Cyclin D1 targets hexokinase 2 to control aerobic glycolysis in myeloma cells.

Cancer cells are characterized by the Warburg effect, a shift from mitochondrial respiration to oxidative glycolysis. We report here the crucial role of cyclin D1 in promoting this effect in a cyclin-dependent kinase (CDK)4/6-independent manner in multiple myeloma (MM) cells. We show that the cyclin D1 oncoprotein targets hexokinase 2 (HK2), a major glycolysis regulator, through two original molecular mechanisms in the cytoplasmic and nuclear compartments. In the cytoplasm, cyclin D1 binds HK2 at the outer mitochondrial membrane, and in the nucleus, it binds hypoxia-inducible factor-1α (HIF1α), which regulates HK2 gene transcription. We also show that high levels of HK2 expression are correlated with shorter event-free survival (EFS) and overall survival (OS) in MM patients. HK2 may therefore be considered as a possible target for antimyeloma therapy.

The specific metabolome profiling of patients infected by SARS-COV-2 supports the key role of tryptophan-nicotinamide pathway and cytosine metabolism.

The biological mechanisms involved in SARS-CoV-2 infection are only partially understood. Thus we explored the plasma metabolome of patients infected with SARS-CoV-2 to search for diagnostic and/or prognostic biomarkers and to improve the knowledge of metabolic disturbance in this infection. We analyzed the plasma metabolome of 55 patients infected with SARS-CoV-2 and 45 controls by LC-HRMS at the time of viral diagnosis (D0). We first evaluated the ability to predict the diagnosis from the metabotype at D0 in an independent population. Next, we assessed the feasibility of predicting the disease evolution at the 7th and 15th day. Plasma metabolome allowed us to generate a discriminant multivariate model to predict the diagnosis of SARS-CoV-2 in an independent population (accuracy > 74%, sensitivity, specificity > 75%). We identified the role of the cytosine and tryptophan-nicotinamide pathways in this discrimination. However, metabolomic exploration modestly explained the disease evolution. Here, we present the first metabolomic study in SARS-CoV-2 patients which showed a high reliable prediction of early diagnosis. We have highlighted the role of the tryptophan-nicotinamide pathway clearly linked to inflammatory signals and microbiota, and the involvement of cytosine, previously described as a coordinator of cell metabolism in SARS-CoV-2. These findings could open new therapeutic perspectives as indirect targets.

[Iron deficiency anaemia: clinical presentation, biological diagnosis and management]. / Anémies ferriprives: signes d'appel, diagnostic et prise en charge.

The iron deficiency is the first cause of anaemia. In healthy young adult, anemia is well tolerated because of its progressive installation. The most common symptoms of anemia are pallor, fatigue and dyspnea. In biological exams, anemia is classically associated with microcytosis and hypochromia. The origins of microcytic anemia are iron deficiency, inflammatory aetiologies, thalassemia and sideroblastic anaemia. The iron-deficiency diagnosis includes two explorations: biological and clinical. The biological exploration is based on interpretation of serum biologics tests as blood iron, ferritin, transferrin with saturation, total iron-binding capacity and its soluble receptors. This interpretation is simple if it is not associated with clinical disorders influencing the internal iron cycle. The clinical exploration must always be followed by a careful assessment of the underlying cause as blood loss. The most common causes in women of reproductive age are gynaecologic. In men and menopausal women, the gastrointestinal tract bleeding is source of anemia. Therapeutic management of anemia is oral iron therapy. Etiological diagnostic of microcytosis is essential before iron therapy. If not, the treatment could be inefficient or it could mask or delay the etiological diagnostic.

High mTORC1 activity drives glycolysis addiction and sensitivity to G6PD inhibition in acute myeloid leukemia cells.

Alterations in metabolic activities are cancer hallmarks that offer a wide range of new therapeutic opportunities. Here we decipher the interplay between mTORC1 activity and glucose metabolism in acute myeloid leukemia (AML). We show that mTORC1 signaling that is constantly overactivated in AML cells promotes glycolysis and leads to glucose addiction. The level of mTORC1 activity determines the sensitivity of AML cells to glycolysis inhibition as switch-off mTORC1 activity leads to glucose-independent cell survival that is sustained by an increase in mitochondrial oxidative phosphorylation. Metabolic analysis identified the pentose phosphate pathway (PPP) as an important pro-survival pathway for glucose metabolism in AML cells with high mTORC1 activity and provided a clear rational for targeting glucose-6-phosphate dehydrogenase (G6PD) in AML. Indeed, our analysis of the cancer genome atlas AML database pinpointed G6PD as a new biomarker in AML, as its overexpression correlated with an adverse prognosis in this cohort. Targeting the PPP using the G6PD inhibitor 6-aminonicotinamide induces in vitro and in vivo cytotoxicity against AML cells and synergistically sensitizes leukemic cells to chemotherapy. Our results demonstrate that high mTORC1 activity creates a specific vulnerability to G6PD inhibition that may work as a new AML therapy.

Persistent decrease in proliferative potential of marrow CD34(+)cells exposed to early-acting growth factors after autologous bone marrow transplantation.

Post-graft hematopoiesis is characterized by long-term quantitative deficiency in marrow progenitor cells in both autologous and allogenic settings. In order to evaluate the function of post-graft progenitor cells, the proliferative capacity of marrow CD34(+) cells was evaluated in 10 patients 6 months after autologous bone marrow transplantation (ABMT) for non-Hodgkin's lymphoma and compared to that of 10 patients before ABMT and 10 normal controls. Immuno-selected CD34(+) cells were cultured for 7 days in liquid serum-free medium with a combination of early-acting GF consisting of stem cell factor, IL-3 and IL-1beta. Clonogenic efficiency of unselected cells for CFU-GM and BFU-E was decreased in post-graft patients compared to pre-graft and control patients. However, clonogenic efficiency of selected CD34(+) cells for CFU-GM was not different in post-graft, pre-graft and control patients but BFU-E values of post-graft patients remained lower than those of control patients. Decreased percentages of CD34(+) CD38(-) cells were observed in both post-graft and pre-graft patients while those of CD34(+) c-kit(+) cells were similar in all three patient groups. After 7-day liquid culture, expansion yields of total progenitor cells were significantly lower in post-graft patients (147 +/- 28%) than in pre-graft (255 +/- 27%) and control patients (246 +/- 23%). Post-graft deficiency in progenitor cell expansion was particularly marked for BFU-E (61 +/- 24%) compared to pre-graft patients (220 +/- 82%) and to controls (349 +/- 82%). These results indicate impaired proliferative potential of marrow CD34(+) cells several months after ABMT involving erythroid progenitor cells and/or commitment towards erythroid lineage from a more immature stage (pre-CFU).

Proliferation of human progenitor cells in a long-term culture system is more efficiently sustained by the addition of Flt-3 ligand or megakaryocyte growth and development factor than by Kit ligand.

INTRODUCTION: We compared the effects of the early-acting growth factors (GF), Flt-3 ligand (FL), c-Kit ligand (KL), and leukemia inhibitory factor (LIF), and the late-acting GF, granulocyte-colony stimulating factor (G-CSF) and megakaryocyte growth and development factor (MGDF), added alone in human long-term marrow culture (LTMC). MATERIALS AND METHODS: The GF were used in primary cultures of mononuclear cells (MNC) and in cocultures of CD34+ cells on murine preestablished MS-5 stromal layers. GF activity was assessed as nonadherent and adherent progenitor cell production and cobblestone area formation at week 5. RESULTS: In this system, only FL, KL, and MGDF significantly stimulated early stages of hematopoiesis, whereas only G-CSF stimulated the proliferation of mature progenitor cells within the granulo-monocyte lineage and no effect was observed with LIF. FL displayed the strongest activity, and MGDF was more efficient than KL, both in primary cultures of MNC and in cocultures of CD34+ cells. However, the stimulatory effects of these GF used alone were dependent on the presence of a stromal layer. CONCLUSION: These LTMC data emphasize the particular roles for FL and MGDF in the stimulation of primitive hematopoiesis.

Changes in the functional capacity of marrow stromal cells after autologous bone marrow transplantation.

Marrow stromal cells were evaluated several months after autologous BMT for their capacity to support both normal hemopoiesis and secrete the main growth factors involved in its control, G-CSF, GM-CSF, IL-3 and SCF. Stromal layers (SL) were obtained by long-term marrow cultures (LTMC) established from 15 patients (9 with hematologic malignancies and 6 with solid tumors) 3 months after autologous BMT and were compared to pre-graft patients. After irradiation, both post-graft and pre-graft SL were recharged with the same inoculum of normal marrow cells. As compared to pre-graft values, CFU-GM production on post-graft SL was significantly increased during the first 2 weeks of culture whereas it was decreased from week 3 to week 8. These findings were only observed in patients with hematologic malignancies and not in patients with solid tumors. Growth factor secretion was evaluated by ELISA in the supernatants of unstimulated and IL-1-stimulated SL from 10 post-graft patients, 13 pre-graft patients and 5 normal controls. In any group of patients, IL-3 was undetectable either spontaneously or after IL-1-stimulation. As compared to controls, secretion by IL-1-stimulated SL was not different for GM-CSF in pre- and post-graft patients but tended to be decreased for G-CSF in post-graft patients. SCF secretion, which was not induced by IL-1, appeared dramatically decreased in both pre- and post-graft patients. The capacity of post-graft SL to support CFU-GM growth in LTMC was correlated at week 1 with G-CSF secretion and from week 3 to week 8 with SCF secretion. These results suggest that microenvironment remains qualitatively damaged several months after BMT involving a decreased capacity both to support early hemopoiesis and to secrete SCF, particularly in patients grafted for hemopoietic malignancies.

Fate of inhaled monoclonal antibodies after the deposition of aerosolized particles in the respiratory system.

Monoclonal antibodies (mAbs) are usually delivered systemically, but only a small proportion of the drug reaches the lung after intravenous injection. The inhalation route is an attractive alternative for the local delivery of mAbs to treat lung diseases, potentially improving tissue concentration and exposure to the drug while limiting passage into the bloodstream and adverse effects. Several studies have shown that the delivery of mAbs or mAb-derived biopharmaceuticals via the airways is feasible and efficient, but little is known about the fate of inhaled mAbs after the deposition of aerosolized particles in the respiratory system. We used cetuximab, an anti-EGFR antibody, as our study model and showed that, after its delivery via the airways, this mAb accumulated rapidly in normal and cancerous tissues in the lung, at concentrations twice those achieved after intravenous delivery, for early time points. The spatial distribution of cetuximab within the tumor was heterogeneous, as reported after i.v. injection. Pharmacokinetic (PK) analyses were carried out in both mice and macaques and showed aerosolized cetuximab bioavailability to be lower and elimination times shorter in macaques than in mice. Using transgenic mice, we showed that FcRn, a key receptor involved in mAb distribution and PK, was likely to make a greater contribution to cetuximab recycling than to the transcytosis of this mAb in the airways. Our results indicate that the inhalation route is potentially useful for the treatment of both acute and chronic lung diseases, to boost and ensure the sustained accumulation of mAbs within the lungs, while limiting their passage into the bloodstream.

The dual mTORC1 and mTORC2 inhibitor AZD8055 has anti-tumor activity in acute myeloid leukemia.

The serine/threonine kinase mammalian target of rapamycin (mTOR) is crucial for cell growth and proliferation, and is constitutively activated in primary acute myeloid leukemia (AML) cells, therefore representing a major target for drug development in this disease. We show here that the specific mTOR kinase inhibitor AZD8055 blocked mTORC1 and mTORC2 signaling in AML. Particularly, AZD8055 fully inhibited multisite eIF4E-binding protein 1 phosphorylation, subsequently blocking protein translation, which was in contrast to the effects of rapamycin. In addition, the mTORC1-dependent PI3K/Akt feedback activation was fully abrogated in AZD8055-treated AML cells. Significantly, AZD8055 decreased AML blast cell proliferation and cell cycle progression, reduced the clonogenic growth of leukemic progenitors and induced caspase-dependent apoptosis in leukemic cells but not in normal immature CD34+ cells. Interestingly, AZD8055 strongly induced autophagy, which may be either protective or cell death inducing, depending on concentration. Finally, AZD8055 markedly increased the survival of AML transplanted mice through a significant reduction of tumor growth, without apparent toxicity. Our current results strongly suggest that AZD8055 should be tested in AML patients in clinical trials.

In vitro effect of stem cell factor on human clonogenic marrow progenitors after myeloablative treatments.

Abnormal hematopoiesis, including a deficiency of marrow progenitors and particularly of erythroid progenitors, has been described after autologous stem cell transplantation (ASCT), persisting for several years. In order to explain this deficiency, a resistance of marrow progenitors to stem cell factor (SCF) after ASCT was investigated. Marrow samples were harvested from pregraft patients at graft collection prior to ASCT, transplanted patients 6-24 months after high-dose therapy and control patients. CD34+ cells were cultured in a serum-free clonogenic assay with increasing doses of SCF. The clonogenic efficiency without SCF was lower for BFU-E in treated groups than in controls, whereas it was not different for CFU-GM. With increasing doses of SCF a dose-dependent effect was found on the numbers of both CFU-GM and BFU-E in all groups, although the maximal number of BFU-E remained lower in treated groups. However, the SCF dose that induced 50% of maximal BFU-E growth (D50) was similar in all groups. Furthermore, a dose-dependent effect on the size of BFU-E was found in all groups, with no difference in the proportion of large colonies. Thus, clonogenic erythroid progenitors from patients who have received myelotoxic treatments remain sensitive to SCF, with no evidence for a chemotherapy-related resistance.

Induction of monocyte tissue factor expression by antibodies to heparin-platelet factor 4 complexes developed in heparin-induced thrombocytopenia.

The pathogenesis of thrombosis in heparin-induced thrombocytopenia (HIT) was studied by investigating whether antibodies to heparin-platelet factor 4 (H-PF4) induced tissue factor (TF) synthesis by monocytes. Plasma from 5 patients with HIT containing IgG to H-PF4 was incubated with peripheral blood mononuclear cells without or with purified PF4 and heparin. Significant TF-dependent procoagulant activity (PCA) expressed by monocytes, measured with a factor Xa-based chromogenic assay, was induced after incubation of each HIT plasma sample. This monocyte PCA required the presence of PF4 and was inhibited by high concentrations of heparin. Furthermore, purified HIT IgG added to whole blood with PF4 and heparin also provoked significant synthesis of TF mRNA by monocytes, demonstrated by RT-PCR, and this effect was not observed with normal IgG. These findings strongly support the hypothesis that antibodies to PF4 developed in HIT trigger the production of tissue factor by monocytes, and this effect could account in vivo for hypercoagulability and thrombotic complications in affected patients.

All-trans-retinoic acid up-regulates CD38 but not c-Kit antigens on human marrow CD34+ cells without recruitment into cell cycle.

Retinoids, especially all-trans-retinoic acid (ATRA), are well known for their differentiating activity on HL-60 cells. Moreover ATRA induces CD38 antigen overexpression on these cells. In this study we examined the effects of ATRA on purified normal CD34+ cells from adult human marrows incubated with ATRA (1 microM) or stem cell factor (SCF) after 7 d liquid cultures in serum-deprived medium. Before and after the incubation, CD34+ cells were studied by flow cytometry to evaluate the cell-surface expression of CD38 and c-Kit antigens and the cycle status of these cells using high-resolution analysis (DNA content v Ki-67 antigen expression) to clarify the functional meaning of antigenic variations. When compared with control cultures, ATRA-treated cells displayed changes in their immunophenotypic profile. Particularly relevant was the up-regulation of CD38 antigen with a mean (+/-SEM) fold increase of 21 +/- 0.1 (P=0.028) for geometric mean fluorescence intensity (GMFI), without modulation of c-Kit expression. SCF only down-regulated expression of c-Kit with a fold decrease of 4.6 +/- 0.9 for GMFI (P=0.043). Unlike SCF, ATRA did not induce CD34+ cells to entry into cell cycle despite increased levels of surface CD38 antigen. Moreover morphological and functional assays did not argue for an ATRA-induced maturation process. Contrary to steady-state cells, CD34+ cells treated with pharmacological doses of ATRA alone displayed CD38 over-expression without change in c-Kit levels and cycle status, suggesting an absence of maturation pressure.

A rapid single-laser flow cytometric method for discrimination of early apoptotic cells in a heterogenous cell population.

A recently reported cytometric method described the possibility of discriminating apoptotic from necrotic cells using FITC-labelled annexin V and propidium iodide (PI). Nevertheless, the brightness of PI-staining and its extensive spectral emission overlap with phycoerythrin (PE) does not permit the study of a subset of a heterogenous cell population with single laser instrumentation. The surface staining of a subset with PE in a heterogenous cell population therefore requires another exclusion dye to detect necrotic cells. We used 7-amino-actinomycin D (7-AAD) that can be excited by the 488 nm argon laser line. 7-AAD emits in the far red range of the spectrum and 7-AAD spectral emission can be separated from the emissions of FITC and PE. The fluorescence parameters allow characterization of necrotic (7-AAD+ annexin V-FITC+ cells), apoptotic (7-AAD-annexin V-FITC+ cells) and viable cells (7-AAD- annexin V-FITC- cells) in a subset of PE+ cells. The value of this method was demonstrated by measuring apoptosis and necrosis in a model of HL-60 cells exposed to different inducers of cell death. The method was validated by fluorescent cell sorting in combination with morphologic examination of the sorted cells. The technique we present is particularly valuable in a clinical setting because it enables rapid multiparameter analysis of necrosis and early apoptosis in combination with cell surface phenotyping with a single laser. We present the effects of haemopoietic growth factor deprivation on myeloid progenitor CD34+ cells as an example of its application.

Evaluation of performance of white blood cell reduction filters: an original flow cytometric method for detection and quantification of cell-derived membrane fragments.

BACKGROUND: Contamination of blood products by white blood cells leads to a risk of transmission of infectious agents, particularly abnormal prion protein, the probable causative agent of new-variant Creutzfeldt-Jakob disease. Blood product filtration could reduce this risk, but the filtration systems might generate potentially infectious membrane fragments. We developed an original flow cytometric method that allows the detection and quantification of membrane fragments in filtered products and the evaluation of the quantity of destroyed cells. METHODS: This method has four technical requirements: cytofluorometric acquisition of forward scatter parameters on a log scale, use of a fluorescent aliphatic reporter molecule (PKH26-GL) to identify membrane fragments, quantification with fluorescent beads, and the drawing up of a standard curve on the basis of cells destroyed by freezing/thawing to generate cell debris (i.e., quantity of membrane fragments measured versus quantity of destroyed cells). RESULTS AND CONCLUSIONS: This original method can be used to test new filtration devices and it allows optimization of the filtration process or comparison of different filtration systems. We tested the method with three commercial white cell removal filters. We demonstrated that it is possible to evaluate the filter quality, particularly the likelihood of fragment removal during the filtration process.