NOX2 control over energy metabolism plays a role in acute myeloid leukaemia prognosis and survival.

Acute myeloid leukaemia (AML) is a highly heterogeneous disease, however the therapeutic approaches have hardly changed in the last decades. Metabolism rewiring and the enhanced production of reactive oxygen species (ROS) are hallmarks of cancer. A deeper understanding of these features could be instrumental for the development of specific AML-subtypes treatments. NADPH oxidases (NOX), the only cellular system specialised in ROS production, are also involved in leukemic metabolism control. NOX2 shows a variable expression in AML patients, so patients can be classified based on such difference. Here we have analysed whether NOX2 levels are important for AML metabolism control. The lack of NOX2 in AML cells slowdowns basal glycolysis and oxidative phosphorylation (OXPHOS), along with the accumulation of metabolites that feed such routes, and a sharp decrease of glutathione. In addition, we found changes in the expression of 725 genes. Among them, we have discovered a panel of 30 differentially expressed metabolic genes, whose relevance was validated in patients. This panel can segregate AML patients according to CYBB expression, and it can predict patient prognosis and survival. In summary, our data strongly support the relevance of NOX2 for AML metabolism, and highlights the potential of our discoveries in AML prognosis.

NOX2 and NOX4 control mitochondrial function in chronic myeloid leukaemia.

Cancer cells are characterised by an elevated metabolic plasticity and enhanced production of reactive oxygen species (ROS), two features acknowledged as hallmarks in cancer, with a high translational potential to the therapeutic setting. These aspects, that have been traditionally studied separately, are in fact intimately intermingled. As part of their transforming activity, some oncogenes stimulate rewiring of metabolic processes, whilst simultaneously promoting increased production of intracellular ROS. In this scenario the latest discoveries suggest the relevance of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOX) to connect ROS production and metabolic control. Here we have analysed the relevance of NOX2 and NOX4 in the regulation of metabolism in chronic myeloid leukaemia (CML), a neoplasia driven by the expression of the breakpoint cluster region-Abelson fusion oncogene (BCR-ABL). Silencing of NOX2 enhances glycolysis and oxidative phosphorylation rates, together with an enhanced production of mitochondrial ROS and a decrease in mitochondrial DNA copy number, which reflects mitochondrial dysfunction. NOX4 expression was upregulated upon NOX2 silencing, and this was required to alter mitochondrial function. Our results support the relevance of NOX2 to regulate metabolism-related signalling pathways downstream of BCR-ABL. Overall we show that NOX2, through the regulation of NOX4 expression, controls metabolism and mitochondrial function in CML cells. This notion was confirmed by transcriptomic analyses, that strongly relate both NOX isoforms with metabolism regulation in CML.

Reactive Oxygen Species and Metabolism in Leukemia: A Dangerous Liaison.

Reactive oxygen species (ROS), previously considered toxic by-products of aerobic metabolism, are increasingly recognized as regulators of cellular signaling. Keeping ROS levels low is essential to safeguard the self-renewal capacity of hematopoietic stem cells (HSC). HSC reside in a hypoxic environment and have been shown to be highly dependent on the glycolytic pathway to meet their energy requirements. However, when the differentiation machinery is activated, there is an essential enhancement of ROS together with a metabolic shift toward oxidative metabolism. Initiating and sustaining leukemia depend on the activity of leukemic stem cells (LSC). LSC also show low ROS levels, but unlike HSC, LSC rely on oxygen to meet their metabolic energetic requirements through mitochondrial respiration. In contrast, leukemic blasts show high ROS levels and great metabolic plasticity, both of which seem to sustain their invasiveness. Oxidative stress and metabolism rewiring are recognized as hallmarks of cancer that are intimately intermingled. Here we present a detailed overview of these two features, sustained at different levels, that support a two-way relationship in leukemia. Modifying ROS levels and targeting metabolism are interesting therapeutic approaches. Therefore, we provide the most recent evidence on the modulation of oxidative stress and metabolism as a suitable anti-leukemic approach.

Granuloma Formation in a Cyba-Deficient Model of Chronic Granulomatous Disease Is Associated with Myeloid Hyperplasia and the Exhaustion of B-Cell Lineage.

Haematopoiesis is a paradigm of cell differentiation because of the wide variety and overwhelming number of mature blood cells produced daily. Under stress conditions, the organism must adapt to a boosted demand for blood cells. Chronic granulomatous disease (CGD) is a genetic disease caused by inactivating mutations that affect the phagocyte oxidase. Besides a defective innate immune system, CGD patients suffer from recurrent hyper-inflammation episodes, circumstances upon which they must face emergency haematopoiesis. The targeting of Cybb and Ncf1 genes have produced CGD animal models that are a useful surrogate when studying the pathophysiology and treatment of this disease. Here, we show that Cyba-/- mice spontaneously develop granuloma and, therefore, constitute a CGD animal model to complement the existing Cybb-/- and Ncf1-/- models. More importantly, we have analysed haematopoiesis in granuloma-bearing Cyba-/- mice. These animals showed a significant loss of weight, developed remarkable splenomegaly, bone marrow myeloid hyperplasia, and signs of anaemia. Haematological analyses showed a sharped decrease of B-cells and a striking development of myeloid cells in all compartments. Collectively, our results show that granuloma inflammatory lesions dramatically change haematopoiesis homeostasis. Consequently, we suggest that besides their defective innate immunity, the alteration of haematopoiesis homeostasis upon granuloma may contribute to the dismal outcome of CGD.

Cyba-deficient mice display an increase in hematopoietic stem cells and an overproduction of immunoglobulins.

The regulation of protein function by reversible oxidation is increasingly recognized as a key mechanism for the control of cellular signaling, modulating crucial biological processes such as cell differentiation. In this scenario, NADPH oxidases must occupy a prominent position. Our results show that hematopoietic stem and progenitor cells express three p22phox-dependent NADPH oxidases members (NOX1, NOX2 and NOX4). By deleting the p22phox coding gene (Cyba), here we have analyzed the importance of this family of enzymes during in vivo hematopoiesis. Cyba-/- mice show a myeloid bias, and an enrichment of hematopoietic stem cell populations. By means of hematopoietic transplant experiments we have also tried to dissect the specific role of the NADPH oxidases. While the absence of NOX1 or NOX2 provides a higher level of reconstitution, a lack of NOX4 rendered the opposite result, suggesting a functional specificity among the different NADPH oxidases. Cyba-/- cells showed a hampered activation of AKT1 and a sharp decrease in STAT5 protein. This is in line with the diminished response to IL-7 shown by our results, which could explain the overproduction of immunoglobulins observed in Cyba-/- mice.

Inhibition of Xanthine Oxidoreductase Enhances the Potential of Tyrosine Kinase Inhibitors against Chronic Myeloid Leukemia.

Chronic myeloid leukemia (CML) is characterized by the expression of the oncogenic kinase BCR-ABL. Although tyrosine kinase inhibitors (TKIs) against BCR-ABL represent the standard therapeutic option for CML, resistances to TKIs can be a serious problem. Thus, the search for novel therapeutic approaches is still needed. CML cells show an increased ROS production, which is required for maintaining the BCR-ABL signaling cascade active. In line with that, reducing ROS levels could be an interesting therapeutic strategy for the clinical management of resistant CML. To analyze the therapeutic potential of xanthine oxidoreductase (XOR) in CML, we tested the effect of XOR inhibitor allopurinol. Here, we show for the first time the therapeutic potential of allopurinol against BCR-ABL-positive CML cells. Allopurinol reduces the proliferation and clonogenic ability of the CML model cell lines K562 and KCL22. More importantly, the combination of allopurinol with imatinib or nilotinib reduced cell proliferation in a synergistic manner. Moreover, the co-treatment arms hampered cell clonogenic capacity and induced cell death more strongly than each single-agent arm. The reduction of intracellular ROS levels and the attenuation of the BCR-ABL signaling cascade may explain these effects. Finally, the self-renewal potential of primary bone marrow cells from CML patients was also severely reduced especially by the combination of allopurinol with TKIs. In summary, here we show that XOR inhibition is an interesting therapeutic option for CML, which can enhance the effectiveness of the TKIs currently used in clinics.

Reactive oxygen species in haematopoiesis: leukaemic cells take a walk on the wild side.

Oxidative stress is related to ageing and degenerative diseases, including cancer. However, a moderate amount of reactive oxygen species (ROS) is required for the regulation of cellular signalling and gene expression. A low level of ROS is important for maintaining quiescence and the differentiation potential of haematopoietic stem cells (HSCs), whereas the level of ROS increases during haematopoietic differentiation; thus, suggesting the importance of redox signalling in haematopoiesis. Here, we will analyse the importance of ROS for haematopoiesis and include evidence showing that cells from leukaemia patients live under oxidative stress. The potential sources of ROS will be described. Finally, the level of oxidative stress in leukaemic cells can also be harnessed for therapeutic purposes. In this regard, the reliance of front-line anti-leukaemia chemotherapeutics on increased levels of ROS for their mechanism of action, as well as the active search for novel compounds that modulate the redox state of leukaemic cells, will be analysed.

NADPH oxidases as therapeutic targets in chronic myelogenous leukemia.

PURPOSE: Cancer cells show higher levels of reactive oxygen species (ROS) than normal cells and increasing intracellular ROS levels are becoming a recognized strategy against tumor cells. Thus, diminishing ROS levels could be also detrimental to cancer cells. We surmise that avoiding ROS generation would be a better option than quenching ROS with antioxidants. Chronic myelogenous leukemia (CML) is triggered by the expression of BCR-ABL kinase, whose activity leads to increased ROS production, partly through NADPH oxidases. Here, we assessed NADPH oxidases as therapeutic targets in CML. EXPERIMENTAL DESIGN: We have analyzed the effect of different NADPH oxidase inhibitors, either alone or in combination with BCR-ABL inhibitors, in CML cells and in two different animal models for CML. RESULTS: NADPH oxidase inhibition dramatically impaired the proliferation and viability of BCR-ABL-expressing cells due to the attenuation of BCR-ABL signaling and a pronounced cell-cycle arrest. Moreover, the combination of NADPH oxidase inhibitors with BCR-ABL inhibitors was highly synergistic. Two different animal models underscore the effectiveness of NADPH oxidase inhibitors and their combination with BCR-ABL inhibitors for CML targeting in vivo. CONCLUSION: Our results offer further therapeutic opportunities for CML, by targeting NADPH oxidases. In the future, it would be worthwhile conducting further experiments to ascertain the feasibility of translating such therapies to clinical practice.