Use of an anti-CD200-blocking antibody improves immune responses to AML in vitro and in vivo.

Treatment of relapsed/resistant acute myeloid leukaemia (AML) remains a significant area of unmet patient need, the outlook for most patients remaining extremely poor. A promising approach is to augment the anti-tumour immune response in these patients; most cancers do not activate immune effector cells because they express immunosuppressive ligands. We have previously shown that CD200 (an immunosuppressive ligand) is overexpressed in AML and confers an inferior overall survival compared to CD200low/neg patients. Here we show that a fully human anti-CD200 antibody (TTI-CD200) can block the interaction of CD200 with its receptor and restore AML immune responses in vitro and in vivo.

LEF-1 drives aberrant ß-catenin nuclear localization in myeloid leukemia cells.

Canonical Wnt/ß-catenin signaling is frequently dysregulated in myeloid leukemias and is implicated in leukemogenesis. Nuclear-localized ß-catenin is indicative of active Wnt signaling and is frequently observed in acute myeloid leukemia (AML) patients; however, some patients exhibit little or no nuclear ß-catenin even where cytosolic ß-catenin is abundant. Control of the subcellular localization of ß-catenin therefore represents an additional mechanism regulating Wnt signaling in hematopoietic cells. To investigate the factors mediating the nuclear-localization of ß-catenin, we carried out the first nuclear/cytoplasmic proteomic analysis of the ß-catenin interactome in myeloid leukemia cells and identified putative novel ß-catenin interactors. Comparison of interacting factors between Wnt-responsive cells (high nuclear ß-catenin) versus Wnt-unresponsive cells (low nuclear ß-catenin) suggested the transcriptional partner, LEF-1, could direct the nuclear-localization of ß-catenin. The relative levels of nuclear LEF-1 and ß-catenin were tightly correlated in both cell lines and in primary AML blasts. Furthermore, LEF-1 knockdown perturbed ß-catenin nuclear-localization and transcriptional activation in Wnt-responsive cells. Conversely, LEF-1 overexpression was able to promote both nuclear-localization and ß-catenin-dependent transcriptional responses in previously Wnt-unresponsive cells. This is the first ß-catenin interactome study in hematopoietic cells and reveals LEF-1 as a mediator of nuclear ß- catenin level in human myeloid leukemia.

TET-2 up-regulation is associated with the anti-inflammatory action of Vicenin-2.

Vicenin-2, a C-glycoside flavone that is present in many plant sources, exerts potent anti-inflammatory effects in a number of cell and animal models of inflammation. Ten-eleven translocation (TET)-2 has recently gained considerable attention due to the role it plays in regulating the inflammasome. We studied the ability of Vicenin-2 (V-2) to regulate a range of lipopolysaccharide (LPS) stimulated inflammatory activities in PMA-differentiated THP-1 cells and human primary mononuclear cells. We also investigated the action of V-2 on the secretion of NLRP3 inflammasome regulated cytokines (IL-1ß and IL-18) by ELISA, and determined if V-2 can regulate the expression of NLRP3, IL-10, IL-1Ra and TET-2. The effect of V-2 on NF-κB signalling was investigated by fluorescence microscopy and gene reporter assay. Additionally, the effect of V-2 on LPS-induced phosphorylation of IKB-α was also investigated by Western blot analysis. V-2 down-regulated LPS-induced secretion of proinflammatory cytokines (TNF-α and IL-1ß), in both THP-1 and primary mononuclear cells. V-2 also decreased the LPS-stimulated secretion of IL-18 in THP-1 cells. V-2 significantly down-regulated TNF-α induced NF-κB reporter activity in HEK293T transfected cells and attenuated IKB-α phosphorylation in THP-1 cells. V-2 treatment also induced enhanced nuclear staining of the p50 subunit and reduced p65 subunit of NF-κB. V-2 treatment alone increased the expression of anti-inflammatory cytokine, IL-10, and the regulator of the inflammasome; IL-1Ra, in the presence of LPS. V-2 also significantly decreased LPS-induced NLRP3 expression while concomitantly increasing TET-2 expression. This study demonstrates that the anti-inflammatory actions of V-2 are associated not only with increased IL-10 and IL-1Ra expression, but also with TET-2 up-regulation. Further work is required to establish if the effects of V-2 can be definitively linked to TET-2 activity and that these actions are mirrored in a range of relevant cell types.

Overproduction of NOX-derived ROS in AML promotes proliferation and is associated with defective oxidative stress 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(+) 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.

Cord Blood-Derived Quiescent CD34+ Cells Are More Transcriptionally Matched to AML Blasts Than Cytokine-Induced Normal Human Hematopoietic CD34+ Cells.

Acute myeloid leukemia (AML) is characterized by developmental arrest, which is thought to arise from transcriptional dysregulation of myeloid development programs. Hematopoietic stem and progenitor cells (HSPCs) isolated from human blood are frequently used as a normal comparator in AML studies. Previous studies have reported changes in the transcriptional program of genes involved in proliferation, differentiation, apoptosis, and homing when HSPCs were expanded ex vivo. The intrinsic functional differences between quiescent and dividing CD34+ HSPCs prompted us to determine whether fresh or cytokine-induced cord blood-derived CD34+ HSPCs are a more appropriate normal control compared to AML blasts. Based on principal component analysis and gene expression profiling we demonstrate that CD34+ HSPCs that do not undergo ex vivo expansion are transcriptionally similar to minimally differentiated AML blasts. This was confirmed by comparing the cell cycle status of the AML blasts and the HSPCs. We suggest that freshly isolated CD34+ HSPCs that do not undergo ex vivo expansion would serve as a better control to identify novel transcriptional targets in the AML blast population.

Factors affecting the nuclear localization of ß-catenin in normal and malignant tissue.

The canonical Wnt signaling pathway has been the focus of intensive research because of its frequent dysregulation in human cancers. Much of this has been directed towards the aberrant expression and/or activity of the central mediator of this pathway, ß-catenin. In particular, the nuclear localization of ß-catenin and subsequent inappropriate activation of TCF/LEF-mediated transcription appears to be an important process in both the establishment and maintenance of cancer stem cells. Despite this, the exact mechanisms controlling ß-catenin nuclear localization in both normal and malignant cells are poorly understood. This prospect article brings together the many mechanisms previously reported to regulate the nuclear localization of ß-catenin and how they are relevant to cancer.

The PDK1 master kinase is over-expressed in acute myeloid leukemia and promotes PKC-mediated survival of leukemic blasts.

PDK1 is a master kinase that activates at least six protein kinase groups including AKT, PKC and S6K and is a potential target in the treatment of a range of malignancies. Here we show overexpression of PDK1 in over 40% of myelomonocytic acute leukemia patients. Overexpression of PDK1 occurred uniformly throughout the leukemic population, including putative leukemia-initiating cells. Clinical outcome analysis revealed PDK1 overexpression was associated with poorer treatment outcome. Primary acute myeloid leukemia blasts over-expressing PDK1 showed improved in vitro survival and ectopic expression of PDK1 promoted the survival of myeloid cell lines. Analysis of PDK1 target kinases revealed that PDK1 overexpression was most closely associated with increased phosphorylation of PKC isoenzymes and inhibition of PKC strongly inhibited the survival advantage of PDK1 over-expressing cells. Membrane localization studies implicated PKCα as a major target for PDK1 in this disease. PDK1 over-expressing blasts showed differential sensitivity to PDK1 inhibition (in the low micromolar range) suggesting oncogene addiction, whilst normal bone marrow progenitors were refractory to PDK1 inhibition at effective inhibitor concentrations. PDK1 inhibition also targeted subpopulations of leukemic blasts with a putative leukemia-initiating cell phenotype. Together these data show that overexpression of PDK1 is common in acute myelomonocytic leukemia and is associated with poorer treatment outcome, probably arising from the cytoprotective function of PDK1. We also show that therapeutic targeting of PDK1 has the potential to be both an effective and selective treatment for these patients, and is also compatible with current treatment regimes.

Reactive oxygen species and its role in pathogenesis and resistance to therapy in acute myeloid leukemia.

Relapse following a short clinical response to therapy is the major challenge for the management of acute myeloid leukemia (AML) patients. Leukemic stem cells (LSC), as the source of relapse, have been investigated for their metabolic preferences and their alterations at the time of relapse. As LSC rely on oxidative phosphorylation (OXPHOS) for energy requirement, reactive oxygen species (ROS), as by-products of OXPHOS, have been investigated for their role in the effectiveness of the standard AML therapy. Increased levels of non-mitochondrial ROS, generated by nicotinamide adenine dinucleotide phosphate oxidase, in a subgroup of AML patients add to the complexity of studying ROS. Although there are various studies presenting the contribution of ROS to AML pathogenesis, resistance, and its inhibition or activation as a target, a model that can clearly explain its role in AML has not been conceptualized. This is due to the heterogeneity of AML, the dynamics of ROS production, which is influenced by factors such as the type of treatment, cell differentiation state, mitochondrial activity, and also the heterogeneous generation of non-mitochondrial ROS and limited available data on their interaction with the microenvironment. This review summarizes these challenges and the recent progress in this field.

Modification of the Antibiotic, Colistin, with Dextrin Causes Enhanced Cytotoxicity and Triggers Apoptosis in Myeloid Leukemia.

Introduction: Acute myeloid leukemia (AML) remains difficult to treat due to its heterogeneity in molecular landscape, epigenetics and cell signaling alterations. Precision medicine is a major goal in AML therapy towards developing agents that can be used to treat patients with different 'subtypes' in combination with current chemotherapies. We have previously developed dextrin-colistin conjugates to combat the rise in multi-drug resistant bacterial infections and overcome dose-limiting nephrotoxicity. Recent evidence of colistin's anticancer activity, mediated through inhibition of intracellular lysine-specific histone demethylase 1 (LSD1/KDM1A), suggests that dextrin-colistin conjugates could be used to treat cancer cells, including AML. This study aimed to evaluate whether dextrin conjugation (which reduces in vivo toxicity and prolongs plasma half-life) could enhance colistin's cytotoxic effects in myeloid leukemia cell lines and compare the intracellular uptake and localization of the free and conjugated antibiotic. Results: Our results identified a conjugate (containing 8000 g/mol dextrin with 1 mol% succinoylation) that caused significantly increased toxicity in myeloid leukemia cells, compared to free colistin. Dextrin conjugation altered the mechanism of cell death by colistin, from necrosis to caspase 3/7-dependent apoptosis. In contrast, conjugation via a reversible ester linker, instead of an amide, had no effect on the mechanism of the colistin-induced cell death. Live cell confocal microscopy of fluorescently labelled compounds showed both free and dextrin-conjugated colistins were endocytosed and co-localized in lysosomes, and increasing the degree of modification by succinoylation of dextrin significantly reduced colistin internalization. Discussion: Whilst clinical translation of dextrin-colistin conjugates for the treatment of AML is unlikely due to the potential to promote antimicrobial resistance (AMR) and the relatively high colistin concentrations required for anticancer activity, the ability to potentiate the effectiveness of an anticancer drug by polymer conjugation, while reducing side effects and improving biodistribution of the drug, is very attractive, and this approach warrants further investigation.

Do reactive oxygen species play a role in myeloid leukemias?

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.

Nuclear factor I-C overexpression promotes monocytic development and cell survival in acute myeloid leukemia.

Nuclear factor I-C (NFIC) belongs to a family of NFI transcription factors that binds to DNA through CAATT-boxes and are involved in cellular differentiation and stem cell maintenance. Here we show NFIC protein is significantly overexpressed in 69% of acute myeloid leukemia patients. Examination of the functional consequences of NFIC overexpression in HSPCs showed that this protein promoted monocytic differentiation. Single-cell RNA sequencing analysis further demonstrated that NFIC overexpressing monocytes had increased expression of growth and survival genes. In contrast, depletion of NFIC through shRNA decreased cell growth, increased cell cycle arrest and apoptosis in AML cell lines and AML patient blasts. Further, in AML cell lines (THP-1), bulk RNA sequencing of NFIC knockdown led to downregulation of genes involved in cell survival and oncogenic signaling pathways including mixed lineage leukemia-1 (MLL-1). Lastly, we show that NFIC knockdown in an ex vivo mouse MLL::AF9 pre-leukemic stem cell model, decreased their growth and colony formation and increased expression of myeloid differentiation markers Gr1 and Mac1. Collectively, our results suggest that NFIC is an important transcription factor in myeloid differentiation as well as AML cell survival and is a potential therapeutic target in AML.

Ras-induced reactive oxygen species promote growth factor-independent proliferation in human CD34+ hematopoietic progenitor cells.

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) oxidative stress response, this failed to induce expression of the cell-cycle inhibitor, p16(INK4A); instead, ROS promoted the expression of D cyclins. These data are the first to show that excessive ROS production in the context of oncogene activation can promote proliferative responses in normal human hematopoietic progenitor cells.

The BCAT1 CXXC Motif Provides Protection against ROS in Acute Myeloid Leukaemia Cells.

The cytosolic branched-chain aminotransferase (BCAT1) has received attention for its role in myeloid leukaemia development, where studies indicate metabolic adaptations due to BCAT1 up-regulation. BCAT1, like the mitochondria isoform (BCAT2), shares a conserved CXXC motif ~10 Å from the active site. This CXXC motif has been shown to act as a 'redox-switch' in the enzymatic regulation of the BCAT proteins, however the response to reactive oxygen species (ROS) differs between BCAT isoforms. Studies indicate that the BCAT1 CXXC motif is several orders of magnitude less sensitive to the effects of ROS compared with BCAT2. Moreover, estimation of the reduction mid-point potential of BCAT1, indicates that BCAT1 is more reductive in nature and may possess antioxidant properties. Therefore, the aim of this study was to further characterise the BCAT1 CXXC motif and evaluate its role in acute myeloid leukaemia. Our biochemical analyses show that purified wild-type (WT) BCAT1 protein could metabolise H2O2 in vitro, whereas CXXC motif mutant or WT BCAT2 could not, demonstrating for the first time a novel antioxidant role for the BCAT1 CXXC motif. Transformed U937 AML cells over-expressing WT BCAT1, showed lower levels of intracellular ROS compared with cells over-expressing the CXXC motif mutant (CXXS) or Vector Controls, indicating that the BCAT1 CXXC motif may buffer intracellular ROS, impacting on cell proliferation. U937 AML cells over-expressing WT BCAT1 displayed less cellular differentiation, as observed by a reduction of the myeloid markers; CD11b, CD14, CD68, and CD36. This finding suggests a role for the BCAT1 CXXC motif in cell development, which is an important pathological feature of myeloid leukaemia, a disease characterised by a block in myeloid differentiation. Furthermore, WT BCAT1 cells were more resistant to apoptosis compared with CXXS BCAT1 cells, an important observation given the role of ROS in apoptotic signalling and myeloid leukaemia development. Since CD36 has been shown to be Nrf2 regulated, we investigated the expression of the Nrf2 regulated gene, TrxRD1. Our data show that the expression of TrxRD1 was downregulated in transformed U937 AML cells overexpressing WT BCAT1, which taken with the reduction in CD36 implicates less Nrf2 activation. Therefore, this finding may implicate the BCAT1 CXXC motif in wider cellular redox-mediated processes. Altogether, this study provides the first evidence to suggest that the BCAT1 CXXC motif may contribute to the buffering of ROS levels inside AML cells, which may impact ROS-mediated processes in the development of myeloid leukaemia.

Protein Kinase C Epsilon Overexpression Is Associated With Poor Patient Outcomes in AML and Promotes Daunorubicin Resistance Through p-Glycoprotein-Mediated Drug Efflux.

The protein kinase C (PKC) family of serine/threonine kinases are pleiotropic signaling regulators and are implicated in hematopoietic signaling and development. Only one isoform however, PKCϵ, has oncogenic properties in solid cancers where it is associated with poor outcomes. Here we show that PKCϵ protein is significantly overexpressed in acute myeloid leukemia (AML; 37% of patients). In addition, PKCϵ expression in AML was associated with a significant reduction in complete remission induction and disease-free survival. Examination of the functional consequences of PKCϵ overexpression in normal human hematopoiesis, showed that PKCϵ promotes myeloid differentiation, particularly of the monocytic lineage, and decreased colony formation, suggesting that PKCϵ does not act as an oncogene in hematopoietic cells. Rather, in AML cell lines, PKCϵ overexpression selectively conferred resistance to the chemotherapeutic agent, daunorubicin, by reducing intracellular concentrations of this agent. Mechanistic analysis showed that PKCϵ promoted the expression of the efflux pump, P-GP (ABCB1), and that drug efflux mediated by this transporter fully accounted for the daunorubicin resistance associated with PKCϵ overexpression. Analysis of AML patient samples also showed a link between PKCϵ and P-GP protein expression suggesting that PKCϵ expression drives treatment resistance in AML by upregulating P-GP expression.

Increased expression of RUNX3 inhibits normal human myeloid development.

RUNX3 is a transcription factor dysregulated in acute myeloid leukemia (AML). However, its role in normal myeloid development and leukemia is poorly understood. Here we investigate RUNX3 expression in both settings and the impact of its dysregulation on myelopoiesis. We found that RUNX3 mRNA expression was stable during hematopoiesis but decreased with granulocytic differentiation. In AML, RUNX3 mRNA was overexpressed in many disease subtypes, but downregulated in AML with core binding factor abnormalities, such as RUNX1::ETO. Overexpression of RUNX3 in human hematopoietic stem and progenitor cells (HSPC) inhibited myeloid differentiation, particularly of the granulocytic lineage. Proliferation and myeloid colony formation were also inhibited. Conversely, RUNX3 knockdown did not impact the myeloid growth and development of human HSPC. Overexpression of RUNX3 in the context of RUNX1::ETO did not rescue the RUNX1::ETO-mediated block in differentiation. RNA-sequencing showed that RUNX3 overexpression downregulates key developmental genes, such as KIT and RUNX1, while upregulating lymphoid genes, such as KLRB1 and TBX21. Overall, these data show that increased RUNX3 expression observed in AML could contribute to the developmental arrest characteristic of this disease, possibly by driving a competing transcriptional program favoring a lymphoid fate.

RUNX3 overexpression inhibits normal human erythroid development.

RUNX proteins belong to a family of transcription factors essential for cellular proliferation, differentiation, and apoptosis with emerging data implicating RUNX3 in haematopoiesis and haematological malignancies. Here we show that RUNX3 plays an important regulatory role in normal human erythropoiesis. The impact of altering RUNX3 expression on erythropoiesis was determined by transducing human CD34+ cells with RUNX3 overexpression or shRNA knockdown vectors. Analysis of RUNX3 mRNA expression showed that RUNX3 levels decreased during erythropoiesis. Functionally, RUNX3 overexpression had a modest impact on early erythroid growth and development. However, in late-stage erythroid development, RUNX3 promoted growth and inhibited terminal differentiation with RUNX3 overexpressing cells exhibiting lower expression of glycophorin A, greater cell size and less differentiated morphology. These results suggest that suppression of RUNX3 is required for normal erythropoiesis. Overexpression of RUNX3 increased colony formation in liquid culture whilst, corresponding RUNX3 knockdown suppressed colony formation but otherwise had little impact. This study demonstrates that the downregulation of RUNX3 observed in normal human erythropoiesis is important in promoting the terminal stages of erythroid development and may further our understanding of the role of this transcription factor in haematological malignancies.

CD200 ectodomain shedding into the tumor microenvironment leads to NK cell dysfunction and apoptosis.

The basis of immune evasion, a hallmark of cancer, can differ even when cancers arise from one cell type such as in the human skin keratinocyte carcinomas: basal and squamous cell carcinoma. Here we showed that the basal cell carcinoma tumor-initiating cell surface protein CD200, through ectodomain shedding, was responsible for the near absence of NK cells within the basal cell carcinoma tumor microenvironment. In situ, CD200 underwent ectodomain shedding by metalloproteinases MMP3 and MMP11, which released biologically active soluble CD200 into the basal cell carcinoma microenvironment. CD200 bound its cognate receptor on NK cells to suppress MAPK pathway signaling that in turn blocked indirect (IFN-γ release) and direct cell killing. In addition, reduced ERK phosphorylation relinquished negative regulation of PPARγ-regulated gene transcription and led to membrane accumulation of the Fas/FADD death receptor and its ligand, FasL, which resulted in activation-induced apoptosis. Blocking CD200 inhibition of MAPK or PPARγ signaling restored NK cell survival and tumor cell killing, with relevance to many cancer types. Our results thus uncover a paradigm for CD200 as a potentially novel and targetable NK cell-specific immune checkpoint, which is responsible for NK cell-associated poor outcomes in many cancers.

Make Your Cake and Eat It: Refueling of Immune Fitness in AML Post Allo-HCT Using Baking Soda.

Although there has been a recent renaissance in the availability of new therapeutic options for patients with acute myeloid leukemia (AML), survival rates remain low coupled with a high incidence of relapse. Enhancing T cell and immune function has become an effective therapeutic approach in hematological malignancies. However, AML cells can modulate the bone marrow microenvironment by changing extracellular nutrient and biochemical availability which can metabolically regulate immune function. Here we review the findings by Uhl et al. showing that T cell metabolism and function can be boosted by treatment with sodium bicarbonate to counteract the metabolic changes induced by lactic acid produced by leukemia cells.

Reactive Oxygen Species Rewires Metabolic Activity in Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is a heterogeneous disease with poor clinical outcomes. We have previously shown that constitutive activation of NADPH oxidase 2 (NOX2), resulting in over-production of reactive oxygen species (ROS), occurs in over 60% of AML patients. We have also shown that increased ROS production promotes increased glucose uptake and proliferation in AML cells, mediated by changes in carbohydrate metabolism. Given that carbohydrate, lipid, and protein metabolisms are all intricately interconnected, we aimed to examine the effect of cellular ROS levels on these pathways and establish further evidence that ROS rewires metabolism in AML. We carried out metabolomic profiling of AML cell lines in which NOX2-derived ROS production was inhibited and conversely in cells treated with exogenous H2O2. We report significant ROS-specific metabolic alterations in sphingolipid metabolism, fatty acid oxidation, purine metabolism, amino acid homeostasis and glycolysis. These data provide further evidence of ROS directed metabolic changes in AML and the potential for metabolic targeting as novel therapeutic arm to combat this disease.