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Purpose: The growth and survival of acute myeloid leukemia (AML) cells are enhanced by the deregulation of signaling pathways such as phosphoinositide 3-kinase (PI3K)/Akt and mammalian target of rapamycin (mTOR). Major efforts have thus been made to develop molecules targeting these activated pathways. The mTOR serine/threonine kinase belongs to two separate complexes: mTORC1 and mTORC2. The mTORC1 pathway is rapamycin sensitive and controls protein translation through the phosphorylation of 4E-BP1 in most models. In AML, however, the translation process is deregulated and rapamycin resistant. Furthermore, the activity of PI3K/Akt and mTOR is closely related, as mTORC2 activates the oncogenic kinase Akt. We therefore tested, in this study, the antileukemic activity of the dual PI3K/mTOR ATP-competitive inhibitor NVP-BEZ235 compound (Novartis).Experimental Design: The activity of NVP-BEZ235 was tested in primary AML samples (n = 21) and human leukemic cell lines. The different signaling pathways were analyzed by Western blotting. The cap-dependent mRNA translation was studied by 7-methyl-GTP pull-down experiments, polysomal analysis, and [3H]leucine incorporation assays. The antileukemic activity of NVP-BEZ235 was tested by analyzing its effects on leukemic progenitor clonogenicity, blast cell proliferation, and survival.Results: The NVP-BEZ235 compound was found to inhibit PI3K and mTORC1 signaling and also mTORC2 activity. Furthermore, NVP-BEZ235 fully inhibits the rapamycin-resistant phosphorylation of 4E-BP1, resulting in a marked inhibition of protein translation in AML cells. Hence, NVP-BEZ235 reduces the proliferation rate and induces an important apoptotic response in AML cells without affecting normal CD34+ survival.Conclusions: Our results clearly show the antileukemic efficiency of the NVP-BEZ235 compound, which therefore represents a promising option for future AML therapies. Clin Cancer Res; 16(22); 5424–35. ©2010 AACR.

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Background In ineffective erythropoiesis, hepcidin synthesis is suppressed by erythroid regulators, namely erythroferrone and growth differentiation factor-15. For the first time, the hypothesis that iron overload in megaloblastic anemia may be related to ineffective erythropoiesis is explored by describing the kinetics of hepcidin, erythroferrone, and growth differentiation factor-15 levels in a patient diagnosed with megaloblastic anemia associated with iron overload. Case presentation An 81-year-old Caucasian male was admitted for fatigue. He had type-2 diabetes previously treated with metformin, ischemic cardiac insufficiency, and stage-3 chronic kidney disease. Vitiligo was observed on both hands. Biological tests revealed normocytic non-regenerative anemia associated with hemolysis, thrombocytopenia, and elevated sideremia, ferritin, and transferrin saturation levels. Megaloblastic anemia was confirmed with undetectable blood vitamin B12 and typical cytological findings like hyper-segmented neutrophils in blood and megaloblasts in bone marrow. The patient received vitamin B12 supplementation. At 3 months, biological parameters reached normal values. Hepcidin kinetics from diagnosis to 3 months inversely correlated with those of erythroferrone and growth differentiation factor-15. Conclusions This case suggests that iron-overload mechanisms of dyserythropoietic anemias may apply to megaloblastic anemias.

Background Chronic myelomonocytic leukemia is a myelodysplastic/myeloproliferative neoplasm characterized by the infiltration of blood and bone marrow by immature monocytes. Cerebral localization of chronic myelomonocytic leukemia has never been described. Case presentation We report the case of a Caucasian 59 year-old man with multiorgan chronic myelomonocytic leukemia infiltration, associated with uncommon brain involvement. There was no evidence of evolution to acute myeloid leukemia. The evidence of cerebral infiltration by chronic myelomonocytic leukemia was made after autopsy. Conclusions The fatal outcome of the patient raises the question of the potential benefit of early specific treatment, such as demethylating agents or intensive chemotherapy. Sharing such images of atypical and rapidly evolving chronic myelomonocytic leukemia and the disease history may help clinical decision-making in the future.

International audience; DNA methylation, a major biological process regulating the transcription, contributes to the pathophysiology of hematologic malignancies, and hypomethylating agents are commonly used to treat myelodysplastic syndromes (MDS) and acute myeloid leukemias (AML). In these diseases, bone marrow mesenchymal stromal cells (MSCs) play a key supportive role through the production of various signals and interactions. The DNA methylation status of MSCs, likely to reflect their functionality, might be relevant to understand their contribution to the pathophysiology of these diseases. Consequently, the aim of our study was to analyze the modifications of DNA methylation profiles of MSCs induced by MDS or AML. MSCs from MDS/AML patients were characterized via 5-methylcytosine quantification, gene expression profiles of key regulators of DNA methylation, identification of differentially methylated regions (DMRs) by methylome array, and quantification of DMR-coupled genes expression. MDS and AML-MSCs displayed global hypomethylation and under-expression of DNMT1 and UHRF1. Methylome analysis revealed aberrant methylation profiles in all MDS and in a subgroup of AML-MSCs. This aberrant methylation was preferentially found in the sequence of homeobox genes, especially from the HOX family (HOXA1, HOXA4, HOXA5, HOXA9, HOXA10, HOXA11, HOXB5, HOXC4, and HOXC6), and impacted on their expression. These results highlight modifications of DNA methylation in MDS/AML-MSCs, both at global and focal levels dysregulating the expression of HOX genes well known for their involvement in leukemogenesis. Such DNA methylation in MSCs could be the consequence of the malignant disease or could participate in its development through defective functionality or exosomal transfer of HOX transcription factors from MSCs to hematopoietic cells.

International audience; No abstract available

Background Shulman’s disease, or eosinophilic fasciitis (EF), is a rare autoimmune disease, characterized by sclerodermic skin lesions with progressive induration and thickening of the soft tissues. Chronic graft-versus-host-disease (GVHD) presenting as EF is a very rare manifestation of cutaneous GVHD. Case presentation We report an unusual case of EF in a 46-year-old Caucasian male patient who had received an allogenic hematopoietic stem cell transplantation in the context of relapsed/refractory multiple myeloma. The diagnosis was challenging, with the patient presenting hepatic dysfunction, normal eosinophils count, and incomplete clinical signs. Magnetic resonance imaging (MRI) and skin biopsy confirmed the diagnosis of EF. Early initiation of specific treatment with corticosteroids and prednisolone achieved complete response. Conclusion In practice, incomplete signs in this rare complication should lead to MRI as it is a major tool to guide decision-making based on the skin biopsy, allowing a rapid diagnosis and the initiation of treatment without delay.

Acute myeloid leukemia (AML) is characterized by the accumulation of undifferentiated blast cells in the bone marrow and blood. In most cases of AML, relapse frequently occurs due to resistance to chemotherapy. Compelling research results indicate that drug resistance in cancer cells is highly dependent on the intracellular levels of reactive oxygen species (ROS). Modulating ROS levels is therefore a valuable strategy to overcome the chemotherapy resistance of leukemic cells. In this study, we evaluated the efficiency of diphenyleneiodonium (DPI)—a well-known inhibitor of ROS production—in targeting AML cells. Results showed that although inhibiting cytoplasmic ROS production, DPI also triggered an increase in the mitochondrial ROS levels, caused by the disruption of the mitochondrial respiratory chain. We also demonstrated that DPI blocks mitochondrial oxidative phosphorylation (OxPhos) in a dose-dependent manner, and that AML cells with high OxPhos status are highly sensitive to treatment with DPI, which synergizes with the chemotherapeutic agent cytarabine (Ara-C). Thus, our results suggest that targeting mitochondrial function with DPI might be exploited to target AML cells with high OxPhos status.

Low reactive oxygen species (ROS) levels are well-established characteristics of mouse hematopoietic stem cells (HSCs). However, little is known about these levels in human HSCs. This study aimed at quantifying ROS levels in human CD34+ CD38low and CD34+ CD38high human progenitors from bone marrow, cord blood and cells mobilized for autologous HSC transplantation. A specifically devised multiparameter flow cytometry method was used to quantify ROS levels (H2 DCFDA staining) in sub-populations of primary cells. Results were confirmed by assessing gene expression level of the ROS scavenger GPX3, a key determinant of HSC self-renewal, in sorted CD34+ CD38low and CD34+ CD38high cells. CD34+ CD38low cells from bone marrow and cord blood displayed significantly lower levels of ROS than CD34+ CD38high cells and other leukocytes. Moreover, the correlation between ROS and GPX3 expression was verified in sorted CD34+ CD38low and CD34+ CD38high cells. These results confirm, in human, data previously reported in mice. Moreover, the flow cytometry assay we developed could allow for a more precise enumeration of repopulating primitive progenitors in the course of HSC transplantation.

The prognostic significance of hypercalcemia in lymphoma has only been studied on small series to date. We conducted a retrospective, monocentric, matched-control study that aimed to compare the outcome of patients diagnosed with any histological subtype of lymphoma associated with hypercalcemia, at diagnosis or relapse, with a group of controls matched for histological and prognostic factors. Sixty-two and 118 comparable patients treated between 2000 and 2016 were included in hypercalcemia and control cohorts, respectively. Hypercalcemia was found mainly at diagnosis (71%) in higher-risk patients (prognosis scores ≥ 3, 76%) and those with diffuse large B cell lymphoma (67.7%), stage III/IV disease (91.9%), and elevated LDH (90.3%). Two-year progression-free survival (PFS) was shorter in the hypercalcemia than control cohort [30.1% (95% confidence interval (95% CI) 18.3–41.9) vs 63.9% (95% CI 5.1–72.7), p

International audience; Influenza virus infection causes considerable morbidity and mortality, but current therapies have limited efficacy. We hypothesized that investigating the metabolic signaling during infection may help to design innovative antiviral approaches. Using bronchoalveolar lavages of infected mice, we here demonstrate that influenza virus induces a major reprogramming of lung metabolism. We focused on mitochondria-derived succinate that accumulated both in the respiratory fluids of virus-challenged mice and of patients with influenza pneumonia. Notably, succinate displays a potent antiviral activity in vitro as it inhibits the multiplication of influenza A/H1N1 and A/H3N2 strains and strongly decreases virus-triggered metabolic perturbations and inflammatory responses. Moreover, mice receiving succinate intranasally showed reduced viral loads in lungs and increased survival compared to control animals. The antiviral mechanism involves a succinate-dependent posttranslational modification, that is, succinylation, of the viral nucleoprotein at the highly conserved K87 residue. Succinylation of viral nucleoprotein altered its electrostatic interactions with viral RNA and further impaired the trafficking of viral ribonucleoprotein complexes. The finding that succinate efficiently disrupts the influenza replication cycle opens up new avenues for improved treatment of influenza pneumonia.

Simple Summary Pesticide exposure is a public health concern. Two recent studies reported that exposure of normal primary bone marrow mesenchymal stem/stromal cells (BM-MSCs) to low-dose pesticide mixture induces deleterious consequences, such as oxidative stress, senescence, accelerated aging and increased sensitivity to oncogenic hits. Here, we show that exposure to this mixture decreases aldehyde dehydrogenase-2 (ALDH2) activity, with a concomitant increase in acetaldehyde level and DNA damage and alters the MSC capacity to support primitive hematopoiesis. Similar abnormalities were observed in bone marrow-MSCs from patients suffering from myelodysplastic syndrome (MDS), suggesting a role of pesticide-induced ALDH2 inhibition in the pathophysiology of this pre-leukemic disease. Abstract (1) Background: The impact of occupational exposure to high doses of pesticides on hematologic disorders is widely studied. Yet, lifelong exposure to low doses of pesticides, and more particularly their cocktail effect, although poorly known, could also participate to the development of such hematological diseases as myelodysplastic syndrome (MDS) in elderly patients. (2) Methods: In this study, a cocktail of seven pesticides frequently present in water and food (maneb, mancozeb, iprodione, imazalil, chlorpyrifos ethyl, diazinon and dimethoate), as determined by the European Food Safety Authority, were selected. Their in vitro effects at low-doses on primary BM-MSCs from healthy volunteers were examined. (3) Results: Exposure of normal BM-MSCs to pesticides for 21 days inhibited cell proliferation and promoted DNA damage and senescence. Concomitantly, these cells presented a decrease in aldehyde dehydrogenase 2 (ALDH2: mRNA, protein and enzymatic activity) and an increase in acetaldehyde levels. Pharmacological inhibition of ALDH2 with disulfiram recapitulated the alterations induced by exposure to low doses of pesticides. Moreover, BM-MSCs capacity to support primitive hematopoiesis was significantly altered. Similar biological abnormalities were found in primary BM-MSCs derived from MDS patients. (4) Conclusions: these results suggest that ALDH2 could participate in the pathophysiology of MDS in elderly people long exposed to low doses of pesticides.

Introduction . In multiple myeloma (MM), the presence of translocation t(4;14) and/or 17p deletion, found in around 10 to 15% of the patients, is considered as high-risk feature associated with adverse survival. Despite recent advances in the treatment of MM, t(4;14) and del(17p) are still associated with poor outcome. The aim of this study was to analyze the trends of survival in patients with newly diagnosed MM harboring t(4;14) and or 17p deletion over the past two decades. Methods . Patients from five French centers with newly-diagnosed MM from 2001 to 2019 and displaying del(17p) and/or t(4;14) were retrospectively included. Cytogenetics abnormalities were detected by interphase fluorescence in situ hybridization (FISH) and del(17p) positivity cut-off was 30%. New agents were defined as: pomalidomide, carfilzomib, ixazomib and anti-CD38 monoclonal antibodies. Results . 246 patients carrying either t(4;14) (n=106 patients), del(17p) (n=121 patients) or both (n=19 patients) were included. Median age was 64 years (range, 35-91). ISS and R-ISS score were 3 in 88 patients (36%). Eighty-seven patients (35%) were diagnosed in 2001-2010 period, and 159 (65%) in 2011-2019 period. Front-line autologous stem-cell transplantation (ASCT) was performed in 121 (49%) patients. Among transplant eligible patients, 112 patients (n=93%) received triplet induction, 78 patients (64%) a consolidation regimen and 15 patients (12%) a maintenance therapy. Double-ASCT was decided in 21 patients (17%). Among transplant ineligible patients, 61 patients (49%) received melphalan-based regimen in first line, 36 (29%) a bortezomib-based and 15 patients (14%) a lenalidomide-based regimen. At any line, 92 patients (37%) received at least one of the new agents with only 12 patients (5%) in frontline therapy. Median follow-up was 41 months (IQR: 21-69). Median overall survival (OS) was 58.4 months (IQR: 50.1-66.5) for the entire cohort, 55.5 months (IQR: 46.6-78.3) for del(17p), 62.5 months (IQR: 54.2-76.1) for t(4;14) and 48.6 months (18.1-not reached) for patients with both (p=0.2). Median of first progression-free survival (PFS1) was 25.6 months (IQR: 22.2-29.8), with no difference between del(17p), t(4;14) or both (p=0.57). Importantly, no improvement of median OS was observed in patients diagnosed between 2001-2010 in comparison with patients diagnosed between 2011-2019 (63.7 versus 53.2 months, p=0.32). In univariate and multivariate analysis: age (continuous and cut-off 71 years-old) and ASCT significantly were associated with risk of death (HR: 1.03, 1.09 and 0.45, respectively). Median OS of patients eligible to ASCT was 76.1 months (IQR: 62.5-90.3) vs 42.5 months (IQR: 36.8-54.6) for patients not eligible (HR 0.45, 95%CI 0.28-0.0.71; p Conclusion . This large multicenter real-world study confirms that patients with newly diagnosed MM carrying del(17p) and/or t(4;14) remain a therapeutic challenge with no significant overall survival improvement in the past decades despite the use of novel agents. The definition of high-risk MM patients is evolving with incorporation of new markers (i.e chromosome 1 abnormalities, PET-imaging). Minimal-residual disease achievement will also re-defined risk stratification. Nonetheless, the need for innovative approaches such as earlier strategies using new agents or immunotherapy (CAR-T cells, bispecific T-cell engager antibodies) may significantly improve outcomes. Figure captions Table 1. Clinical, biological characteristics, treatment and survival of the 246 included patients based on period of diagnosis. IQR: interquartile range; FISH: fluorescence in situ hybridization; ISS: international score system; LDH: lactate dehydrogenase; ASCT: autologous stem cell transplantation; len: lenalidomide; Poma: pomalidomide; Carfil: carfilzomib; Ixa: ixazomib; mAb: monoclonal antibodies; OS: overall survival Figure 1. Kaplan-Meier curves for overall survival for the 246 included patients based on period of diagnosis. Figure 1 Disclosures Moreau: Amgen: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Celgene/Bristol-Myers Squibb: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Novartis: Honoraria; Takeda: Honoraria. Touzeau:Takeda: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses; Sanofi: Honoraria, Research Funding; Abbvie: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Research Funding; Amgen: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses; GlaxoSmithKline: Honoraria, Research Funding.

The bone marrow (BM) niche impacts the progression of acute myeloid leukemia (AML) by favoring the chemoresistance of AML cells. Intimate interactions between leukemic cells and BM mesenchymal stromal cells (BM-MSCs) play key roles in this process. Direct intercellular communications between hematopoietic cells and BM-MSCs involve connexins, components of gap junctions. We postulated that blocking gap junction assembly could modify cell–cell interactions in the leukemic niche and consequently the chemoresistance. The comparison of BM-MSCs from AML patients and healthy donors revealed a specific profile of connexins in BM-MSCs of the leukemic niche and the effects of carbenoxolone (CBX), a gap junction disruptor, were evaluated on AML cells. CBX presents an antileukemic effect without affecting normal BM-CD34+ progenitor cells. The proapoptotic effect of CBX on AML cells is in line with the extinction of energy metabolism. CBX acts synergistically with cytarabine (Ara-C) in vitro and in vivo. Coculture experiments of AML cells with BM-MSCs revealed that CBX neutralizes the protective effect of the niche against the Ara-C-induced apoptosis of leukemic cells. Altogether, these results suggest that CBX could be of therapeutic interest to reduce the chemoresistance favored by the leukemic niche, by targeting gap junctions, without affecting normal hematopoiesis.

The coexistence of dual hematological neoplasms is very rare. Sequential or synchronous neoplasms in hematology are an uncommon and complex clinical situation. The aim of the Hemo2 study was to describe the clinical characteristics and analyze the outcome of these patients. We performed a retrospective review of all patients diagnosed with sequential or synchronous hematological malignancies in the university hospital of Tours, between 2007 and 2018. We identified 49 patients in our study, with a prevalence of 0.89%. Sequential and synchronous combinations were found in 36 (73%) and 13 (27%) patients, respectively. One patient presented three sequential neoplasms. The median cumulative incidence was 6 years (95% CI 3–7). Among all neoplasms diagnosed (n = 99), we found 79 lymphoid neoplasms (LNs) (80%) and 20 myeloid neoplasms (MNs) (20%). Sex ratio was 1.88 with 65% of males and 35% of females. The most common LNs were Hodgkin lymphoma (n = 16; 16%) and multiple myeloma (n = 11; 11%). The most frequent MN was essential thrombocythemia (n = 5; 5%). The most common combination was Hodgkin lymphoma and follicular lymphoma in five (10%) patients. The overall survival from the first diagnosis (OS1) at 5 years was 82.4% (95% CI 72.1–94.3). The median overall survival from the second diagnosis (OS2) was 98 months (95% CI 44–NR) and 5-year OS2 was 58.7% (95% CI 45.5–75.7). Median progression-free survival from the second diagnosis (PFS) was 47 months (95% CI 27–NR) with 5-year PFS of 49% (95% CI 35.9–67). OS and PFS did not statistically differ between synchronous and sequential dual neoplasms. In this cohort, that the death relative risk (RR) was significantly lower if the second neoplasm appeared after more than 4 years following the first diagnosis (OR 0.37 (95% CI 0.16–0.90)). The Hemo2study confirmed the rarity of dual hematological neoplasms. In this cohort, HL and FL were the most frequent combinations. Our results may support that synchronous and sequential dual neoplasms bear the same prognosis. Further studies are needed to better characterize these uncommon clinical situations.

The general population is chronically exposed to multiple environmental contaminants such as pesticides. We have previously demonstrated that human mesenchymal stem cells (MSCs) exposed in vitro to low doses of a mixture of seven common pesticides showed a permanent phenotype modification with a specific induction of an oxidative stress‐related senescence. Pesticide mixture also induced a shift in MSC differentiation toward adipogenesis. Thus, we hypothesized that common combination of pesticides may induce a premature cellular aging of adult MSCs. Our goal was to evaluate if the prolonged exposure to pesticide mixture could accelerate aging‐related markers and in particular deteriorate the immunosuppressive properties of MSCs. MSCs exposed to pesticide mixture, under long‐term culture and obtained from aging donor, were compared by bulk RNA sequencing analysis. Aging, senescence, and immunomodulatory markers were compared. The protein expression of cellular aging‐associated metabolic markers and immune function of MSCs were analyzed. Functional analysis of the secretome impacts on immunomodulatory properties of MSCs was realized after 21 days' exposure to pesticide mixture. The RNA sequencing analysis of MSCs exposed to pesticide showed some similarities with cells from prolonged culture, but also with the MSCs of an aged donor. Changes in the metabolic markers MDH1, GOT and SIRT3, as well as an alteration in the modulation of active T cells and modifications in cytokine production are all associated with cellular aging. A modified functional profile was found with similarities to aging process. stem cells 2019;37:1083–1094, Mesenchymal stem cells (P3) after 21 days' exposure to mixture of pesticides showed some similarities with cells from prolonged culture (P14), but also with the mesenchymal stem cells of aged donors. Modification of metabolic markers for MDH1, GOT, and SIRT3, associated with physiological aging, altered active T‐cell regulation and cytokine production (interleukin‐10, interleukin‐6, CCL2, transforming growth factor‐β) define a modified functional profile with similarities to accelerated cellular aging process.

Bone marrow mesenchymal stromal cells (MSCs) play an essential role in the regulation of normal and leukemic hematopoiesis. Their multipotent potential of differentiation also makes them an interesting therapeutic tool. Among factors involved in the regulation of MSCs, energy metabolism plays a key role in their proliferation and differentiation. Seahorse Bioscience introduced extracellular flux technology to the life sciences market in 2006. This methodology allows, in living cells and in real time, the concomitant determination of basal oxygen consumption, glycolysis rates, ATP production, and respiratory capacity in a single experiment. Here we describe the protocol used to study concomitantly the respiratory and glycolytic metabolism of primary MSCs from the determination of oxygen consumption (OCR) and extracellular acidification (ECAR) rates.

The incidence of myelodysplastic syndrome increases with aging and the early diagnosis enables optimal care of these diseases. The DxH 800 hematology analyzer measures and calculates 126 cytological parameters, but only 23 are used for routine CBC assessment. The goal of this study was to use the 103 unexploited &ldquo, research parameters&rdquo, to develop an algorithm allowing for an early detection of subclinical MDS patients by triggering morphological analysis. Blood sample parameters from 101 MDS patients and 88 healthy volunteers were analyzed to identify the critical &ldquo, with: (i) the most significant differences between MDS patients and healthy volunteers, (ii) the best contributions to principal component analysis (PCA), first axis, and (iii) the best correlations with PCA, first two axes (cos2 &gt, 0.6). Ten critical &ldquo, of white blood cells were identified, allowing for the calculation of an MDS-likelihood score (MDS-LS), based on logistic regression. Automatic calculation of the MDS-LS is easily implementable on the middleware system of the DxH 800 to generate a flag for blood smear review, and possibly early detection of MDS patients in the general population.

The bone marrow (BM) microenvironment plays a crucial role in the development and progression of leukemia (AML). Intracellular reactive oxygen species (ROS) are involved in the regulation of the biology of leukemia-initiating cells, where the antioxidant enzyme GPx-3 could be involved as a determinant of cellular self-renewal. Little is known however about the role of the microenvironment in the control of the oxidative metabolism of AML cells. In the present study, a coculture model of BM mesenchymal stromal cells (MSCs) and AML cells (KG1a cell-line and primary BM blasts) was used to explore this metabolic pathway. MSC-contact, rather than culture with MSC-conditioned medium, decreases ROS levels and inhibits the Nrf-2 pathway through overexpression of GPx3 in AML cells. The decrease of ROS levels also inactivates p38MAPK and reduces the proliferation of AML cells. Conversely, contact with AML cells modifies MSCs in that they display an increased oxidative stress and Nrf-2 activation, together with a concomitant lowered expression of GPx-3. Altogether, these experiments suggest that a reciprocal control of oxidative metabolism is initiated by direct cell&ndash, cell contact between MSCs and AML cells. GPx-3 expression appears to play a crucial role in this cross-talk and could be involved in the regulation of leukemogenesis.

Clonal hematopoiesis (CH) of indeterminate potential has been described in blood samples from large series of patients. Its prevalence and consequences are still not well understood because sequencing methods vary and because most studies were performed in cohorts comprising individuals with nonhematologic diseases. Here, we investigated the frequency of CH in 82 paired bone marrow and blood samples from carefully selected healthy adult volunteers. Forty-one genes known to be mutated in myeloid malignancies were sequenced with a 1% threshold of detection. In bone marrow samples, clones were found in almost 40% of healthy volunteers more than 50 years old. The most frequent mutations were found in DNMT3A and TET2, with 1 individual carrying 3 variants. Variant allele frequencies were highly concordant between blood and bone marrow samples. Blood parameters were normal except for those in 2 individuals: 1 had a mild macrocytosis and 1 had a mild thrombocytosis. Furthermore, no morphologic abnormalities or dysplasia were detected when bone marrow smears were carefully evaluated. Individuals with CH differed from others by age (62.8 vs 38.6 years; P < .0001) and platelet count (294 vs 241 ×109/L; P = .0208), the latter being no more significant when removing the 2 individuals who carried the JAK2 p.V617F mutation. These results confirm that CH is a very common condition in healthy adults over 50 years old. Consequently, the detection of driver myeloid mutations should be interpreted with caution in the absence of cytologic abnormalities in the blood and/or the bone marrow.

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 impact of pesticides on health is a major public health concern. A higher risk to develop chronic lymphoid malignancies has been demonstrated to be associated with occupational pesticide exposure (OPE). By contrast, little is known of the impact of OPE on the occurrence of myeloid malignancies especially acute myeloid leukemia (AML). The purpose of this meta-analysis is to summarize data on the association between OPE and AML. A relevant dataset of case–control studies was extracted. Among 6784 references extracted, 14 were selected, representing 3,955 AML patients and 9,948 control subjects diagnosed between 1976 and 2010. An adverse association was found between OPE and AML (OR = 1.51; 95%CI: 1.10–2.08), not affected by sensitivity analyses. Funnel plot asymmetry suggested a publication bias underestimating OR. Stratified analysis showed the association to be driven by studies with: (1) monocentric AML patients and hospital-based control population, (2) Newcastle–Ottawa scale > 6 and the group of studies identified as with the lowest risk, (3) exposure assessment through peer-to-peer interview, (4) diagnosis in North America and Asia and after 1995, (5) restriction to de novo AML. Moreover, the association between OPE and AML was significant with insecticides. These findings broaden the spectrum of pesticide toxicity to myeloid malignancies.

Metabolic flexibility is the ability of a cell to adapt its metabolism to changes in its surrounding environment. Such adaptability, combined with apoptosis resistance provides cancer cells with a survival advantage. Mitochondrial voltage-dependent anion channel 1 (VDAC1) has been defined as a metabolic checkpoint at the crossroad of these two processes. Here, we show that the hypoxia-induced cleaved form of VDAC1 (VDAC1-&Delta, C) is implicated in both the up-regulation of glycolysis and the mitochondrial respiration. We demonstrate that VDAC1-&Delta, C, due to the loss of the putative phosphorylation site at serine 215, concomitantly with the loss of interaction with tubulin and microtubules, reprograms the cell to utilize more metabolites, favoring cell growth in hypoxic microenvironment. We further found that VDAC1-&Delta, C represses ciliogenesis and thus participates in ciliopathy, a group of genetic disorders involving dysfunctional primary cilium. Cancer, although not representing a ciliopathy, is tightly linked to cilia. Moreover, we highlight, for the first time, a direct relationship between the cilium and cancer cell metabolism. Our study provides the first new comprehensive molecular-level model centered on VDAC1-&Delta, C integrating metabolic flexibility, ciliogenesis, and enhanced survival in a hypoxic microenvironment.

International audience; Multiple myeloma (MM) is a plasma cell neoplasm that remains incurable due to innate or acquired resistance. Although MM cells produce high intracellular levels of reactive oxygen species (ROS), we hypothesised that they could remain sensitive to ROS unbalance. We tested if the inhibition of ROS, on one hand, or the overproduction of ROS, on the other, could (re)sensitise cells to bortezomib (BTZ). Two drugs were used in a panel of MM cell lines with various responses to BTZ: VAS3947 (VAS), an inhibitor of NADPH oxidase and auranofin (AUR), an inhibitor of thioredoxin reductase (TXNRD1), an antioxidant enzyme overexpressed in MM cells. We used several culture models: in suspension, on a fibronectin layer, in coculture with HS-5 mesenchymal cells, and/or in 3-D culture (or spheroids) to study the response of MM primary cells and cell lines. Several MM cell lines were sensitive to VAS but the combination with BTZ showed antagonistic or additive effects at best. By contrast, in all culture systems studied, the combined AUR/BTZ treatment showed synergistic effects on cell lines, including those less sensitive to BTZ and primary cells. MM cell death is due to the activation of apoptosis and autophagy. Modulating the redox balance of MM cells could be an effective therapy for refractory or relapse post-BTZ patients.

International audience; Manganese Superoxide dismutase 2 (SOD2) plays a crucial role in antioxidant defense but there are no data suggesting its role in genetic instability in CML. We evaluated the effects of SOD2 silencing in human UT7 cell line expressing either non-mutated or T315I-mutated BCR-ABL. Array-CGH experiments detected in BCR-ABL-expressing cells silenced for SOD2 a major genetic instability within several chromosomal loci, especially in regions carrying the glypican family (duplicated) and β-defensin genes (deleted). In a large cohort of patients with chronic myeloid leukemia (CML), a significant decrease of SOD2 mRNA was observed. This reduction appeared inversely correlated with leukocytosis and Sokal score, high-risk patients showing lower SOD2 levels. The analysis of anti-oxidant gene expression analysis revealed a specific down-regulation of the expression of PRDX2 in UT7-BCR-ABL and UT7-T315I cells silenced for SOD2 expression. Gene set enrichment analysis performed between the two SOD2-dependent classes of CML patients revealed a significant enrichment of Reactive Oxygen Species (ROS) Pathway. Our data provide the first evidence for a link between SOD2 expression and genetic instability in CML. Consequently, SOD2 mRNA levels should be analyzed in prospective studies as patients with low SOD2 expression could be more prone to develop a mutator phenotype under TKI therapies.

INTRODUCTION Cytokines are involved in many processes, including hematopoiesis and inflammation. Aging is associated with the onset of clonal hematopoiesis (CH) of indeterminate potential, putatively associated with a higher risk of progression to hematological malignancies such as myelodysplastic syndromes or acute myeloid leukemia. Moreover, CH may participate to create a pro-inflammatory environment contributing to the pathogenesis of age-related diseases, such as cardiovascular diseases. This is likely to be driven by or translated in changes in bone marrow (BM) and/or peripheral blood (PB) soluble factors for which reference values still remain unclear, because BM cytokines levels have never been determined in strictly selected healthy people. Indeed, control BM samples classically used in studies are from subjects undergoing surgeries for non-hematologic causes, such as total hip replacement or cardiac surgery, patients suffering from immune thrombocytopenic purpura, brain death patients or allogeneic BM donors. In this study, the BM and PB plasma concentrations of 49 hematopoietic and inflammatory cytokines were measured in a representative panel of 94 healthy adult volunteers and the results were analyzed considering their age and presence of CH. METHOD Ninety-four healthy donors aged from 18.6 to 80.1 years old (yo), including 58 women were recruited for this study (HEALTHOX protocol, CPP Tours, AFSSAPS identifier ID-RCB: 2016-A00571-50 and ClinicalTrials.gov # NCT02789839). The presence or absence of CH (>1% of variant allele frequency) in this cohort is already known (Guermouche H, Ravalet N et al, Blood Adv 2020;4(15):3550-3557). BM samples were obtained through sternal aspiration using a classical procedure in France, and PB sampling was performed at the same time by venipuncture. Samples were collected on sodium heparin or EDTA, centrifuged twice (1200 g, 10 min, 20 C°), aliquoted and stored at -80°C. A 48-plex human cytokine panel assay was used to quantify 48 human cytokines in PB and BM plasmas [beta-NGF, CCL2, CCL3, CCL4, CCL5, CCL7, CCL11, CCL27, CLEC11A, CXCL1, CXCL8, CXCL9, CXCL10, CXCL12, FGF-2, G-CSF, GM-CSF, HGF, IFN-alpha-2, IFN-gamma, IL-1 alpha, IL-1 beta, IL-1ra, IL-2, IL-2-RA, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12, IL-12B, IL-13, IL-15, IL-16, IL-17A, IL-18, KITLG, LIF, LT-alpha, M-CSF, MIF, PDGF subunit B, TNF, TNFSF10, VEGF-A]. MIF and FLT3L quantification were performed by ELISA. Regarding CH, the controls were subjects older than 50 yo without CH. Statistical analyses were performed with the R (3.6.3) and Rstudio version 1.2.5042 (www.rstudio.org) software. Comparisons were computed by Wilcoxon and Kruskal-Wallis tests. All pairwise multiple comparisons were performed using Dunn's-test for multiple comparisons of independent samples (PMCMR package). Correlations between cytokine levels in PB and BM were tested with Pearson and Spearman methods. Correlation matrices were plotted using the "corrplot" package. RESULTS CH was detected in 16 volunteers, mostly in individuals over 50 yo. BM and PB plasma samples were studied in 3 age-groups: 18-40, 40-60 and 60-80 yo. With aging, variations were observed for 18 BM cytokine levels, with 7 increasing (FLT3L, CXCL9, HGF, FGF-2, CCL27, IL-16, IL-18) and 8 decreasing (G-CSF, TNF, IL-2, IL-15, IL-17a, IL-4, LT-alpha, IL-1 alpha). In PB, 10 cytokines significantly increased with age (CXCL9, FLT3L, CCL27, CXCL10, HGF, CCL11, IL-16, IL-6, IL-1 beta, CCL2). CH was associated with significantly higher BM levels of MIF and IL-1 beta, lower BM levels of IL-9 and IL-5 and higher PB levels of IL-15, VEGF-A, IL-2, CXCL8, CXCL1 and G-CSF (Table). CONCLUSION In this study and for the first time, we concomitantly analyzed BM and PB concentrations of a panel of hematopoietic and inflammatory cytokines in a cohort of strictly selected healthy volunteers. In addition to the establishment of reference values, useful for various biological studies, and correlations between blood and BM levels, we identify variations in the BM of key cytokines according to age and CH. The differences in these cytokine concentrations, either as causes or as consequences, may shape both the BM microenvironment and hematopoietic processes, eventually leading to the beginning of age-related myeloid malignancies or inflammatory conditions. Figure 1 Figure 1. Disclosures Hirsch: Daiichi Sankyo Oncology: Consultancy; Novartis Pharma: Consultancy. Suner: Sanofi - Genzyme: Consultancy. Delhommeau: Celgene: Consultancy; BMS: Consultancy; Novartis: Consultancy.

Although it has been well-demonstrated that bone marrow mesenchymal stromal cells (MSCs) from CML patients do not belong to the Ph1-positive clone, there is growing evidence that they could play a role in the leukemogenesis process or the protection of leukemic stem cells from the effects of tyrosine kinase inhibitors (TKIs). The aim of the present study was to identify genes differentially expressed in MSCs isolated from CML patients at diagnosis (CML-MSCs) as compared to MSCs from healthy controls. Using a custom gene-profiling assay, we identified six genes over-expressed in CML-MSCs (BMP1, FOXO3, MET, MITF, NANOG, PDPN), with the two highest levels being documented for PDPN (PODOPLANIN) and NANOG. To determine whether this aberrant signature persisted in patients in deep molecular response induced by TKIs, we analyzed MSCs derived from such patients (MR-MSCs). This analysis showed that, despite the deep molecular responses, BMP1, MET, MITF, NANOG, and PDPN mRNA were upregulated in MR-MSCs. Moreover, BMP1, MITF, and NANOG mRNA expressions in MR-MSCs were found to be intermediate between control MSCs and CML-MSCs. These results suggest that CML-MSCs exhibit an abnormal gene expression pattern which might have been established during the leukemogenic process and persist in patients in deep molecular response.

Background Solitary plasmacytoma of the liver is a very rare and aggressive form of plasma cell dyscrasia. To the best of our knowledge, very few cases have been reported without systemic disease. We reported a rare case of hepatic solitary plasmacytoma that successfully responded to fractionated stereotactic radiotherapy. Case presentation A 64-year-old white French man had monoclonal gammopathy of the immune globulin G lambda type; he developed a cholestasis and cytolysis with the discovery of a subscapular nodule. A biopsy showed plasma cells and, for several reasons, the decision was made to use the fractionated stereotactic radiotherapy strategy. After 20 months, he is asymptomatic and the immune globulin G component has completely disappeared. Conclusion We suggest considering Cyberknife® radiosurgery as an option for the treatment of hepatic solitary plasmacytoma.

Mesenchymal stem cells (MSCs)-derived spheroid models favor maintenance of stemness, ex vivo expansion and transplantation efficacy. Spheroids may also be considered as useful surrogate models of the hematopoietic niche. However, accessibility to primary cells, from bone marrow (BM) or adipose tissues, may limit their experimental use and the lack of consistency in methods to form spheroids may affect data interpretation. In this study, we aimed to create a simple model by examining the ability of cell lines, from human (HS-27a and HS-5) and murine (MS-5) BM origins, to form spheroids, compared to primary human MSCs (hMSCs). Our protocol efficiently allowed the spheroid formation from all cell types within 24 hours. Whilst hMSCs-derived spheroids began to shrink after twenty-four hours, the size of spheroids derived from cell lines remained constant during three weeks. The difference was partially explained by the balance between proliferation and cell death, which could be triggered by hypoxia and induced oxidative stress. Our results demonstrate that, unlike hMSCs, MSC cell lines make reproductible spheroids that are easily handled. Thus, this model could help in understanding mechanisms involved in MSC functions and may provide a simple model by which to study cell interactions in the BM niche.

Low reactive oxygen species (ROS) levels are well-established characteristics of mouse hematopoietic stem cells (HSCs). However, little is known about these levels in human HSCs. This study aimed at quantifying ROS levels in human CD34

Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal stem cell disorders with an inherent tendency for transformation in secondary acute myeloid leukemia. This study focused on the redox metabolism of bone marrow (BM) cells from 97 patients compared with 25 healthy controls. The level of reactive oxygen species (ROS) was quantified by flow cytometry in BM cell subsets as well as the expression level of 28 transcripts encoding for major enzymes involved in the antioxidant cellular response. Our results highlight increased ROS levels in BM nonlymphoid cells and especially in primitive CD34posCD38low progenitor cells. Moreover, we identified a specific antioxidant signature, dubbed “antioxidogram,” for the different MDS subgroups or secondary acute myeloblastic leukemia (sAML). Our results suggest that progression from MDS toward sAML could be characterized by 3 successive molecular steps: (1) overexpression of enzymes reducing proteic disulfide bonds (MDS with

International audience; Erythropoiesis-stimulating agents are generally the first line of treatment of anemia in patients with lower-risk myelodysplastic syndrome. We prospectively investigated the predictive value of somatic mutations, and biomarkers of ineffective erythropoiesis including the flow cytometry RED score, serum growth-differentiation factor-15, and hepcidin levels. Inclusion criteria were no prior treatment with erythropoiesis-stimulating agents, low- or intermediate-1-risk myelodysplastic syndrome according to the International Prognostic Scoring System, and a hemoglobin level 4 (P=0.05) and a hepcidin:ferritin ratio 2000 pg/mL and a hepcidin:ferritin ratio

// Sophie Bustany 1, * , Jerome Bourgeais 2, * , Guergana Tchakarska 1, 4 , Simon Body 1 , Olivier Herault 2, 3 , Fabrice Gouilleux 2 , Brigitte Sola 1 1 Universite de Caen Normandie, EA4652 (MILPAT), MICAH Team, Caen, France 2 Universite Francois Rabelais, CNRS UMR 7292 (GICC), LNox Team, Tours, France 3 Service d'Hematologie Biologique, CHRU Tours, Tours, France 4 Present address: Cytogenetics Laboratory, Research Institute, McGill University Health Centre, Montreal, Canada * Co-first authors Correspondence to: Brigitte Sola, email: brigitte.sola@unicaen.fr Keywords: reactive oxygen species, p44/42 mitogen-activated protein kinase, pomalidomide, carfilzomib, NADPH oxidase Received: February 01, 2016 Accepted: May 28, 2016 Published: June 07, 2016 ABSTRACT The interactions of multiple myeloma (MM) cells with their microenvironment are crucial for pathogenesis. MM cells could interact differentially with their microenvironment depending on the type of cyclin D they express. We established several clones that constitutively express cyclin D1 from the parental RPMI8226 MM cell line and analyzed the impact of cyclin D1 expression on cell behavior. We performed a gene expression profiling study on cyclin D1-expressing vs . control cells and validated the results by semi-quantitative RT-PCR. The expression of cyclin D1 altered the transcription of genes that control adhesion and migration. We confirmed that cyclin D1 increases cell adhesion to stromal cells and fibronectin, stabilizes F-actin fibers, and enhances chemotaxis and inflammatory chemokine secretion. Both control and cyclin D1-expressing cells were more resistant to acute carfilzomib treatment when cultured on stromal cells than in suspension. However, this resistance was specifically reduced in cyclin D1-expressing cells after pomalidomide pre-treatment that modifies tumor cell/microenvironment interactions. Transcriptomic analysis revealed that cyclin D1 expression was also associated with changes in the expression of genes controlling metabolism. We also found that cyclin D1 expression disrupted the redox balance by producing reactive oxygen species. The resulting oxidative stress activated the p44/42 mitogen-activated protein kinase (or ERK1/2) signaling pathway, increased cell adhesion to fibronectin or stromal cells, and controlled drug sensitivity. Our results have uncovered a new function for cyclin D1 in the control of redox metabolism and interactions of cyclin D1-expressing MM cells with their bone marrow microenvironment.

Background. Acute myeloid leukemia (AML) with unfavorable cytogenetics remains a therapeutic challenge. We previously established that ex vivo exposure of AML blasts to eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) or fish oil emulsion (FO) induces Nrf2 pathway activation, metabolic switch and cell death (Picou et al., Pharmacol Res 2018;136:45-55). Objectives. The FILO group launched a pilot clinical study to evaluate the feasibility, safety and efficacy of the adjunction of a commercial FO emulsion (OMEGAVEN) to 3+7 in untreated AML with unfavorable cytogenetics. Patients and methods. The FAMYLY trial was a multicenter single-arm phase II study. Eligible patients had a diagnosis of untreated AML with unfavorable cytogenetics, 18 years old or above. For patients with WBC < 30 G/L, FO was administered at 2 mL/kg/d IV 2 days before 3+7 induction chemotherapy (daunorubicin 60 mg/m² days 1-3 and cytarabine 200 mg/m²/d as a continuous infusion days 1-7), until day 7 of induction. For patients with WBC >= 30 G/L, FO and 3+7 were started concomitantly, and FO was administered until day 9. The primary objective was complete response (CR) on the BM aspirate at the end of induction. Data from 75 patients with adverse cytogenetics and treated with daunorubicin and cytarabine in LAM2001 study (Lioure et al., Blood 2012) was used as a historical control. Serial blood samples were collected before treatment, and on days 1 and 3 after start of treatment, to evaluate the expression of NRF-2 target genes and antioxidant enzymes by RT-qPCR. Results. Thirty patients were included. The median age was 54 years (range: 30-64 years), and only 3 patients were hyperleukocytic. FO administration raised the plasma levels of EPA and DHA (p Conclusions. Altogether, FO emulsion adjunction to 3+7 is feasible and safe. Although CR rate improvement was not shown in this cohort of high-risk patients, further research aiming at finding the sufficient dose to trigger an NRF-2 response in vivo should be considered. ClinicalTrials.gov identifier: NCT01999413. Disclosures No relevant conflicts of interest to declare. OffLabel Disclosure: OMEGAVEN (fish oil emulsion) in Acute Myeloid Leukemia