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1.
Blood Adv ; 4(7): 1464-1477, 2020 04 14.
Article in English | MEDLINE | ID: mdl-32282884

ABSTRACT

Murine-based cellular models have provided and continue to provide many useful insights into the fundamental mechanisms of erythropoiesis, as well as insights into the pathophysiology of inherited and acquired red cell disorders. Although detailed information on many aspects of these cell models is available, comprehensive proteomic data are lacking. This is a critical knowledge gap, as proteins are effectors of most biologic processes. To address this critical unmet need, proteomes of the murine cell lines Friend erythroleukemia (MEL), GATA1 erythroid (G1ER), and embryonic stem cell-derived erythroid progenitor (MEDEP) and proteomes of cultured murine marrow-derived erythroblasts at different stages of terminal erythroid differentiation were analyzed. The proteomes of MEDEP cells and primary murine erythroid cells were most similar, whereas those of MEL and G1ER cells were more distantly related. We demonstrated that the overall cellular content of histones does not decrease during terminal differentiation, despite strong chromatin condensation. Comparison of murine and human proteomes throughout terminal erythroid differentiation revealed that many noted transcriptomic changes were significantly dampened at the proteome level, especially at the end of the terminal differentiation process. Analysis of the early events associated with induction of terminal differentiation in MEDEP cells revealed divergent alterations in associated transcriptomes and proteomes. These proteomic data are powerful and valuable tools for the study of fundamental mechanisms of normal and disordered erythropoiesis and will be of broad interest to a wide range of investigators for making the appropriate choice of various cell lines to study inherited and acquired diseases of the erythrocyte.


Subject(s)
Leukemia, Erythroblastic, Acute , Proteomics , Animals , Erythroblasts , Erythroid Cells , Erythropoiesis , Humans , Mice
3.
Haematologica ; 104(5): 907-918, 2019 05.
Article in English | MEDLINE | ID: mdl-30309849

ABSTRACT

AMP-activated protein kinase (AMPK) is a heterotrimeric complex containing α, ß, and γ subunits involved in maintaining integrity and survival of murine red blood cells. Indeed, Ampk α1-/- , Ampk ß1-/- and Ampk γ1-/- mice develop hemolytic anemia and the plasma membrane of their red blood cells shows elasticity defects. The membrane composition evolves continuously along erythropoiesis and during red blood cell maturation; defects due to the absence of Ampk could be initiated during erythropoiesis. We, therefore, studied the role of AMPK during human erythropoiesis. Our data show that AMPK activation had two distinct phases in primary erythroblasts. The phosphorylation of AMPK (Thr172) and its target acetyl CoA carboxylase (Ser79) was elevated in immature erythroblasts (glycophorin Alow), then decreased conjointly with erythroid differentiation. In erythroblasts, knockdown of the α1 catalytic subunit by short hairpin RNA led to a decrease in cell proliferation and alterations in the expression of membrane proteins (band 3 and glycophorin A) associated with an increase in phosphorylation of adducin (Ser726). AMPK activation in mature erythroblasts (glycophorin Ahigh), achieved through the use of direct activators (GSK621 and compound 991), induced cell cycle arrest in the S phase, the induction of autophagy and caspase-dependent apoptosis, whereas no such effects were observed in similarly treated immature erythroblasts. Thus, our work suggests that AMPK activation during the final stages of erythropoiesis is deleterious. As the use of direct AMPK activators is being considered as a treatment in several pathologies (diabetes, acute myeloid leukemia), this observation is pivotal. Our data highlighted the importance of the finely-tuned regulation of AMPK during human erythropoiesis.


Subject(s)
AMP-Activated Protein Kinases/metabolism , Cell Differentiation , Erythroblasts/cytology , Erythropoiesis , Gene Expression Regulation, Enzymologic , AMP-Activated Protein Kinases/antagonists & inhibitors , AMP-Activated Protein Kinases/genetics , Adult , Animals , Apoptosis , Autophagy , Cells, Cultured , Enzyme Activation , Erythroblasts/metabolism , Humans , Mice , Mice, Knockout , Phosphorylation , RNA, Small Interfering/genetics
4.
Blood Adv ; 2(20): 2646-2657, 2018 10 23.
Article in English | MEDLINE | ID: mdl-30327373

ABSTRACT

Reticulocytes produced in the bone marrow undergo maturation in the bloodstream to give rise to erythrocytes. Although the proteome of circulating red cells has been the subject of several reports, the cellular populations used for these studies were never completely devoid of reticulocytes. In our current study, we used highly purified erythrocyte and reticulocyte populations to quantify the absolute expression levels of the proteins in each cell population. Erythrocytes and reticulocytes were purified in a multistep process involving cellulose chromatography, Percoll gradient centrifugation, and fluorescence cell sorting after thiazole orange labeling. Proteins were analyzed by mass spectrometry from whole cells and erythrocyte plasma membrane (ghosts), leading to the identification and quantification of 2077 proteins, including 654 that were reticulocyte-specific. Absolute quantifications of these proteins were made using the mean corpuscular hemoglobin content of the cells as a standard. For each protein, we calculated the percentage loss during the terminal stages of reticulocyte maturation and the percentage of association with the plasma membrane. In addition, we used modified adenosine triphosphate and adenosine diphosphate molecules that enable the transfer of a biotin molecule to the catalytic sites of kinases to isolate active kinases in the erythrocytes and determined the absolute expression of 75 protein kinases and the modification of their expression during reticulocyte maturation. Our findings represent the first absolute quantification of proteins that are specifically expressed in normal erythrocytes with no detectable contamination by reticulocytes. Our findings thus represent a reference database for the future proteomic analysis of pathological erythrocytes.


Subject(s)
Erythrocytes/metabolism , Proteome/metabolism , Reticulocytes/metabolism , Humans
5.
Oncotarget ; 9(5): 6478-6489, 2018 Jan 19.
Article in English | MEDLINE | ID: mdl-29464086

ABSTRACT

Acute myeloid leukemia (AML) with t(8;21) and inv(16), together referred as core binding factor (CBF)-AML, are recognized as unique entities. Both rearrangements share a common pathophysiology, the disruption of the CBF, and a relatively good prognosis. Experiments have demonstrated that CBF rearrangements were insufficient to induce leukemia, implying the existence of cooperating events. To explore these aberrations, we performed single nucleotide polymorphism (SNP)-array in a well-annotated cohort of 198 patients with CBF-AML. Excluding breakpoint-associated lesions, the most frequent events included loss of a sex chromosome (53%), deletions at 9q21 (12%) and 7q36 (9%) in patients with t(8;21) compared with trisomy 22 (13%), trisomy 8 (10%) and 7q36 deletions (12%) in patients with inv(16). SNP-array revealed novel recurrent genetic alterations likely to be involved in CBF-AML leukemogenesis. ZBTB7A mutations (20% of t(8;21)-AML) were shown to be a target of copy-neutral losses of heterozygosity (CN-LOH) at chromosome 19p. FOXP1 focal deletions were identified in 5% of inv(16)-AML while sequence analysis revealed that 2% carried FOXP1 truncating mutations. Finally, CCDC26 disruption was found in both subtypes (4.5% of the whole cohort) and possibly highlighted a new lesion associated with aberrant tyrosine kinase signaling in this particular subtype of leukemia.

6.
Oncoimmunology ; 6(12): e1307491, 2017.
Article in English | MEDLINE | ID: mdl-29209559

ABSTRACT

NKp46 is a major determinant of natural killer (NK) cell function and it is implicated in tumor immune surveillance in acute myeloid leukemia (AML). The purpose of this study was to investigate the prognostic significance of NKp46 expression in an independent cohort of patients with AML, and to investigate the impact of NKp46 on clinical outcome after allogeneic stem cell transplantation (allo-SCT). NKp46 expression was assessed at diagnosis on NK cells by flow cytometry (N = 180 patients). Clinical outcome was evaluated with regard to NKp46 expression. Patients with NKp46high phenotype at diagnosis had better progression-free survival (PFS) and overall survival (OS) than patients with NKp46low phenotype (74.3% vs. 46.6%, p = 0.014; 82.6% vs. 57.1%, p = 0.010, respectively). In multivariate analysis, high NKp46 was an independent factor for improved OS (HR = 0.409, p = 0.010) and PFS (HR = 0.335, p = 0.011). Subgroup analysis revealed that allo-SCT had a favorable impact on PFS in patients with NKp46high phenotype (p = 0.025). By contrast, allo-SCT did not impact PFS in patients with low NKp46 expression (p = 0.303). In conclusion, we validate the prognostic value of NKp46 expression at diagnosis in AML. However, the prognostic value of NKp46 expression is limited to patients treated with allo-SCT, thus suggesting that NKp46 status may be predictive for allo-SCT responsiveness.

7.
Front Immunol ; 8: 573, 2017.
Article in English | MEDLINE | ID: mdl-28611767

ABSTRACT

Accumulating evidence highlights natural killer (NK) cell parameters as potential prognostic factors in cancer patients, which provides a strong rationale for developing therapeutic strategies aiming at restoring NK cell. However, reaching this point warrants better characterization of tumor-induced NK cell alterations. Our group recently reported heterogeneous NK maturation in acute myeloid leukemia (AML) patients. However, the clinical significance of such observations remained to be assessed on a larger cohort of patients. NK maturation based on expression of CD56, CD57, and KIR was assessed by flow cytometry in newly diagnosed AML patients (N = 87 patients from GOELAMS-LAM-IR-2006 multicenter trial). Clinical outcome was evaluated with regard to NK maturation profiles. Unsupervised integrated analysis of NK maturation markers confirmed the existence of three distinct groups of patients [hypomaturation (24.1%), intermediate maturation (66.7%), and hypermaturation (9.2%)]. In univariate analysis, significant differences in overall survival (OS) (P = 0.0006) and relapse-free survival (RFS) (P < 0.0001) were observed among these different groups. Patients with hypomaturation profile had reduced OS, with 3-year OS rates of 12.5 vs 57.1 and 57.4% for patients with intermediate and hypermaturation, respectively. Consistently, patients with hypomaturation profile had reduced RFS, with 3-year RFS rates of 0 vs 52.6 and 73.3% for patients with intermediate and hypermaturation, respectively. In multivariate Cox regression models, NK hypomaturation remained significantly associated with reduced OS and RFS, independent of other factors [hazard ratio (HR) = 4.15, P = 0.004 and HR = 8.23, P = 0.003, respectively]. NK maturation defects were further explored by mass cytometry and revealed that NK hypomaturation profile is associated with a reduced frequency of memory-like NK cells. In conclusion, besides classical alterations of NK triggering and inhibitory receptors expression in AML, we confirm that the homeostasis of NK maturation can be modified in the context of AML, notably with a deep maturation blockade in almost 10% patients.

8.
Fungal Biol ; 121(6-7): 529-540, 2017.
Article in English | MEDLINE | ID: mdl-28606348

ABSTRACT

Wood rot fungi form one of the main classes of phytopathogenic fungus. The group includes many species, but has remained poorly studied. Many species belonging to the Ganoderma genus are well known for causing decay in a wide range of tree species around the world. Ganoderma boninense, causal agent of oil palm basal stem rot, is responsible for considerable yield losses in Southeast Asian oil palm plantations. In a large-scale sampling operation, 357 sporophores were collected from oil palm plantations spread over peninsular Malaysia and Sumatra and genotyped using 11 SSR markers. The genotyping of these samples made it possible to investigate the population structure and demographic history of G. boninense across the oldest known area of interaction between oil palm and G. boninense. Results show that G. boninense possesses a high degree of genetic diversity and no detectable genetic structure at the scale of Sumatra and peninsular Malaysia. The fact that few duplicate genotypes were found in several studies including this one supports the hypothesis of spore dispersal in the spread of G. boninense. Meanwhile, spatial autocorrelation analysis shows that G. boninense is able to disperse across both short and long distances. These results bring new insight into mechanisms by which G. boninense spreads in oil palm plantations. Finally, the use of approximate Bayesian computation (ABC) modelling indicates that G. boninense has undergone a demographic expansion in the past, probably before the oil palm was introduced into Southeast Asia.


Subject(s)
Arecaceae/microbiology , Ganoderma/classification , Ganoderma/isolation & purification , Genetic Variation , Plant Diseases/microbiology , Ganoderma/genetics , Gene Flow , Genotyping Techniques , Indonesia , Malaysia
9.
Oncotarget ; 8(30): 49548-49563, 2017 Jul 25.
Article in English | MEDLINE | ID: mdl-28548938

ABSTRACT

Cytogenetics and European Leukemia Net (ELN) genetic classification predict patients at increased risk of relapse in acute myeloid leukemia (AML) except in the intermediate risk group for which further prognostic determinants are required. We have previously shown that Natural Killer (NK) cell defects in AML are predictors of poor overall survival (OS). This study aimins at validating NKp30, a receptor that mediates NK activation, as a prognostic biomarker for AML patients with intermediate prognosis.NKp30 expression was prospectively assessed at diagnosis on NK cells from peripheral blood by flow cytometry (N = 201 patients). Clinical outcome was evaluated with regard to NKp30 status.In patients with intermediate cytogenetic (N = 162), NKp30high phenotype at diagnosis was predictive of better OS (HR = 0.26; 95%CI = [0.14-0.50]; P < 0.0001) and relapse-free survival (RFS) (HR = 0.21; 95%CI = [0.08-0.52]; P = 0.0007). In patients with intermediate ELN (N = 116), NKp30high phenotype at diagnosis was predictive of better OS (HR = 0.33; 95%CI = [0.16-0.67]; P = 0.0019) and RFS (HR = 0.24; 95%CI = [0.08-0.67]; P = 0.0058). In multivariate analysis, high NKp30 expression independently predicted improved OS (HR = 0.56, P = 0.046) and RFS (HR = 0.37, P = 0.048). Consistently, cumulative incidence of relapse (CIR) was lower in patients with high NKp30 expression (HR = 0.37, P = 0.026).In conclusion, we propose NKp30 status as a simple and early prognostic biomarker that identifies intermediate-risk patients with poor prognosis who otherwise may not be identified with existing risk stratification systems.


Subject(s)
Gene Expression , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/mortality , Natural Cytotoxicity Triggering Receptor 3/genetics , Adult , Biomarkers , Bone Marrow/pathology , Female , Gene Expression Regulation, Leukemic , Genetic Testing , Humans , Kaplan-Meier Estimate , Killer Cells, Natural/immunology , Killer Cells, Natural/metabolism , Leukemia, Myeloid, Acute/diagnosis , Leukemia, Myeloid, Acute/immunology , Ligands , Male , Middle Aged , Prognosis , Proportional Hazards Models
10.
Cell Rep ; 16(5): 1470-1484, 2016 08 02.
Article in English | MEDLINE | ID: mdl-27452463

ABSTRACT

Mass spectrometry-based proteomics now enables the absolute quantification of thousands of proteins in individual cell types. We used this technology to analyze the dynamic proteome changes occurring during human erythropoiesis. We quantified the absolute expression of 6,130 proteins during erythroid differentiation from late burst-forming units-erythroid (BFU-Es) to orthochromatic erythroblasts. A modest correlation between mRNA and protein expression was observed. We identified several proteins with unexpected expression patterns in erythroid cells, highlighting a breakpoint in the erythroid differentiation process at the basophilic stage. We also quantified the distribution of proteins between reticulocytes and pyrenocytes after enucleation. These analyses identified proteins that are actively sorted either with the reticulocyte or the pyrenocyte. Our study provides the absolute quantification of protein expression during a complex cellular differentiation process in humans, and it establishes a framework for future studies of disordered erythropoiesis.


Subject(s)
Erythropoiesis/physiology , Proteome/metabolism , Cell Differentiation , Cells, Cultured , Erythroblasts/metabolism , Erythroblasts/physiology , Erythroid Precursor Cells/metabolism , Erythroid Precursor Cells/physiology , Humans , Proteomics/methods , RNA, Messenger/metabolism
11.
Blood ; 127(20): 2451-9, 2016 05 19.
Article in English | MEDLINE | ID: mdl-26980726

ABSTRACT

Acute myeloid leukemia (AML) with t(8;21) or inv(16) have been recognized as unique entities within AML and are usually reported together as core binding factor AML (CBF-AML). However, there is considerable clinical and biological heterogeneity within this group of diseases, and relapse incidence reaches up to 40%. Moreover, translocations involving CBFs are not sufficient to induce AML on its own and the full spectrum of mutations coexisting with CBF translocations has not been elucidated. To address these issues, we performed extensive mutational analysis by high-throughput sequencing in 215 patients with CBF-AML enrolled in the Phase 3 Trial of Systematic Versus Response-adapted Timed-Sequential Induction in Patients With Core Binding Factor Acute Myeloid Leukemia and Treating Patients with Childhood Acute Myeloid Leukemia with Interleukin-2 trials (age, 1-60 years). Mutations in genes activating tyrosine kinase signaling (including KIT, N/KRAS, and FLT3) were frequent in both subtypes of CBF-AML. In contrast, mutations in genes that regulate chromatin conformation or encode members of the cohesin complex were observed with high frequencies in t(8;21) AML (42% and 18%, respectively), whereas they were nearly absent in inv(16) AML. High KIT mutant allele ratios defined a group of t(8;21) AML patients with poor prognosis, whereas high N/KRAS mutant allele ratios were associated with the lack of KIT or FLT3 mutations and a favorable outcome. In addition, mutations in epigenetic modifying or cohesin genes were associated with a poor prognosis in patients with tyrosine kinase pathway mutations, suggesting synergic cooperation between these events. These data suggest that diverse cooperating mutations may influence CBF-AML pathophysiology as well as clinical behavior and point to potential unique pathogenesis of t(8;21) vs inv(16) AML.


Subject(s)
Chromosome Inversion , Chromosomes, Human, Pair 16/genetics , Chromosomes, Human, Pair 21/genetics , Chromosomes, Human, Pair 8/genetics , Core Binding Factors/genetics , DNA, Neoplasm/genetics , Leukemia, Myeloid, Acute/genetics , Mutation , Translocation, Genetic , Adolescent , Adult , Alleles , Cell Cycle Proteins/genetics , Child , Child, Preschool , Chromatin/genetics , Chromatin/ultrastructure , Chromosomal Proteins, Non-Histone/genetics , Core Binding Factor Alpha 2 Subunit/genetics , DNA Mutational Analysis , Female , Genetic Association Studies , High-Throughput Nucleotide Sequencing , Humans , Infant , Male , Middle Aged , Oncogene Proteins, Fusion/genetics , Prognosis , RUNX1 Translocation Partner 1 Protein , Young Adult , Cohesins
12.
Sci Adv ; 1(8): e1500221, 2015 Sep.
Article in English | MEDLINE | ID: mdl-26601252

ABSTRACT

Fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) is frequently detected in acute myeloid leukemia (AML) patients and is associated with a dismal long-term prognosis. FLT3 tyrosine kinase inhibitors provide short-term disease control, but relapse invariably occurs within months. Pim protein kinases are oncogenic FLT3-ITD targets expressed in AML cells. We show that increased Pim kinase expression is found in relapse samples from AML patients treated with FLT3 inhibitors. Ectopic Pim-2 expression induces resistance to FLT3 inhibition in both FLT3-ITD-induced myeloproliferative neoplasm and AML models in mice. Strikingly, we found that Pim kinases govern FLT3-ITD signaling and that their pharmacological or genetic inhibition restores cell sensitivity to FLT3 inhibitors. Finally, dual inhibition of FLT3 and Pim kinases eradicates FLT3-ITD(+) cells including primary AML cells. Concomitant Pim and FLT3 inhibition represents a promising new avenue for AML therapy.

13.
Biosci Rep ; 35(6)2015 Oct 23.
Article in English | MEDLINE | ID: mdl-26500282

ABSTRACT

The oncogenic Pim2 kinase is overexpressed in several haematological malignancies, such as multiple myeloma and acute myeloid leukaemia (AML), and constitutes a strong therapeutic target candidate. Like other Pim kinases, Pim2 is constitutively active and is believed to be essentially regulated through its accumulation. We show that in leukaemic cells, the three Pim2 isoforms have dramatically short half-lives although the longer isoform is significantly more stable than the shorter isoforms. All isoforms present a cytoplasmic localization and their degradation was neither modified by broad-spectrum kinase or phosphatase inhibitors such as staurosporine or okadaic acid nor by specific inhibition of several intracellular signalling pathways including Erk, Akt and mTORC1. Pim2 degradation was inhibited by proteasome inhibitors but Pim2 ubiquitination was not detected even by blocking both proteasome activity and protein de-ubiquitinases (DUBs). Moreover, Pyr41, an ubiquitin-activating enzyme (E1) inhibitor, did not stabilize Pim2, strongly suggesting that Pim2 was degraded by the proteasome without ubiquitination. In agreement, we observed that purified 20S proteasome particles could degrade Pim2 molecule in vitro. Pim2 mRNA accumulation in UT7 cells was controlled by erythropoietin (Epo) through STAT5 transcription factors. In contrast, the translation of Pim2 mRNA was not regulated by mTORC1. Overall, our results suggest that Pim2 is only controlled by its mRNA accumulation level. Catalytically active Pim2 accumulated in proteasome inhibitor-treated myeloma cells. We show that Pim2 inhibitors and proteasome inhibitors, such as bortezomib, have additive effects to inhibit the growth of myeloma cells, suggesting that Pim2 could be an interesting target for the treatment of multiple myeloma.


Subject(s)
Multiple Myeloma/drug therapy , Multiple Myeloma/genetics , Proteasome Inhibitors/administration & dosage , Protein Serine-Threonine Kinases/biosynthesis , Proto-Oncogene Proteins/biosynthesis , Cell Line, Tumor , Gene Expression Regulation, Leukemic/drug effects , Humans , Mechanistic Target of Rapamycin Complex 1 , Multiple Myeloma/pathology , Multiprotein Complexes/genetics , Protein Isoforms/biosynthesis , Protein Isoforms/genetics , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/genetics , Protein Stability/drug effects , Proteolysis/drug effects , Proto-Oncogene Proteins/antagonists & inhibitors , Proto-Oncogene Proteins/genetics , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , STAT5 Transcription Factor/genetics , TOR Serine-Threonine Kinases/genetics
14.
Blood ; 126(11): 1346-56, 2015 Sep 10.
Article in English | MEDLINE | ID: mdl-26186940

ABSTRACT

Cancer cells require glutamine to adapt to increased biosynthetic activity. The limiting step in intracellular glutamine catabolism involves its conversion to glutamate by glutaminase (GA). Different GA isoforms are encoded by the genes GLS1 and GLS2 in humans. Herein, we show that glutamine levels control mitochondrial oxidative phosphorylation (OXPHOS) in acute myeloid leukemia (AML) cells. Glutaminase C (GAC) is the GA isoform that is most abundantly expressed in AML. Both knockdown of GLS1 expression and pharmacologic GLS1 inhibition by the drug CB-839 can reduce OXPHOS, leading to leukemic cell proliferation arrest and apoptosis without causing cytotoxic activity against normal human CD34(+) progenitors. Strikingly, GLS1 knockdown dramatically inhibited AML development in NSG mice. The antileukemic activity of CB-839 was abrogated by both the expression of a hyperactive GAC(K320A) allele and the addition of the tricarboxyclic acid cycle product α-ketoglutarate, indicating the critical function of GLS1 in AML cell survival. Finally, glutaminolysis inhibition activated mitochondrial apoptosis and synergistically sensitized leukemic cells to priming with the BCL-2 inhibitor ABT-199. These findings show that targeting glutamine addiction via GLS1 inhibition offers a potential novel therapeutic strategy for AML.


Subject(s)
Glutamine/metabolism , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/metabolism , Proto-Oncogene Proteins c-bcl-2/antagonists & inhibitors , Animals , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Benzeneacetamides/pharmacology , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Citric Acid Cycle/drug effects , Enzyme Inhibitors/pharmacology , Gene Knockdown Techniques , Glutaminase/antagonists & inhibitors , Glutaminase/genetics , Glutaminase/metabolism , Humans , Leukemia, Myeloid, Acute/genetics , Mice , Mitochondria/metabolism , Oxidative Phosphorylation/drug effects , Oxygen Consumption/drug effects , Sulfonamides/pharmacology , Thiadiazoles/pharmacology , Xenograft Model Antitumor Assays
15.
Cell Rep ; 11(9): 1446-57, 2015 Jun 09.
Article in English | MEDLINE | ID: mdl-26004183

ABSTRACT

AMPK is a master regulator of cellular metabolism that exerts either oncogenic or tumor suppressor activity depending on context. Here, we report that the specific AMPK agonist GSK621 selectively kills acute myeloid leukemia (AML) cells but spares normal hematopoietic progenitors. This differential sensitivity results from a unique synthetic lethal interaction involving concurrent activation of AMPK and mTORC1. Strikingly, the lethality of GSK621 in primary AML cells and AML cell lines is abrogated by chemical or genetic ablation of mTORC1 signaling. The same synthetic lethality between AMPK and mTORC1 activation is established in CD34-positive hematopoietic progenitors by constitutive activation of AKT or enhanced in AML cells by deletion of TSC2. Finally, cytotoxicity in AML cells from GSK621 involves the eIF2α/ATF4 signaling pathway that specifically results from mTORC1 activation. AMPK activation may represent a therapeutic opportunity in mTORC1-overactivated cancers.


Subject(s)
AMP-Activated Protein Kinases/metabolism , Antineoplastic Agents/pharmacology , Enzyme Activation/drug effects , Imidazoles/pharmacology , Leukemia, Myeloid, Acute/metabolism , Multiprotein Complexes/agonists , Pyrimidinones/pharmacology , Animals , Fluorescent Antibody Technique , Heterografts , Humans , Mechanistic Target of Rapamycin Complex 1 , Mice , Mice, Nude , Microscopy, Electron, Transmission , Oligonucleotide Array Sequence Analysis , Polymerase Chain Reaction , RNA Interference , Signal Transduction/drug effects , TOR Serine-Threonine Kinases
16.
Curr Opin Hematol ; 22(3): 193-8, 2015 May.
Article in English | MEDLINE | ID: mdl-25767952

ABSTRACT

PURPOSE OF REVIEW: The type 1 transferrin receptor (TfR1) is well known as a key player in erythroid differentiation through its role in iron uptake. Recently, it has been demonstrated that TfR1 could also have signaling functions in erythroid cells. Moreover, the second transferrin receptor, TfR2, whose signaling functions in hepatic cells are well established, was recently shown to be a partner of the erythropoietin receptor (EpoR) and thereby likely to play a role in erythroid differentiation. RECENT FINDINGS: This review reports recent findings regarding the specificities of the regulation of TfR1 expression and iron uptake in erythroblasts. The newly discovered noncanonical actions of TfR1 and TfR2 in erythroid cells are also discussed. SUMMARY: Erythrocytes contain more than 60% of the iron of the body and each day, differentiating erythroid cells uptake around 20 mg of iron for heme synthesis. Accordingly, TfR1 is one of the most abundant membrane proteins of the erythroblasts and it is not surprising that specific regulations regarding both its expression and its mechanism of action operate in erythroblasts. The signaling functions of both TfR1 and TfR2 in erythroid cells were unexpected and these recent findings open a new field of research regarding the last steps of erythroid differentiation and their regulation.


Subject(s)
Erythropoiesis/physiology , Receptors, Transferrin/metabolism , Cell Differentiation , Humans , Iron/metabolism
17.
Haematologica ; 100(4): 458-65, 2015 Apr.
Article in English | MEDLINE | ID: mdl-25637053

ABSTRACT

Transferrin receptor-2 is a transmembrane protein whose expression is restricted to hepatocytes and erythroid cells. Transferrin receptor-2 has a regulatory function in iron homeostasis, since its inactivation causes systemic iron overload. Hepatic transferrin receptor-2 participates in iron sensing and is involved in hepcidin activation, although the mechanism remains unclear. Erythroid transferrin receptor-2 associates with and stabilizes erythropoietin receptors on the erythroblast surface and is essential to control erythrocyte production in iron deficiency. We identified a soluble form of transferrin receptor-2 in the media of transfected cells and showed that cultured human erythroid cells release an endogenous soluble form. Soluble transferrin receptor-2 originates from a cleavage of the cell surface protein, which is inhibited by diferric transferrin in a dose-dependent manner. Accordingly, the shedding of the transferrin receptor-2 variant G679A, mutated in the Arginine-Glycine-Aspartic acid motif and unable to bind diferric transferrin, is not modulated by the ligand. This observation links the process of transferrin receptor-2 removal from the plasma membrane to iron homeostasis. Soluble transferrin receptor-2 does not affect the binding of erythropoietin to erythropoietin receptor or the consequent signaling and partially inhibits hepcidin promoter activation only in vitro. Whether it is a component of the signals released by erythropoiesis in iron deficiency remains to be investigated. Our results indicate that membrane transferrin receptor-2, a sensor of circulating iron, is released from the cell membrane in iron deficiency.


Subject(s)
Cell Membrane/metabolism , Iron/metabolism , Receptors, Transferrin/metabolism , Antigens, CD/metabolism , Cell Line , Erythroid Cells/metabolism , Erythropoietin/metabolism , Gene Expression , Hepcidins/genetics , Humans , Mutation , Promoter Regions, Genetic , Protein Binding , Protein Transport , Proteolysis , Receptors, Erythropoietin/genetics , Receptors, Erythropoietin/metabolism , Receptors, Transferrin/blood , Receptors, Transferrin/genetics , Transcriptional Activation , Transferrin/metabolism
18.
Blood ; 124(9): 1445-9, 2014 Aug 28.
Article in English | MEDLINE | ID: mdl-24973361

ABSTRACT

Acute myeloid leukemia (AML) with t(8;21) (q22;q22) is considered to have favorable risk; however, nearly half of t(8;21) patients are not cured, and recent studies have highlighted remarkable genetic heterogeneity in this subset of AML. Here we identify somatic mutations in additional sex combs-like 2 (ASXL2) in 22.7% (25/110) of patients with t(8;21), but not in patients with inv(16)/t(16;16) (0/60) or RUNX1-mutated AML (0/26). ASXL2 mutations were similarly frequent in adults and children t(8;21) and were mutually exclusive with ASXL1 mutations. Although overall survival was similar between ASXL1 and ASXL2 mutant t(8;21) AML patients and their wild-type counterparts, patients with ASXL1 or ASXL2 mutations had a cumulative incidence of relapse of 54.6% and 36.0%, respectively, compared with 25% in ASXL1/2 wild-type counterparts (P = .226). These results identify a high-frequency mutation in t(8;21) AML and identify the need for future studies to investigate the clinical and biological relevance of ASXL2 mutations in this unique subset of AML.


Subject(s)
Core Binding Factor Alpha 2 Subunit/genetics , Leukemia, Myeloid, Acute/genetics , Mutation , Oncogene Proteins, Fusion/genetics , Repressor Proteins/genetics , Adolescent , Adult , Child , Child, Preschool , Chromosomes, Human, Pair 21/genetics , Chromosomes, Human, Pair 8/genetics , Female , Gene Frequency , Humans , Leukemia, Myeloid, Acute/classification , Leukemia, Myeloid, Acute/drug therapy , Male , Middle Aged , Neoplasm, Residual/genetics , RUNX1 Translocation Partner 1 Protein , Translocation, Genetic , Young Adult
19.
Blood ; 122(20): 3521-32, 2013 Nov 14.
Article in English | MEDLINE | ID: mdl-24014241

ABSTRACT

Cancer cells require nutrients and energy to adapt to increased biosynthetic activity, and protein synthesis inhibition downstream of mammalian target of rapamycin complex 1 (mTORC1) has shown promise as a possible therapy for acute myeloid leukemia (AML). Glutamine contributes to leucine import into cells, which controls the amino acid/Rag/mTORC1 signaling pathway. We show in our current study that glutamine removal inhibits mTORC1 and induces apoptosis in AML cells. The knockdown of the SLC1A5 high-affinity transporter for glutamine induces apoptosis and inhibits tumor formation in a mouse AML xenotransplantation model. l-asparaginase (l-ase) is an anticancer agent also harboring glutaminase activity. We show that l-ases from both Escherichia coli and Erwinia chrysanthemi profoundly inhibit mTORC1 and protein synthesis and that this inhibition correlates with their glutaminase activity levels and produces a strong apoptotic response in primary AML cells. We further show that l-ases upregulate glutamine synthase (GS) expression in leukemic cells and that a GS knockdown enhances l-ase-induced apoptosis in some AML cells. Finally, we observe a strong autophagic process upon l-ase treatment. These results suggest that l-ase anticancer activity and glutamine uptake inhibition are promising new therapeutic strategies for AML.


Subject(s)
Glutamine/antagonists & inhibitors , Leukemia, Myeloid, Acute/drug therapy , Adult , Aged , Aged, 80 and over , Amino Acid Transport System ASC/antagonists & inhibitors , Amino Acid Transport System ASC/genetics , Animals , Apoptosis/drug effects , Asparaginase/isolation & purification , Asparaginase/pharmacology , Autophagy/drug effects , Bacterial Proteins/pharmacology , Biological Transport/drug effects , Cell Line, Tumor/drug effects , Cell Line, Tumor/metabolism , Dickeya chrysanthemi/enzymology , Drug Screening Assays, Antitumor , Escherichia coli Proteins/pharmacology , Female , Glutaminase/isolation & purification , Glutaminase/pharmacology , Glutamine/metabolism , Humans , Leukemia, Myeloid, Acute/metabolism , Leukemia, Myeloid, Acute/pathology , Leukemia, Myelomonocytic, Acute/drug therapy , Leukemia, Myelomonocytic, Acute/metabolism , Male , Mechanistic Target of Rapamycin Complex 1 , Mice , Mice, Nude , Middle Aged , Minor Histocompatibility Antigens , Multiprotein Complexes/antagonists & inhibitors , Protein Biosynthesis/drug effects , RNA Interference , RNA, Small Interfering/pharmacology , RNA, Small Interfering/therapeutic use , Signal Transduction/drug effects , TOR Serine-Threonine Kinases/antagonists & inhibitors , Xenograft Model Antitumor Assays , Young Adult
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