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Key Points• More than 60% of primary AML blasts constitutively produce high levels of NOXderived reactive oxygen species (ROS), which drives AML proliferation.• High ROS AMLs show depleted antioxidant defenses but evade the oxidative stress response through suppression of p38 MAPK signaling.Excessive production of reactive oxygen species (ROS) is frequently observed in cancer and is known to strongly influence hematopoietic cell function. Here we report that extracellular ROS production is strongly elevated (mean >10-fold) in >60% of acute myeloid leukemia (AML) patients and that this increase is attributable to constitutive activation of nicotinamide adenine dinucleotide phosphate oxidases (NOX). In contrast, overproduction of mitochondrial ROS was rarely observed. Elevated ROS was found to be associated with lowered glutathione levels and depletion of antioxidant defense proteins. We also show for the first time that the levels of ROS generated were able to strongly promote the proliferation of AML cell lines, primary AML blasts, and, to a lesser extent, normal CD34 1 cells, and that the response to ROS is limited by the activation of the oxidative stress pathway mediated though p38 MAPK. Consistent with this, we observed that p38 MAPK responses were attenuated in patients expressing high levels of ROS. These data show that overproduction of NOX-derived ROS can promote the proliferation of AML blasts and that they also develop mechanisms to suppress the stress signaling that would normally limit this response. Together these adaptations would be predicted to confer a competitive advantage to the leukemic clone. (Blood. 2013;122(19):3322-3330)

Reactive oxygen species (ROS) are a heterogeneous group of molecules that are generated by mature myeloid cells during innate immune responses, and are also implicated in normal intracellular signaling. Excessive production of ROS (and/or a deficiency in antioxidant pathways) can lead to oxidative stress, a state that has been observed in several hematopoietic malignancies including acute and chronic myeloid leukemias (AML and CML). Currently it is unclear what the cause of oxidative stress might be and whether oxidative stress contributes to the development, progression, or maintenance of these diseases. This article reviews the current evidence suggesting a role for ROS both in normal hematopoiesis and in myeloid leukemogenesis, and discusses the usefulness of therapeutically targeting oxidative stress in myeloid malignancy. (Blood. 2011;117(22):5816-5826) IntroductionOver the past 15 years, there has been a growing appreciation that reactive oxygen species (ROS) production plays an important role in a variety of cellular processes, in addition to their antimicrobial role during phagocytosis by cells of the innate immune system. Specifically, ROS generated by the mitochondria or nicotinamide adenine dinucleotide phosphate (NADPH) oxidases have been shown to influence cell-cycle progression, cell motility, and growth factor signaling in a variety of normal cell types. 1 Many pathologic states are accompanied by excessive cellular ROS production and/or a deficiency in antioxidant defenses, leading to a state known as oxidative stress. 2 Evidence for chronic oxidative stress has been found in many cancers, both in solid tumors such as prostate carcinoma 3 and melanoma 4 and in several hematopoietic malignancies including acute lymphoblastic leukemia (ALL), 5 myelodysplastic syndrome (MDS), 6 and myeloid leukemias including chronic myeloid leukemia (CML) and acute myeloid leukemia (AML). 7 The importance of the association between oxidative stress and malignancy is not currently clear; however, there is evidence that tumor-derived ROS may promote cell survival, 8-10 migration and metastasis, 11,12 proliferation, 13,14 and even drug-resistance, 15 depending on the origin of the cancer. These observations suggest that oxidative stress may subvert the normal roles of ROS so as to benefit the malignant clone.In the context of AML, a recent report indicates that relapse in this disease is associated with increased markers of oxidative stress within the leukemic blasts, suggesting that ROS production may be an important factor in AML progression. 16 Indeed, recent published data from our group suggest that constitutively active Ras (one of the most common abnormalities detected in AML) is capable of driving ROS production in CD34 ϩ human hematopoietic progenitor cells, and this significantly contributes to the mutant Ras phenotype. 17 Furthermore, ROS production appears to contribute to proliferation and migration of hematopoietic cells expressing a variety of oncogenic tyrosine kinases. 18 Thus, increased oxidative s...

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Upregulation of the immunosuppressive cell surface glycoprotein, CD200, is a common feature of acute myeloid leukemia (AML) and is associated with poor patient outcome. We investigated whether CD200 overexpression on AML cells could specifically compromise patient natural killer (NK) cell anti-tumor responses. We found that CD200hi patients showed a 50% reduction in the frequency of activated NK cells (CD56dimCD16+) compared with CD200lo patients. Additionally, NK receptor expression (NKp44 and NKp46) on these cells was also significantly downregulated in CD200hi patients. To assess whether NK cell activity was directly influenced by CD200 expression, we examined the effect of ectopic expression of CD200. These assays revealed that both NK cell cytolytic activity and interferon-γ response were significantly reduced toward CD200+ leukemic targets and that these targets showed increased survival compared with CD200− cells. Similarly, NK cells isolated from AML patients were less functionally active toward CD200hi autologous blasts from both cytolytic and immunoregulatory perspectives. Finally, blocking CD200 alone was sufficient to recover a significant proportion of NK cell cytolytic activity. Together, these findings provide the first evidence that CD200 has a direct and significant suppressive influence on NK cell activity in AML patients and may contribute to the increased relapse rate in CD200+ patients.

Honey is used as a therapy to aid wound healing. Previous data indicate that honey can stimulate cytokine production from human monocytes. The present study further examines this phenomenon in manuka honey. As inflammatory cytokine production in innate immune cells is classically mediated by pattern recognition receptors in response to microorganisms, bacterial contamination of honey and the effect of blocking TLR2 and -4 on stimulatory activity were assessed. No vegetative bacteria were isolated from honey; however, bacterial spores were cultured from one-third of samples, and low levels of LPS were detected. Blocking TLR4 but not TLR2 inhibited honey-stimulated cytokine production significantly. Cytokine production did not correlate with LPS levels in honey and was not inhibited by polymyxin B. Further, the activity was reduced significantly following heat treatment, indicating that component(s) other than LPS are responsible for the stimulatory activity of manuka honey. To identify the component responsible for inducing cytokine production, honey was separated by molecular weight using microcon centrifugal filtration and fractions assessed for stimulatory activity. The active fraction was analyzed by MALDI-TOF mass spectroscopy, which demonstrated the presence of a number of components of varying molecular weights. Additional fractionation using miniaturized, reverse-phase solid-phase extraction resulted in the isolation of a 5.8-kDa component, which stimulated production of TNF-alpha via TLR4. These findings reveal mechanisms and components involved in honey stimulation of cytokine induction and could potentially lead to the development of novel therapeutics to improve wound healing for patients with acute and chronic wounds.

Excessive production of reactive oxygen species (ROS) is a feature of human malignancy and is often triggered by activation of oncogenes such as activated Ras. ROS act as second messengers and can influence a variety of cellular process including growth factor responses and cell survival. We have examined the contribution of ROS production to the effects of N-Ras G12D and H-Ras G12V on normal human CD34 ؉ progenitor cells. Activated Ras strongly up-regulated the production of both superoxide and hydrogen peroxide through the stimulation of NADPH oxidase (NOX) activity, without affecting the expression of endogenous antioxidants or the production of mitochondrially derived ROS. Activated Ras also promoted both the survival and the growth factor-independent proliferation of CD34 ؉ cells. Using oxidase inhibitors and antioxidants, we found that excessive ROS production by these cells did not contribute to their enhanced survival; rather, ROS promoted their growth factor-independent proliferation. Although Ras-induced ROS production specifically activated the p38 MAPK IntroductionReactive oxygen species (ROS) are a heterogeneous group of inorganic molecules and free radicals, with a wide spectrum of life span and reactivity. In physiologic systems, ROS formation begins with the univalent reduction of diatomic oxygen to produce superoxide radicals. There are 2 main sources of cellular superoxide; the first is the mitochondrial electron transport chain, where incomplete reduction of oxygen to water can result in superoxide formation. The other major source of superoxide is professional oxidases (exemplified by the NADPH oxidase [NOX] protein family), 1 which are expressed and functional throughout hematopoietic development. 2 These proteins form part of a membrane-bound complex that transfers single electrons from cytosolic nicotinamide adenine dinucleotide phosphate via flavin adenine dinucleotide to extracellular oxygen, producing superoxide. Superoxide is a shortlived radical but can dismutate forming hydrogen peroxide (H 2 O 2 ), a relatively long-lived species that lies at the hub of a variety of potential chemical reactions and is the main molecule from which all other physiologic ROS molecules are derived. ROS levels are regulated by a complex network of antioxidant molecules and enzymes that detoxify ROS. 3 For example, superoxide generation is balanced by the actions of superoxide dismutases (SODs), which convert superoxide to H 2 O 2 . Subsequently, H 2 O 2 is destroyed during oxidation reactions involving glutathione or members of the peroxiredoxin family.Although ROS production can have adverse consequences causing lipid peroxidation and DNA damage, it is also clear that ROS act as cell-signaling molecules. In recent years, it has become clear that H 2 O 2 reacts with a variety of proteins sensitive to thiol oxidation. 4 In particular, H 2 O 2 can inhibit phosphatases via oxidation of cysteine at the active site and may consolidate growth factor signaling by preventing futile cycles of phosphorylation a...

The t(8;21)(q22;q22) occurs frequently in acute myelogenous leukaemia and gives rise to the transcription factor fusion protein, RUNX1-RUNX1T1 (also known as AML1-ETO). To identify the genes dysregulated by the aberrant transcriptional activity of RUNX1-RUNX1T1, we used microarrays to determine the effect of this mutation on gene expression in human progenitor cells and during subsequent development. Gene signatures of these developmental subsets were very dissimilar indicating that effects of RUNX1-RUNX1T1 are highly context dependent. We focused on gene changes associated with the granulocytic lineage and identified a clinically relevant subset of these by comparison with 235 leukaemia patient transcriptional signatures. We confirmed the overexpression of a number of significant genes (Sox4, IL-17BR, CD200 and c-catenin). Further, we show that overexpression of CD200 and c-catenin is also associated with the inv(16) abnormality which like RUNX1-RUNX1T1 disrupts core binding factor activity. We investigated the functional significance of CD200 and c-catenin overexpression in normal human progenitor cells. The effect of IL17 on growth was also assessed. Individually, none of these changes were sufficient to recapitulate the effects of RUNX1-RUNX1T1 on normal development. These data provide the most comprehensive and pertinent assessment of the effect of RUNX1-RUNX1T1 on gene expression and demonstrate the highly context-dependent effects of this fusion gene.