CSF) but increased secretion of mitotic inhibitor TNF-

hypersensitive to a variety of extracellular cytokines, including interferon-

hematopoiesis in FA (Bagby *et al.,* 2003; Fagerlie *et al.,* 2001; Tischkowitz *et al.,* 2003).

*et al.,* 1996).

*al.,* 2000).

tumor necrosis factor

(TNF-

**2. FA hematopoiesis** 


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 crosslink) lesions and to maintain genome stability.

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 development of mutant clones, which could be transformed to leukemia (Cumming *et al.,* 1996, 2001; Fagerlie *et al.,* 2001; Haneline *et al.,* 1998, 1999, 2003; Koh *et al.,* 1999; Li X *et al.,* 2004; Li Y *et al.,* 1997; Maciejewski *et al.,* 1995; Nakata *et al.,* 2004; Pang *et al.,* 2001a, 2001b, 2002; Rathbun *et al.,* 1997, 2000; Si *et al.,* 2006; Walsh *et al.,* 1994; Wang *et al.,* 1998; Whitney *et al.,* 1996).
