**Abstract**

Reactive oxygen species (ROS) is the collective term for several oxygen containing free radicals, such as hydrogen peroxide. ROS is important in innate immunity, protein folding in the endoplasmic reticulum and as a cell signalling molecule involved in cellular proliferation, survival, differentiation, and gene expression. ROS has been implicated in both hematopoietic stem cell quiescence and hematopoietic differentiation. Consequently, ROS is of considerable interest as a therapeutic target, with both pro-oxidant and anti-oxidant cellular modulation being explored. Recently, it has been established that increased ROS production in acute myeloid leukemia (AML) leads to increased glycolysis and metabolic reprogramming. It is often stated as a key tenet of the Warburg effect, that transformed cells, including AML, show increased aerobic glycolysis accompanied by increased cellular glucose uptake and lactate secretion. This review will summarize ROS state of the art in acute leukemia and how these reactive molecules re-wire metabolism in cancer cells. The review will focus on what are ROS? What are the sources of ROS in hematopoietic cells and their function and how this relates to the Warburg effect and regulation of metabolic pathways in acute leukemias.

**Keywords:** NADPH, NOX, ROS, hematopoiesis, HSC, AML

#### **1. Introduction**

To maintain hemostasis, new blood cells must be constantly generated to replace those lost through injury, disease, or age. Hematopoiesis, is the process where hematopoietic stem cells (HSC) differentiate into mature blood cells and is tightly regulated by the bone marrow (BM) micro-environment (or stem cell niche; reviewed in [1]), signal transduction pathways (reviewed in [2]), cytokines (reviewed in [3]), transcription factors (reviewed in [4]), epigenetics, (reviewed in [5]) and metabolic pathways (reviewed in [6]). HSCs are rare, constituting only 0.001% of peripheral blood (PB) and 0.05% of BM cells, but are responsible for producing a lifetime supply of blood cells. HSCs are cells that able to durably self-renew whilst also being multipotent. This differentiation is generally considered to occur via several intermediate progenitor cells, ultimately terminating in the specific mature blood cell through a process termed fate restriction or lineage commitment.

The compartmentalization of HSC, their progenitors and terminally differentiated blood cells, into different stages of differentiation, is traditionally based on the expression of cell surface proteins (**Figure 1**). The recent emergence of single cell technologies such as fluorescent in situ hybridization, high-throughput single-cell quantitative PCR, single cell mass spectrometry and mass cytometry however, have led to re-analysis of these models of hematopoietic differentiation [7]. Discrete progenitor cell populations, as determined by cell surface markers, have been shown to consist of heterogenous populations with different fates [8]. Recently, a study by Velten *et al*., 2017, using a combination of single cell technologies and xenotransplantation as functional validation, proposed that early hematopoiesis consists of, a cellular continuum of low-primed undifferentiated (CLOUD) hematopoietic stem progenitor cells (HSPC), with simultaneous lineage gene expression for multiple fates [9]. This study suggested that early discrete stable progenitors do not exist, with any lineage determination occurring further downstream than originally presumed.

Regardless of provenance, leukemogenesis is characterized by a block in differentiation and an accumulation of immature white blood cell blasts with a rapid increase in these blasts, characteristic of the acute leukemias. Acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) are heterogenous diseases with a block in lymphoid or myeloid differentiation, respectively. They occur due to one or more genetic insults. Whilst ALL is predominantly a disease of children (80%), with a greater than 90% 5 y survival rate [10], in adults long term survival stands at only 30–40% [11]. AML in contrast is primarily a disease of the elderly, and like adult ALL it's 5 y survival rate is around 30%, however this falls in the over

#### **Figure 1.**

*Human hematopoiesis. Schematic diagram showing classical model of hematopoietic lineage commitment, with phenotypical cell surface markers (red), transcription factors determining differentiation (green box) and growth factors involved in myelopoiesis (blue). Hematopoietic stem cell (HSC), cluster of differentiation (CD), hematopoietic progenitor cell (HPC), common myeloid progenitor (CMP), common lymphoid progenitor (CLP), interleukin (IL), granulocyte macrophage (GM) colony-stimulating-factor (CSF), stem cell factor (SCF), thrombopoietin (TPO), erythropoietin (EPO), granulocyte myeloid progenitor (GMP), runt-related transcription factor 1 (RUNX1), transcription factor stem cell leukemia (SCL), ccaat enhancer binding proteins (C/EBP), friend of GATA protein 1 (FOG-1).*

**37**

*(NO•*

**Figure 2.**

*superoxide (O2*

*) with O2 •−.*

*Reactive Oxygen Species and Metabolic Re-Wiring in Acute Leukemias*

60's to a particularly bleak 10% [12]. In ALL, recent advances for example in the use of tyrosine kinase inhibitors and CAR-T cell therapy, have started to suggest improvements to overall survival [10]. However, in patients fit enough to tolerate chemotherapy, the standard treatment for AML since 1973 has been a seven-day continuous intravenous infusion of cytarabine (Ara-C) (100–200 mg/m<sup>2</sup>

autologous stem cell transplantation, and despite some recent advances (reviewed in [13, 14]), current treatments appear to have reached their efficacious limits and

One potential therapeutic opportunity involves exploiting the metabolic differences that exist between malignant and non-malignant cells [15]. Differences that, in AML at least, appear exacerbated by cellular levels of reactive oxygen species

ROS is the collective term for several oxygen containing free radicals and other reactive molecules, such as hydrogen peroxide (H2O2). Physiologically, ROS are initially generated via the univalent reduction of molecular oxygen which generates superoxide (O2•−). Superoxide (t1/2 = 1 μs) subsequently dismutates to H2O2 (t1/2 = 1 ms) [17], either spontaneously or via the catalytic action of the enzyme superoxide dismutase (SOD), or reacts with other ROS molecules, forming a variety of other ROS (**Figure 2**). Functionally, ROS is important in innate immunity,

*Formation of reactive oxygen species (ROS). Diatomic oxygen (O2) is univalently reduced by peroxisomes (PO), xanthine oxidase (XO), the electron transport chain (ETC), or NADPH oxidase (NOX) to generate* 

*spontaneously or through the enzymatic action of superoxide dismutase (SOD). Hydroxyl radicals (OH•*

*may then be formed from H2O2 via the formation of hypochlorous radical (HOCl) in the PO, or via Fenton chemistry. Reactive nitrogen species (RNS) may also be formed through the reaction of nitric oxide radical* 

*•− may then dismutate to H2O2 either* 

*)* 

*•−). PO may also reduce O2 directly to form H2O2. O2*

) and 3

), sometimes followed by allogeneic or

*DOI: http://dx.doi.org/10.5772/intechopen.94829*

daily doses of daunorubicin (45–90 mg/m2

new therapies are required.

**2. Reactive oxygen species**

(ROS) [16].

#### *Reactive Oxygen Species and Metabolic Re-Wiring in Acute Leukemias DOI: http://dx.doi.org/10.5772/intechopen.94829*

60's to a particularly bleak 10% [12]. In ALL, recent advances for example in the use of tyrosine kinase inhibitors and CAR-T cell therapy, have started to suggest improvements to overall survival [10]. However, in patients fit enough to tolerate chemotherapy, the standard treatment for AML since 1973 has been a seven-day continuous intravenous infusion of cytarabine (Ara-C) (100–200 mg/m<sup>2</sup> ) and 3 daily doses of daunorubicin (45–90 mg/m2 ), sometimes followed by allogeneic or autologous stem cell transplantation, and despite some recent advances (reviewed in [13, 14]), current treatments appear to have reached their efficacious limits and new therapies are required.

One potential therapeutic opportunity involves exploiting the metabolic differences that exist between malignant and non-malignant cells [15]. Differences that, in AML at least, appear exacerbated by cellular levels of reactive oxygen species (ROS) [16].

#### **2. Reactive oxygen species**

*Acute Leukemias*

presumed.

The compartmentalization of HSC, their progenitors and terminally differentiated blood cells, into different stages of differentiation, is traditionally based on the expression of cell surface proteins (**Figure 1**). The recent emergence of single cell technologies such as fluorescent in situ hybridization, high-throughput single-cell quantitative PCR, single cell mass spectrometry and mass cytometry however, have led to re-analysis of these models of hematopoietic differentiation [7]. Discrete progenitor cell populations, as determined by cell surface markers, have been shown to consist of heterogenous populations with different fates [8]. Recently, a study by Velten *et al*., 2017, using a combination of single cell technologies and xenotransplantation as functional validation, proposed that early hematopoiesis consists of, a cellular continuum of low-primed undifferentiated (CLOUD) hematopoietic stem progenitor cells (HSPC), with simultaneous lineage gene expression for multiple fates [9]. This study suggested that early discrete stable progenitors do not exist, with any lineage determination occurring further downstream than originally

Regardless of provenance, leukemogenesis is characterized by a block in differentiation and an accumulation of immature white blood cell blasts with a rapid increase in these blasts, characteristic of the acute leukemias. Acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) are heterogenous diseases with a block in lymphoid or myeloid differentiation, respectively. They occur due to one or more genetic insults. Whilst ALL is predominantly a disease of children (80%), with a greater than 90% 5 y survival rate [10], in adults long term survival stands at only 30–40% [11]. AML in contrast is primarily a disease of the elderly, and like adult ALL it's 5 y survival rate is around 30%, however this falls in the over

*Human hematopoiesis. Schematic diagram showing classical model of hematopoietic lineage commitment, with phenotypical cell surface markers (red), transcription factors determining differentiation (green box) and growth factors involved in myelopoiesis (blue). Hematopoietic stem cell (HSC), cluster of differentiation (CD), hematopoietic progenitor cell (HPC), common myeloid progenitor (CMP), common lymphoid progenitor (CLP), interleukin (IL), granulocyte macrophage (GM) colony-stimulating-factor (CSF), stem cell factor (SCF), thrombopoietin (TPO), erythropoietin (EPO), granulocyte myeloid progenitor (GMP), runt-related transcription factor 1 (RUNX1), transcription factor stem cell leukemia (SCL), ccaat enhancer binding* 

**36**

**Figure 1.**

*proteins (C/EBP), friend of GATA protein 1 (FOG-1).*

ROS is the collective term for several oxygen containing free radicals and other reactive molecules, such as hydrogen peroxide (H2O2). Physiologically, ROS are initially generated via the univalent reduction of molecular oxygen which generates superoxide (O2•−). Superoxide (t1/2 = 1 μs) subsequently dismutates to H2O2 (t1/2 = 1 ms) [17], either spontaneously or via the catalytic action of the enzyme superoxide dismutase (SOD), or reacts with other ROS molecules, forming a variety of other ROS (**Figure 2**). Functionally, ROS is important in innate immunity,

#### **Figure 2.**

*Formation of reactive oxygen species (ROS). Diatomic oxygen (O2) is univalently reduced by peroxisomes (PO), xanthine oxidase (XO), the electron transport chain (ETC), or NADPH oxidase (NOX) to generate superoxide (O2 •−). PO may also reduce O2 directly to form H2O2. O2 •− may then dismutate to H2O2 either spontaneously or through the enzymatic action of superoxide dismutase (SOD). Hydroxyl radicals (OH• ) may then be formed from H2O2 via the formation of hypochlorous radical (HOCl) in the PO, or via Fenton chemistry. Reactive nitrogen species (RNS) may also be formed through the reaction of nitric oxide radical (NO• ) with O2 •−.*

protein folding in the endoplasmic reticulum and as a cell signalling molecule involved in cellular proliferation, survival, differentiation and gene expression [18].

There are several sources of cellular ROS, including the mitochondria, the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase family of enzymes (NOX), the cytochrome P450 enzymes, peroxisomes and the metabolic enzyme xanthine oxidase (XO).

#### **2.1 Sources of ROS: electron transport chain**

Generation of ROS by the mitochondria is primarily a function of 'electron leakage' from the electron transport chain (ETC), however, mitochondrial ROS may also be generated as a result of numerous enzymes including monoamine oxidase, cytochrome b5 reductase, glycerol-3-phosphate dehydrogenase, aconitase, pyruvate dehydrogenase and α-ketoglutarate dehydrogenase (reviewed in [19]). Mitochondrial ROS production resulting from the ETC generates O2 •−, and is thought to occur as result of one of three mechanisms. The first mechanism is a consequence of a high NADH/NAD+ ratio, and results from oxygen interacting with fully reduced FMN. Mitochondrial ROS generated by this mechanism has been observed due to mitochondrial mutation, physiological damage such as ischemia or aging, and only small amounts of ROS are thought to be generated via these mechanisms in normally respiring cells [20]. The second mechanism occurs when there is a high level of reduced co-enzyme Q (CoQH2) in complex II, which in the presence of a high proton motive force generated by the proton pump, force electrons back into complex I in a process known as reverse electron transport (RET). Whilst RET generated ROS has also been implicated in diseases such as ischemia, it is now also thought to be involved as a cell signalling molecule in metabolic adaptation, myeloid differentiation and response to bacterial infection [21]. The third mechanism of ROS generation by the ETC occurs at complex III and has also been implicated in ROS signalling. The formation of O2 •− occurs at the ubiquinol oxidation centre (Qo) site of the cytochrome bc1 complex, in which fully oxidized CoQ supports formation of O2 •−, through the transfer of electrons from reduced heme b1 to molecular oxygen [22]. Generation of O2 •− by complex I and II occurs exclusively in the mitochondrial matrix, whereas O2 •− generated by complex III also occurs in the intermembrane space. O2 •− generated in the mitochondrial matrix is rapidly converted to H2O2 by mitochondrial SOD (Mn-SOD), whereas O2 •− generated in the intermembrane space travels through the outer mitochondrial membrane prior to conversion to H2O2 by cytosolic SOD (Cu/Zn-SOD).

#### **2.2 Sources of ROS: nicotinamide adenine dinucleotide phosphate (NADPH) oxidase family of enzymes (NOX)**

Whilst mitochondrial oxidative phosphorylation is a major source of intracellular ROS, the main source of extracellular ROS involves the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase family of enzymes (NOX). The NOX family of enzymes comprise of seven members, NOX1–5 and dual oxidase (DUOX) 1 and 2. NOX enzymes are transmembrane proteins that transfer electrons from NADPH to molecular oxygen, generating O2 •− (or H2O2), which can then be converted to other forms of ROS. Different NOX isoforms share conserved structural features comprising of six helical transmembrane domains (TM) (with helix III and helix V containing two heme-binding histidines), and a C-terminus cytosolic domain (DH), which allows binding of FAD and NADPH (**Figure 3**). Difficulties in obtaining suitable levels of NOX proteins mean that to date relatively little crystal structure data is available. However, a recently published report [23], has

**39**

generates O2

**Figure 3.**

*(Rac2).*

*Generation of superoxide (O2*

•− (**Figure 3**).

protein family of aquaporins [25, 26].

**3. Role of ROS on normal hematopoiesis**

tive, that extracellular H2O2 (which is rapidly formed from O2

*Reactive Oxygen Species and Metabolic Re-Wiring in Acute Leukemias*

elucidated the structure of the TM and DH domains (common to all NOX isoforms) of *Cylindrospermum stagnale* NOX5 in complex with FAD. In this structure, the six transmembrane helices of TM domain form a pyramidal shape with the base on the cytosolic side, a N-terminus α-helix runs parallel to the cytosolic side of the membrane and the heme groups sit in cavities formed by helices II–V, so that one is positioned near the cytosolic side of the membrane (heme 1) and the other on the outer side (heme 2). The DH domain, located on the cytosolic side, contains two pockets, one for FAD binding and one for NADPH binding. The FAD is positioned so that the flavin is in direct contact with heme 1 of the TM, to promote interdomain electron transfer. The proposed mechanism of electron transfer then involves NADPH donating its electron to FAD, which in turn donates an electron to heme 1 and then to heme 2 via Trp378 (equating to Phe 215 in human NOX2, Phe 200 in human NOX4 and Val 362 in human NOX5) of the loop between helix II and III of the TM, before reduction of molecular oxygen, via a final electron transfer step

*structural features of NOX2, it's activation by phosphorylation (P) of p67phoxand p47phox and the assembly of the major subunits of the NOX complex, and the generation of superoxide via electron transfer from NADPH to flavin adenine dinucleotide (FAD) to heme groups to diatomic oxygen. Guanosine triphosphate (GTP), guanosine diphosphate (GDP), homology domain (DH), RAS-related C3 botulinum toxin substrate 2* 

*•−) by NADPH Oxidase (NOX). Schematic diagram showing the major* 

From a metabolic perspective, one source of NOX2 activation results when cells experience intermittent hypoxia. Under this condition activation of the metabolic enzyme XO, an enzyme important in the catabolism of purines and a major source of cellular ROS, occurs [24]. XO activation leads to increased ROS, which induces Ca2+ activation of protein kinase C, an enzyme important in cell signalling, migration of p47phox and p67phox to the cell membrane, resulting in activation of the NOX2 complex (**Figure 3**). Finally it is important to note, from a cell signalling perspec-

ported across the cell membrane via the transmembrane water permeable channel

ROS has been implicated in both HSC quiescence and hematopoietic differentiation. HSC reside in the bone marrow and their quiescence is known to be negatively regulated by ROS. Forkhead box O (FOXO) transcription factors are involved in

•−) is readily trans-

*DOI: http://dx.doi.org/10.5772/intechopen.94829*

*Reactive Oxygen Species and Metabolic Re-Wiring in Acute Leukemias DOI: http://dx.doi.org/10.5772/intechopen.94829*

#### **Figure 3.**

*Acute Leukemias*

xanthine oxidase (XO).

**2.1 Sources of ROS: electron transport chain**

a consequence of a high NADH/NAD+

implicated in ROS signalling. The formation of O2

conversion to H2O2 by cytosolic SOD (Cu/Zn-SOD).

converted to H2O2 by mitochondrial SOD (Mn-SOD), whereas O2

to molecular oxygen [22]. Generation of O2

**oxidase family of enzymes (NOX)**

NADPH to molecular oxygen, generating O2

in the mitochondrial matrix, whereas O2

supports formation of O2

the intermembrane space. O2

protein folding in the endoplasmic reticulum and as a cell signalling molecule involved in cellular proliferation, survival, differentiation and gene expression [18]. There are several sources of cellular ROS, including the mitochondria, the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase family of enzymes (NOX), the cytochrome P450 enzymes, peroxisomes and the metabolic enzyme

Generation of ROS by the mitochondria is primarily a function of 'electron leakage' from the electron transport chain (ETC), however, mitochondrial ROS may also be generated as a result of numerous enzymes including monoamine oxidase, cytochrome b5 reductase, glycerol-3-phosphate dehydrogenase, aconitase, pyruvate dehydrogenase and α-ketoglutarate dehydrogenase (reviewed in [19]). Mitochondrial ROS production resulting from the ETC generates O2

is thought to occur as result of one of three mechanisms. The first mechanism is

with fully reduced FMN. Mitochondrial ROS generated by this mechanism has been observed due to mitochondrial mutation, physiological damage such as ischemia or aging, and only small amounts of ROS are thought to be generated via these mechanisms in normally respiring cells [20]. The second mechanism occurs when there is a high level of reduced co-enzyme Q (CoQH2) in complex II, which in the presence of a high proton motive force generated by the proton pump, force electrons back into complex I in a process known as reverse electron transport (RET). Whilst RET generated ROS has also been implicated in diseases such as ischemia, it is now also thought to be involved as a cell signalling molecule in metabolic adaptation, myeloid differentiation and response to bacterial infection [21]. The third mechanism of ROS generation by the ETC occurs at complex III and has also been

tion centre (Qo) site of the cytochrome bc1 complex, in which fully oxidized CoQ

intermembrane space travels through the outer mitochondrial membrane prior to

**2.2 Sources of ROS: nicotinamide adenine dinucleotide phosphate (NADPH)** 

Whilst mitochondrial oxidative phosphorylation is a major source of intracellular ROS, the main source of extracellular ROS involves the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase family of enzymes (NOX). The NOX family of enzymes comprise of seven members, NOX1–5 and dual oxidase (DUOX) 1 and 2. NOX enzymes are transmembrane proteins that transfer electrons from

verted to other forms of ROS. Different NOX isoforms share conserved structural features comprising of six helical transmembrane domains (TM) (with helix III and helix V containing two heme-binding histidines), and a C-terminus cytosolic domain (DH), which allows binding of FAD and NADPH (**Figure 3**). Difficulties in obtaining suitable levels of NOX proteins mean that to date relatively little crystal structure data is available. However, a recently published report [23], has

•−, and

ratio, and results from oxygen interacting

•− occurs at the ubiquinol oxida-

•− generated in the

•− by complex I and II occurs exclusively

•− generated by complex III also occurs in

•− (or H2O2), which can then be con-

•−, through the transfer of electrons from reduced heme b1

•− generated in the mitochondrial matrix is rapidly

**38**

*Generation of superoxide (O2 •−) by NADPH Oxidase (NOX). Schematic diagram showing the major structural features of NOX2, it's activation by phosphorylation (P) of p67phoxand p47phox and the assembly of the major subunits of the NOX complex, and the generation of superoxide via electron transfer from NADPH to flavin adenine dinucleotide (FAD) to heme groups to diatomic oxygen. Guanosine triphosphate (GTP), guanosine diphosphate (GDP), homology domain (DH), RAS-related C3 botulinum toxin substrate 2 (Rac2).*

elucidated the structure of the TM and DH domains (common to all NOX isoforms) of *Cylindrospermum stagnale* NOX5 in complex with FAD. In this structure, the six transmembrane helices of TM domain form a pyramidal shape with the base on the cytosolic side, a N-terminus α-helix runs parallel to the cytosolic side of the membrane and the heme groups sit in cavities formed by helices II–V, so that one is positioned near the cytosolic side of the membrane (heme 1) and the other on the outer side (heme 2). The DH domain, located on the cytosolic side, contains two pockets, one for FAD binding and one for NADPH binding. The FAD is positioned so that the flavin is in direct contact with heme 1 of the TM, to promote interdomain electron transfer. The proposed mechanism of electron transfer then involves NADPH donating its electron to FAD, which in turn donates an electron to heme 1 and then to heme 2 via Trp378 (equating to Phe 215 in human NOX2, Phe 200 in human NOX4 and Val 362 in human NOX5) of the loop between helix II and III of the TM, before reduction of molecular oxygen, via a final electron transfer step generates O2 •− (**Figure 3**).

From a metabolic perspective, one source of NOX2 activation results when cells experience intermittent hypoxia. Under this condition activation of the metabolic enzyme XO, an enzyme important in the catabolism of purines and a major source of cellular ROS, occurs [24]. XO activation leads to increased ROS, which induces Ca2+ activation of protein kinase C, an enzyme important in cell signalling, migration of p47phox and p67phox to the cell membrane, resulting in activation of the NOX2 complex (**Figure 3**). Finally it is important to note, from a cell signalling perspective, that extracellular H2O2 (which is rapidly formed from O2 •−) is readily transported across the cell membrane via the transmembrane water permeable channel protein family of aquaporins [25, 26].

#### **3. Role of ROS on normal hematopoiesis**

ROS has been implicated in both HSC quiescence and hematopoietic differentiation. HSC reside in the bone marrow and their quiescence is known to be negatively regulated by ROS. Forkhead box O (FOXO) transcription factors are involved in

cell-cycle arrest and apoptosis and are activated in response to oxidative stress whereupon they translocate to the nucleus [27]. Translocation of FOXO4 to the nucleus has been shown to be a function of redox signalling, where oxidation of cys-239 by ROS mediates the formation of disulphide bonds with nuclear import receptor transportin-1, which in turn allows nuclear localization [28]. FOXO deactivation occurs as a result of phosphorylation in response to activation of the regulatory cell cycle PI3K/AKT/mTOR pathway, resulting in their export from the nucleus and subsequent degradation in the cytoplasm [29]. Studies in murine HSC have shown that deletion of FOXO3a, which upregulates transcription of Mn-SOD [30], results in decreased HSC renewal [31] which is mediated by the tumor suppressor protein ataxia-telangiectasia mutated (ATM) and is accompanied by elevated ROS levels and myeloid lineage expansion [32]. Deletion of ATM in mice resulted in BM failure which was restored following treatment with antioxidants [33]. In a different study, isolation of murine HSC into ROS high and ROS low populations showed that the ROS low population maintained self-renewal capacity following serial transplantations, whilst the self-renewal capacity of the ROS high population was exhausted following the third serial transplantation. Treatment of the ROS high HSC with the antioxidant N-acetyl cysteine (NAC), the p38 inhibitor SB203508 or rapamycin (a mTOR inhibitor), restored self-renewal activity [34]. Interestingly, the ROS high population in this study also exhibited a decreased ability to adhere to cells containing calcium sensing receptors, whilst NOX generated ROS has additionally been implicated in osteoclast differentiation in human mesenchymal cells, further emphasizing a potential regulatory role of ROS, in the BM niche [35].

Whilst these increased ROS levels are associated with HSC losing quiescence, it has also been shown, in the human megakaryocytic cell line MO7e, that hematopoietic cytokines, such as granulocyte macrophage-colony stimulating factor, interleukin-3, stem cell factor and thrombopoietin all increase ROS levels [36]. In megakaryopoiesis, ROS has been shown to increase platelet production and maturation in the chronic myeloid leukemia (CML) cell line MEG-01 and primary human megakaryocytes [37], which in murine models is mediated by the transcription factor NF-E2 [38]. Following lineage commitment, megakaryocyte progenitors undergo endomitosis (chromosomal replication in the absence of cell division), which in murine cells is potentially mediated by NOX1-derived ROS [39]. In human HSC, NOX-derived ROS has also been shown to be crucial for megakaryocyte differentiation via activation of ERK, AKT and JAK2 signalling pathways [40], whilst another study revealed the importance of cytochrome P450 2E1-generated ROS in megakaryocyte differentiation in human HSC [41]. As noted above, increased ROS in HSC has been associated with expanded myelopoiesis. Interestingly, a recent study using murine CMP, showed that higher levels of ROS impeded megakaryopoiesis, instead directing differentiation of CMP into GMP [42]. Finally, ROS has also been shown to induce differentiation of the promonocytic cell line, U937, into macrophages [43], and the differentiation of primary human monocytes into dendritic cells [44].

#### **4. Role of ROS on solid tumors and leukemia development**

One of the first studies implicating ROS in carcinogenesis was performed in mice subcutaneously injected with C3H mouse fibroblasts, that had been previously cultured *in vitro* with neutrophils stimulated with 12-O-tetradecanoylphorbol-13 acetate (TPA) stimulated or unstimulated or with the ROS generating enzyme XO and hypoxanthine. In this study approximately 20% of mice treated with these cells developed tumors within 13–22 weeks compared to none of the control mice [45].

**41**

activation.

*Reactive Oxygen Species and Metabolic Re-Wiring in Acute Leukemias*

in tumorigenesis is complex and multifaceted (reviewed in [51]).

In 1991, analysis of H2O2 production in human melanoma, colon, pancreatic, neuroblastoma, breast and ovarian cancer cell lines, revealed constitutively active H2O2 production over a 4 h period, generating H2O2 levels similar to those observed in TPA stimulated neutrophils, suggesting increased ROS production may be a feature of transformation [46]. Later, studies in patients with liver disease suggested ROS plays a part in hepatocarcinogenesis [47], and levels of Cu/Zn-SOD are significantly lower in hepatoma tissue than normal human liver tissue [48]. Further, homozygous deletion of Cu/Zn-SOD in mice results in decreased lifespan, with 70% developing hepatocarcinoma or benign nodular hyperplasia [49], whilst homozygous deletion of Mn-SOD in mice is lethal within two weeks of birth [50]. In the same study, heterozygous deletion of Mn-SOD resulted in increased incidence of hemangioma and adenocarcinoma and significant increases in the incidence of lymphoma. Currently, elevated ROS levels have been reported in many solid tumors and the role they play

In leukemia, a study which collected blood samples from ALL and CML patients samples and compared them with normal blood samples showed elevated levels of ROS in both ALL and CML patients [52], whilst elevated levels of NOX generated ROS, are observed, alongside increased proliferation in both AML models and AML patient samples when compared with healthy controls [53]. Reactions of ROS with DNA can generate numerous oxidised bases, including 8-hydroxy-2-deoxyguanosine (8-OHdG) which causes G:C to T:A DNA transversions (reviewed in [54]). Increased levels of 8-OHdG have been observed in patients with breast cancer [55], gastric carcinomas [56], lung cancer [57] and colorectal cancer [58]. In leukemia, a study of 116 Chinese children with either ALL or AML revealed significantly elevated levels of 8-OHdG, whilst 8-OHdG levels were also significantly elevated in

As a signalling molecule, ROS can lead to hyperactivation of the PI3K pathway,

Broadly defined, cellular metabolism involves a series of catabolic or anabolic chemical reactions which generate or use energy as part of this process. In chemotrophs this energy is obtained through the oxidation of nutrients, with the energy typically stored in the form of ATP. Whilst in higher organisms a plethora of enzymatically catalyzed metabolic reactions occur, which are all part of different interconnecting metabolic pathways with multitudinous feedback mechanisms. These pathways are evolutionarily highly conserved with the citric acid cycle, for example, essentially a feature in all terrestrial life. There are three main classes of molecules involved in metabolism; carbohydrates, proteins and lipids that are either catabolized to generate energy or energy stores or used by anabolic pathways

a common feature of many cancers, resulting in increased cell survival, VEGF production, secretion of MMP (reviewed in [60]) and inactivation of FOXO [32]. In AML, constitutive activation of the PI3K/AKT pathway is frequently observed [61, 62], however the role of FOXO is less clear. A recent study revealed that FOXO1 expression in osteoblasts mediated β-catenin initiated AML [63], whilst a study of AML patient samples showed that 40% exhibited FOXO activation, that upon inhibition resulted in myeloid differentiation and AML cell death [64]. Additionally, in both CML and AML the BCR-ABL fusion protein and FMS-like tyrosine kinase receptor 3 internal tandem duplications (FLT3-ITD) have been shown to lead to phosphorylation of AKT resulting in increased activation of NOX, and increased ROS production (reviewed in [65]), which may in turn reinforce PI3K/AKT

*DOI: http://dx.doi.org/10.5772/intechopen.94829*

relapsed AML adult patients [59].

**5. Metabolism and cancer**

#### *Reactive Oxygen Species and Metabolic Re-Wiring in Acute Leukemias DOI: http://dx.doi.org/10.5772/intechopen.94829*

*Acute Leukemias*

cell-cycle arrest and apoptosis and are activated in response to oxidative stress whereupon they translocate to the nucleus [27]. Translocation of FOXO4 to the nucleus has been shown to be a function of redox signalling, where oxidation of cys-239 by ROS mediates the formation of disulphide bonds with nuclear import receptor transportin-1, which in turn allows nuclear localization [28]. FOXO deactivation occurs as a result of phosphorylation in response to activation of the regulatory cell cycle PI3K/AKT/mTOR pathway, resulting in their export from the nucleus and subsequent degradation in the cytoplasm [29]. Studies in murine HSC have shown that deletion of FOXO3a, which upregulates transcription of Mn-SOD [30], results in decreased HSC renewal [31] which is mediated by the tumor suppressor protein ataxia-telangiectasia mutated (ATM) and is accompanied by elevated ROS levels and myeloid lineage expansion [32]. Deletion of ATM in mice resulted in BM failure which was restored following treatment with antioxidants [33]. In a different study, isolation of murine HSC into ROS high and ROS low populations showed that the ROS low population maintained self-renewal capacity following serial transplantations, whilst the self-renewal capacity of the ROS high population was exhausted following the third serial transplantation. Treatment of the ROS high HSC with the antioxidant N-acetyl cysteine (NAC), the p38 inhibitor SB203508 or rapamycin (a mTOR inhibitor), restored self-renewal activity [34]. Interestingly, the ROS high population in this study also exhibited a decreased ability to adhere to cells containing calcium sensing receptors, whilst NOX generated ROS has additionally been implicated in osteoclast differentiation in human mesenchymal cells, further

emphasizing a potential regulatory role of ROS, in the BM niche [35].

**4. Role of ROS on solid tumors and leukemia development**

One of the first studies implicating ROS in carcinogenesis was performed in mice subcutaneously injected with C3H mouse fibroblasts, that had been previously cultured *in vitro* with neutrophils stimulated with 12-O-tetradecanoylphorbol-13 acetate (TPA) stimulated or unstimulated or with the ROS generating enzyme XO and hypoxanthine. In this study approximately 20% of mice treated with these cells developed tumors within 13–22 weeks compared to none of the control mice [45].

Whilst these increased ROS levels are associated with HSC losing quiescence, it has also been shown, in the human megakaryocytic cell line MO7e, that hematopoietic cytokines, such as granulocyte macrophage-colony stimulating factor, interleukin-3, stem cell factor and thrombopoietin all increase ROS levels [36]. In megakaryopoiesis, ROS has been shown to increase platelet production and maturation in the chronic myeloid leukemia (CML) cell line MEG-01 and primary human megakaryocytes [37], which in murine models is mediated by the transcription factor NF-E2 [38]. Following lineage commitment, megakaryocyte progenitors undergo endomitosis (chromosomal replication in the absence of cell division), which in murine cells is potentially mediated by NOX1-derived ROS [39]. In human HSC, NOX-derived ROS has also been shown to be crucial for megakaryocyte differentiation via activation of ERK, AKT and JAK2 signalling pathways [40], whilst another study revealed the importance of cytochrome P450 2E1-generated ROS in megakaryocyte differentiation in human HSC [41]. As noted above, increased ROS in HSC has been associated with expanded myelopoiesis. Interestingly, a recent study using murine CMP, showed that higher levels of ROS impeded megakaryopoiesis, instead directing differentiation of CMP into GMP [42]. Finally, ROS has also been shown to induce differentiation of the promonocytic cell line, U937, into macrophages [43], and the differentiation of primary human monocytes into

**40**

dendritic cells [44].

In 1991, analysis of H2O2 production in human melanoma, colon, pancreatic, neuroblastoma, breast and ovarian cancer cell lines, revealed constitutively active H2O2 production over a 4 h period, generating H2O2 levels similar to those observed in TPA stimulated neutrophils, suggesting increased ROS production may be a feature of transformation [46]. Later, studies in patients with liver disease suggested ROS plays a part in hepatocarcinogenesis [47], and levels of Cu/Zn-SOD are significantly lower in hepatoma tissue than normal human liver tissue [48]. Further, homozygous deletion of Cu/Zn-SOD in mice results in decreased lifespan, with 70% developing hepatocarcinoma or benign nodular hyperplasia [49], whilst homozygous deletion of Mn-SOD in mice is lethal within two weeks of birth [50]. In the same study, heterozygous deletion of Mn-SOD resulted in increased incidence of hemangioma and adenocarcinoma and significant increases in the incidence of lymphoma. Currently, elevated ROS levels have been reported in many solid tumors and the role they play in tumorigenesis is complex and multifaceted (reviewed in [51]).

In leukemia, a study which collected blood samples from ALL and CML patients samples and compared them with normal blood samples showed elevated levels of ROS in both ALL and CML patients [52], whilst elevated levels of NOX generated ROS, are observed, alongside increased proliferation in both AML models and AML patient samples when compared with healthy controls [53]. Reactions of ROS with DNA can generate numerous oxidised bases, including 8-hydroxy-2-deoxyguanosine (8-OHdG) which causes G:C to T:A DNA transversions (reviewed in [54]). Increased levels of 8-OHdG have been observed in patients with breast cancer [55], gastric carcinomas [56], lung cancer [57] and colorectal cancer [58]. In leukemia, a study of 116 Chinese children with either ALL or AML revealed significantly elevated levels of 8-OHdG, whilst 8-OHdG levels were also significantly elevated in relapsed AML adult patients [59].

As a signalling molecule, ROS can lead to hyperactivation of the PI3K pathway, a common feature of many cancers, resulting in increased cell survival, VEGF production, secretion of MMP (reviewed in [60]) and inactivation of FOXO [32]. In AML, constitutive activation of the PI3K/AKT pathway is frequently observed [61, 62], however the role of FOXO is less clear. A recent study revealed that FOXO1 expression in osteoblasts mediated β-catenin initiated AML [63], whilst a study of AML patient samples showed that 40% exhibited FOXO activation, that upon inhibition resulted in myeloid differentiation and AML cell death [64]. Additionally, in both CML and AML the BCR-ABL fusion protein and FMS-like tyrosine kinase receptor 3 internal tandem duplications (FLT3-ITD) have been shown to lead to phosphorylation of AKT resulting in increased activation of NOX, and increased ROS production (reviewed in [65]), which may in turn reinforce PI3K/AKT activation.
