**6. The FA syndrome links inflammatory ROS to leukemogenesis**

Certain chronic inflammatory conditions have long been known to link to cancer. There is compelling evidence that chronic inflammation increases the risk of human cancers such as hepatocellular carcinoma, colon and bladder cancers, B cell lymphomas, and visceral malignancies (Kuper *et al.,* 2000; Mackay *et al.,* 2001; Martin *et al.,* 2011; Suematsu *et al.,* 2003; Umeda *et al.,* 2002; Ziech *et al.,* 2010), probably through the unbalanced machinery between DNA damage and repair (Fig. 3.).

Inflammatory ROS in Fanconi Anemia Hematopoiesis and Leukemogenesis 49

*al.,* 2007). Alterations of pro-inflammatory cytokine expression such as reduced IL-6 and

microenvironment changes such as growth factor deprivation or constant exposure to mitogenic inhibitors. These alterations may subsequently cause deregulation of cellular homeostasis in FA (de Cremoux *et al.,* 1996; Dufour *et al.,* 2003; Rosselli *et al.,* 1992, 1994; Schultz *et al.,* 1993; Stark *et al.,* 1993) at least partially through upregulation of ROS

ROS induce a variety of responses in HSCs, including cellular proliferation and apoptosis (Nakamura *et al.,* 1997; Nakata *et al.,* 2004). ROS can also cause DNA damage and drive HSCs into cell division, which is essential for DNA repair processes (Wilson A *et al.,* 2008). There is strong evidence that HSCs are activated and thus functionally exhausted by oxidative stress. Mice with mutations in the ATM or FOXO genes, as well as various DNA repair genes exhibit premature exhaustion of HSCs due to accumulation of ROS or DNA damage, indicating that cellular balance between ROS and antioxidant defense as well as DNA repair is crucial for the maintenance of HSC self-renewal and hematopoietic function (Rossi *et al.,*

considered as one important pathological factor involved in the abnormal hematopoiesis in FA. Extensive evidence demonstrated that excessive apoptosis of FA hematopoietic cells induced by TNF-, may contribute to at least partially the pathophysiology of BM failure in

*al.,* 2004; Ma *et al.,* 2009; Ventura *et al.,* 2004). The JNK kinase can be activated by TNF-

induced ROS. This activation then in turn leads to more ROS production, and sustained JNK activation in NF-B-deficient cells was suggested to depend on ROS. It has been shown that

cause mutation and cancer (Aggarwal *et al.,* 2003; Kryston *et al.,* 2011; Martin *et al.,* 2011 Sedelnikova *et al.,* 2010; Suematsu *et al.,* 2003; Wajant *et al.,* 2003; Ziech *et al.,* 2010). One possible mechanism is through Oxidation of bases and generation of DNA strand interruptions. However, the accurate measurement of oxidative stress is a hallmark of disease diagnosis as well as treatment. Recently, HPLC associated with tandem mass spectrometry (MS/MS) or electrochemical detector (ECD) together with optimized DNA extraction conditions has been developed as a relevant analytical approach for measuring oxidatively base damage in cellular DNA (Cadet *et al.,* 2006, 2010). Our recent studies demonstrated the inflammatory ROS-mediated hematopoietic suppression and increased chromosomal aberrations in *Fancc-/-* mice, which is associated with impaired oxidative DNA-damage repair, implicating a role of FA pathway in maintaining genomic stability

apoptotic signal suppressing FA hematopoietic progenitor activity, but also promotes leukemic transformation of FA hematopoietic stem/progenitor cells (Li *et al.,* 2007). Therefore, FA disease progression to leukemia is governed not only by genetic changes intrinsic to the FA cells, but also by epigenetic and environmental factors and that TNF-

mediated inflammation is one of the most important epigenetic and environmental factors contributing to FA leukemogenesis. Recent study indicate that FA hematopoietic cells are prone to clonal hematopoiesis and malignancy, which is associated with increased

induced ROS production at inflammatory sites causes DNA damage and therefore

(Sejas *et al.,* 2007; Zhang *et al.,* 2007). Further studies indicated that TNF-

FA. The c-JUN NH2-terminal kinase (JNK) and nuclear factor-kappa B (NF-

which are often found in FA patient cells, may account for BM

which is overproduced in FA patients, has been


not only is a pro-


B) pathways


increased TNF-

production.

TNF-

2007; Nijnik *et al.,* 2007).

The inflammatory cytokine TNF-

are two well-established pathway involved in TNF-

Fig. 3. Possible mechanisms for induction of oxidative stress and DNA damage and the roles in carcinogenesis. Intracellular stress or exogenous insults induces ROS production, which damages DNA, lipids and proteins. Over-produced ROS leads to cell death and activates cell defense machinery, including DNA repair and other cellular signaling pathways to maintain genome stability. Insufficient DNA repair or apoptosis causes mutagenesis, which results in cancer development.

Oxidative stress is considered as an important pathogenic factor in leukemia-prone bone marrow diseases like FA (Bogliolo *et al.,* 2002; Cohen–Haguenauer *et al.,* 2006; Cumming *et al.,* 1996; Futaki *et al.,* 2002; Hadjur *et al.,* 2001; Joenje *et al.,* 1987; Kruyt *et al.,* 1998; Mukhopadhyay *et al.,* 2006; Pagano *et al.,* 2005; Park *et al.,* 2004; Saadatzadeh *et al.,* 2004; Schindler *et al.,* 1988; Zhang *et al.,* 2005a, 2005b). The expression of inflammatory mediators, particularly the pro-inflammatory cytokines TNF-, interleukin-1beta (IL-1, and IL-6 in these patients is often associated with increased production of ROS either as a component of their immune response or as a consequence of increased metabolism (Macciò *et al.,* 1998; Mantovani *et al.,* 1997; Mantovani *et al.,* 2002; Tischkowitz *et al.,* 2004). Many studies have shown a correlation between elevated circulating pro-inflammatory cytokines and anemia in patients with leukemia-related BM diseases but direct evidence for the mechanistic link between inflammation and leukemia is lacking.

Normal hematopoiesis is maintained by dynamic interactions between HSCs and the bone marrow microenvironment, which is a complex system consisting of a variety of cell types, including stromal cells of nonhematopoietic, mesenchymal origin as well as hematopoietically derived stromal macrophages producing extracellular matrix components and hematopoietic growth factors (Bhatia *et al.,* 1995; Konopleva & Michael, 2007; Marina *et*

Fig. 3. Possible mechanisms for induction of oxidative stress and DNA damage and the roles in carcinogenesis. Intracellular stress or exogenous insults induces ROS production, which damages DNA, lipids and proteins. Over-produced ROS leads to cell death and activates cell defense machinery, including DNA repair and other cellular signaling pathways to maintain genome stability. Insufficient DNA repair or apoptosis causes mutagenesis, which

Oxidative stress is considered as an important pathogenic factor in leukemia-prone bone marrow diseases like FA (Bogliolo *et al.,* 2002; Cohen–Haguenauer *et al.,* 2006; Cumming *et al.,* 1996; Futaki *et al.,* 2002; Hadjur *et al.,* 2001; Joenje *et al.,* 1987; Kruyt *et al.,* 1998; Mukhopadhyay *et al.,* 2006; Pagano *et al.,* 2005; Park *et al.,* 2004; Saadatzadeh *et al.,* 2004; Schindler *et al.,* 1988; Zhang *et al.,* 2005a, 2005b). The expression of inflammatory mediators,

these patients is often associated with increased production of ROS either as a component of their immune response or as a consequence of increased metabolism (Macciò *et al.,* 1998; Mantovani *et al.,* 1997; Mantovani *et al.,* 2002; Tischkowitz *et al.,* 2004). Many studies have shown a correlation between elevated circulating pro-inflammatory cytokines and anemia in patients with leukemia-related BM diseases but direct evidence for the mechanistic link

Normal hematopoiesis is maintained by dynamic interactions between HSCs and the bone marrow microenvironment, which is a complex system consisting of a variety of cell types, including stromal cells of nonhematopoietic, mesenchymal origin as well as hematopoietically derived stromal macrophages producing extracellular matrix components and hematopoietic growth factors (Bhatia *et al.,* 1995; Konopleva & Michael, 2007; Marina *et*

, interleukin-1beta (IL-1

, and IL-6 in

results in cancer development.

particularly the pro-inflammatory cytokines TNF-

between inflammation and leukemia is lacking.

*al.,* 2007). Alterations of pro-inflammatory cytokine expression such as reduced IL-6 and increased TNF-which are often found in FA patient cells, may account for BM microenvironment changes such as growth factor deprivation or constant exposure to mitogenic inhibitors. These alterations may subsequently cause deregulation of cellular homeostasis in FA (de Cremoux *et al.,* 1996; Dufour *et al.,* 2003; Rosselli *et al.,* 1992, 1994; Schultz *et al.,* 1993; Stark *et al.,* 1993) at least partially through upregulation of ROS production.

ROS induce a variety of responses in HSCs, including cellular proliferation and apoptosis (Nakamura *et al.,* 1997; Nakata *et al.,* 2004). ROS can also cause DNA damage and drive HSCs into cell division, which is essential for DNA repair processes (Wilson A *et al.,* 2008). There is strong evidence that HSCs are activated and thus functionally exhausted by oxidative stress. Mice with mutations in the ATM or FOXO genes, as well as various DNA repair genes exhibit premature exhaustion of HSCs due to accumulation of ROS or DNA damage, indicating that cellular balance between ROS and antioxidant defense as well as DNA repair is crucial for the maintenance of HSC self-renewal and hematopoietic function (Rossi *et al.,* 2007; Nijnik *et al.,* 2007).

The inflammatory cytokine TNF- which is overproduced in FA patients, has been considered as one important pathological factor involved in the abnormal hematopoiesis in FA. Extensive evidence demonstrated that excessive apoptosis of FA hematopoietic cells induced by TNF-, may contribute to at least partially the pathophysiology of BM failure in FA. The c-JUN NH2-terminal kinase (JNK) and nuclear factor-kappa B (NF-B) pathways are two well-established pathway involved in TNF--induced ROS production (Nakata *et al.,* 2004; Ma *et al.,* 2009; Ventura *et al.,* 2004). The JNK kinase can be activated by TNF- induced ROS. This activation then in turn leads to more ROS production, and sustained JNK activation in NF-B-deficient cells was suggested to depend on ROS. It has been shown that TNF-induced ROS production at inflammatory sites causes DNA damage and therefore cause mutation and cancer (Aggarwal *et al.,* 2003; Kryston *et al.,* 2011; Martin *et al.,* 2011 Sedelnikova *et al.,* 2010; Suematsu *et al.,* 2003; Wajant *et al.,* 2003; Ziech *et al.,* 2010). One possible mechanism is through Oxidation of bases and generation of DNA strand interruptions. However, the accurate measurement of oxidative stress is a hallmark of disease diagnosis as well as treatment. Recently, HPLC associated with tandem mass spectrometry (MS/MS) or electrochemical detector (ECD) together with optimized DNA extraction conditions has been developed as a relevant analytical approach for measuring oxidatively base damage in cellular DNA (Cadet *et al.,* 2006, 2010). Our recent studies demonstrated the inflammatory ROS-mediated hematopoietic suppression and increased chromosomal aberrations in *Fancc-/-* mice, which is associated with impaired oxidative DNA-damage repair, implicating a role of FA pathway in maintaining genomic stability (Sejas *et al.,* 2007; Zhang *et al.,* 2007). Further studies indicated that TNF- not only is a proapoptotic signal suppressing FA hematopoietic progenitor activity, but also promotes leukemic transformation of FA hematopoietic stem/progenitor cells (Li *et al.,* 2007). Therefore, FA disease progression to leukemia is governed not only by genetic changes intrinsic to the FA cells, but also by epigenetic and environmental factors and that TNF- mediated inflammation is one of the most important epigenetic and environmental factors contributing to FA leukemogenesis. Recent study indicate that FA hematopoietic cells are prone to clonal hematopoiesis and malignancy, which is associated with increased

Inflammatory ROS in Fanconi Anemia Hematopoiesis and Leukemogenesis 51

**7. Functional interaction between the FA proteins and other oxidative stress** 

Recent findings of a reduction of the HSC pool and a deficient repopulating capacity in Foxo3a knockout animals (Miyamoto *et al.,* 2007) indicate that FOXO3a plays essential regulatory roles in HSC maintenance through a mechanism of regulating ROS. This is consistent with our recent finding that FANCD2 forms complex with FOXO3a in response to oxidative stress (Li *et al.,* 2010). In addition, we observed several hematopoietic defects in FA mice deficient for Foxo3a (unpublished data). These results suggest that the FA proteins functionally interplay with other oxidative stress response pathways. Indeed, our preliminary results with primary BM cells from FA-A patients show that certain genes functioning in anti-oxidant defense and ROS metabolism fail to respond to oxidative stress (unpublished data). This suggests that one critical function of FA proteins under oxidative stress is to safeguard the expression of these anti-oxidant defense genes through DNA damage repair or gene promoter protection. While these observations indicate that the FA pathway functionally interacts with other cellular oxidative stress response pathways, the molecular mechanisms by which FA proteins function to modulate physiologic oxidative stress remain to be elucidated. Further investigation into the roles of FA proteins in oxidative DNA-damage response and repair, and the functional relationship between inflammatory ROS and genomic instability during FA leukemogenesis not only will advance our understanding of the function of FA proteins in hematopoiesis but also may suggest new targets for therapeutic prevention and treatment of BM failure and cancer

Given other known genomic instability syndromes such as ataxia telangiectasia, Nijmegen breakage syndrome, xeroderma pigmentosum, and Werner syndrome rarely develop BM failure and leukemia, FA has been considered an excellent disease model for studying oxidative stress response in cancer development. Further investigation into the function of FA proteins in oxidative damage response and repair will help shed new light on the role of FA proteins in the maintenance of normal hematopoiesis under conditions of oxidative stress, and yield valuable information on whether targeting components of FA-related oxidative stress signaling pathways may be therapeutically useful in the prevention and treatment of FA BMF and leukemia. In addition, while FA is a rare disease, understanding functional interaction between FA proteins and other critical oxidative stress signaling pathways provides a unique opportunity to mechanistically comprehend and potentially

Aggarwal, B.B. (2003) Signaling pathways of the TNF superfamily: a double-edged sword.

Ames, B.N., Gold, L.S., & Willett WC. (1995) The causes and prevention of cancer. *Proc Natl* 

Aubé, M., Lafrance, M., Charbonneau, C., Goulet, I. & Carreau, M. (2002) Hematopoietic

response to growth factors. *Stem Cells*. 20(5): 438-447.

stem cells from fancc(-/-) mice have lower growth and differentiation potential in

**response pathways** 

progression of the disease.

intervene in these physiologically important processes.

*Nature Rev Immunol* 3: 745–756.

*Acad Sci USA.* 92: 5258–5265.

**8. Conclusion** 

**9. References** 

cytogenetic abnormalities and myeloid malignancies in *Fancc-/-* BM cells (Haneline *et al.,* 1998, 1999, 2003; Li X *et al.,* 2004; Si *et al.,* 2006). While the role of FA proteins in the regulation of TNF--induced ROS production remains to be elucidated, several hypotheses have been proposed, including that FA proteins protect chromosomal DNA from ROS attack or facilitate the repair of oxidative DNA damage, which in turn downstream ROS signaling. It is also possible that FA proteins can regulate the biosynthesis of ROS metabolic molecules, such as glutathione and the expression of antioxidant enzymes (such as glutathione *S*transferases and catalase). However, there is no direct evidence for any of these assumptions so far. Another potential target is the redox-sensitive transcription factor NF- B, a major player involved in transcription regulating during differentiation and inflammation (Dhar *et al.,* 2006). The activation of NF-B is known to enhance inflammation and promote cancer (Coussens *et al.,* 2002; Fiers *et al.,* 1999; Macdougal *et al.,* 2002). In addition, chronic exposure of FA BM cells to proinflammatory cytokine TNF- creates an environment selects for somatically mutated preleukemic stem cell clones which are apoptosis-resistant and acquire proliferative advantage (Li *et al.,* 2007). Patients with these TNF--resistant BM cells may advance to MDS and AML via a mechanism involving genomic instability, coupled with inflammation driven by high NF-B transcriptional activity (Fig. 4).

Fig. 4. The pro-inflammatory cytokines and their potential role in FA pathophysiology. Overproduced pro-inflammatory cytokines (TNF-, IL-6, IL-1 etc.) plays roles in not only pro-apoptotic signal suppressing FA hematopoietic progenitor activity, but also promoting leukemic transformation of FA HSC/P cells, which lead to typical phenotype of FA patients.
