Pathogen and human NDPK-proteins promote AML cell survival via monocyte NLRP3-inflammasome activation.

A history of infection has been linked with increased risk of acute myeloid leukaemia (AML) and related myelodysplastic syndromes (MDS). Furthermore, AML and MDS patients suffer frequent infections because of disease-related impaired immunity. However, the role of infections in the development and progression of AML and MDS remains poorly understood. We and others previously demonstrated that the human nucleoside diphosphate kinase (NDPK) NM23-H1 protein promotes AML blast cell survival by inducing secretion of IL-1ß from accessory cells. NDPKs are an evolutionary highly conserved protein family and pathogenic bacteria secrete NDPKs that regulate virulence and host-pathogen interactions. Here, we demonstrate the presence of IgM antibodies against a broad range of pathogen NDPKs and more selective IgG antibody activity against pathogen NDPKs in the blood of AML patients and normal donors, demonstrating that in vivo exposure to NDPKs likely occurs. We also show that pathogen derived NDPK-proteins faithfully mimic the catalytically independent pro-survival activity of NM23-H1 against primary AML cells. Flow cytometry identified that pathogen and human NDPKs selectively bind to monocytes in peripheral blood. We therefore used vitamin D3 differentiated monocytes from wild type and genetically modified THP1 cells as a model to demonstrate that NDPK-mediated IL-1ß secretion by monocytes is NLRP3-inflammasome and caspase 1 dependent, but independent of TLR4 signaling. Monocyte stimulation by NDPKs also resulted in activation of NF-κB and IRF pathways but did not include the formation of pyroptosomes or result in pyroptotic cell death which are pivotal features of canonical NLRP3 inflammasome activation. In the context of the growing importance of the NLRP3 inflammasome and IL-1ß in AML and MDS, our findings now implicate pathogen NDPKs in the pathogenesis of these diseases.

Combined bezafibrate, medroxyprogesterone acetate and valproic acid treatment inhibits osteosarcoma cell growth without adversely affecting normal mesenchymal stem cells.

Drug repurposing is a cost-effective means of targeting new therapies for cancer. We have examined the effects of the repurposed drugs, bezafibrate, medroxyprogesterone acetate and valproic acid on human osteosarcoma cells, i.e., SAOS2 and MG63 compared with their normal cell counterparts, i.e. mesenchymal stem/stromal cells (MSCs). Cell growth, viability and migration were measured by biochemical assay and live cell imaging, whilst levels of lipid-synthesising enzymes were measured by immunoblotting cell extracts. These drug treatments inhibited the growth and survival of SAOS2 and MG63 cells most effectively when used in combination (termed V-BAP). In contrast, V-BAP treated MSCs remained viable with only moderately reduced cell proliferation. V-BAP treatment also inhibited migratory cell phenotypes. MG63 and SAOS2 cells expressed much greater levels of fatty acid synthase and stearoyl CoA desaturase 1 than MSCs, but these elevated enzyme levels significantly decreased in the V-BAP treated osteosarcoma cells prior to cell death. Hence, we have identified a repurposed drug combination that selectively inhibits the growth and survival of human osteosarcoma cells in association with altered lipid metabolism without adversely affecting their non-transformed cell counterparts.

Muscle length and joint angle influence spinal but not corticospinal excitability to the biceps brachii across forearm postures.

Forearm rotation (supination/pronation) alters corticospinal excitability to the biceps brachii, but it is unclear whether corticospinal excitability is influenced by joint angle, muscle length, or both. Thus the purpose of this study was to separately examine elbow joint angle and muscle length on corticospinal excitability. Corticospinal excitability to the biceps and triceps brachii was measured using motor evoked potentials (MEPs) elicited via transcranial magnetic stimulation. Spinal excitability was measured using cervicomedullary motor evoked potentials (CMEPs) elicited via transmastoid electrical stimulation. Elbow angles were manipulated with a fixed biceps brachii muscle length (and vice versa) across five unique postures: 1) forearm neutral, elbow flexion 90°; 2) forearm supinated, elbow flexion 90°; 3) forearm pronated, elbow flexion 90°; 4) forearm supinated, elbow flexion 78°; and 5) forearm pronated, elbow flexion 113°. A musculoskeletal model determined biceps brachii muscle length for postures 1-3, and elbow joint angles (postures 4-5) were selected to maintain biceps length across forearm orientations. MEPs and CMEPs were elicited at rest and during an isometric contraction of 10% of maximal biceps muscle activity. At rest, MEP amplitudes to the biceps were largest during supination, which was independent of elbow joint angle. CMEP amplitudes were not different when the elbow was fixed at 90° but were largest in pronation when muscle length was controlled. During an isometric contraction, there were no significant differences across forearm postures for either MEP or CMEP amplitudes. These results highlight that elbow joint angle and biceps brachii muscle length can each independently influence spinal excitability. NEW & NOTEWORTHY Changes in upper limb posture can influence the responsiveness of the central nervous system to artificial stimulations. We established a novel approach integrating neurophysiology techniques with biomechanical modeling. Through this approach, the effects of elbow joint angle and biceps brachii muscle length on corticospinal and spinal excitability were assessed. We demonstrate that spinal excitability is uniquely influenced by joint angle and muscle length, and this highlights the importance of accounting for muscle length in neurophysiological studies.

Malonate as a ROS product is associated with pyruvate carboxylase activity in acute myeloid leukaemia cells.

BACKGROUND: The role of anaplerotic nutrient entry into the Krebs cycle via pyruvate carboxylase has been the subject of increased scrutiny and in particular whether this is dysregulated in cancer. Here, we use a tracer-based NMR analysis involving high-resolution (1)H-(13)C-HSQC spectra to assess site-specific label incorporation into a range of metabolite pools, including malate, aspartate and glutamate in the acute myeloid leukaemia cell line K562. We also determine how this is affected following treatment with the redeployed drug combination of the lipid-regulating drug bezafibrate and medroxyprogesterone (BaP). RESULTS: Using the tracer-based approach, we assessed the contribution of pyruvate carboxylase (PC) vs. pyruvate dehydrogenase (PDH) activity in the derivation of Krebs cycle intermediates. Our data show that PC activity is indeed high in K562 cells. We also demonstrate a branched entry to the Krebs cycle of K562 cells with one branch running counterclockwise using PC-derived oxaloacetate and the other clockwise from the PDH activity. Finally, we show that the PC activity of K562 cells exclusively fuels the ROS-induced decarboxylation of oxaloacetate to malonate in response to BaP treatment; resulting in further Krebs cycle disruption via depletion of oxaloacetate and malonate-mediated inhibition of succinate dehydrogenase (SDH) resulting in a twofold reduction of fumarate. CONCLUSIONS: This study extends the interest in the PC activity in solid cancers to include leukaemias and further demonstrates the value of tracer-based NMR approaches in generating a more accurate picture of the flow of carbons and metabolites within the increasingly inappropriately named Krebs cycle. Moreover, our studies indicate that the PC activity in cancer cells can be exploited as an Achilles heel by using treatments, such as BaP, that elevate ROS production.

Tracer-Based Metabolic NMR-Based Flux Analysis in a Leukaemia Cell Line.

High levels of reactive oxygen species (ROS) have a profound impact on acute myeloid leukaemia cells and can be used to specifically target these cells with novel therapies. We have previously shown how the combination of two redeployed drugs, the contraceptive steroid medroxyprogesterone and the lipid-regulating drug bezafibrate exert anti-leukaemic effects by producing ROS. Here we report a 13C-tracer-based NMR metabolic study to understand how these drugs work in K562 leukaemia cells. Our study shows that [1,2-13C]glucose is incorporated into ribose sugars, indicating activity in oxidative and non-oxidative pentose phosphate pathways alongside lactate production. There is little label incorporation into the tricarboxylic acid cycle from glucose, but much greater incorporation arises from the use of [3-13C]glutamine. The combined medroxyprogesterone and bezafibrate treatment decreases label incorporation from both glucose and glutamine into α-ketoglutarate and increased that for succinate, which is consistent with ROS-mediated conversion of α-ketoglutarate to succinate. Most interestingly, this combined treatment drastically reduced the production of several pyrimidine synthesis intermediates.

Knockdown of AKR1C3 exposes a potential epigenetic susceptibility in prostate cancer cells.

BACKGROUND: The aldo-keto reductase 1C3 (AKR1C3) has been heavily implicated in the propagation of prostate malignancy. AKR1C3 protein is elevated within prostate cancer tissue, it contributes to the formation of androgens and downstream stimulation of the androgen receptor (AR). Elevated expression of AKR1C3 is also reported in acute myeloid leukemia but the target nuclear receptors have been identified as members of the peroxisome-proliferator activated receptor (PPARs) subfamily. Thus, AKR1C3 cancer biology is likely to be tissue dependent and hormonally linked to the availability of ligands for both the steroidogenic and non-steroidogenic nuclear receptors. METHODS: In the current study we investigated the potential for AKR1C3 to regulate the availability of prostaglandin-derived ligands for PPARg mainly, prostaglandin J2 (PGJ2). Using prostate cancer cell lines with stably reduced AKR1C3 levels we examined the impact of AKR1C3 upon proliferation mediated by PPAR ligands. RESULTS: These studies revealed knockdown of AKR1C3 had no effect upon the sensitivity of androgen receptor independent prostate cancer cells towards PPAR ligands. However, the reduction of levels of AKR1C3 was accompanied by a significantly reduced mRNA expression of a range of HDACs, transcriptional co-regulators, and increased sensitivity towards SAHA, a clinically approved histone deacetylase inhibitor. CONCLUSIONS: These results suggest a hitherto unidentified link between AKR1C3 levels and the epigenetic status in prostate cancer cells. This raises an interesting possibility of a novel rational to target AKR1C3, the utilization of AKRIC3 selective inhibitors in combination with HDAC inhibition as part of novel epigenetic therapies in androgen deprivation therapy recurrent prostate cancer.

Tracer-Based Metabolic NMR-Based Flux Analysis in a Leukaemia Cell Line.

High levels of reactive oxygen species (ROS) have a profound impact on acute myeloid leukaemia cells and can be used to specifically target these cells with novel therapies. We have previously shown how the combination of two redeployed drugs, the contraceptive steroid medroxyprogesterone and the lipid-regulating drug bezafibrate exert anti-leukaemic effects by producing ROS. Here we report a 13 C-tracer-based NMR metabolic study to understand how these drugs work in K562 leukaemia cells. Our study shows that [1,2-13 C]glucose is incorporated into ribose sugars, indicating activity in oxidative and non-oxidative pentose phosphate pathways alongside lactate production. There is little label incorporation into the tricarboxylic acid cycle from glucose, but much greater incorporation arises from the use of [3-13 C]glutamine. The combined medroxyprogesterone and bezafibrate treatment decreases label incorporation from both glucose and glutamine into α-ketoglutarate and increased that for succinate, which is consistent with ROS-mediated conversion of α-ketoglutarate to succinate. Most interestingly, this combined treatment drastically reduced the production of several pyrimidine synthesis intermediates.

The case for extracellular Nm23-H1 as a driver of acute myeloid leukaemia (AML) progression.

Studies in the 1990s identified a link between extracellular Nm23 proteins and acute myeloid leukaemia (AML). Confidence in the importance of these observations was undermined by a lack of appreciation that extracellular Nm23 proteins were relevant to either normal or pathophysiology coupled with the lack of demonstrable activity of Nm23 proteins against human AML cell lines. However, independent studies have highlighted the importance of Nm23-H1 in AML and have identified an elaborate Nm23-H1-mediated cross talk between cells within the AML clone. In other studies, roles for Nm23-H1 have now also been implicated in the maintenance of the stem cell state of embryonic stem (ES) cells and induced pluripotent stem (IPS) cells. In this review, we have generated a unifying model of the action of Nm23-H1 in AML, including previously unpublished data from our laboratory, and provide arguments as to why we consider this role to be distinct from that in ES and IPS cells.

Bezafibrate and medroxyprogesterone acetate target resting and CD40L-stimulated primary marginal zone lymphoma and show promise in indolent B-cell non-Hodgkin lymphomas.

B cell non-Hodgkin lymphomas (B-NHLs) are the most common adult hematological cancers and many remain incurable. Development of chemotherapy regimens is confounded by the prevalence of B-NHL in older, frailer patients and the chemo-protective tumor microenvironment. Although biological therapies such as rituximab have significantly improved outcomes and selective kinase inhibitors are showing promise, the rate of new drug discovery remains disappointing, the treatments expensive and long-term benefits uncertain. An alternative strategy is redeployment of available, inexpensive and non-toxic drugs. Here, we demonstrate the antiproliferative and mitochondrial superoxide (MSO) driven pro-apoptotic activities of bezafibrate (BEZ) and medroxyprogesterone acetate (MPA) against B-NHL cells, with a bias toward MZL, in the presence and absence of the microenvironmental signal CD40L. Our study is the first to confirm the presence of CD40L within the lymph node of B-NHL and its capacity to drive B-NHL proliferation. These findings implicate BEZ + MPA as a potential therapeutic strategy in B-NHL.

Proton NMR-based metabolite analyses of archived serial paired serum and urine samples from myeloma patients at different stages of disease activity identifies acetylcarnitine as a novel marker of active disease.

BACKGROUND: Biomarker identification is becoming increasingly important for the development of personalized or stratified therapies. Metabolomics yields biomarkers indicative of phenotype that can be used to characterize transitions between health and disease, disease progression and therapeutic responses. The desire to reproducibly detect ever greater numbers of metabolites at ever diminishing levels has naturally nurtured advances in best practice for sample procurement, storage and analysis. Reciprocally, since many of the available extensive clinical archives were established prior to the metabolomics era and were not processed in such an 'ideal' fashion, considerable scepticism has arisen as to their value for metabolomic analysis. Here we have challenged that paradigm. METHODS: We performed proton nuclear magnetic resonance spectroscopy-based metabolomics on blood serum and urine samples from 32 patients representative of a total cohort of 1970 multiple myeloma patients entered into the United Kingdom Medical Research Council Myeloma IX trial. FINDINGS: Using serial paired blood and urine samples we detected metabolite profiles that associated with diagnosis, post-treatment remission and disease progression. These studies identified carnitine and acetylcarnitine as novel potential biomarkers of active disease both at diagnosis and relapse and as a mediator of disease associated pathologies. CONCLUSIONS: These findings show that samples conventionally processed and archived can provide useful metabolomic information that has important implications for understanding the biology of myeloma, discovering new therapies and identifying biomarkers potentially useful in deciding the choice and application of therapy.

The haematopoietic stem cell niche: new insights into the mechanisms regulating haematopoietic stem cell behaviour.

The concept of the haematopoietic stem cell (HSC) niche was formulated by Schofield in the 1970s, as a region within the bone marrow containing functional cell types that can maintain HSC potency throughout life. Since then, ongoing research has identified numerous cell types and a plethora of signals that not only maintain HSCs, but also dictate their behaviour with respect to homeostatic requirements and exogenous stresses. It has been proposed that there are endosteal and vascular niches within the bone marrow, which are thought to regulate different HSC populations. However, recent data depicts a more complicated picture, with functional crosstalk between cells in these two regions. In this review, recent research into the endosteal/vascular cell types and signals regulating HSC behaviour are considered, together with the possibility of a single subcompartmentalised niche.

Hypoxia triggers major metabolic changes in AML cells without altering indomethacin-induced TCA cycle deregulation.

Our previous studies have shown that the nonsteroidal anti-inflammatory drug indomethacin exhibits antileukemic activity in vitro and can inhibit the aldo-keto reductase AKR1C3, which we identified as a novel target in acute myeloid leukemia. However, the antileukemic actions of indomethacin are likely to be complex and extend beyond inhibition of either AKR1C3 or cycloxygenases. To further understand the antileukemic activity of indomethacin we have used untargeted nuclear magnetic resonance-based metabolic analysis to characterize the responses of KG1a and K562 cell lines in both normal culture conditions and in hypoxia, which better represents the tumor environment in vivo. Hypoxia induced dramatic metabolic changes in untreated KG1a and K562, including adaptation of both phospholipid and glycolytic metabolism. Despite these changes, both cell lines sustained relatively unaltered mitochondrial respiration. The administration of indomethacin induced similar metabolic responses regardless of the oxygen level in the environment. Notable exceptions included metabolites associated with de novo fatty acid synthesis and choline phospholipid metabolism. Collectively, these results suggest that leukemia cells have the inherent ability to tolerate changes in oxygen tension while maintaining an unaltered mitochondrial respiration. However, the administration of indomethacin significantly increased oxidative stress in both KG1a and K562, inducing mitochondrial dysfunction, regardless of the oxygenation conditions. These findings emphasize the particular pertinence of the tricarboxylic acid cycle to the survival of cancer cells and may explain why some antileukemic drugs have been discovered and developed successfully despite the use of culture conditions that do not reflect the hypoxic environment of cancer cells in vivo.

Nm23-h1 indirectly promotes the survival of acute myeloid leukemia blast cells by binding to more mature components of the leukemic clone.

Nm23-H1 plays complex roles in the development of diverse cancers including breast carcinoma, high-grade lymphomas, and acute myeloid leukemia (AML). In the case of AML and lymphomas, serum Nm23-H1 protein is elevated with the highest levels correlating with poorest prognosis. A recent study identified that this association is most likely causal in AML and that Nm23-H1 acts as an AML cell survival factor. In this study, we report heterogeneity in the ability of AML samples to bind and respond to Nm23-H1, and we offer evidence that binding is essential for improved survival. Further, we show that the subset of AMLs that bind Nm23-H1 do not do so through the putative Nm23-H1 receptor MUC1*. Although rNm23-H1 promoted the survival of the most primitive blasts within responding AMLs, it was not these cells that actually bound the protein. Instead, rNm23-H1 bound to more mature CD34(lo)/CD34(-) and CD11b(+) cells, revealing an indirect survival benefit of Nm23-H1 on primitive blasts. In support of this finding, the survival of purified blast cells was enhanced by medium conditioned by more mature cells from the clone that had been stimulated by rNm23-H1. Levels of interleukin 1ß (IL1ß) and IL6 in rNm23-H1 conditioned medium mirrored the potency of the conditioned media to promote blast cell survival. Furthermore, Nm23-H1 expression was significantly associated with IL1ß and IL6 expression in primary uncultured AML samples. These findings have implications for the role of Nm23-H1 in AML and its use as a prognostic marker. Additionally, they offer the first evidence of novel cross-talk between cell populations within the tumor clone.

Elevated NCOR1 disrupts PPARalpha/gamma signaling in prostate cancer and forms a targetable epigenetic lesion.

The loss of anti-proliferative responsiveness in prostate cancer cell lines toward ligands for vitamin D receptor, retinoic acid receptors/retinoid X receptors and peroxisome proliferator activated receptor (PPAR)alpha/gamma may entail underlying epigenetic events, as ligand insensitivity reflects significantly altered messenger RNA expression of corepressors and histone-modifying enzymes. Expression patterns were dependent on phases of the cell cycle and associated with repressed basal gene expression of vitamin D receptor and PPARalpha/gamma target genes, for example CDKN1A [encodes p21((waf1/cip1))]. Elevated nuclear corepressor 1 (NCOR1) and nuclear corepressor 2/silencing mediator of retinoic acid and thyroid hormone receptor protein levels were detected in prostate cancer cell lines compared with non-malignant counterparts. Knockdown of the corepressor NCOR1 significantly elevated basal expression of a cohort of target genes, including CDKN1A. Both chemical [histone deacetylases inhibitor (HDACi)] and NCOR1 knockdown targeting enhanced anti-proliferative sensitivity toward PPARalpha/gamma ligands in prostate cancer cell lines. Pursuing PPARalpha/gamma signaling, microarray approaches were undertaken to identify pathways and genes regulated uniquely by a combination of PPARalpha/gamma activation and HDAC inhibition. Again, HDACi and knockdown approaches demonstrated that elevated NCOR1 expression and activity distorted PPARalpha/gamma gene targets centered on, for example cell cycle control, including CDKN1A and TGFBRAP1. Quantitative real time polymerase chain reaction validation and chromatin immunoprecipitation assays both confirmed that elevated NCOR1 disrupted the ability of PPARalpha/gamma to regulate key target genes (CDKN1A and TGFBRAP1). Interrogation of these relationships in prostate cancer samples using principal component and partial correlation analyses established significant interdependent relationships between NCOR1-PPARalpha/gamma and representative target genes, independently of androgen receptor expression. Therefore, we conclude that elevated NCOR1 distorts the actions of PPARalpha/gamma selectively and generates a potential epigenetic lesion with diagnostic and prognostic significance.

Characterization of two novel aldo-keto reductases from Arabidopsis: expression patterns, broad substrate specificity, and an open active-site structure suggest a role in toxicant metabolism following stress.

Aldo-keto reductases (AKRs) are widely distributed in nature and play numerous roles in the metabolism of steroids, sugars, and other carbonyls. They have also frequently been implicated in the metabolism of exogenous and endogenous toxicants, including those stimulated by stress. Although the Arabidopsis genome includes at least 21 genes with the AKR signature, very little is known of their functions. In this study, we have screened the Arabidopsis thaliana genomic sequence for genes with significant homology to members of the mammalian AKR1 family and identified four homologues for further study. Following alignment of the predicted protein sequences with representatives from the AKR superfamily, the proteins were ascribed not to the AKR1 family but to the AKR4C subfamily, with the individual designations of AKR4C8, AKR4C9, AKR4C10, and AKR4C11. Expression of two of the genes, AKR4C8 and AKR4C9, has been shown to be coordinately regulated and markedly induced by various forms of stress. The genes have been overexpressed in bacteria, and recombinant proteins have been purified and crystallized. Both enzymes display NADPH-dependent reduction of carbonyl compounds, typical of the superfamily, but will accept a very wide range of substrates, reducing a range of steroids, sugars, and aliphatic and aromatic aldehydes/ketones, although there are distinct differences between the two enzymes. We have obtained high-resolution crystal structures of AKR4C8 (1.4 A) and AKR4C9 (1.25 A) in ternary complexes with NADP(+) and acetate. Three extended loops, present in all AKRs and responsible for defining the cofactor- and substrate-binding sites, are shorter in the 4C subfamily compared to other AKRs. Consequently, the crystal structures reveal open and accommodative substrate-binding sites, which correlates with their broad substrate specificity. It is suggested that the primary role of these enzymes may be to detoxify a range of toxic aldehydes and ketones produced during stress, although the precise nature of the principal natural substrates remains to be determined.

Analysis of the role of COP9 Signalosome (CSN) subunits in K562; the first link between CSN and autophagy.

BACKGROUND: The COP9/signalosome (CSN) is a highly conserved eight subunit complex that, by deneddylating cullins in cullin-based E3 ubiquitin ligases, regulates protein degradation. Although studied in model human cell lines such as HeLa, very little is known about the role of the CSN in haemopoietic cells. RESULTS: Greater than 95% knockdown of the non-catalytic subunit CSN2 and the deneddylating subunit CSN5 of the CSN was achieved in the human myeloid progenitor cell line K562. CSN2 knockdown led to a reduction of both CSN5 protein and mRNA whilst CSN5 knockdown had little effect on CSN2. Both knockdowns inhibited CSN deneddylase function as demonstrated by accumulation of neddylated Cul1. Furthermore, both knockdowns resulted in the sequential loss of Skp2, Cdc4 and beta-TrCP F-box proteins. These proteins were rescued by the proteasome inhibitor MG132, indicating the autocatalytic degradation of F-box proteins upon loss of CSN2 or CSN5. Interestingly, altered F-box protein gene expression was also observed in CSN2 and CSN5 knockdowns, suggesting a potential role of the CSN in regulating F-box protein transcription.Loss of either CSN subunit dramatically reduced cell growth but resulted in distinct patterns of cell death. CSN5 knockdown caused mitotic defects, G2/M arrest and apoptotic cell death. CSN2 knockdown resulted in non-apoptotic cell death associated with accumulation of both the autophagy marker LC3-II and autophagic vacuoles. Treatment of vector control K562 cells with the autophagy inhibitors 3-methyladenine and bafilomycin A1 recapitulated the growth kinetics, vacuolar morphology and LC3-II accumulation of CSN2 knockdown cells indicating that the cellular phenotype of CSN2 cells arises from autophagy inhibition. Finally, loss of CSN2 was associated with the formation of a CSN5 containing subcomplex. CONCLUSION: We conclude that CSN2 is required for CSN integrity and the stability of individual CSN subunits, and postulate that CSN2 loss results in a phenotype distinct from that of cells lacking CSN5 possibly as a consequence of altered CSN5 activity within a resultant CSN subcomplex. Our data present the first evidence for the sequential loss of F-box proteins upon CSN manipulation and are the first to identify a potential link between CSN function and autophagy.

Metabolomic profiling of drug responses in acute myeloid leukaemia cell lines.

Combined bezafibrate (BEZ) and medroxyprogesterone acetate (MPA) exert unexpected antileukaemic activities against acute myeloid leukaemia (AML) and these activities are associated with the generation of reactive oxygen species (ROS) within the tumor cells. Although the generation of ROS by these drugs is supported by preceding studies including our own, the interrelationship between the cellular effects of the drugs and ROS generation is not well understood. Here we report the use of NMR metabolomic profiling to further study the effect of BEZ and MPA on three AML cell lines and to shed light on the underlying mechanism of action. For this we focused on drug effects induced during the initial 24 hours of treatment prior to the onset of overt cellular responses and examined these in the context of basal differences in metabolic profiles between the cell lines. Despite their ultimately profound cellular effects, the early changes in metabolic profiles engendered by these drugs were less pronounced than the constitutive metabolic differences between cell types. Nonetheless, drug treatments engendered common metabolic changes, most markedly in the response to the combination of BEZ and MPA. These responses included changes to TCA cycle intermediates consistent with recently identified chemical actions of ROS. Notable amongst these was the conversion of alpha-ketoglutarate to succinate which was recapitulated by the treatment of cell extracts with exogenous hydrogen peroxide. These findings indicate that the actions of combined BEZ and MPA against AML cells are indeed mediated downstream of the generation of ROS rather than some hitherto unsuspected mechanism. Moreover, our findings demonstrate that metabolite profiles represent highly sensitive markers for genomic differences between cells and their responses to external stimuli. This opens new perspectives to use metabolic profiling as a tool to study the rational redeployment of drugs in new disease settings.

Optimized metabolite extraction from blood serum for 1H nuclear magnetic resonance spectroscopy.

Blood serum is commonly used for clinical diagnostics because its protein composition bears a wealth of information about the health of an organism. More recently the analysis of the small molecule composition, the metabolome, has received increased attention because the metabolite composition is influenced by many diseases, by the administration of drugs and toxins, and by the diet and life style of an individual. When nuclear magnetic resonance spectroscopy is used as an analytical tool it is often preferable to remove catalytically active proteins, in particular for longer measurements, because metabolite concentrations are otherwise in constant flux. Here we have compared different protocols for the separation of proteins and metabolites, including precipitation methods and ultrafiltration. Whereas most extraction methods involving protein precipitation deplete some metabolites, ultrafiltration is superior in retaining metabolite concentrations and offers excellent reproducibility. We also describe a new method to recover the hydrophobic fraction for ultrafiltration with good reproducibility.

The serotonin transporter (SLC6A4) is present in B-cell clones of diverse malignant origin: probing a potential anti-tumor target for psychotropics.

Following our previous description of the serotonin transporter (SERT) acting as a conduit to 5-hydroxytryptamine (5-HT)-mediated apoptosis, specifically in Burkitt's lymphoma, we now detail its expression among a broad spectrum of B cell malignancy, while exploring additional SERT substrates for potential therapeutic activity. SERT was readily detected in derived B cell lines with origins as diverse as B cell precursor acute lymphoblastic leukemia, mantle cell lymphoma, diffuse large B cell lymphoma, and multiple myeloma. Concentration and timecourse kinetics for the antiproliferative and proapoptotic activities of the amphetamine derivatives fenfluramine (an appetite suppressant) and 3,4-methylenedioxymethamphetamine (MDMA; "Ecstasy") revealed them as being similar to the endogenous indoleamine. A tricyclic antidepressant, clomipramine, instead mirrored the behavior of the selective serotonin reuptake inhibitor fluoxetine, both being effective in the low micromolar range. A majority of neoplastic clones were sensitive to one or more of the serotonergic compounds. Dysregulated bcl-2 expression, either by t(14;18)(q32;q21) translocation or its introduction as a constitutively active transgene, provided protection from proapoptotic but not antiproliferative outcomes. These data indicate a potential for SERT as a novel anti-tumor target for amphetamine analogs, while evidence is presented that the seemingly more promising antidepressants are likely impacting malignant B cells independently of the transporter itself.