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1
Fadini, G. P. "Circulating CD34+ cells, metabolic syndrome, and cardiovascular risk." European Heart Journal 27, no. 18 (August 9, 2006): 2247–55. http://dx.doi.org/10.1093/eurheartj/ehl198.
2
Kochi, Yu, Yoshikane Kikushige, Toshihiro Miyamoto, and Koichi Akashi. "Identification of ASCT1 As a Candidate Molecule Enhancing Antioxidant Activity in Primary Human AML Cells." Blood 128, no. 22 (December 2, 2016): 1674. http://dx.doi.org/10.1182/blood.v128.22.1674.1674.
Анотація:
Abstract Introduction: Recent studies have shown that the specific alteration of metabolic pathways are involved in the regulation of function of normal hematopoietic stem cells (HSCs) and leukemic stem cells (LSCs) in acute myeloid leukemia (AML). However, little is known about the features of metabolic activity in human HSCs and LSCs. To reveal the metabolic pathway alterations in primary AML cells, we performed the comprehensive metabolome analysis by comparing normal human CD34+ hematopoietic stem/progenitor cells (HSPCs)(n=5) and CD34+ primitive AML cells containing LSCs (n=16) using highly sensitive CE-tandem mass spectrometry. Method: Metabolome analysis Metabolites were extracted from primitive CD34+ AML cells (n=16) and normal CD34+ bone marrow cells (n=4) and cord blood cells (n=1). Metabolome analysis was conducted by the C-SCOPE package of HMT using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS) for cation analysis, and capillary electrophoresis-tandem mass spectrometry (CE-MS/MS) for anion analysis. 116 metabolites were targeted for analysis in this study. Oxygen consumption rates and extracellular acidification rate O2 consumption rates (OCR) and extracellular acidification rate (ECAR) were measured by the Seahorse XF96 extracellular flux analyzer. Three replicate wells of 400,000 leukemic or normal cells per well were seeded in 96-well XF96 well plates coated with BD Cell-Tak (BD Biosciences) in serum-free unbuffered DMEM. Analyses were performed both at basal conditions and after injection of OLI (1 mg/ml), FCCP (1 mM), Antimycin A (5 mM). Result: We detected 101 metabolites involved in central carbon and energy metabolism. In glucose metabolism, the level of lactate, an end-product of aerobic glycolysis, were lower in CD34+ AML cells than normal HSPCs, whereas the level of pyruvate, a precursor of lactate, was not different. Thus, CD34+ AML cells had a significantly high pyruvate/lactate ratio as compared to normal HSPCs, suggesting that aerobic respiration is preferentially utilized in CD34+ AML cells. To confirm this observation, we directly measured the O2 consumption rate (OCR) and extracellular acidification rate (ECAR) of CD34+ AML cells (n=4) and normal HSPCs (n=5) by XF96 extracellular flux analyzer. OCR reflects the activity of aerobic respiration, whereas ECAR reflects lactate generation and correlates with anaerobic respiration activity. Therefore, OCR/ECAR ratio is a good index for the discrimination of aerobic and anaerobic respiration pattern. We found that the OCR/ECAR ratio of CD34+ primitive AML cells was significantly high as compared to that of HSPCs, suggesting that CD34+ AML cells predominantly utilized aerobic respiration. Although the aerobic respiration resulted in the production of reactive oxygen species (ROS), the intracellular ROS level was not different between CD34+ AML (n=7) cells and normal HSPCs (n=3), suggesting that the antioxidant activity should be strongly enhanced in CD34+ AML cells. Consistent with the observation, we found that CD34+ AML cells had a much higher level of glutathione (GSH), a primary intracellular antioxidant, than normal HSPCs. To clarify the molecular mechanisms how CD34+ primitive AML cells could maintain high GSH level, we analyzed the expression of cysteine transporters, because cysteine uptake is the rate-limiting step of GSH synthesis. In human, three amino acid transporters including ASCT1, ASC1 and xCT are known as cystine/cysteine transporters. Interestingly, ASCT1 was significantly highly expressed in CD34+ AML cells (n=10) as compared to normal CD34+ HSPCs (n=3). Of note, normal CD34+CD38- HSCs completely lacked ASCT1 expression, whereas CD34+CD38-LSCs expressed at a high level, indicating the possibility that the high expression of ASCT1 should be a LSC specific machinery for enhanced GSH synthesis. Thus, human AML cells predominantly utilize aerobic respiration that is supported by a high level of GSH, and AML specific ASCT1 expression presumably contributes to the high level of GSH. These data suggest that ASCT1 should be a promising molecule to specifically target AML stem/progenitor cells. Disclosures Akashi: Celgene: Research Funding; Astellas Pharma: Research Funding; Shionogi & Co., Ltd: Research Funding; Asahi Kasei Pharma Corporation: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Bristol Meyers Squibb: Research Funding; Kyowa Hakko Kirin: Consultancy, Research Funding; Sunitomo Dainippon Pharma: Consultancy.
3
Nakasone, Hideki, Misato Kikuchi, Yu Akahoshi, Koji Kawamura, Miki Sato, Kazuki Yoshimura, Yukiko Misaki, et al. "The Expression of CD83 Would be Increased in CD34-Positive Monocytes Detected in Peripheral Blood Mobilized By G-CSF in Humans." Blood 132, Supplement 1 (November 29, 2018): 2063. http://dx.doi.org/10.1182/blood-2018-99-112084.
Анотація:
Abstract [Background] CD34-positive monocytes (CD34+mono) have recently been identified following mobilization by granulocyte-colony stimulating factor (G-CSF), and have been suggested to have a potential to modulate immune functions in animal models. However, the biological feature of CD34+mono in humans still remains unclear. Thus, we explored the difference between CD34+mono, CD34+cells, and monocytes through the analyses of gene expression profiles (GEP). [Methods] CD34+mono (Lin-CD34+CD33+CD14+CD11b+, Figure1), CD34+cells (Lin-CD34+CD33-CD14-CD11b- ), and monocytes (Lin-CD34-CD33+CD14+CD11b+) were directly sorted into tubes from cryopreserved grafts from three healthy donors. After the extraction of total RNA, microarray analyses were performed with GeneChip™ 3' IVT Pico Kit and analyzed according to the algorithm of Transcriptome Analysis Console (Thermo Fisher Scientific). Condition false discovery rate (FDR) F-test was used for filtering genes, and the threshold was <0.0005 for hierarchical clustering without considering fold changes. In addition, enrichment analyses were performed for gene ontology (GO) and pathways by the Kyoto Encyclopedia of Genes and Genomes (KEGG) through Database for Annotation, Visualization and Integrated Discovery (DAVID). The differentially expressed genes with <0.05 of F-test values and 2 or more fold changes in CD34+mono compared with CD34+cells and monocytes were selected for the enrichment analyses. Thereafter, the protein-protein interaction (PPI) network in CD34+mono was constructed through Search Tool for the Retrieval of Interacting Genes/Proteins (STRING), which is a biological database. This study was approved by the institutional review board of Jichi Medical University and all subjects gave their written informed consent for the cryopreservation and analysis of the blood samples in accordance with the Helsinki Declaration. [Results] CD34+mono resembled con-mono in the appearance, although it might look slightly immature. In fact, the number of differential expressed genes was smaller in the pair of CD34+mono vs. monocytes (n=605) compared with the other pair (n=2136), suggesting that CD34+mono might stand nearby monocytes. However, a principle component analysis of GEP demonstrated that CD34+mono would be categorized into an independent cell type entity. In addition, the hierarchical clustering heat map suggested that CD34+mono would have the interim gene expressions between CD34+ cells and monocytes (Figure 2). GO analysis was also performed using the 203 genes which were differentially expressed in CD34+mono compared with CD34+cells and monocytes. The differentially expressed genes seemed to be significantly involved in the "immune system process" of biological process, followed by "locomotion", "metabolic process" and "response to stimulus", because pro-inflammatory genes in CD34+mono like IL6, CCL3, IL8, VEGFA, and IL1A were increased. In addition, PPI analyses indicated that 128 of the 203 differentially expressed genes in CD34+mono had close connections with each other. Especially, IL6, VEGFA, IL8, NFkB1, EGR1, CDKN1A (p21), and CYCS could be considered as a hub gene in CD34+mono. Of them, IL6, IL8, and VEGFA are considered to be pro-inflammatory cytokines. NFkB1, and EGR1 are transcriptional factors, and CDKN1A, and CYCS are considered to regulate cell cycle or apoptosis. Furthermore, simultaneously focusing on the difference in the fold changes of gene expressions between CD34+mono vs. monocytes, CD83 (a membrane protein and immunoglobulin superfamily that regulates antigen presentation) and FOSL1 (a kind of regulators of cell proliferation, differentiation, and transformation) were specifically increased in CD34+mono (Figure3). The increased expression of CD83 strongly suggests antigen presenting cells like mature dendritic cells. CD34+mono might be one of progenitors of dendritic cells, or might have a potential of antigen presentation itself. [Conclusion] In summary, the differentially expressed genes in CD34+mono would be mapped in the immune process, especially against infectious pathogens. In addition, the increased CD83 suggested that CD34+mono might play a role of antigen presentation in the immune response. Further investigations would be necessary to confirm the clinical roles of CD34+mono after transplantation. Disclosures Nakasone: Phizer: Honoraria; Novartis: Honoraria; Kyowa Hakko Kirin: Honoraria; Celgene: Honoraria; Bristol-Myers Squibb: Honoraria; Janssen: Honoraria; Takeda: Honoraria. Kimura:Astellas: Honoraria; Pfizer: Honoraria; Sumitomo Dainippon Pharma: Honoraria; MSD: Other: Investigator in the institute; Nippon Kayaku: Honoraria; Celgene: Honoraria; Kyowa Hakko Kirin: Honoraria; Takeda: Honoraria. Kako:Takeda Pharmaceutical Company Limited.: Honoraria; Takeda Pharmaceutical Company Limited.: Honoraria; Celgene K.K.: Honoraria; Bristol-Myers Squibb: Honoraria; Sumitomo Dainippon Pharma Co., Ltd.: Honoraria; Chugai Pharmaceutical Co., Ltd.: Honoraria; Otsuka Pharmaceutical Co., Ltd.: Honoraria; Ono Pharmaceutical Co., Ltd.: Honoraria; Janssen Pharmaceutical K.K.: Honoraria. Kanda:Eisai: Consultancy, Honoraria, Research Funding; Dainippon-Sumitomo: Consultancy, Honoraria, Research Funding; MSD: Research Funding; Nippon-Shinyaku: Research Funding; Pfizer: Research Funding; Kyowa-Hakko Kirin: Consultancy, Honoraria, Research Funding; Chugai: Consultancy, Honoraria, Research Funding; Otsuka: Research Funding; Taisho-Toyama: Research Funding; CSL Behring: Research Funding; Tanabe-Mitsubishi: Research Funding; Astellas: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria; Ono: Consultancy, Honoraria, Research Funding; Taiho: Research Funding; Shionogi: Consultancy, Honoraria, Research Funding; Sanofi: Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Asahi-Kasei: Research Funding; Novartis: Research Funding; Celgene: Consultancy, Honoraria; Mochida: Consultancy, Honoraria; Alexion: Consultancy, Honoraria; Takara-bio: Consultancy, Honoraria.
4
Desterke, Christophe, Estelle Balducci, Xavier Fund, Claire Borie, Annelise Bennaceur-Griscelli, and Ali G. Turhan. "A Novel Metabolic Transcriptome Identified in Myelodysplastic Syndromes (MDS) Correlates with OMS Classification and Poor Prognosis." Blood 132, Supplement 1 (November 29, 2018): 5495. http://dx.doi.org/10.1182/blood-2018-99-110678.
Анотація:
Abstract Myelodysplastic syndromes (MDS) are clonal malignancies of the hematopoietic stem cell leading to an ineffective hematopoiesis with a complex and poorly understood pathophysiology combining increased apoptosis and propensity to transformation associated with immune dysregulation. Despite a major improvement of the classification of MDS in terms of diagnosis and prognosis according to OMS recommendations, a significant fraction of MDS remains unclassified. In this study, we wished to determine if the integrative analysis of global MDS transcriptome associated with single cell experiments performed on CD34+ hematopoietic progenitors using novel bioinformatics tools could identify novel signaling pathways associated with different subtypes of MDS. This transcriptomic analysis included a large cohort of of MDS patients (n=323) as compared to healthy controls (n=86). We concentrated our analysis in the pathways involved in metabolomics as there was an increase of expression of genes involved in metabolic pathways in MDS transcriptome as compared to hematopoietic progenitors from healthy donors. Metabolic meta-analysis on HP transcriptome (datasets GSE15061 and GSE58831) were subsequently analyzed by creating a software "gene2bcp.sh" (https://github.com/cdesterke/gene2bcp) based on metabolic database "BioCycPathway". Forty two genes involved in metabolism were found to be upregulated in MDS samples (LIMMA algorithm, FDR adjust p-value < 0.05 and fold change > 2 in both dataset). A multivariate model was built by selecting the best 17 genes and in order to validate these findings an independent cohort including 11 healthy donors and 55 MDS patients (pts) were studied. This latter group included 18 patients with refractory anemia (RA), 19 pts with RA with ring sideroblasts (RARS) and 18 pts with RA with excess of blasts (RAEB) (GSE4619). This analysis (fig1A) allowed to predict MDS diagnosis as compared to healthy donors in a robust way (p=0.0013). Similarly with this unique metabolic transcriptome, we could stratified MDS patients correctly according to their OMS classification (p=0.00065). Metabolic transcriptome also clearly identified patients with MDS with cytogenetic abnormalities, in particular MDS with 5q- syndromes as compared to MDS without cytogenetic abnormalities (p=0.0023). We then applied these results to the RNAseq single-cell sequencing with cytometric analysis of a MDS patient with RAEB2 and monosomy 7 (GSE99095). Single cell trajectories were built based on 39 metabolic genes identified from previous meta-analysis. Monocle2 algorithm with metabolic gene profile allowed to discriminate CD34+CD38- from CD34+CD38+ cells after "tSNE" clustering (fig1B). These results were submitted to a pseudotime analysis which allows the analysis of transitional states in cell signaling pathways. Nine transition stages were found with stage 5 enriched in CD34+CD38- and stages 1 and 7 were found enriched in CD34+CD38+ (fig1C), differential test on this metabolic trajectory stratified genes in 6 clusters (gig 1D), with an enriched high expression of ACOT4 (Acyl-CoA Thioesterase 4), EARS2 (Glutamyl-TRNA Synthetase 2), PLD6 (Phospholipase D Family Member 6), PSAT1 (Phosphoserine Aminotransferase 1) in sub-compartment CD34+CD38- of this RAEB2 MDS patient. Overall, we show here for the first time a novel metabolic transcriptome correlating with classical MDS subtypes and prognosis. We describe a primitive metabolic trajectory in single bone marrow cells able to reconstitute the clonal architecture. The genes that we identified could be of use as additional markers of diagnosis and prognosis in future studies. Disclosures No relevant conflicts of interest to declare.
5
Perrone, Olivia, Tiziana Coppola, James Bartram, Waseem Nasr, Juying Xu, and Marie-Dominique Filippi. "The Effect of SCD-1 Inhibition on Human Hematopoietic Stem Cell Mitochondrial Metabolism, Cell Proliferation, and Differentiation Potential." Blood 142, Supplement 1 (November 28, 2023): 1308. http://dx.doi.org/10.1182/blood-2023-185260.
Анотація:
Hematopoietic stem cells (HSC) give rise to all blood lineages and sustain the production of blood cells throughout life. Due to their inherently high regenerative potential, HSC are used in a variety of clinical settings, including bone marrow transplantation (BMT) directly to cure a variety of hematologic and oncologic disorders often with gene therapy. During regeneration, HSC are activated into cycle. For this, HSC undergo drastic mitochondrial and metabolic remodeling to meet the bioenergetic and biosynthetic needs of activated HSC. A growing body of evidence indicates that HSC sustain injury during activation. This remodeling is important for optimal HSC function but is permanently changed after HSC activating stress. It is thus important to identify the metabolic needs of activated HSC to improve HSC functions for therapeutic purposes. In both murine and human HSC, numerous metabolic enzymes get upregulated during activation, including those involved in de novo lipid synthesis. This metabolism is poorly understood in human HSC. Our overarching question is to understand the metabolic needs of HSC within the human system. Stearoyl-co-A-desaturase 1 (SCD-1), an enzyme responsible for conversion of stearic to oleic acid within the de novo lipid synthesis pathway, is suspected to play a role in metabolic reprogramming after stress. Using CD34+ mobilized human peripheral blood, we analyzed the effect of SCD-1 inhibition on HSC and progenitor proliferation, mitochondrial metabolism, and lineage differentiation potential. Cells were cultured either in vehicle control conditions or with SCD-1 inhibitor (SCDi). Subsequently, cells were counted and analyzed by flow cytometry on Days 4-5 and 10-15. More primitive or stem cell markers were utilized on Days 0 and 5 whereas differentiation markers were utilized on Day 10-15. Both tetramethylrhodamine-ethyl ester dye (TMRE) and MitoSOX reagents were used to assess mitochondrial membrane potential and mitochondrial ROS, respectively. Our results demonstrate that the size of the HSC population generated by SCDi-treated CD34+ cells within 5 days of culture was similar to vehicle treated cells and was composed of similar proportion of CD34+CD38+, CD34+CD38-, and CD38+CD34- cells, indicating that SCD-1 inhibition does not alter HSC expansion under these culture conditions. However, TMRE levels were lower in all HSC populations whereas mitoSOX levels were unchanged by SCD-1 inhibition compared to vehicle. In differentiation conditions, SCDi cultures were composed of a larger proportion of myeloid progenitors and a smaller proportion of erythroid cells after 10-15 days, compared to vehicle-treated cultures. These findings suggest that de novo lipid synthesis is necessary for HSC differentiation but dispensable for proliferation/expansion in vitro. We then used a xenotransplant model to assess the effect of SCDi on HSC regenerative potential in vivo. CD34+ mobilized human peripheral blood cells were cultured for 3 days under conditions that maintain HSC functions in vitro and transplanted into immunodeficient, sublethally irradiated NSG mice. Peripheral blood of xenotransplanted mice was analyzed monthly for human cell chimerism and mature blood lineage potential from 1 to 6 months post-transplant. Bone marrow analysis was performed at the 6 month time point as well, assessing for both HSC markers and lineage markers. Mice that received SCDi-treated cells exhibited human cell chimerism at a level similar to mice that were transplanted with vehicle treated cells, at about 3 to 30%. However, SCDi-treated cells gave rise to a lymphoid-biased graft, in particular T cells, both in the peripheral blood and in the bone marrow of xenotransplanted mice, compared to a more balanced myeloid-lymphoid graft from vehicle treated cells. In conclusion, these findings suggest that de novo lipid synthesis is critically important for HSC lineage fate and balanced differentiation in vitro and in vivo. This is especially clinically relevant as this work may implicate a possible therapeutic target as improving de novo lipid synthesis may aid in patients who suffer from persistent cytopenias after BMT and graft failure.
6
Devaraj, Sridevi, and Ishwarlal Jialal. "Dysfunctional Endothelial Progenitor Cells in Metabolic Syndrome." Experimental Diabetes Research 2012 (2012): 1–5. http://dx.doi.org/10.1155/2012/585018.
Анотація:
The metabolic syndrome (MetS) is highly prevalent and confers an increased risk of diabetes and cardiovascular disease. A key early event in atherosclerosis is endothelial dysfunction. Numerous groups have reported endothelial dysfunction in MetS. However, the measurement of endothelial function is far from optimum. There has been much interest recently in a subtype of progenitor cells, termed endothelial progenitor cells (EPCs), that can circulate, proliferate, and dfferentiate into mature endothelial cells. EPCs can be characterized by the assessment of surface markers, CD34 and vascular endothelial growth factor receptor-2, VEGFR-2 (KDR). The CD34+KDR+phenotype has been demonstrated to be an independent predictor of cardiovascular outcomes. MetS patients without diabetes or cardiovascular diseases have decreased EPC number and functionality as evidenced by decreased numbers of colony forming units, decreased adhesion and migration, and decreased tubule formation. Strategies that have been shown to upregulate and enhance EPC number and functionality include statins, angiotensin converting enzyme inhibitors, angiotensin receptor blockers, and peroxisome-proliferator-activating-receptor gamma agonists. Mechanisms by which they affect EPC number and functionality need to be studied. Thus, EPC number and/or functionality could emerge as novel cellular biomarkers of endothelial dysfunction and cardiovascular disease risk in MetS.
7
Dalloul, Ali H., Claire Patry, Jean Salamero, Bruno Canque, Fernanda Grassi, and Christian Schmitt. "Functional and Phenotypic Analysis of Thymic CD34+CD1a− Progenitor-Derived Dendritic Cells: Predominance of CD1a+ Differentiation Pathway." Journal of Immunology 162, no. 10 (May 15, 1999): 5821–28. http://dx.doi.org/10.4049/jimmunol.162.10.5821.
Анотація:
Abstract Whether thymic dendritic cells (DC) are phenotypically and functionally distinct from the monocyte lineage DC is an important question. Human thymic progenitors differentiate into T, NK, and DC. The latter induce clonal deletion of autoreactive thymocytes and therefore might be different from their monocyte-derived counterparts. The cytokines needed for the differentiation of DC from thymic progenitors were also questioned, particularly the need for GM-CSF. We show that various cytokine combinations with or without GM-CSF generated DC from CD34+CD1a− but not from CD34+CD1a+ thymocytes. CD34+ thymic cells generated far fewer DC than their counterparts from the cord blood. The requirement for IL-7 was strict whereas GM-CSF was dispensable but nonetheless improved the yield of DC. CD14+ monocytic intermediates were not detected in these cultures unless macrophage-CSF (M-CSF) was added. Cultures in M-CSF generated CD14−CD1a+ DC precursors but also CD14+CD1a− cells. When sorted and recultured in GM-CSF, CD14+ cells down-regulated CD14 and up-regulated CD1a. TNF-α accelerated the differentiation of progenitors into DC and augmented MHC class II transport to the membrane, resulting in improved capacity to induce MLR. The trafficking of MHC class II molecules was studied by metabolic labeling and immunoprecipitation. MHC class II molecules were transported to the membrane in association with invariant chain isoforms in CD14+ (monocyte)-derived and in CD1a+ thymic-derived DC but not in monocytes. Thus, thymic progenitors can differentiate into DC along a preferential CD1a+ pathway but have conserved a CD14+ maturation capacity under M-CSF. Finally, CD1a+-derived thymic DC and monocyte-derived DC share very close Ag-processing machinery.
8
Nishida, Yuki, Edward Ayoub, Darah Scruggs, Shayaun Khazaei, Faryal Munir, Lauren B. Ostermann, Po Yee Mak, et al. "Stem-Cell Enriched Cellular Hierarchy of TP53 Mutant Acute Myeloid Leukemia Is Vulnerable to Targeted Protein Degradation of c-MYC." Blood 142, Supplement 1 (November 28, 2023): 583. http://dx.doi.org/10.1182/blood-2023-174938.
Анотація:
Deregulation of MYC genes occurs in up to 70% of all human cancers and is associated with hallmarks of cancer including mitochondrial and ribosomal biogenesis, cell cycle progression, and metabolic abnormalities. TP53 regulates MYC while MYC suppresses TP53, suggesting counteracting negative feedback loops. Therefore, MYC or its function can be activated when TP53 is not functional. TP53 mutations occur in 30% of relapsed/refractory acute myeloid leukemias (AMLs) patients' survival is dismal, and there are no effective therapies for these patients. Compared to TP53 wild-type (TP53wt), TP53 mutant (TP53mut) AMLs have lower percentages and numbers of leukemia blasts with increased immature CD34+ cells and resistance to chemo- or molecularly targeted therapies. However, the exact cellular hierarchy of TP53mut AML has not been elucidated. We observed significantly increased MYC mRNA levels in (TP53mut), as compared to TP53wt AML, and also increased levels in TP53mut versus TP53wt AML leukemia stem cell (LSC) fractions. This finding was confirmed in a dataset from the Munich Leukemia Laboratory (N = 732). We found significantly upregulated MYC pathways in TP53mut compared to TP53wt AML LSC. We confirmed the increased MYC mRNA levels at the protein level in TP53mut AML by single-cell mass cytometry (CyTOF). To dissect the cellular hierarchy in TP53mut AML, we performed single-cell RNA sequencing of 32 BM samples from healthy donors (N = 3), newly diagnosed, high-risk TP53wt (N = 7) and TP53mut (N = 22) AML patients, with 6,685, 10,687 and 10,687 cells from normal, TP53wt and TP53mut AML bone marrow (BM) cells, respectively. We found highly enriched HSC-like cells and reduced progenitor- and GMP-like cells in TP53mut AML compared to normal BM (NBM) and TP53wt AML samples. We overlayed MYC expression levels on the mapping of cellular components and found higher MYC levels in HSC-like cells in TP53mut AML compared to HSC and HSC-like cells in NBM and TP53wt AML samples, suggesting enrichment of immature HSC-like cells and increased activity of MYC in TP53mut AML LSCs ( Fig. A). To target c-MYC and MYC signaling, we utilized GT19715, the first-in-class cereblon modulator (CELMoD) for c-MYC protein (Nishida, ASH 2022). CyTOF confirmed the presence of much increased c-MYC protein levels in primary CD34+ AML than in CD34+ NBM hematopoietic stem cells (HSCs). Notably, CD34+ AML cells showed much greater sensitivity to GT19715 compared to CD34+ NBM cells. Data suggest on-target activity of GT19715 against c-MYC in LSCs with a therapeutic window between LSCs vs. NBM HSCs. Intriguingly, c-MYC protein levels are higher in CD34+CD38+ than in CD34+CD38- AML cells, suggesting that c-MYC drives the proliferation of AML progenitor cells differentiating from quiescent LSCs. Consequently, CD34+CD38+ AML progenitor cells exhibited greater sensitivity to GT19715 compared to CD34+CD38- LSCs. Using paired, TP53 null HL-60 GT19715-sensitive and -resistant cells generated through chronic exposure to GT19715, we interrogated the impact of GT19715 on metabolic changes. GT19715 induced pronounced reductions in basal and maximal oxygen consumption rates (OCRs) in GT19715-sensitive HL-60 cells. GT19715 reduced both basal, glutamine- and glucose-dependent extracellular acidification rates (ECARs) in GT19715-sensitive cells while no inhibition in OCRs or ECARs was observed in GT19715-resistant cells. GT19715 severely inhibited ECARs even after adding glucose to GT19715-sensitive cells, suggesting irreversible inhibition of glycolysis as one of the mechanisms of action of GT19715. GT19715 profoundly reduced AML blasts in TP53mut AML samples (N = 3) ( Fig. B), and in a very aggressive patient-derived xenograft (PDX) TP53mut AML model established from a patient with TP53 p.Y220C and p.P151A mutations along with MECOM rearrangement and K/NRAS mutations. In humanized Crbn I391V mice, where the Crbn-mediated protein degradation is operational, GT19715 only reduced WBC counts along with minimal body weight loss. GT19715 but did not reduce total mouse BM CD45+ cells, suggesting favorable toxicity profiles of GT19715. In conclusion, TP53mut AML comprised highly enriched LSC populations compared to TP53wt AML and targeting of c-MYC protein is highly effective in TP53mut AML in vitro and in vivo with a therapeutic window between AML LSC and normal hematopoietic cells.
9
Rai, Richa, Foramben Patel, Stella Melana, Jonathan Feld, Shyamala C. Navada, Rosalie Odchimar-Reissig, Erin P. Demakos, E. Premkumar Reddy, and Lewis R. Silverman. "Rigosertib in Combination with Azacitidine Impacts Metabolic and Differentiation Pathways in the MDS-L Cell Line." Blood 136, Supplement 1 (November 5, 2020): 35–36. http://dx.doi.org/10.1182/blood-2020-142908.
Анотація:
Background Myelodysplastic Syndrome (MDS) is characterized by ineffective clonal hematopoiesis with peripheral blood cytopenias, leading to death from infection or bleeding. Azacitidine (AZA), a hypomethylating agent (HMA) is the standard of care for treatment of MDS patients (pts) with higher-risk MDS [Silverman LR, The Myelodysplastic Syndrome in Cancer Medicine, Editors: R.J. Bast, et al. 2017]. Responses to AZA occur in 50% of pts with significant effects on hematopoiesis ranging from improvement in a single lineage to complete restoration of blood counts and transfusion independence [Silverman LR, et al. Leukemia, 1993]. AZA treatment is associated with global DNA hypomethylation, including human endogenous retroviruses (HEV) which further activates innate immune signaling [Chiappinelli KB,et al. Cell, 2015]. The exact mechanism by which AZA improves hematopoiesis is unknown. AZA improves overall survival of pts, yet despite this, 100% of pts ultimately fail treatment with worsening cytopenias or transformation to leukemia [Silverman LR, et al. B. J Clin Oncol, 2002; Cancer, 2011]. Thus, understanding the mechanism of resistance and identification of targets which can reverse HMA failure and improve hematopoiesis in MDS pts is critical. Our clinical data demonstrate that AZA combined with Rigosertib (RIGO), a novel Ras mimetic that inhibits Ras/Raf signaling [Athuluri-Divakar SK, et al. Cell, 2016], yields a response rate of 54% of pts who were HMA failures [Navada SC, et al. EHA 2019]. The response was associated with significant improvement in hematopoiesis and represents a critical observation in overcoming the epigenetic clinical resistance phenotype. The precise mechanism that leads to reversal of the resistance phenotype is poorly understood. Methods: We investigated the differential protein expression in response to different treatment (AZA, RIGO alone and sequential combinations RIGO/AZA; AZA/RIGO) in vitro in MDS-L cell line by Reverse phase protein array (RPPA). Further, we also studied the functional role of these treatments on differentiation in the cell line by growing cells on semi-solid media as well as by flow cytometry using various stem cell and differentiation markers (CD34, CD38, CD45, CD123). Results: RPPA analysis indicated a discrete responses to treatment in the MDS-L cell line. The response was prominent with differential expression of 43 proteins specifically in MDS-L cells treated with RIGO/AZA that include PIK3R1, AKT1, mTOR, p38 MAPK, PTEN, RPS6KA1 (Fold change (FC) < -2). All of these proteins are downregulated which suggests inhibition of PI3K and mTOR signaling. Proteins belonging to metabolic pathways, including ACC1 and ACLY, were found to be downregulated, whereas proteins related to mitochondrial function and oxidative phosphorylation (OXPHOS) were upregulated (FC > 1.5/< -1.5) in cells treated with RIGO/AZA. In addition, we found that MDS-L cells represented both CD34+CD38+ and CD34+CD38- populations by FACS analysis (Fig 1). AZA increases the percentage of CD34+CD38+, indicative of differentiation, whereas RIGO alone increased the percentage of CD34+CD38- cells, representing a primitive stem cell population (Table in Fig 1). Based on the combination of differentiation markers (illustrated in fig 1), we observed that RIGO alone, and sequenced as combination RIGO/AZA, impacts different progenitors such as granulocyte-macrophage progenitor (GMP), megakaryocyte erythroid progenitor (MEP), and multipotent progenitor (MPP). RIGO/AZA treatment showed a decrease in GMP, while the % of MEP was increased, as compared to other treatments. Moreover, we found a remarkable reduction in the number of colony forming units on differentiation media in response to RIGO (83%) and RIGO/AZA (90%). Conclusions: These data demonstrate that in addition to inhibition of the PI3/AKT/mTOR pathway, the RIGO/AZA combination also impacts metabolic and differentiation pathways of MDS-L cells. RIGO alone appears to promote maintenance of a primitive stem cell population, while the RIGO/AZA sequenced combination appears to push the cells toward a cycling stage with increased expression of genes associated with OXPHOS. In comparison, when treated with RIGO, cells remain in a less differentiated stage. Further studies are underway to determine the effect of metabolic changes on differentiation and maintenance of hematopoietic stem cells. Figure Disclosures Navada: Onconova Therapeutics Inc: Research Funding. Reddy:Onconova Therapeutics Inc: Research Funding. Silverman:Celgene: Research Funding; Medimmune: Research Funding; Onconova Therapeutics Inc: Patents & Royalties, Research Funding.
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Forte, Dorian, Roberto Maria Pellegrino, Francesco Fabbri, Ivan Vannini, Samantha Bruno, Giulia Corradi, Rafael J. Argüello, et al. "Circulating Extracellular Vesicles from Acute Myeloid Leukemia Patients Drive Distinct Metabolic Profile of Leukemic Cells and Reveal Crucial Lipidomic Biomarkers." Blood 138, Supplement 1 (November 5, 2021): 3471. http://dx.doi.org/10.1182/blood-2021-150339.
Анотація:
Abstract Background. Extracellular vesicles (EVs) are submicron vesicles released from various cell types including blood cells with pleiotropic effects on cell signalling and metabolism. EV cargos are enriched in nucleic acids, proteins, and lipids that can be delivered to target cells to influence surrounding microenvironment. Thus, EVs represent a powerful tool for liquid biopsy in hematological malignancies including acute myeloid leukemia (AML). AML is an aggressive disease with high relapse rate and less invasive tools are urgently needed to investigate disease (metabolic) dynamics. Accumulating evidence has reported a key role for EVs in shaping the AML bone marrow niche. However, at present, the metabolic function and the lipidomic signature driven by circulating EVs have yet to fully emerge. Methods. Peripheral blood (PB) and bone marrow (BM) were collected from AML patients at diagnosis (n=40) and PB from age/sex-matched healthy donors (HD, n=20). EVs were purified from platelet-poor plasma by size exclusion chromatography and quantified using the NanoSight technology. Immunomagnetically isolated CD34+ cells from umbilical cord blood (CB) or AML patients were characterized by analyzing the hematopoietic stem/progenitor cell (HSPC)-specific cluster of differentiation marker expression, redox metabolic profiling (using CellROX, glutathione detection reagent and MitoTracker) after 24 hours co-culture with EVs. Quantitative lipidomic profiling of circulating EVs was performed by Liquid Chromatography coupled with High-Resolution Mass Spectrometry (LC/HRMS). Seahorse extracellular flux analyses were performed in leukemia cell lines (including KG-1, KASUMI-1, MOLM-13, THP-1 and OCI-AML3). To functionally define the metabolic reprogramming of leukemic cellular components within their microenvironment, leukemic stem cell subsets were assessed by flow cytometry-based SCENITH (Single Cell ENergetic metabolism by profilIng Translation inHibition) method in both whole blood and BM samples (n=4). Results. In our work, plasma-derived EVs from AML patients showed a significant increase in the size and protein amounts compared to HD counterparts. To explore the metabolic perturbation triggered by EVs, we developed a co-culture system with circulating EVs from either HD or AML patients with CB or AML CD34+. We found a reduction in the frequency of AML CD34+ with high ROS levels in the presence of AML EVs without affecting the ROS levels in normal CB CD34+. In parallel, AML EVs increased the frequency of AML CD34+ with both high mitochondrial activity and glutathione, a key antioxidant molecule involved in many metabolic pathways. Similar metabolic profiles were also confirmed in human leukemic cell lines tested. Specifically, Seahorse flux analysis revealed that EVs induced a cell energy phenotype consistent with quiescent and chemoresistant state in human leukemic cell lines, showing a more glycolytic state in MOLM-13. Interestingly, both CD34+ and CD34+/CD38- leukemic fractions from whole blood and BM of the same AML patients were analyzed by SCENITH after co-cultures with HD/AML EVs. Remarkably, PB CD34+/CD38- leukemic fractions were more dependent on mitochondrial activity in the presence of AML EVs, suggesting a metabolic shift triggered by leukemic EV that apparently occur in the leukemic fractions out of the BM niche. In addition, to give insights into lipidomic signatures of EVs as disease biomarkers, we detected a total of 25 (out of 200) independent lipid species significantly different between AML-derived EVs and HD (n=20, respectively). We reported the abundance of both glycerolipid and fatty acids species in AML EVs. Also, through a multivariate statistical analysis of EV lipidomic profile, we revealed that AML EVs were depleted in sphingomyelin classes, a class of lipids that are interconnected to HSC metabolism. Finally, according to the 2017 ELN risk stratification system, we observed the depletion in important modulators of EV release and formation as ether-linked phosphatidylethanolamine and phosphatidylethanolamine species in adverse-risk AML patients. Conclusion. Overall, our study provides the basis for further investigations on the metabolic alterations trigger by EVs within the BM microenvironment and suggests prognostic biomarkers for leukemic patients that might reveal novel metabolic vulnerabilities in AML scenario. Disclosures Cavo: Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: TRAVEL, ACCOMMODATIONS, EXPENSES, Speakers Bureau; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel Accommodations, Speakers Bureau; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Adaptive Biotechnologies: Consultancy, Honoraria; GlaxoSmithKline: Consultancy, Honoraria; Bristol-Myers Squib: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Curti: Jazz Pharma: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees.
11
Rattazzi, Marcello, Sabina Villalta, Silvia Galliazzo, Laura Del Pup, Alessandra Sponchiado, Elisabetta Faggin, Elisa Bertacco, et al. "Low CD34+ cells, high neutrophils and the metabolic syndrome are associated with an increased risk of venous thromboembolism." Clinical Science 125, no. 4 (May 1, 2013): 211–22. http://dx.doi.org/10.1042/cs20120698.
Анотація:
The relationship between MetS (metabolic syndrome), levels of circulating progenitor/immune cells and the risk of VTE (venous thromboembolism) has not yet been investigated. We studied 240 patients with previous VTE and 240 controls. The presence of MetS was identified according to NCEP ATP III guidelines and flow cytometry was used to quantify circulating CD34+ cells. VTE patients showed higher BMI (body mass index), waist circumference, triacylglycerol (triglyceride) levels, blood glucose, hs-CRP (high-sensitivity C-reactive protein) and lower HDL-C (high-density lipoprotein cholesterol) levels. The prevalence of MetS was significantly higher in VTE (38.3%) than in control individuals (21.3%) with an adjusted OR (odds ratio) for VTE of 1.96 (P=0.002). VTE patients had higher circulating neutrophils (P<0.0001), while the CD34+ cell count was significantly lower among patients with unprovoked VTE compared with both provoked VTE (P=0.004) and controls (P=0.003). Subjects were also grouped according to the presence/absence of MetS (MetS+ or MetS−) and the level (high/low) of both CD34+ cells and neutrophils. Very high adjusted ORs for VTE were observed among neutrophils_high/MetS+ (OR, 3.58; P<0.0001) and CD34+_low/MetS+ (OR, 3.98; P<0.0001) subjects as compared with the neutrophils_low/MetS− and CD34+_high/MetS− groups respectively. In conclusion, low CD34+ blood cell count and high circulating neutrophils interplay with MetS in raising the risk for venous thromboembolic events.
12
Subedi, Amit, Qiang Liu, David Sharon, Severine Cathelin, Gary D. Bader, Changjiang Xu, Veronique Voisin, et al. "Nicotinamide Phosphoribosyltransferase Inhibitors Induce Apoptosis of AML Stem Cells through Dysregulation of Lipid Metabolism." Blood 136, Supplement 1 (November 5, 2020): 25–26. http://dx.doi.org/10.1182/blood-2020-142404.
Анотація:
Current chemotherapeutic regimens for acute myeloid leukemia (AML) often fail to eliminate leukemic stem cells (LSCs) which contribute to disease relapse. A key step towards the development of more effective therapies is the identification of vulnerabilities that are unique to LSCs. Here, we sought to identify LSC-specific metabolic dependencies by performing a flow cytometry-based screen of 110 metabolically-focused drugs against a primary human AML sample. This sample harbored distinct subsets defined by CD34 and CD38 expression, and LSC activity assayed by xenotransplantation was restricted to the CD34+CD38- fraction. Through this screen, we found that inhibitors of nicotinamide phosphoribosyltransferase (NAMPT), which catalyzes the rate-limiting step in the NAD+ salvage pathway, preferentially depleted CD34+CD38- cells, implicating NAMPT inhibitors as potential anti-LSC agents. To evaluate the therapeutic potential of NAMPT inhibitors, we focused on KPT-9274, a small-molecule NAMPT inhibitor currently under clinical development for other cancer types. Treatment with KPT-9274 depleted the CD34+CD38- fraction across multiple primary human AML samples through induction of apoptosis. The preferential sensitivity of CD34+CD38- cells to NAMPT inhibition correlated with a lower basal level of intracellular NAD+ and greater dependency on NAMPT activity for NAD+ generation relative to the other fractions. In contrast, normal CD34+ HSPCs were largely resistant to the cytotoxic effects of KPT-9274 due to their capacity to utilize the Preiss-Handler pathway for NAD+ generation. Consistent with the in vitro findings, KPT-9274 treatment significantly reduced LSC activity as determined by secondary engraftment potential in 2 of 3 patient-derived xenograft (PDX) models of human AML and had minimal impact on normal HSC activity in mice engrafted cord blood cells. To gain mechanistic insights into how NAMPT inhibition induces cell death, we performed transcriptomic analysis of sorted CD34+CD38- cells treated with KPT-9274. This analysis revealed a striking upregulation of genes involved in cholesterol and lipid synthesis including the stearoyl-CoA desaturase (SCD) gene. The upregulated genes were highly enriched for known targets of the sterol regulatory element binding protein (SREBP) transcription factors. Functional studies demonstrated that this transcriptional response was protective against the cytotoxic effect of NAMPT inhibition in AML cells. To uncover the metabolic basis of this protective effect, we performed global metabolomic profiling of AML cells treated with KPT-9274 and observed a decrease in the ratio of monounsaturated fatty acids (MUFAs) to saturated fatty acids (SFAs) upon drug treatment. This drop in MUFA:SFA ratio reflected a reduction in SCD activity which catalyzes the desaturation of SFAs to MUFAs in a NADPH-dependent reaction. Since depletion of intracellular MUFAs could trigger apoptosis, we hypothesized that the SREBP response might protect against cell death through upregulation of SCD activity and consequent increase in MUFA synthesis. In line with this hypothesis, we found that exogenous oleic acid, a MUFA, completely rescued cell death induced by KPT-9274, while treatment with SCD inhibitors sensitized AML cells to the cytotoxic effects of NAMPT inhibition. To explore the translational application of our findings, we tested whether dipyridamole (DP), a clinically approved anti-platelet agent with inhibitory activity against SREBP signaling, can be repurposed to enhance the anti-leukemic effects of KPT-9274. We showed that treatment with DP, at non-toxic concentrations, potentiated the cytotoxicity of KPT-9274 against AML cells in vitro. Importantly, in vivo combination treatment with KPT-9274 and DP effectively targeted LSC activity in a PDX model that was refractory to KPT-9274 as single agent. In summary, our findings demonstrate that LSCs are preferentially dependent on NAMPT activity for survival over non-LSCs and normal HSCs. We further uncovered that NAMPT inhibition results in dysregulation of lipid homeostasis and induces a lipogenic response coordinated by SREBPs that protects AML cells against NAD+ depletion. These findings offer insights into drug combination strategies to enhance the efficacy of NAMPT inhibitors and provide the rationale for testing NAMPT inhibitors in the treatment of AML in clinical trials. Disclosures Dick: Bristol-Myers Squibb/Celgene: Research Funding. Wang:Trilium therapeutics: Patents & Royalties: There is an existing license agreement between TTI and University Health Network and J.C.Y.W. may be entitled to receive financial benefits further to this license and in accordance with UHN's intellectual property policies. .
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Díaz-Flores, Lucio, Ricardo Gutiérrez, Maria Pino García, Miriam González-Gómez, Rosa Rodríguez-Rodriguez, Nieves Hernández-León, Lucio Díaz-Flores, and José Luís Carrasco. "Cd34+ Stromal Cells/Telocytes in Normal and Pathological Skin." International Journal of Molecular Sciences 22, no. 14 (July 8, 2021): 7342. http://dx.doi.org/10.3390/ijms22147342.
Анотація:
We studied CD34+ stromal cells/telocytes (CD34+SCs/TCs) in pathologic skin, after briefly examining them in normal conditions. We confirm previous studies by other authors in the normal dermis regarding CD34+SC/TC characteristics and distribution around vessels, nerves and cutaneous annexes, highlighting their practical absence in the papillary dermis and presence in the bulge region of perifollicular groups of very small CD34+ stromal cells. In non-tumoral skin pathology, we studied examples of the principal histologic patterns in which CD34+SCs/TCs have (1) a fundamental pathophysiological role, including (a) fibrosing/sclerosing diseases, such as systemic sclerosis, with loss of CD34+SCs/TCs and presence of stromal cells co-expressing CD34 and αSMA, and (b) metabolic degenerative processes, including basophilic degeneration of collagen, with stromal cells/telocytes in close association with degenerative fibrils, and cutaneous myxoid cysts with spindle-shaped, stellate and bulky vacuolated CD34+ stromal cells, and (2) a secondary reactive role, encompassing dermatitis—e.g., interface (erythema multiforme), acantholytic (pemphigus, Hailey–Hailey disease), lichenoid (lichen planus), subepidermal vesicular (bullous pemphigoid), psoriasiform (psoriasis), granulomatous (granuloma annulare)—vasculitis (leukocytoclastic and lymphocytic vasculitis), folliculitis, perifolliculitis and inflammation of the sweat and sebaceous glands (perifolliculitis and rosacea) and infectious dermatitis (verruca vulgaris). In skin tumor and tumor-like conditions, we studied examples of those in which CD34+ stromal cells are (1) the neoplastic component (dermatofibrosarcoma protuberans, sclerotic fibroma and solitary fibrous tumor), (2) a neoplastic component with varying presentation (fibroepithelial polyp and superficial myxofibrosarcoma) and (3) a reactive component in other tumor/tumor-like cell lines, such as those deriving from vessel periendothelial cells (myopericytoma), epithelial cells (trichoepithelioma, nevus sebaceous of Jadassohn and seborrheic keratosis), Merkel cells (Merkel cell carcinoma), melanocytes (dermal melanocytic nevi) and Schwann cells (neurofibroma and granular cell tumor).
14
Wu, Andrew, Katharina Rothe, Min Chen, Hanyang Lin, Artem Babaian, Ryan Yen, Donna L. Forrest, and Xiaoyan Jiang. "Inhibition of the MiR-185-PAK6-Mediated Survival and Metabolic Pathways Selectively Targets Drug-Resistant CML Stem/Progenitor Cells." Blood 134, Supplement_1 (November 13, 2019): 4138. http://dx.doi.org/10.1182/blood-2019-127826.
Анотація:
Overcoming drug resistance and targeting leukemic stem cells (LSCs) remain major challenges for curative treatment of human leukemia, including chronic myeloid leukemia (CML). Indeed, most patients with CML require life-long therapy with ABL1 tyrosine kinase inhibitors (TKIs), due to the persistence of residual LSCs that maintain the potential for relapse. Increasing evidence also indicates that LSCs are susceptible to cellular metabolic changes and seem to have a greater dependence on mitochondrial oxidative phosphorylation (OXPHOS) for survival. Previously, through global transcriptome profiling, we identified a key microRNA (miRNA), miR-185 as a predictive biomarker and it is also required for CML LSC survival. Its expression was significantly reduced in CD34+treatment-naïve CML cells and predictive of therapy response. Conversely, restored expression of miR-185 by lentiviral transduction in CD34+TKI-nonresponder cells significantly impaired survival of these cells, sensitizing them to TKIs in vitroand in pre-clinical xenotransplantation models, indicating that miR-185 acts as a tumor suppressor and is critical in regulating TKI response/resistance of CML stem/progenitor cells. PAK6, a serine/threonine-protein kinase, was identified as a target gene of miR-185; it is upregulated in CD34+TKI-nonresponder cells vs. TKI-responders, correlating with reduced miR-185 expression. To further investigate the molecular and biological roles of the miR-185-PAK6 axisin the regulation of survival of drug-resistant cells, including LSCs, we performed RNA-seq and gene set enrichment analysis (GSEA) in the same CD34+patient cells where miR-185 and PAK6 were identified as being differentially expressed. Interestingly, this analysis has now identified a significant gene set enrichment of OXPHOS, reactive oxygen species (ROS), and adipogenesis pathways in CD34+CML cells compared to healthy CD34+cells (Normalized Enrichment Scores (NES): 2.44, 1.65 and 1.8). Moreover, these changes were significantly higher in TKI-nonresponder cells than in TKI-responders (NES: 1.73, 0.49 and 0.34). We have thus hypothesized that the miR-185-PAK6 axis may contribute to the perturbation of specific metabolic pathways in TKI-nonresponder LSC/progenitor cells and confer therapy-resistance to these cells. Indeed, a pre-clinically validated pan-PAK inhibitor (PF-3758309) alone, or in combination with a TKI, greatly reduced mitochondrial activity in TKI-nonresponder cells, in MitoTracker analysis, an effect that was not seen in the same cells treated with a TKI. Notably, ROS production was also significantly reduced in these cells treated with PF-3758309 and further reduction was observed with a combination of PF-3758309 and TKIs. Notably, PF-3758309 significantly reduced the growth of IM-resistant cell lines (IC50 25-70 nM) and CD34+TKI-nonresponder cells, as assessed by viability and colony-forming cell assays, and increased their apoptosis; these effects were greatly enhanced by TKIs (2-fold, P<0.05). These results were further confirmed in TKI-resistant cells using a lentiviral CRISPR/Cas9 knockout system that specifically targets PAK6. In addition, specific molecular changes associated with PF-3758309 treatment were also investigated using the PharmacoDB database and PharmacoGx R-package. Several candidates were identified, including growth factor independent 1B transcriptional repressor (GFI1B), a myeloid-enriched transcription factor. Its expression was reduced by PF-3758309 treatment and significantly increased in CML compared to healthy controls (>2-fold). Interestingly, expression of GFI1B is further upregulated in CD34+TKI-nonresponders compared to responders. Taken together, these findings indicate that dual targeting of miR-185-PAK6-mediated survival and metabolic pathways, along with BCR-ABL, selectively eradicates therapy-resistant LSC/progenitors, providing a valuable therapeutic strategy for improved treatment and care. Disclosures No relevant conflicts of interest to declare.
15
Kuntz, Elodie Marie, Pablo Baquero, Tessa L. Holyoake, Eyal Gottlieb, and G. Vignir Helgason. "Therapy Resistant CML Stem Cells Are Dependent on Mitochondrial Oxidative Metabolism for Their Survival." Blood 128, no. 22 (December 2, 2016): 932. http://dx.doi.org/10.1182/blood.v128.22.932.932.
Анотація:
Abstract We and others have shown that tyrosine kinase inhibitors (TKIs), such as imatinib, fail to eliminate primitive chronic myeloid leukaemia (CML) stem cells (LSCs), suggesting that combination of TKIs with other targeted agents will be required to eradicate the LSC-pool. Therefore, identification of targetable pathways that selectively maintain CML LSC survival is critical. Metabolic reprogramming is a core feature of cancer cells making them susceptible to manipulation in a selective manner. Indeed, in recent years numerous studies have shown that targeting abnormal aspects of metabolism can be of therapeutic value. The aim of this study was to identify and target metabolic dependencies in CML LSCs using stem cell-enriched (CD34+) primary cells isolated from CML patients and healthy donors. Although CML represents a highly suitable model for cancer stem cell studies, investigation of LSCs metabolism has so far been restricted by technical limitations. Therefore, we have applied improved protocols for metabolomics using liquid chromatography-mass spectrometry (LC-MS) and functional assays. Initially we cultured leukaemic cells in the presence of uniformly-labelled glucose with stable (heavy) 13C isotope (13C6-glucose) and compared isotopic enrichment in CD34+ versus CD34- cells isolated from the same CML patient. Our results showed that CD34+ cells contained an increased proportion of isotopologues with 2 or more labelled carbons in tricarboxylic acid (TCA) cycle metabolites (such as citrate, α-ketoglutarate and malate) when compared with CD34- cells, indicating an increase in flux of glucose through the TCA cycle in more primitive CML cells. In contrast, CML CD34+ cells contained reduced levels of glucose-derived lactate when compared with patient-matched CD34- cells, suggesting that more primitive CML cells utilise mitochondrial metabolism rather than glycolysis to supply their energy demands. In line with this, CML CD34+ cells displayed more than a two-fold increase in their mitochondrial oxygen consumption rates (OCR) when compared with CD34- cells (p≤0.05), confirming that mitochondrial metabolism is enhanced in stem cell-enriched CML cells. Next we traced 13C6-glucose in CD34+ cells from healthy donors and compared this with isotopic enrichment in CML CD34+ cells. This revealed that 13C enrichment in TCA cycle metabolites was significantly higher in CML CD34+ cells when compared with their normal counterparts. This correlated with a significant increase in mitochondrial respiration (p≤0.001) and mitochondrial membrane potential in primitive CML cells, including CD34+38- cells (p≤0.001), suggesting that CML LSCs may be selectively sensitive to inhibition of mitochondrial metabolism. Of clinical significance, we show that the antibiotic tigecycline, an inhibitor of mitochondrial translation, reduced this aberrant oxidative metabolism and selectively induced death in primitive CML cells at a clinically achievable concentration. More precisely, in vitro treatment with tigecycline as a single agent, decreased the number of CML CD34+ cell-derived colonies in comparison with untreated conditions (p≤0.001), and combining tigecycline with imatinib resulted in a 50% decrease in colony number when compared to tigecycline or imatinib alone (p≤0.01). Importantly, this drug combination had no effect on colony formation potential of CD34+ cells derived from healthy donors. Moreover, we show that tigecycline alone, or in combination with imatinib, reduced the number of colonies in a long-term culture-initiating cell assay (p≤0.001) while imatinib, as a single agent, did not have any significant effect. To examine the effect on transplantable CML LSCs in vivo, human CML CD34+ cells were injected into irradiated NSG mice. Following confirmation of engraftment mice were treated with imatinib, tigecycline alone and in combination with imatinib. Remarkably, 4-week combination treatment with tigecycline and imatinib led to near complete elimination of CML LSCs measured by the number of human CD45+ and CD34+38- cells in the bone marrow. We conclude that CML LSC are dependent on oxidative phosphorylation for their survival and tigecycline-mediated inhibition of mitochondrial metabolism, combined with TKI treatment, shows potential as a novel therapeutic strategy to selectively target these cells to enhance cure rates. Disclosures Holyoake: Bristol Myers Squib: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.
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Thomas, Geethu, Laura Garcia Prat, Marcela Gronda, Rose Hurren, Neil MacLean, Xiaoming Wang, Botham Aaron, John E. Dick, and Aaron D. Schimmer. "The Metabolic Enzyme Hexokinase 2 Localizes to the Nucleus in AML and Normal Hematopoietic Stem/Progenitor Cells to Maintain Stemness." Blood 132, Supplement 1 (November 29, 2018): 2795. http://dx.doi.org/10.1182/blood-2018-99-110021.
Анотація:
Abstract Hematopoietic cells are arranged in a hierarchy where stem and progenitor cells differentiate into mature blood cells. Likewise, AML (Acute Myeloid Leukemia) is also hierarchical with leukemic stem and progenitor cells giving rise to more mature and differentiated blasts. Recent studies have shown that mitochondrial enzymes such as IDH2 can regulate AML stemness by altering metabolites that affect epigenetic marks. However, it is unknown whether mitochondrial metabolic enzymes can directly localize to the nucleus to regulate stemness in AML and normal hematopoietic cells. Here, we show that the mitochondrial enzyme, Hexokinase 2, localizes to the nucleus in AML and normal hematopoietic stem cells to maintain stemness. We sought to identify mitochondrial metabolic enzymes that localize to the nucleus of stem cells, by evaluating the stem and bulk fractions from 8227 leukemia cells. 8227 leukemia cells are arranged in a hierarchy with functionally defined stem cells present in the CD34+CD38- fraction. We separated 8227 cells into CD34+CD38- and CD34-CD38+ populations by FACS sorting and prepared lysates of the nuclear and cytoplasmic fractions from each population. Using immunoblotting, we measured levels of mitochondrial enzymes in the subcellular fractions of each population. We discovered that the metabolic enzyme Hexokinase 2 (HK2) was increased in the nuclear fraction of 8227 stem cells compared to bulk cells. In contrast, other mitochondrial enzymes such as Aconitase 2 and Succinate Dehydrogenase B were not detected in the nuclear fractions. HK2 is an outer mitochondrial membrane protein that phosphorylates glucose to glucose-6-phosphate, thereby initiating glycolysis and the entry of glucose metabolites into the TCA cycle in the mitochondria. The nuclear localization of HK2 in mammalian cells has not been previous reported. We confirmed that 8227 cells have nuclear HK2 by confocal fluorescent microscopy and also demonstrated nuclear HK2 in AML cell lines (OCI-AML2, NB4, K562, and MV411) and primary AML samples. We also FACS sorted normal cord blood into populations of stem/progenitor (HSC, MPP, MLP, CMP, GMP and MEP) and differentiated (B cells, T cells, NK cells, Monocytes and Granulocytes) cells. The localization of HK2 in these cell fractions was measured by immunofluorescence and quantified by Metamorph and Imaris. Nuclear HK2 was detected in the stem/progenitor cells and progressively declined to minimal levels as the cells matured (Fig 1A). The mitochondrial localization of HK2 is dependent on AKT-mediated phosphorylation of Thr-473 and inhibited by dephosphorylation by the phosphatase PHLPP1. We asked whether phosphorylation of HK2 regulates the nuclear abundance of HK2. Using AML2 cells, we showed that knockdown of PHLPP1 decreased the abundance of nuclear HK2, while inhibition of AKT increased HK2 in the nucleus. Finally, we tested whether the nuclear localization of HK2 was functionally important to maintain stemness. We over-expressed HK2 tagged with nuclear localizing signals (PKKKRKV and PAAKRVKLD) in 8227 and NB4 leukemia cells. We confirmed the selective over-expression of HK2 in the nucleus of these cells by immunoblotting and immunofluorescence. Increasing nuclear HK2 did not alter the proliferation of the cells under basal conditions. However, increasing nuclear HK2 enhanced clonogenic growth and blocked retinoic acid-mediated cell differentiation. In summary, we discovered that the unphosphorylated form of the metabolic enzyme HK2 localizes to the nucleus in malignant and normal hematopoietic stem cells and is functionally important to maintain stem/progenitor state. Thus, we define a new role for mitochondrial enzymes in the regulation of stemness and differentiation. Disclosures Schimmer: Medivir AB: Research Funding; Jazz Pharmaceuticals: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Otsuka Pharmaceuticals: Consultancy.
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Pershina, Pakhomova, Widera, Ermakova, Epanchintsev, Pan, Krupin, et al. "Gender Differences in the Pharmacological Actions of Pegylated Glucagon-Like Peptide-1 on Endothelial Progenitor Cells and Angiogenic Precursor Cells in a Combination of Metabolic Disorders and Lung Emphysema." International Journal of Molecular Sciences 20, no. 21 (October 30, 2019): 5414. http://dx.doi.org/10.3390/ijms20215414.
Анотація:
In clinical practice, the metabolic syndrome (MetS) is often associated with chronic obstructive pulmonary disease (COPD). Although gender differences in MetS are well documented, little is known about sex-specific differences in the pathogenesis of COPD, especially when combined with MetS. Consequently, it is not clear whether the same treatment regime has comparable efficacy in men and women diagnosed with MetS and COPD. In the present study, using sodium glutamate, lipopolysaccharide, and cigarette smoke extract, we simulated lipid metabolism disorders, obesity, hyperglycemia, and pulmonary emphysema (comorbidity) in male and female C57BL/6 mice. We assessed the gender-specific impact of lipid metabolism disorders and pulmonary emphysema on angiogenic precursor cells (endothelial progenitor cells (EPC), pericytes, vascular smooth muscle cells, cells of the lumen of the nascent vessel), as well as the biological effects of pegylated glucagon-like peptide 1 (pegGLP-1) in this experimental paradigm. Simulation of MetS/COPD comorbidity caused an accumulation of EPC (CD45−CD31+CD34+), pericytes, and vascular smooth muscle cells in the lungs of female mice. In contrast, the number of cells involved in the angiogenesis decreased in the lungs of male animals. PegGLP-1 had a positive effect on lipids and area under the curve (AUC), obesity, and prevented the development of pulmonary emphysema. The severity of these effects was stronger in males than in females. Furthermore, PegGLP-1 stimulated regeneration of pulmonary endothelium. At the same time, PegGLP-1 administration caused a mobilization of EPC (CD45−CD31+CD34+) into the bloodstream in females and migration of precursors of angiogenesis and vascular smooth muscle cells to the lungs in male animals. Gender differences in stimulatory action of pegGLP-1 on CD31+ endothelial lung cells in vitro were not observed. Based on these findings, we postulated that the cellular mechanism of in vivo regeneration of lung epithelium was at least partly gender-specific. Thus, we concluded that a pegGLP-1-based treatment regime for metabolic disorder and COPD should be further developed primarily for male patients.
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Michurova, Marina Sergeevna, Victor Yur'evich Kalashnikov, Olga Michailovna Smirnova, Olga Nikolaevna Ivanova, and Sergey Anatol'evich Terekhin. "Mobilization of endothelial progenitor cells after endovascular interventions in patients with type 2 diabetes mellitus." Diabetes mellitus 17, no. 4 (December 8, 2014): 35–42. http://dx.doi.org/10.14341/dm2014435-42.
Анотація:
Aim. To investigate the mobilisation of endothelial progenitor cells (EPC) in patients with type 2 diabetes mellitus (T2DM) after endovascular interventions for coronary and peripheral arteries. Materials and Methods. The levels of EPC in peripheral blood were determined by flow cytometry in 42 patients prior to endovascular intervention and 2?4 days after surgery. EPC were defined as CD34+ VEGFR2+ CD45- and CD34+ CD133+CD45- cells. Twenty-three patients with T2DM were included in group 1, and 19 patients without metabolic disorders were included in group 2. Results. The levels of EPC in the peripheral blood of patients with T2DM before and after endovascular interventions were not significantly different. In the subgroup of patients without TDM2, the levels of CD34+VEGFR2 +CD45- cells increased after surgery to 55,5% (p
19
Krüger, Karsten, Rainer Klocke, Julia Kloster, Sigrid Nikol, Johannes Waltenberger, and Frank C. Mooren. "Activity of daily living is associated with circulating CD34+/KDR+ cells and granulocyte colony-stimulating factor levels in patients after myocardial infarction." Journal of Applied Physiology 116, no. 5 (March 1, 2014): 532–37. http://dx.doi.org/10.1152/japplphysiol.01254.2013.
Анотація:
The study aimed to investigate whether the extent of activities of daily living (ADL) of patients after myocardial infarction affect numbers of circulating CD34+/KDR+ and CD45+/CD34+ cells, which are supposed to protect structural and functional endothelial integrity. In a cross-sectional study, 34 male coronary artery disease patients with a history of myocardial infarction were assessed for times spent per week for specific physical ADL, including basic activities (instrumental ADL), leisure time activities, and sport activities, using a validated questionnaire. Individual specific activity times were multiplied with respective specific metabolic equivalent scores to obtain levels of specific activities. Numbers of circulating CD34+/KDR+ and CD45+/CD34+ cells were analyzed by flow cytometry. Furthermore, the colony-forming capacity of CD34+ cells and the level of granulocyte colony-stimulating factor (G-CSF) in serum were measured. Analysis revealed that the extent of total activities and basic activities, as well as total activity time, were positively correlated with numbers of circulating CD34+/KDR+ cells ( r = 0.60, 0.56, and 0.55, P < 0.05). Higher levels of total activity were also associated with increased colony-forming capacity of CD34+ cells ( r = 0.54, P < 0.05) and with higher systemic levels of G-CSF ( r = 0.44, P < 0.05). These findings indicate that even ADL-related activities of coronary artery disease patients after myocardial infarction exert stimulating effects on CD34+/KDR+ cell mobilization, potentially mediated by increased G-CSF levels. This, in turn, potentially contributes to the beneficial effects of exercise on the diseased cardiovascular system.
20
Guo, Bin, Xinxin Huang та Hal E. Broxmeyer. "Antagonizing PPARγ Expands Human Hematopoietic Stem and Progenitor Cells By Switching on FBP1-Repressed Glycolysis and Preventing Differentiation". Blood 130, Suppl_1 (7 грудня 2017): 709. http://dx.doi.org/10.1182/blood.v130.suppl_1.709.709.
Анотація:
Abstract Allogeneic hematopoietic cell transplantation (HCT) is widely used as a life-saving treatment for malignant and non-malignant blood disorders. Hematopoietic stem cells (HSCs) are a major contributing cell population for a successful HCT. While cord blood (CB) is an acceptable source of HSCs for clinical HCTbecause of its many advantages including prompt availability, lower incidence of GvHD and virus infection, CB HCT is usually associated with slower time to engraftment especially in adult patients when compared with other cell sources; this is partly due to limiting numbers of HSCs in single cord units. In order to overcome this limitation, ex vivo expansion of CB HSCs has been evaluated in preclinical and clinical studies for improvement of the clinical efficacy of CB HCT. While a number of different ways have been evaluated to ex-vivo expand human HSCs, little is known about the mechanisms involved, and whether efficient expansion of CB HSCs could be achieved by metabolic reprogramming. In a compound screen for potential candidates which could promote ex vivo expansion of CB HSCs, we found that PPARγ antagonist GW9662 treatment significantly enhanced ex vivo expansion of CB phenotypic HSCs (~5 fold) and progenitor cells (HPCs) (~6.8 fold) in RPMI-1640 medium containing 10% fetal bovine serum (FBS) and cytokines (SCF, FL, TPO) when compared with vehicle control. GW9662 significantly increased numbers of CB colony-forming unit (CFU) granulocyte/macrophage (GM) (~1.8 fold) and granulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM) (~3.2 fold) progenitors after 4 days ex vivo culture. To assess whether the ex vivo expanded CB HSCs enhanced by the PPARγ antagonist were functional in vivo, we performed both primary and secondary transplantation in immunocompromised NSG mice. Engraftment of CB CD34+ cells in primary recipients was significantly increased (~3 fold) both in bone marrow (BM) and peripheral blood (PB) by the cultured cells treated with GW9662. The percentages of both myeloid and lymphoid lineages were enhanced in BM of primary recipients transplanted with GW9662-treated CB CD34+ cells. We also transplanted CB CD34+ cells transfected with control shRNA or PPAR γ shRNA into NSG mice, and consistently found that both myeloid and lymphoid chimerism was enhanced in BM of recipients which were infused with PPAR γ shRNA transfected-CD34+ cells compared with control shRNA transfected-CD34+ cells. Long term reconstituting and self-renewing capability of GW9662-treated CB CD34+ cells with both enhanced myeloid and lymphoid chimerism, was confirmed in PB and BM in secondary recipients. Limiting dilution analysis was performed to calculate SCID-repopulating cells (SRC), a measure of the number of functional human HSCs. The SRC frequency of GW9662-cultured CB CD34+ cells was 4 fold greater than that of day 0 uncultured CD34+ cells, and 5 fold increased above that of vehicle-treated CD34+ cells with cytokines alone. To gain mechanistic insight into how PPARγ antagonism enhances expansion of human CB HSCs and HPCs, we performed RNA-seq analysis. Antagonizing PPARγ in CB CD34+ cells resulted in downregulation of a number of differentiation associated genes, including CD38, CD1d, HIC1, FAM20C, DUSP4, DHRS3 and ALDH1A2, which suggests that PPARγ antagonist may maintain stemness of CB CD34+ cells partly by preventing differentiation. Of interest, we found that FBP1, encoding fructose 1, 6-bisphosphatase, a negative regulator of glycolysis, was significantly down-regulated by GW9662, which was further confirmed by RT-PCR, western blot and flow cytometry analysis. GW9662 significantly enhanced glucose metabolism in CB HSCs and HPCs without compromising mitochondrial respiration. Enhanced expansion of CB HSCs by antagonizing PPARγ was totally suppressed by removal of glucose or by inhibition of glycolysis. Importantly, suppression of FBP1 greatly promoted glycolysis and ex vivo expansion of long-term repopulating CB HSCs (~3.2 fold). Overexpression of FBP1 significantly suppressed enhancedexpansion and engraftment of CB HSCs by PPARγ antagonist. Our study demonstrates that PPARγ antagonism drives ex vivo expansion of human CB HSCs and HPCs by switching on FBP1 repressed glucose metabolism and by preventing differentiation. This provides new insight into human HSC self-renewal, and suggests a novel and simple means by which metabolic reprogramming may improve the efficacy of CB HCT. Disclosures No relevant conflicts of interest to declare.
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Forte, Dorian, Filippo Maltoni, Samantha Bruno, Paulina Garcia-Gonzalez, Gianluca Cristiano, Chiara Sartor, Simona Soverini, et al. "Single-Cell Metabolic Profiling Integrated with Extracellular Vesicle Analysis Reveals Novel Metabolic Vulnerabilities and Prognostic Biomarkers in Acute Myeloid Leukemia." Blood 142, Supplement 1 (November 28, 2023): 1598. http://dx.doi.org/10.1182/blood-2023-185909.
Анотація:
Introduction Acute myeloid leukemia (AML) is an aggressive disease for which less invasive tools are needed to explore disease dynamics. Changes in cell metabolism and metabolic adaptation are hallmark features for AML, thus emerging as promising therapeutic targets. However, it is still poorly elucidated whether and which extrinsic microenvironment signals may regulate the metabolic reprogramming occurring in AML. In this regard, Extracellular Vesicles (EVs) represent a promising tool for non-invasive liquid biopsy and may reveal a novel role in energy metabolism as well as clinically relevant biomarkers for AML. Along with EV profiling from peripheral blood (PB), we mapped the metabolic signature of leukemic stem cells (LSCs) and immune cell subsets to identify metabolic vulnerabilities and novel prognostic biomarkers. Methods PB (and paired bone marrow) were collected from AML patients at diagnosis (n=98) and healthy donors (HD, n=12). EVs were purified from platelet-free PB plasma of AML patients by size-exclusion chromatography followed by ultrafiltration. We have submitted all relevant data of our EV experiments to the EV-TRACK knowledgebase (EV-TRACK ID: EV230004). MACSPlex Exosome Kit was used for screening up to 37 surface EV markers. Western blot analysis was also performed. Energy cell metabolism was explored by Single Cell ENergetic metabolism by profilIng Translation inHibition (SCENITH TM) in LSC-like cells (CD34 +CD38 low/-)and immune cell subsets (CD3 +/CD4 +/CD8 +/T regulatory cells) using conventional/spectral flow-cytometry. Results Here, we developed a novel method exploiting both LSCs and EVs from AML patients using fresh whole blood for metabolic exploration. Firstly, we observed that LSC-like cells display low reactive oxygen species (ROS) levels with both high glutathione (GSH, as antioxidant molecule) levels and mitochondrial functionality suggesting a more resistant phenotype. Consistently, poor clinical outcomes were experienced by AML patients treated with conventional chemotherapy and with high GSH levels and mitochondrial potential in LSC-like cells (log-rank p=0.04). We also found high dependence on glucose and a preferential skewing toward glycolysis in LSC-like cells revealing a distinct metabolic status for LSCs in circulation. However, according to European LeukemiaNet (ELN) 2022 risk classification, a more prominent mitochondrial dependence on LSC-like cells was reported for intermediate- or adverse-risk AML patients revealing a prognostic relevance. By contrast, exploring the immunometabolic reprogramming in circulation, we revealed a significant decrease in glucose dependence for immune cells (CD4 + and CD8 + T cells) in comparison to LSCs proving the presence of a complex metabolic network in AML. Interestingly, we investigated the potential metabolic effects of EVs on LSCs. Indeed, CD34 + LSC-like cells co-cultured with circulating EVs enhanced their GSH levels and mitochondrial functionality towards a more aggressive phenotype. Accordingly, we discovered the presence of the antioxidant enzyme, glutathione peroxidases 3 (GPx-3), in AML EVs. Then, we profiled the surface proteins of isolated EVs to detect their cell origins and provide a reference map for clinical prognosis. We found that circulating AML EVs were significantly enriched in CD4 (helper T cell marker), CD14 (monocyte marker), CD40 (B-cell associated marker), CD44 and CD133-1 (stem cell markers), CD105 (stromal cell marker) and HLA-II (antigen-presenting cell marker) compared to HD EVs. In particular, we found that intermediate-risk AML patients showed a strong depletion in the expression of EV markers including T cell markers (CD2 and CD8) and mesenchymal markers (CD49e and CD146) in comparison to favorable and adverse-risk patients. Interestingly, AML patients who had been treated not intensively and showed low expression of CD40 (HR=0.23, 95% CI, 0.05−0,99, p=0.04) or CD62P (platelet activation marker, HR=0.24, 95% CI, 0.05−1.02, p=0.05) on EVs experienced poor outcomes suggesting that dedifferentiated phenotype on EVs might be associated with worse prognosis. Conclusion Overall, towards a new redefined ‘metabolic’ stratification for AML patients, we identified novel prognostic biomarkers for AML patients that might reveal novel metabolic vulnerabilities by exploiting in parallel single-cell metabolic profile and EV-based liquid biopsy.
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Pierre-Louis, Olivier, Denis Clay, Bernadette Guerton, Christophe Desterke, Aurelie Chabanon, Jean-Valer Malfuson, Jean-Jacques Lataillade, and Marie-Caroline Le Bousse-Kerdiles. "A New Multiparametric Flow Cytometry Technique Based on Combined Side Population (SP) and Aldehyde Deshydrogenase (ALDH) Functionalities Identifies a Hierarchy within the Human Hematopoietic Stem/Progenitor Compartment." Blood 110, no. 11 (November 16, 2007): 2226. http://dx.doi.org/10.1182/blood.v110.11.2226.2226.
Анотація:
Abstract Hematopoietic stem cells (HSC) are characterized by their potential to reconstitute in vivo haematopoiesis in NOD/SCID mice and to give rise to differentiated cells of all haematopoietic lineages. They are usually defined by a CD34+CD38−CD90+ antigenic profile; however taking into account the versatility of antigen membrane expression (Lataillade et al., J. Leukoc. Biol. 2005), more reliable methods based on their selective functional/metabolic activities such as ABCG2 activity and Aldehyde Deshydrogenase expression have been developed. Actually, HSC can be purified on basis of Hoechst 33342 die exclusion property due to the membrane ABCG2/BRCP1 transporter expression (SP cells). SP cells could be isolated from a wide variety of human and mammalian tissues and in many cases have been shown to contain multipotent stem cells; SP cells are heterogeneous since the lowest Hoechst fluorescent profile exhibited the highest primitive hierarchical level. Hematopoietic progenitors also expressed ALDH enzymatic activity and the use of BODIPY-AminoAcetAldehyde (BAAA), a fluorescent substrate of the ALDH enzyme, allows analyzing its expression by using Fluorescence Activated Cell Sorting (FACS). It has been reported that there is a close association between high ALDH expression (ALDHbr) and high HSC activity. Based on the hypothesis that the more primitive HSC should express both ALDH high expression level and SP profile, we developed a new multiparamatric flow cytometric assay combining an ALDH/SP co-labelling to identify different haematopoietic progenitor subpopulations. Our strategy allows identifying six lineage negative (Lin−) haematopoietic sub-populations from human bone marrow cells according to the co-expression of ALDH levels and SP phenotype: SP+ALDHBr, SP−ALDHBr, SP+ALDHMid, SP−ALDHMid, SP+ALDHLo and SP-ALDHLo. We showed that these different sub-populations exhibited specific antigenic patterns and demonstrated variable differentiation properties in long term cultures and clonogenic assays. Whatever the ALDH expression level, SP+ and SP−cells expressed the CD34 antigen, however, the SP phenotype identify a higher proportion of CD34+CD38− cells. Furthermore, in contrast to the other subpopulations, Lin-SP+ALDHBr cells were highly enriched with CD34+CD38−CD90+ progenitor cells and demonstrated a higher amplification and high clonogenic efficiency in liquid LTC-IC cultures than SP-ALDHBr cells. This subpopulation allowed obtaining all myeloid haematopoietic lineage differentiated cells after 30 days culture in liquid culture, including osteoclastic cells. Interestingly, in contrast to ALDHBr cells, ALDHLo sub-populations were highly enriched with megakaryocytic CD34+CD41+ progenitors that gave rise to CD41+CD42a+ cells. SP+ALDHMid and SP-ALDHMid have intermediate amplification, clonogenic and differentiation abilities as compared with the other subpopulations. In conclusion, our results show that the combined SP/ALDH technology allows discriminating a hierarchy within the human hematopoietic stem/progenitor compartment, the most primitive one being SP+ALDHBr. The stemness of these subpopulations is currently explored by testing their capability to reconstitute long term haematopoiesis in NOD/SCID mice.
23
Mendler, Jason H., Marlene Balys, Umayal Sivagnanalingam, Allison Eberhardt, Korinne Thorne, Tzu-Chieh Ho, Mark W. LaMere, et al. "Distinct Properties of Leukemia Stem Cells in Primary Refractory Acute Myeloid Leukemia." Blood 126, no. 23 (December 3, 2015): 685. http://dx.doi.org/10.1182/blood.v126.23.685.685.
Анотація:
Abstract Introduction: Acute Myeloid Leukemia (AML) patients who are refractory to induction chemotherapy (RF) have a dismal prognosis. Properties of AML cell populations that cause the refractory phenotype are poorly understood. It is postulated that the leukemia stem cell (LSC) pool promotes chemotherapy resistance in AML; yet whether differences exist in this pool as a function of eventual remission status, and thus may account for induction failure, is unknown. Our group has recently found that in AML patients who initially achieve complete remission (CR) and then relapse, the LSC pool evolves to become larger and more immunophenotypically diverse at the time of relapse [Blood 2013; 122(21): abstract #883]. Since relapsed AML patients are often refractory to salvage chemotherapy, we postulated that diagnostic specimens from RF AML patients would have enlarged, immunophenotypically-diverse LSC pools relative to AML patients achieving CR, implicating these properties in the refractory phenotype. Defining unique properties of LSCs in RF AML might provide greater insight into the mechanisms responsible for the treatment-resistant phenotype. Methods: RF AML patients were defined by having ≥ 6% leukemic blasts in their bone marrow or peripheral blood after 1 or 2 cycles of anthracycline/cytarabine-based induction chemotherapy. All studies were conducted with pre-treatment AML specimens on approved IRB protocols at the University of Rochester Medical Center. LSC frequency was determined by limiting dilution analysis and xenotransplantation into sublethally irradiated NOD scid gamma (NSG) mice. To determine immunophenotypically-defined cell populations harboring functional LSC activity, AML specimens were sorted into four distinct populations defined by CD34 and CD38 staining. The presence of LSC activity in the four resultant populations (CD34+/CD38-, CD34+/CD38+, CD34-/CD38+, and CD34-/CD38-) was determined by the ability of each sorted population to engraft NSG mice in primary and secondary transplantation experiments. RNA-seq analysis was conducted on LSC-enriched cell populations. Results: Specimens from RF AML patients were more likely to engraft NSG mice relative to those from AML patients achieving CR (6/6 vs. 13/29 in RF vs. CR patients, respectively; P=0.02). The LSC pool from six RF AML patients was studied in detail. LSC frequency ranged from 1/19 to 1/326,123. Four of the 6 RF specimens harbored LSC frequencies 6- to 7,700-fold higher than those previously reported in primary AML [Sarry et al. J Clin Invest 2011; 121(1384-395]. In 5/6 RF specimens, LSCs were present in more than one immunophenotypically-defined cell population. Relative to specimens from AML patients achieving CR, specimens from RF patients were more likely to harbor LSCs in both CD34+ and CD34- populations (P=0.04). Treatment of NSG mice xenografted with specimens from RF patients with anthracycline/cytarabine-based chemotherapy failed to eradicate disease from engrafted mice; thus, therapeutic outcome of NSG mice engrafted with specimens from RF patients resembles that seen in patients. To identify genes potentially driving the refractory LSC phenotype, we compared gene expression profiles of CD34+ cells from RF patients to those from AML patients achieving long-term remission and to those from normal donors. Ninety-nine genes were uniquely deregulated in RF LSCs, including numerous Homeobox transcription factors and components of the Wnt and Hedgehog signaling pathways, all implicated in the maintenance of stemness. Pathway analysis revealed that amino acid metabolic pathways critical to other treatment-resistant cancers and molecules involved in IL-1 signaling and implicated in metastasis of solid tumors are dysregulated in primary refractory LSCs. Conclusions: This study is the first to systematically analyze the LSC pool in primary refractory AML. Similar to our findings in relapsed AML, RF AML patients harbor enlarged, phenotypically diverse LSC pools relative to AML patients achieving CR. Challenge of leukemic mice with an anthracycline/cytarabine treatment regimen mimics the effect seen in patients, facilitating the development of patient-derived xenograft models of RF AML. We identify a refractory LSC gene signature, opening the door to future mechanistic investigations and novel therapeutic approaches for this AML patient population in great need. Disclosures Calvi: Fate Therapeutics: Patents & Royalties. Becker:Millenium: Research Funding.
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Kikushige, Yoshikane, Toshihiro Miyamoto, Takahiro Maeda, and Koichi Akashi. "Human Acute Leukemia Is Addicted to Branched-Chain Amino Acid Metabolism to Maintain Leukemia Stemness." Blood 134, Supplement_1 (November 13, 2019): 2516. http://dx.doi.org/10.1182/blood-2019-129372.
Анотація:
Hematopoietic stem cells (HSCs) have capabilities to self-renew, maintaining an undifferentiated status, as well as to proliferate and mature into blood cells. Similarly, cancer stem cells (CSCs) self-renew and propagate to form cancer tissues. In human acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL), CSC-like populations that can reconstitute human leukemia in immuno-deficient mice have been found, and are called leukemic stem cells or leukemia-initiating cells (LICs). In order to obtain "cure" by eradicating LICs, it should be critical to understand molecular machineries as to how LICs self-renew and expand to exert their cancer stemness properties. Recent studies revealed that several specific metabolism pathways actively contribute to the maintenance of stemness in several types of stem cells including ES/iPS cells. However, little is known about the LICs-specific metabolic activity. To clarify metabolic features of human LICs, we comprehensively analyzed 116 cellular metabolites of human CD34+ normal hematopoietic stem progenitor cells (HSPCs) (n=10), CD34+ AML (n=30) and CD34+ ALL cells (n=18). The metabolome analysis revealed that CD34+ AML and ALL cells commonly contain extremely high levels of branched chain amino acids (BCAA) as compared to normal CD34+ HSPCs. AML and ALL cells, but not normal HSCsexpressed BCAA-metabolism related enzymes as well as BCAA-transporters at high levels, and actively transport BCAAs into cytoplasm. Enzymatic inhibition of BCAA metabolism induced apoptosis in LICs but not in normal HSCs. Furthermore, deprivation of BCAA from daily diet in mice xeno-transplanted with human LICs caused significant inhibition of their reconstitution activities in vivo. Serial transplantation experiments revealed that hCD34+AML/ALL cells from BCAA-free-dieted mice exhibited extremely impaired reconstitution of human AML/ALL in secondary recipients fed with conventional diet, but those from mice with control diet developed AML/ALL with a high leukemia burden. To clarify how BCAA metabolism pathway regulates the leukemia-initiating activity, the global gene expression of primary CD34+AML /ALL cells and cell lines including THP-1(AML), Kasumi-9(B-ALL), Jurkat cells (T-ALL) were compared before and after pharmacological BCAA-metabolism inhibition and BCAA-deprivation. Gene set enrichment analysis (GSEA) revealed BCAA metabolism inhibition induced the expression of ES-related PRC2 target genes, which should be suppressed in ES cells via H3K27me3 histone modification, and ChIP-Seq analysis confirmed the significantly decreased H3K27me3 level around thetranscription start site ofPRC2 target genes both in CD34+ AML and ALL cells. Furthermore, BCAA metabolism inhibition or BCAA-deprivation resulted in the down regulation of EZH2 and EED, critical components of PRC2, at mRNA and protein level in human AML/ALL cells. Thus, BCAA-metabolism activity is a common metabolic machinery to maintain PRC2 function through potentiating EZH2 and EED expression in AML/ALL. We also found that human AML/ALL cells, but not normal HSPCs are dependent on BCAA metabolism for maintaining energy production through TCA cycle activity, leading to the adequate supply of alpha ketoglutarate (α-KG), an intermediate metabolite of TCA cycle. Since BCAT1, a catalytic enzyme for BCAA, requires α-KGas a substrate, the active energy production driven by BCAA metabolism contributes to prevent α-KG exhaustion to maintain the high BCAA catabolism activity in human AML/ALL. Consistent with persistent α-KG supply through enhanced BCAA metabolism, primary CD34+AML/ALL cells, which highly express BCAT1 did not exhibit decreased cellular α-KG level as compared to normal HSPCs in our metabolome analysis. Thus, the energy production and regulation of PRC2 function driven by enhanced BCAA metabolism constitute a vicious molecular cycle to maintain the leukemia stemness through the maintaining α-KG level in human acute leukemia. In summary, the current study demonstrates that human LICs are addicted to the activated BCAA metabolism to maintain their leukemia stemness irrespective of their lineage origin, and that the BCAA metabolic pathway is a generic therapeutic target in human acute leukemias. Disclosures Akashi: Sumitomo Dainippon, Kyowa Kirin: Consultancy; Celgene, Kyowa Kirin, Astellas, Shionogi, Asahi Kasei, Chugai, Bristol-Myers Squibb: Research Funding.
25
Qiu, Jiajing, Jana Gjini, Tasleem Arif, Kateri Moore, Miao Lin, and Saghi Ghaffari. "Using mitochondrial activity to select for potent human hematopoietic stem cells." Blood Advances 5, no. 6 (March 12, 2021): 1605–16. http://dx.doi.org/10.1182/bloodadvances.2020003658.
Анотація:
Abstract Hematopoietic cell transplantation is a critical curative approach for many blood disorders. However, obtaining grafts with sufficient numbers of hematopoietic stem cells (HSCs) that maintain long-term engraftment remains challenging; this is due partly to metabolic modulations that restrict the potency of HSCs outside of their native environment. To address this, we focused on mitochondria. We found that human HSCs are heterogeneous in their mitochondrial activity as measured by mitochondrial membrane potential (MMP) even within the highly purified CD34+CD38−CD45RA−CD90+CD49f+ HSC population. We further found that the most potent HSCs exhibit the lowest mitochondrial activity in the population. We showed that the frequency of long-term culture initiating cells in MMP-low is significantly greater than in MMP-high CD34+CD38−CD45RA−CD90+ (CD90+) HSCs. Notably, these 2 populations were distinct in their long-term repopulating capacity when transplanted into immunodeficient mice. The level of chimerism 7 months posttransplantation was >50-fold higher in the blood of MMP-low relative to MMP-high CD90+ HSC recipients. Although more than 90% of both HSC subsets were in G0, MMP-low CD90+ HSCs exhibited delayed cell-cycle priming profile relative to MMP-high HSCs. These functional differences were associated with distinct mitochondrial morphology; MMP-low in contrast to MMP-high HSCs contained fragmented mitochondria. Our findings suggest that the lowest MMP level selects for the most potent, likely dormant, stem cells within the highly purified HSC population. These results identify a new approach for isolating highly potent human HSCs for further clinical applications. They also implicate mitochondria in the intrinsic regulation of human HSC quiescence and potency.
26
Antonova, E. I., D. I. Omarova, N. V. Firsova, and K. A. Krasnikova. "The Role of Liver Progenitor Cells in Postembryonic Development of <i>Rana terrestris</i> under Normal Physiological Conditions." Uchenye Zapiski Kazanskogo Universiteta Seriya Estestvennye Nauki 166, no. 1 (March 15, 2024): 38–65. http://dx.doi.org/10.26907/2542-064x.2024.1.38-65.
Анотація:
The liver plays an essential role in the metabolism of animals, acting as a central hub for metabolic reactions. It serves as a “peripheral integrator” and balances the body’s energy needs. Its regenerative capacity is remarkably high and is maintained by the proliferation of hepatocytes, as well as hematopoietic and regional liver progenitor cells (LPC). This study investigated LPC-driven liver regeneration during postembryonic development in Rana terrestris under normal physiological conditions. The analysis of intrahepatic and hematopoietic markers by immunohistochemistry and flow cytometry revealed that progenitor cells with the immunophenotypes of CK19+ (intrahepatic progenitor cells), CD34+CD45+ (hematopoietic progenitor cell population), and CD34+CD45– (hemangioblast population) equally promote liver regeneration during the first year of postembryonic development. However, in the second and third years of postembryonic development, liver regeneration was found to be primarily associated with CK19+-positive cells, with a smaller contribution from CD34+CD45– cells. The results obtained were largely determined by the habitat of the amphibians, thermoregulation, and the completion of morphogenetic processes in the third year of postembryonic development. It is also noteworthy that the liver of the examined specimens remained the major hematopoietic organ throughout all observed stages of postembryonic development.
27
Chu, Su, Tinisha McDonald, and Ravi Bhatia. "Enhanced Phosphorylation and Altered Localization Lead to Impairment of p27kip Activity in CML Progenitor Cells Despite Enhanced Protein Translation and Expression." Blood 110, no. 11 (November 16, 2007): 999. http://dx.doi.org/10.1182/blood.v110.11.999.999.
Анотація:
Abstract The cyclin-dependent kinase inhibitor p27kip is a key regulator of hematopoietic progenitor proliferation and pool size. The activity of p27 can be regulated by modulation of its expression and localization in different cellular compartments. The levels of p27 protein expression are reported to be reduced in BCR-ABL expressing cell lines. In contrast p27 levels have been reported to be elevated in BCR/ABL expressing CML progenitor cells. However, CML progenitors paradoxically demonstrate enhanced proliferation despite having elevated levels of p27. Therefore the regulation of p27 protein expression and activity in primary CML progenitor cells is not well understood and requires further investigation. We have therefore performed a careful assessment of p27 expression, phosphorylation and localization in CD34+ cells from CML patients as well as in cord blood CD34+ cells transduced with retroviral vectors to ectopically express the BCR-ABL gene. We observed that p27 protein levels were markedly increased in both primary CML CD34+ cells and BCR/ABL transduced cells. CML CD34+ cells and BCR/ABL transduced CD34+ cells also demonstrated increased phosphorylation of p27 on T157 (an Akt-dependent phosphorylation site) and T187 (a cdk2-dependent site) on western blotting as well as increased tyrosine phosphorylation of p27 in immunoprecipitation studies. Immunofluorescence microscopy analysis of cells labeled with anti-p27 antibodies demonstrated primarily nuclear localization of p27 in normal CD34+ cells, but markedly reduced nuclear and increased cytoplasmic localization of p27 in CML CD34+ cells and BCR/ABL transduced CD34+ cells. Cdk2 activity was maintained, cdk4 activity was increased and phosphorylated RB levels were increased in BCR-ABL expressing hematopoietic cells, indicating CDKI activity was reduced in these cells despite increased total p27 levels. Consistent with this BCR-ABL expressing CD34+ cells demonstrated enhanced cell cycling compared to controls. Mutation of the Y177 residue in BCR-ABL (BCR-ABL-Y177F), which abrogates Grb2 binding and reduces Ras and Akt activation by BCR-ABL, reversed BCR-ABL induced abnormalities in p27 expression, phosphorylation and localization. Treatment of BCR-ABL transduced CD34+ cells with the PI-3K inhibitor LY294002 resulted in reduced phosphorylation of p27 on T157 but not T187, and restored nuclear localization of p27, further indicating an important role for AKT signaling in abnormal cytoplasmic localization of p27. Quantitative reverse transcription PCR analysis indicated that p27 mRNA levels were similar in BCR/ABL expressing and control CD34+ cells, suggesting that p27 expression is regulated mainly at the posttranscriptional level. Metabolic labeling of cells with S35-methionine showed that p27 translation was increased in BCR/ABL expressing cells, explaining the observed increase in total p27 protein levels. We conclude that p27 CDKI activity is reduced in BCR-ABL expressing progenitors as a result of altered p27 phosphorylation at important regulatory sites and a shift in cellular localization of p27 from the nucleus to the cytoplasm. The enhanced level of total p27 in primary CML progenitors reflects enhanced protein translation, which may be a compensatory response to reduced p27 CDKI activity. These results provide important insights into p27 deregulation in CML progenitors and its potential role in the expansion of the hematopoietic progenitor pool size in CML.
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Peng, Ching-Tien. "Metabolic Reprogramming of Human Mitochondrial NAD(P)+-Dependent-Malic Enzyme 2 in Acute Myeloid Leukemia." Blood 134, Supplement_1 (November 13, 2019): 5168. http://dx.doi.org/10.1182/blood-2019-123339.
Анотація:
Yu-Nan Huang1, Kang-Hsi Wu4, Te-fu Weng4, Su-Ching Liu4, Hui-Chih Hung1*, Ching-Tien Peng4,5* FLT3 internal tandem duplication (FLT3-ITD) mutations in patients with acute myeloid leukemia (AML) are usually associated with other mutations resulting in unfavorable outcome. Tyrosine kinase inhibitors (TKI) have shown promising responses, however, these responses are almost transient in therapy-resistant AML. Here, we show that human mitochondrial NAD(P)+-dependent-malic enzyme 2 (ME2) have significantly increased in CD34+ cell of patients with AML. To determine how ME2 establish metabolic reprogramming of leukemogenesis, we performed a comprehensive analysis of metabolism in CRISPR-mediated ME2 knockout leukemic cells (THP-1 and MV4-11) and purified leukemic blast cells (CD34+) derived from patients with AML. We demonstrate that disrupting ME2 signaling exerts potent activities against proliferation, reduced oxidative metabolism and lactate metabolism. We also show that genetic inhibition of RUNX1/FLT3/ME2 markedly repressed AML cell leukemogenesis. In conclusion, our findings provide a rationale for clinical development of this strategy for treating RUNX1 and FLT3-mutated leukemic patients. Disclosures No relevant conflicts of interest to declare.
29
Irifune, Hidetoshi, Yu Kochi, Masayasu Hayashi, Yoshikane Kikushige, Toshihiro Miyamoto, and Koichi Akashi. "Identification of GPAT1 As a Novel Therapeutic Target for Acute Leukemia By Inhibiting Leukemia Specific Metabolism." Blood 134, Supplement_1 (November 13, 2019): 1384. http://dx.doi.org/10.1182/blood-2019-125661.
Анотація:
With the development of mass spectrometer technology, recent studies revealed the critical roles of cancer-specific metabolism for tumor propagation in several types of cancers. In leukemia, many studies have been conducted to elucidate a leukemia-specific metabolism, and several effective treatments such as IDH1/2 inhibitors targeting acute myeloid leukemia (AML) with IDH1/2 mutation have been developed. To identify the new metabolic pathways on which acute leukemia cells depend, we purified water-soluble metabolites from CD34+ hematopoietic stem and progenitor cells (HSPCs) of healthy donors, AML and acute lymphoblastic leukemia (ALL) patients, and we comprehensively measured 116 metabolites using mass spectrometer analysis. From this experiment, we found that the cellular content of glycerol 3-phosphate (G3P) in CD34+ AML and ALL cells was lower than that of normal CD34+ HSPCs. G3P is an intermediate metabolite in the glycolysis metabolic pathway and is utilized as a substrate for phospholipids synthesis. The initial and rate-limiting step of phospholipids synthesis is the synthesis of lysophosphatidic acid (LPA) from G3P and acyl-CoA mediated by glycerol 3-phosphate acyltransferases (GPATs). Since CD34+ acute leukemia cells contained significantly lower level of G3P, we hypothesized that leukemia cells actively consumed G3P and synthesized LPA by GPATs. GPATs are classified into four isoforms based on intracellular localization and substrate preference. GPAT1 and GPAT2 are mitochondrial GPATs that are localized to the mitochondrial outer membrane, but on the other hand, GPAT3 and GPAT4 are microsomal GPATs that are localized to the endoplasmic reticulum membrane, each encoded by independent genes. GPAT1 is identified as an essential gene for the growth of leukemia cells by RNAi screen analysis in the public database (DepMap). We found that CD34+ immature AML cells exhibited higher GPAT1 expression as compared to CD34- more differentiated AML cells and normal T cells. GPAT1 knockdown inhibited the proliferation of several acute leukemia cell lines including THP-1 and Kasumi-1 in vitro and in vivo. Moreover, a mitochondrial GPATs specific inhibitor (FSG67), which was originally developed as a drug to treat obesity and diabetes, suppressed the growth of the leukemia cell lines through the induction of G1 cell cycle arrest. Growth inhibition was rescued by exogenous administration of LPA, suggesting that the synthetic activity mediated by mitochondrial GPATs should be required for acute leukemia growth. Furthermore, FSG67 induced the apoptosis of leukemia cells derived from AML and ALL patients without affecting normal CD34+ HSPCs at least in vitro. We also confirmed that the injection of FSG67 resulted in the suppression of AML and ALL propagation in vivo using patient-derived xenograft models (see figure). GPAT1 regulates the mitochondrial function by producing LPA which is an essential metabolite for maintaining mitochondrial fusion. Actually, the amount of LPA was decreased in GPAT1 knockdown acute leukemia cells. We next examined mitochondrial energy production by extracellular flux assay, and found that GPAT1 knockdown as well as FSG67 significantly suppressed oxygen consumption rate of acute leukemia cells. Consistent with the impaired mitochondrial function, FSG67 suppressed the mitochondrial membrane potential, indicating that GPAT1 should play a pivotal role in maintaining leukemia-specific mitochondrial function. These results collectively suggest that the synthesis of LPA from G3P catalyzed by GPAT1 has a critical role in propagation of acute leukemia cells irrespective of their lineage origin. Thus, GPAT1 is a novel and common therapeutic target for human acute leukemia through suppressing leukemia-specific mitochondrial function. Figure Disclosures Akashi: Celgene, Kyowa Kirin, Astellas, Shionogi, Asahi Kasei, Chugai, Bristol-Myers Squibb: Research Funding; Sumitomo Dainippon, Kyowa Kirin: Consultancy.
30
Mantel, Charlie, Steven Messina-Graham, Akira Moh, Xin-Yuan Fu, and Hal E. Broxmeyer. "The Earliest Stages of Loss of Stem Cell Self-Renewal in-Vivo Is Linked to Upregulated Biosynthesis of “Quiet” Mitochondria and Is Influenced by CXCR4 Activation and STAT3 Gene Deletion." Blood 114, no. 22 (November 20, 2009): 2546. http://dx.doi.org/10.1182/blood.v114.22.2546.2546.
Анотація:
Abstract Abstract 2546 Poster Board II-523 One attribute of all stem cells is self-renewal (SR). Progress has been made in identifying genes/proteins involved in stem cell SR mechanisms, especially in embryonic stem cells, but very little is known in adult stem cells such as hematopoietic stem cells (HSCs). Mitochondria (Mt) have recently been considered more than an ATP generator and mediator of apoptosis. They act as a kind of scaffold for integrating numerous signals to and from the nucleus and from the extracellular environment, and signals involved in differentiation and cell cycle. They also stabilize microtubules of the mitotic spindle and suppress rotation of the mitotic division plane, potentially influencing asymmetric/symmetric stem cell divisions. Populations of primitive hematopoietic cells with low Mt activity (i.e. Rho123low) are highly enriched in long-term repopulating (LTR)-HSC. Because Mt can suppress mitotic spindle rotation by stabilizing spindle microtubules, potentially influencing asymmetric divisions, we hypothesized that Mt biogenesis/function could be important for mechanisms of SR loss and early-stage differentiation of mouse HSC. We examined Mt mass and Mt membrane potential in primitive mouse CD34lo/neg LINnegSca-1posc-kitpos (CD34-LSK) cells, CD34+LSK cells, and related populations. We note that as the small CD34-LSK cells begin to express CD34, Mt mass significantly increases (2×), while Mt membrane potential does not change in these CD34+LSK cells. The CD34+LSK cells then increase in size without further increases in Mt mass, but with upregulated Mt membrane potential. Because CD34 expression in LSK cells is associated with loss of LTR-HSC (i.e. self-renewal) and retention of only short-term repopulating ability, we propose this previously uncharacterized population of LSK cells with increased numbers/mass of “quiet” Mt may be an important component during initial stages of loss of SR and asymmetric HSC division. We found that primitive LSK cells from mice constitutively expressing an SDF-1α/CXCL12 transgene (TG mice) have shifted ratios of these various mitochondrially distinct populations that may be related to CXCR4 signaling and its known influence on glycolysis and STAT3 activity, both of which can influence Mt metabolism. LSK cells in TG mouse total bone marrow significantly increased from .039+/_.01% to .085+/_.02% (n=6), but they were predominantly CD34+LSK cells. These CD34+LSK cells were composed of two distinct types of cells; small/high-Mt mass and large/high-Mt mass cells. The small LSK cells have lower Mt membrane potential while the large LSK cells had increased Mt membrane potential. Because CXCR4 can signal through STAT3, and because STAT3 can bind to Mt and influence their metabolic activities (i.e. membrane potential), we hypothesized that mice with a tissue-specific targeted gene deletion of STAT3 in hematopoietic cells might result in LSK cells that are defective in upregulating Mt membrane potential, which could shift the proportions of the three mitochondrially distinct populations of LSK cells toward the smaller, Mt mass-low, Mt membrane potential-low type of LSK cell. Bone marrow from targeted STAT3-deleted had LSK numbers increased 50%, but they were composed almost entirely of small, Mt mass-high, membrane potential-low, CD34+ type of LSK cells. This is consistent with our hypothesis and suggests that Mt membrane potential may be important for latter stages of CD34-LSK cell differentiation to more lineage-committed progenitor cells but may not be required for initial stages of SR loss. We propose a model where Mt biogenesis occurs before/during loss of SR in CD34-LSK cells. These Mt are metabolically quiescent (“quiet”), attach to spindle microtubules of dividing cells, and influence spindle orientation resulting in loss of SR (small, Mt mass high, Mt potential low, CD34+LSK cells). Before further differentiation, these cells upregulate Mt metabolism/membrane potential and increase in size to facilitate further expansion and differentiation (large, Mt mass high, Mt potential high, CD34+LSK cells). This model could help explain the observed phenotypes of SDF-1 α/CXCL12 transgenic mice and hematopoietic cell-targeted STAT3 deleted mice. This information may also be useful in the search for strategies to prevent loss of SR capacity of HSCs in culture and expansion of HSCs in-vitro. Disclosures: No relevant conflicts of interest to declare.
31
Gueller, Saskia, Martina Komor, Julian C. Desmond, Oliver G. Ottmann, Dieter Hoelzer, H. Phillip Koeffler, and Wolf-Karsten Hofmann. "Identification of Putative New Tumor Suppressor Genes in Highly Purified CD34+ Bone Marrow Cells from Patients with Myelodysplastic Syndromes." Blood 104, no. 11 (November 16, 2004): 204. http://dx.doi.org/10.1182/blood.v104.11.204.204.
Анотація:
Abstract Activation of transcription of DNA by demethylation and hyperacetylation is known to cause hematologic improvement in patients with myelodysplastic syndromes (MDS). In this study we discriminated genes not expressed in CD34+ cells from untreated patients with MDS but activated by in vitro demethylation (2-aza-5-deoxycytidine, Decitabine) and hyperacetylation (suberoylanilide hydroxamic acid, SAHA). Highly purified CD34+ cells from normal individuals (n=3) and patients with low (n=3) and high (n=3) risk MDS were cultured with SCF (50 ng/ml), IL-3 (10 ng/ml) and GM-CSF (10 ng/ml). The cells were treated with 5 μmol Decitabine on day 1 and supplemented with 2.5 μmol SAHA on day 4 of culture. On day 5, global gene expression in these cells was compared to untreated cells (HG-U133A, Affymetrix, Santa Clara, CA). We identified 50 genes which are not expressed in untreated MDS CD34+ cells but 3-fold induced in all MDS samples by Decitabine and SAHA. Thirty-one of these genes were found to be expressed in normal CD34+ cells underlining the importance of such genes for normal hematopoiesis. This set of genes includes two genes for growth arrest and DNA damage control, the inducible protein beta (GADD45B), a regulator of growth and apoptosis and neural cell adhesion molecule 1 (NCAM1) that plays an important role in cell migration. Furthermore, hematological and neurological expressed 1 (HN1) which was not expressed in MDS CD34+ cells is known to have an anti-proliferative effect on tumor cell lines. N-myc downstream regulated 3 (NDRG3) is up-regulated during normal cell differentiation and suppressed in several tumor cells. In normal CD34+ cells, after in vitro treatment with Decitabine and SAHA we have discriminated 52 genes to be 3-fold up-regulated compared to untreated cells. Thirty-eight of these genes (73 %) were not inducible by demethylation and hyperacetylation in MDS CD34+ cells. These genes include chemokine receptor 3 (CCR3), a receptor for a C-C type chemokine involved in signal transduction, integrin beta-7 (ITGB7) that plays a role in adhesive interactions of leukocytes, preferentially expressed antigen in melanoma (PRAME) which is frequently expressed in human solid cancers and acute leukemia and tumor necrosis factor receptor superfamily member 1B (TNFRSF1B) that recruits apoptotic suppressors and mediates most of the metabolic effects of TNF-alpha. The silencing of these genes is independent of methylation and acetylation state and might be due to other mechanisms. This study shows that in CD34+ cells from MDS patients several genes are suppressed by methylation and hypoacetylation but can be activated by treatment with Decitabine and SAHA. Some of these genes are present in normal untreated CD34+ cells which leads to the assumption that they might function as tumor suppressor genes. Low or absent expression of these genes may contribute to the clonal expansion of MDS CD34+ which can be overcome by treatment with Decitabine or SAHA. Furthermore, the knowledge about these target genes may enable a more specific evaluation of the mechanisms of action of demethylating/hyperacetylating agents.
32
Zarou, Martha M., Kevin Rattigan, Zuzana Brabcova, Amy Dawson, David Sumpton, Alexei Vazquez, and Vignir Helgason. "Inhibition of Folate Metabolism Drives Autophagy-Dependent Differentiation and Reduces Survival of Therapy-Resistant Leukaemic Stem Cells." Blood 138, Supplement 1 (November 5, 2021): 2543. http://dx.doi.org/10.1182/blood-2021-149664.
Анотація:
Abstract Metabolic rewiring is an important hallmark of cancer. The folate metabolism pathway, also known as one-carbon (1C) metabolism, allows for transfer of 1C units through folate intermediates for biosynthetic processes, including precursors for DNA synthesis. Recent studies have shown that enzymes involved in the mitochondrial arm of 1C metabolism are overexpressed in a subset of aggressive cancers and that their expression affects responses to anti-metabolite drug treatments. However, the role of 1C metabolism in therapy resistant leukemic stem cells (LSCs) is currently unknown. Therefore, we aimed to investigate the activity and the impact of genetic and pharmacological inhibition of folate enzymes in primitive chronic myeloid leukaemia (CML) cells. We initially performed transcriptomic analysis of CD34+38- cells, from individuals with chronic phase CML (E-MTAB-2581). This revealed a significant upregulation of folate metabolism genes in CML LSCs, including serine hydroxymethyltransferase (SHMT2; p≤0.05), a key mitochondrial enzyme. To assess the activity of 1C metabolism in primitive cells we performed gas chromatography-mass spectrometry-mediated secretomic analysis using patient-derived, c-KIT enriched CML cells, which revealed a significant increase in the exchange rate of formate (folate intermediate necessary for purine synthesis) in CML cells, when compared to the secretome of normal counterparts (p<0.05). This reinforced the idea that 1C metabolism may be a metabolic dependency in CML. Following CRISPR-Cas9-mediated SHMT2 knockout (KO) in CML cell line, we observed a significant decrease in growth rate, together with a decrease in glycolytic capacity and oxygen consumption rate (p<0.01), suggesting impairment in proliferation and central carbon metabolism. Further metabolic characterisation of CML SHMT2 KO cells using liquid chromatography-mass spectrometry demonstrated a significant increase in AICAR, a purine biosynthesis intermediate and an AMP activated kinase (AMPK) activator. This prompted us to investigate the effect of 1C metabolism inhibition on AMPK. We found that AMPK phosphorylation on the conserved Thr 172 (a site that is phosphorylated under energy stress) was increased in SHMT2 KO cells, with similar effect seen following pharmacological inhibition of both SHMT2 and its cytosolic counterpart SHMT1 using SHIN1,which also promoted AMPK-dependent phosphorylation of the autophagy-inducing kinase ULK1 and downstream ULK1 target ATG13. Moreover, analysis of mitochondrial fraction revealed accumulation of mitochondrial fission related protein DRP1 and the mitophagy receptor NIX on mitochondria, hinting towards cellular interplay between 1C metabolism and mitochondrial homeostasis. Phenotypically, both pharmacological and genetic inhibition of SHMT1/2 induced the expression of erythropoiesis markers CD71 and Glycophorin A, which was reversed following formate supplementation. CRISPR-Cas9-mediated double AMPKα1/α2 KO revealed that the increased expression of these erythropoiesis markers following SHMT1/2 inhibition was independent of AMPK activity. Conversely, while NIX KO had no effect, pharmacological inhibition of ULK1 kinase activity, or genetic inhibition of ULK1 and ATG7 (protein important for autophagosome formation), prevented increased expression of CD71/Glycophorin A following SHMT1/2 inhibition. We next investigated the effect of 1C metabolism inhibition on differentiation and survival of primary CML cells. Of clinical relevance, pharmacological inhibition of SHMT1/2 promoted erythroid maturation of CD34+ CML cells (measured by expression of CD71, CD44, CD36 and Glycophorin A) when challenged with erythropoietin, which sensitises primitive cells to erythroid lineage commitment. Lastly, pharmacological inhibition of 1C metabolism decreased the colony formation capacity of CD34+ CML by 50%, with minimum effect on normal CD34+ cells. Moreover, combination treatment of SHIN1 with imatinib, a frontline treatment for CML patients, further increased the sensitivity of primary CML cells to imatinib by 40%. Overall, our novel findings indicate that disruption of the folate metabolism pathway inhibits central carbon metabolism in CML cells, promotes autophagy dependent, but AMPK independent maturation phenotype and has detrimental effect on the survival of primitive CML cells. Disclosures No relevant conflicts of interest to declare.
33
Redondo Monte, Enric, Anja Wilding, Georg Leubolt, Luise Hartmann, Sayantanee Dutta, Wolfgang Hiddemann, Lingping Chen-Wichmann, Christian Wichmann, and Philipp A. Greif. "Loss of ZBTB7A Facilitates RUNX1/RUNX1T1-Dependent Clonal Expansion and Sensitizes for Metabolic Inhibition." Blood 132, Supplement 1 (November 29, 2018): 1499. http://dx.doi.org/10.1182/blood-2018-99-114789.
Анотація:
Abstract ZBTB7A is a transcription factor involved in the regulation of metabolism and hematopoietic linage fate decisions. Recently, we found ZBTB7A mutated in 23% of Acute Myeloid Leukemia (AML) patients with t(8;21) translocation (Hartmann et al., 2016, Nat Commun). However, the oncogenic collaboration between ZBTB7A alterations and the RUNX1/RUNX1T1 fusion in AML t(8;21) remains poorly understood. To study ZBTB7A mutations in the context of RUNX1/RUNX1T1-dependent transformation, we used human CD34+ cells co-transduced with a truncated form of RUNX1/RUNX1T1 and ZBTB7A wild-type (WT) or its mutants (R402C and A175fs). We then followed the evolution of fluorescence marker positive cells over a period of 60 days. While expression of RUNX1/RUNX1T1 alone caused clonal expansion, co-expression of ZBTB7A WT impaired the outgrowth of CD34+ cells (Figure 1a). In contrast, the anti-proliferative effect of ZBTB7A was lost for both of its mutants tested resulting in a rescue of the clonal expansion (Figure 1b). To investigate the effect of ZBTB7A mutations on tumor metabolism, we used CRISPR/Cas9 to knockout (KO) ZBTB7A in the myeloid K562 cell line. As ZBTB7A is a known negative regulator of glycolysis, we treated KO and control cells with the glycolysis inhibitor 2-deoxy-d-glucouse (2-DG). KO cells were more sensitive to 2-DG compared to control cells (mean IC50: 3.06 vs 6.82 mM; p-value=0.087) (Figure 1c). These results are in line with the observed upregulation of glycolytic genes in ZBTB7A-mutant AML t(8;21) and suggest that these patients may benefit from the treatment with metabolic inhibitors. To learn more about deregulation of ZBTB7A target genes we are currently performing RNA-Seq analysis of WT vs KO K562. Moreover, we used the K562 KO model to investigate the impact of loss of ZBTB7A on myeloid differentiation. The baseline expression of the erythroid marker CD235a was reduced in KO cells. Ectopic expression of ZBTB7A WT in the KO cells restored the CD235a levels to a control level, while expression of mutants or vector showed no effect. These findings are in agreement with previous reports of ZBTB7A involvement in erythroid differentiation. To study the effect of ZBTB7A mutations on granulopoiesis, we established HL60 cells stably expressing WT or mutant ZBTB7A. We then differentiated the cells into granulocytes through all-trans retinoic acid (ATRA) treatment. Expression of WT, but not the mutants, resulted in a 4-fold increase of the granulocytic marker CD11b. Additionally, we induced monocytic differentiation through Phorbol 12-myristate 13-acetate (PMA) treatment. The mutant expressing cells showed similar levels of the monocytic marker CD14 as control cells. WT overexpressing cells had a 50% decrease in the number of monocytes. We then used CRISPR/Cas9 to establish ZBTB7A KO HL60, which exhibited a 5.5-fold increase in CD14 compared to control cells (Figure 1d). This data supports a previously unknown negative regulatory role of ZBTB7A in monocytic differentiation. With regards to potential therapeutic applications, we tested the PMA sensitivity of HL60 and found a significantly lower IC50 in absence of ZBTB7A (mean: 124.5 vs 269.8 pM; p-value=0.001). Hence, loss of ZBTB7A may facilitate the pharmacological differentiation of leukemia cells. In conclusion, ZBTB7A mutations in AML t(8;21) display a loss-of-function phenotype. Inactivating mutations of ZBTB7A allow for hCD34+ RUNX1/RUNX1T1-mediated expansion and deregulated tumor metabolism. Finally, loss of ZBTB7A expression perturbs myeloid development and thus may complement the block of differentiation induced by RUNX1/RUNX1T1. These findings open up avenues to novel therapies for ZBTB7A mutated patients including metabolic inhibition and pharmacological differentiation. Disclosures Hiddemann: F. Hoffman-La Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Consultancy, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.
34
Bosman, Matthieu C. J., Jan J. Schuringa, Wim J. Quax та Edo Vellenga. "Identification of the TAK1-NF-κB Axis As Critical Regulator of AML Stem and Progenitor Cell Survival." Blood 120, № 21 (16 листопада 2012): 2982. http://dx.doi.org/10.1182/blood.v120.21.2982.2982.
Анотація:
Abstract Abstract 2982 A small population of leukemic stem cells is resistant to chemotherapy and is responsible for the leukemic out-growth and relapse in acute myeloid leukemia (AML) patients. Evasion of apoptosis might be one of the essential mechanisms involved in this process. In order to gain more insight into the differences in the apoptotic programming between normal and leukemic (stem) cells, we recently performed gene array analysis by comparing CD34+ AML cells versus CD34+ normal bone marrow (NBM) cells. Gene ontology (GO) analysis of the differentially expressed genes between AML and NBM cells revealed differences in GO terms metabolic processes and apoptosis. In order to characterize differences in apoptotic programming in more detail 429 apoptotic related genes were selected and cluster analysis showed that CD34+ AML and CD34+ NBM cells could be separated into two distinct groups. In particular TGF-β activated kinase 1 (TAK1)/MAP3K7 was one of the apoptosis-related genes that was significantly higher expressed in CD34+ AML cells compared to CD34+ NBM cells (p = 1.8e−7). This increased expression of TAK1 could be confirmed by Q-PCR, showing an increase of on average 5.8 fold in TAK1 expression in the studied CD34+ AML cells. In mice it has been demonstrated that TAK1 is required for the survival of hematopoietic cells which is largely dependent on TNFR1 and TNFR2. In accordance with these data, we showed that TAK1 is also necessary in human hematopoiesis. Colony-forming cell (CFC) assays showed that inhibition of TAK1 in human cord blood CD34+cells, either by shRNAs targeting TAK1 or the TAK1 inhibitor 5z–7-oxozeaenol, resulted in a 2 fold reduction in CFU-GM and BFU-E frequencies compared to control cells. The efficacy was strongly further enhanced by the addition of TNFα, which resulted in a 9.4 fold decrease in CFC colonies upon TAK1 inhibition. Subsequently, we questioned whether TAK1 inhibition would affect CD34+ AML cell survival. Treatment of the AML cell lines MOLM13, OCI-M3 and HL60 with the TAK1 inhibitor 5z–7-oxozeaenol alone only induced modest effects, but in combination with TNFα for 24 hrs a strong induction of apoptosis was observed (IC50 respectively = 23nM, 215nM and 60 nM). Comparable results were observed in HL60 cells transduced with shRNAs targeting TAK1 whereby a downmodulation of TAK1 resulted in a 5.4 fold increase in Annexin V+ cells upon TNFα addition. In accordance with previous data, Western blot analysis showed that TAK1 inhibition reduced the levels of p-IκBα, p-p38, p-ERK and p-C-JUN. To test which of these pathways would be important for cell survival, AML cell lines were treated with either the p38 inhibitor SB203580, MEK/ERK inhibitor U0126, JNK inhibitor SP600125 and the NF-κB inhibitor BMS-345541, alone or in combination with TNFα. Addition of the NF-κB inhibitor BMS-345541 induced apoptosis in OCI-M3 and MOLM13 which was significantly increased in combination with TNFα (2.4 fold, p = 0.02). In contrast, inhibition of p38, MEK/ERK and JNK, either alone or in combination with TNFα, did not induce cell death in the AML cell lines. These data suggest that cell death induced by TAK1 inhibition is mainly due to inhibition of the NF-κB pathway. To determine the effects of TAK1 inhibition on primary AML cells, long-term expansion of the leukemic stem cell enriched CD34+ AML cell fraction was evaluated in MS5 stromal co-cultures in the absence or presence of TAK1 inhibitor and/or TNFα. Combined treatment for a period of 2 weeks completely abrogated the out-growth of CD34+ AML cells, indicating that both leukemic progenitors as well as leukemic stem cells were targeted. In contrast, addition of the single agents did not efficiently reduce cell growth. Similarly, downmodulation of TAK1 using shRNAs strongly sensitized primary CD34+ AML cells for TNFα-induced apoptosis, showing a 6 fold increase in Annexin V+ cells compared to control cells. Results on the in vivo efficacy of TAK1 inhibition on primary AML cells are in progress. In conclusion, our results show that TAK1 is frequently overexpressed in CD34+ AML cells, and that inhibition of TAK1 in combination with TNFα is highly efficient in inducing apoptosis of leukemic stem/progenitor cells in a NF-κB-dependent manner. Disclosures: No relevant conflicts of interest to declare.
35
Carter, Bing Z., Po Yee Mak, Wenjing Tao, Mark Warmoes, Philip L. Lorenzi, Duncan Mak, Vivian Ruvolo, et al. "Mcl-1/CDK9 Targeting By AZD5991/AZD4573 Overcomes Intrinsic and Acquired Venetoclax Resistance in Vitro and In Vivo in PDX Model of AML through Modulation of Cell Death and Metabolic Functions." Blood 132, Supplement 1 (November 29, 2018): 768. http://dx.doi.org/10.1182/blood-2018-99-113491.
Анотація:
Abstract Mcl-1 and Bcl-2 are two major anti-apoptotic Bcl-2 proteins frequently overexpressed in malignant cells. They cooperatively support cell survival and are associated with therapy resistance. ABT-199 (venetoclax), a highly selective Bcl-2 inhibitor, showed potent preclinical activity but limited clinical efficacy in AML as a single agent. Mcl-1 is induced by and a major resistance factor to ABT-199. Mcl-1 was recently found to also positively regulate mitochondrial oxidative phosphorylation that induces cancer stem cells and promotes chemoresistance. Mcl-1 is essential for the development of AML and for the survival of AML cells and stem cells. Increased mitochondrial oxidative phosphorylation has been demonstrated in these cells. First we found that Mcl-1 overexpressing (OE)/knockdown (KD) AML cells were markedly more resistant/sensitive to ABT-199 than were corresponding control cells, supporting the notion of Mcl-1 as a resistance factor to ABT-199. Inhibition of Mcl-1 by the selective Mcl-1 inhibitor AZD5991 or the CDK9 inhibitor AZD4573, which down-regulates short-lived proteins such as Mcl-1, induced apoptosis and showed strong synergy when combined with ABT-199 in AML cell lines, primary AML blasts, and stem/progenitor cells from patients. Importantly, combinations of AZD5991 or AZD4573 with ABT-199 synergistically induced apoptosis in OCI-AML3 and Mcl-1 OE cells intrinsically resistant to ABT-199 and in AML cell lines and primary patient cells with acquired resistance to ABT-199. Although OE/KD Mcl-1 in AML cells did not show obvious alterations in baseline cell viability, NSGS mice harboring Mcl-1 OE/KD OCI-AML3 cells survived significantly shorter/longer than those transplanted with control cells, supporting additional, non-apoptogenic functions of Mcl-1 in AML. We observed that genetic modulation of Mcl-1 alters cellular mitochondrial respiration and ROS levels. AML cells with Mcl-1 OE/KD increased/decreased O2 consumption and mitochondrial ATP and ROS generation. Consistent with this finding, inhibition of Mcl-1 by AZD5991 or AZD4573 decreased O2 consumption and ATP generation in AML cells and also in MV4-11 cells with acquired ABT-199 resistance. Mass spectrometry-based stable isotope tracing experiments using 1,2-13C-glucose showed that both genetic and pharmacological inhibition of Mcl-1 decreased flux of glucose carbon through glycolysis, the TCA cycle, and the pentose phosphate pathway, suggesting a role for Mcl-1 in cellular respiration and redox metabolism. To further assess the efficacy of combined Mcl-1 and Bcl-2 inhibition in primary AML cells resistant to ABT-199, we developed a PDX model using cells from an AML patient who initially responded to ABT-199/demethylating agent and then relapsed. NSGS mice engrafted with these PDX cells were treated with ABT-199 (50 mg/kg, oval gavage qd), AZD5991 (60 mg/kg, i.v. weekly), AZD4573 (15 mg/kg, i.p. bid with 2 h interval for two consequent days/week), ABT-199+AZD5991, or ABT-199+AZD4573 for 6 wks. Flow cytometric analysis of circulating human CD45+ cells on day 18 of therapy showed that each agent significantly decreased leukemia burden and that the combinations were significantly more effective (P<0.01) than each single agent. CyTOF analysis of BM cells (day 25) showed that both combinations markedly reduced (P<0.001) human CD45+ cells and, more importantly, human CD34+CD38+/CD38- and CD34+CD38+/CD38-CD123+ cells. Those combination treatments also decreased Mcl-1, Bcl-2, b-catenin, c-Myc, and FAK protein expression in CD34+CD38-CD123+ cells. Interestingly, AZD5991, AZD4573, or their combinations with ABT-199 greatly decreased CXCR4 in all cell populations. Ultimately, each single agent only marginally prolonged survival, whereas ABT-199+AZD4573 and even more so ABT-199+AZD5991 markedly improved survival in this highly ABT-199 resistant PDX model (Fig). Conclusion: we demonstrate that Mcl-1 has metabolic functions in AML and that inhibition of Mcl-1 enhances ABT-199 apoptogenic activity and overcomes intrinsic and acquired ABT-199 resistance in vitro and in vivo in a PDX murine model of AML, suggesting that inhibition of Mcl-1 improves the efficacy of ABT-199, and overcomes established resistance to Bcl-2 inhibition. Suppressing metabolic activity and CXCR4 inhibition may also contribute to the efficacy of this combination against AML stem cells in the BM microenvironment. Figure. Figure. Disclosures Carter: AstraZeneca: Research Funding; novartis: Research Funding. Lorenzi:Erytech Pharma: Consultancy; NIH: Patents & Royalties. Cidado:AstraZeneca: Employment, Equity Ownership. Drew:AstraZeneca: Employment. Andreeff:AstraZeneca: Research Funding.
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Alt, Ruediger, Thomas Riemer, Oliver Fiehn, Dietger Niederwieser, and Michael Cross. "Evidence for Restricted Glycolytic Metabolism in Primary CD133+ Cells." Blood 106, no. 11 (November 16, 2005): 1726. http://dx.doi.org/10.1182/blood.v106.11.1726.1726.
Анотація:
Abstract The slow cycling, location and hypoxia-resistance of hematopoietic stem cells are suggestive of a restricted metabolism. We propose that HSC metabolism is adapted to unique metabolic conditions supplied by the stem cell niche, and that a combination of the metabolic and signalling environments acts to support stem cell amplification and to limit it to a narrowly-defined and physiologically rare set of sites. To investigate this possibility, we have established moderate throughput, small scale cultures to examine the metabolic characteristics of primary CD133+ cells isolated from umbilical cord blood. A screen of carbon and energy sources revealed that pyruvate (but neither fatty acids nor amino acids) can replace glutamine as a major substrate. The fact that pyruvate contributes significantly to the cellular metabolism even in the presence of glucose suggests that CD133+ cells employ an unusually low level of glycolysis. Flow cytometric analysis of surface markers before and after culture confirmed that the addition of glucose (0mM, 5mM and 25mM) or insulin (0μg/mL, 4 μg/mL) increased the overall cell yield, but had no effect on the proliferation of early cells (CD133+, CD34+ and c-kit+). In parallel, metabolic profiling of undifferentiated and differentiated FDCPmix cells using gas chromatography and mass spectrometry techniques revealed an accumulation of glucose in the self-renewing (undifferentiated) population. Taken together, these observations suggest that glycolysis makes little contribution to stem cell metabolism, and that hematopoietic stem cells (as has been suggested for germ cells) may instead use glycolytic products supplied by stromal cells. Furthermore, our studies have revealed an unexpected effect of osmolarity on both glucose metabolism and self-renewal, in that an increase in osmolarity from 0,32 Osm/Kg to 0,36 Osm/kg reduced the rate of glucose-dependent proliferation of CD133+ cells without reducing the yield of early (CD133+ CD34+, ckit+) cells. Similarly, both the proportion of self-renewing cells in FDCPmix cultures and the recruitment of these cells to active self-renewal in semi-solid media in a colony forming assay were found to be increased at higher osmolarity. This suggests that high osmolarities suppreses both glycolytic metabolism and proliferation rate, but favour the maintenance of “early” progenitors. Finally, we have applied our assay to test a range of different growth factor combinations (of SCF, Flt3-ligand, TPO, IL3, IL6, IL7, IL11 and TGF-b) in 18% and 1% O2, and found that hypoxia extends markedly the range and magnitude of the proliferation response. Taken together, our results suggest that hematopoietic stem cells are indeed adapted to a rare metabolic microenvironment which includes (but is probably not limited to) low oxygen and glucose concentrations, and that the metabolic environment is likely to influence strongly the response to growth factors. A thorough understanding of stem cell metabolism may therefore provide a basis for more controlled manipulation of HSC in vitro.
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Orchard, Paul, Glen D. Raffel, Carolyn H. Condon, Catherine A. Monaghan, Demetra Vernet, Suzanne Tracey Sheirr, Jennifer Braun, et al. "Preliminary Phase 2 Results Demonstrate Engraftment with Minimal Neutropenia with MGTA-456, a CD34+ Expanded Cord Blood (CB) Product in Patients Transplanted for Inherited Metabolic Disorders (IMD)." Blood 132, Supplement 1 (November 29, 2018): 3467. http://dx.doi.org/10.1182/blood-2018-99-115102.
Анотація:
Abstract Background: IMDs including mucopolysaccharidosis type IH (MPS1/Hurler Syndrome), metachromatic leukodystrophy (MLD), globoid cell leukodystrophy (GLD) and cerebral adrenoleukodystrophy (cALD) are progressive, fatal diseases affecting the central nervous system which are treatable through allogeneic hematopoietic stem cell transplantation (HSCT). CB, in the absence of a matched donor, is the preferred source of stem cells as it is rapidly available and allows greater flexibility in allele matching. As a result of low cell doses, CB transplants in IMD are associated with prolonged periods of neutropenia and reported graft failure rates in up to ~20% (Lum et al 2017 Bone Marrow Transplant 52:846-53; Mallhi et al 2017 BBMT 23:119-25). MGTA-456 is a first-in-class cell therapy produced from a single CB unit using an aryl hydrocarbon receptor antagonist in a 15-day expansion culture of CD34+ cells. In previous phase 1/2 studies, 24 adult and 3 pediatric patients with hematologic malignancies treated with myeloablative conditioning (MAC) and MGTA-456 demonstrated a median 324-fold expansion of CD34+ cells, all patients engrafted, and the time to neutrophil recovery was significantly reduced by a median of 9 days compared to historical controls (Wagner et al 2016 Cell Stem Cell 18:144-55; Wagner et al 2017 Blood 130 supp:662 abstr). Furthermore, higher CD34+ dose has been correlated with improved engraftment and outcomes in IMD transplant patients (Prasad et al 2008 Blood 112:2979-89). Based on these promising data, we postulate the increased CD34+ dose provided by MGTA-456 would reduce the length of neutropenia and risk of graft failure in IMD patients. Patients and Methods: A Phase 2, open-label trial (NCT03406962) initiated in Feb 2018 is enrolling up to ~12 patients <16 yo with a diagnosis of MPS1, cALD, MLD, and GLD lacking a non-carrier matched related donor. Eligible CB units were matched at ≥ 6 of 8 HLA loci (A, B, C and DRB1) using allele-based typing. The reduced toxicity MAC regimen consists of anti-thymocyte globulin (days -9 to -6) followed by fludarabine (40 mg/m2 days -5 to -2) and busulfan (total exposure 21,000 to 22,000 μM/min/L-1 days -5 to -2). Immunoprophylaxis consists of cyclosporin and methylprednisolone. Results: Four patients have been treated thus far (Table 1). The MGTA-456 process provided a marked expansion of CD34+ cells (median 482-fold, Figure 1) with a median infused CD34+ cell dose of 84 x 106 cells/kg and median total nucleated cell (TNC) dose from the expanded fraction of 19.6 x 107 (Table 1). TNC dosing was capped at 27.0 x 107 cells/kg per protocol. The only infusion-related reaction noted was grade 3 nausea in 1 patient. Two patients had no days of neutropenia and 2 patients had 1 and 4 days respectively (mean 1.25 days) in contrast to a mean of 7.8 days for a historical cohort of 27 IMD patients undergoing CB transplantation at the same institution with identical conditioning (Figure 2). Myeloid chimerism (CD33+/66+) achieved ≥98% donor by day +14 in all patients. For the 3 patients with data at time of reporting, days to discharge after transplant were 17, 12 and 18. No patients experienced acute or chronic GVHD. One patient developed autoimmune cytopenia (not related to MGTA-456) which is a known complication reported in 20-56% of IMD patients undergoing HSCT that resulted in death at day +143 (Page et al 2008 BBMT 14:1108-17; Khalil et al 2014 Sci World J 581657). Long-term disease specific outcome measures including enzymatic activity are being collected and will be reported over time. Conclusions: Preliminary results of transplantation in IMD patients with MGTA-456, containing highly expanded CD34+ cell doses, demonstrated early and robust engraftment in all patients with marked reduction in days of neutropenia (to 0-4 days or mean 1.25 days) in comparison to a historical cohort. MGTA-456 was well tolerated with one infusion-related event of grade 3 nausea. These data, in combination with the previous 27 hematologic malignancy patients treated, suggest that MGTA-456 substantially enhances the engraftment potential of CB. Based on these promising data, MGTA-456 has potential to improve transplant-related outcomes in patients undergoing HSCT and increasing the availability of well-matched CB units that may have been previously excluded due to inadequate CD34+ dose. Disclosures Orchard: Magenta Therapeutics: Research Funding. Raffel:Magenta Therapeutics: Employment, Equity Ownership. Condon:Magenta Therapeutics: Employment, Equity Ownership. Monaghan:Magenta Therapeutics: Employment, Equity Ownership. Vernet:Magenta Therapeutics: Employment. Sheirr:Magenta Therapeutics: Employment, Equity Ownership. Braun:Magenta Therapeutics: Research Funding. Shanley:Magenta Therapeutics: Research Funding. Lund:Magenta Therapeutics: Research Funding. Boitano:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Cooke:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Davis:Magenta Therapeutics: Employment, Equity Ownership. Wagner:Novartis: Research Funding; Magenta Therapeutics: Consultancy, Research Funding.
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Mistry, Jayna J., Charlotte Hellmich, Jamie A. Moore, Christopher R. Marlein, Genevra Pillinger, Angela Collins, Kristian M. Bowles, and Stuart A. Rushworth. "Daratumumab Inhibits AML Metabolic Capacity and Tumor Growth through Inhibition of CD38 Mediated Mitochondrial Transfer from Bone Marrow Stromal Cells to Blasts in the Leukemic Microenvironment." Blood 134, Supplement_1 (November 13, 2019): 1385. http://dx.doi.org/10.1182/blood-2019-128592.
Анотація:
Background Acute myeloid leukemia (AML) is dependent on the bone marrow microenvironment, where bone marrow stromal cells (BMSCs) are an important tumor supporting cell type. We have previously demonstrated that, contrary to the Warburg hypothesis, AML blasts rely on oxidative phosphorylation for survival and are dependent on increased mitochondrial levels compared to non-malignant CD34+ progenitor cells. Moreover, we found that AML blasts meet their high metabolic demands by transferring in mitochondria from surrounding BMSC. We have also recently described how mitochondria are transferred from BMSC to myeloma cells in a pro-tumoral, CD38 dependent, mechanism. As the leukemia-initiating cells in AML may reside within the CD34+/CD38+ compartment, we examined the mitochondrial transfer and the resultant metabolic and functional consequences of inhibiting CD38 using daratumumab in the setting of the AML microenvironment. Methods Primary AML blasts and primary AML BMSC were isolated from patients bone marrow in accordance with the Declaration of Helsinki. BMSC were separated by adherence and then characterised using flow cytometry for expression of CD90+, CD73+, CD105+ and CD45-. Mitochondrial transfer was assessed in vitro using qPCR and MitoTracker staining based methods. In vivo experiments using an NSG AML xenograft model were carried out with darartumumab (or control) treatment given on days 9 and 16 post AML transplant. Tumor engraftment and growth were monitored weekly by live animal in vivo imaging. Post transplantation, AML mitochondrial content and transfer were assessed by evaluation of murine mitochondrial DNA in human AML blasts by species specific PCR analysis. Post transplantation mitochondrial function was measured by TMRM and Seahorse analysis. Results In-vitro experiments using MitoTracker Green demonstrate that daratumumab inhibits the transfer of mitochondria from BMSC to AML. In-vivo, daratumumab treatment significantly reduced tumor growth in human xenograft mouse model. Furthermore, we found that two doses of daratumumab resulted in reduced mitochondrial potential and oxygen consumption rate in the AML cells derived from the BM microenvironment of the AML engrafted NSG mice. Finally, examination of human AML cells sorted from NSG mouse bone marrow confirmed that mouse mitochondrial DNA content in the human AML blasts was reduced from animals treated with daratumumab compared to animals with AML treated with vehicle control. Conclusion Daratumumab treatment inhibits mitochondrial transfer from BMSC to AML in the BM microenvironment, resulting in a reduction of pro-tumoral oxidative phosphorylation in the blasts and subsequent reduced leukemia growth, which is associated with improved animal survival. While it is likely that daratumumab functions through a number of mechanisms of action, here we show in the NSG mouse model (which lacks functional B cells, T cells and NK cells and where macrophages and dendritic cells are defective) that inhibition of mitochondrial transfer in AML can be added to the list of mechanisms of action for daratumumab. These data support the further investigation of daratumumab as a therapeutic approach for the treatment of this mitochondrial dependent tumor. Disclosures Bowles: Janssen: Research Funding; Abbvie: Research Funding. Rushworth:Abbvie: Research Funding; Janssen: Research Funding.
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Thomas, Geethu Emily, Grace Egan, Laura Garcia Prat, Botham Aaron, Veronique Voisin, Elias Orouji, Jordan M. Chin, et al. "The Metabolic Enzyme Hexokinase 2 Localizes to the Nucleus in AML and Normal Hematopoietic Stem/Progenitor Cells to Maintain Stemness." Blood 136, Supplement 1 (November 5, 2020): 1–2. http://dx.doi.org/10.1182/blood-2020-135858.
Анотація:
Abstract Mitochondrial metabolites affect epigenetic marks, but it is largely unknown whether mitochondrial metabolic enzymes can directly localize to the nucleus to regulate stem cell function in AML. Here, we discovered that the mitochondrial enzyme, Hexokinase 2 (HK2), localizes to the nucleus in AML and normal hematopoietic stem cells to maintain stem cell function. We searched for mitochondrial enzymes moonlighting in the nucleus using 8227 AML cells, a low passage primary AML culture model arranged in a hierarchy with functionally defined stem cells in the CD34+CD38-fraction. By immunoblotting and confocal microscopy, we detected HK2 in the nucleus of 8227 cells with higher expression in the nucleus of stem cells vs bulk cells. HK2 is the first and rate-limiting enzyme in glycolysis and phosphorylates glucose. In contrast, other metabolic enzymes including phosphofructokinase, fumarase, pyruvate kinase 2, glucose phosphate isomerase, enolase1, citrate synthase, aconitase 2, and succinate dehydrogenase were not detected in the nucleus of these cells. We also detected HK2, but not these other metabolic enzymes, in the nucleus of OCI-AML2, U937, NB4 and TEX leukemia as well as 8 of 9 primary AML samples. Next, we tested whether nuclear HK2 was functionally important to maintain stem cell function in AML. We over-expressed HK2 tagged with nuclear localizing signals (PKKKRKV and PAAKRVKLD) in 8227 and NB4 leukemia cells. We confirmed selective over-expression of HK2 in the nucleus of these cells without increasing levels in the cytoplasm or mitochondria. Over-expression of nuclear HK2 increased clonogenic growth and inhibited retinoic acid-mediated cell differentiation without changing basal proliferation. Over expression of HK2 also increased engraftment of 8227 cells into mouse marrow. We evaluated the selective inhibition of nuclear HK2 by over-expressing HK2 with an outer mitochondrial localization signal while knocking down total endogenous HK2 with shRNA targeting the 3'UTR of HK2. Selective depletion of nuclear HK2 reduced clonogenic growth, increased AML differentiation after treatment with retinoic, and decreased the percentage of CD34+CD38- 8227 stem cells without changing basal proliferation. To determine whether nuclear HK2 maintains stemness through its kinase activity, we over-expressed a kinase dead double mutant of nuclear HK2(D209A D657A). Nuclear kinase dead HK2 increased clonogenic growth and inhibited differentiation after retinoic acid treatment, demonstrating that HK2 maintains stemness independent of its kinase function. To understand nuclear functions of HK2, we used proximity-dependent biotin labeling (BioID) and mass spectrometry to identify proteins that interact with nuclear HK2 and identified proteins related to chromatin organization and regulation. Therefore, we examined the impact of nuclear HK2 on chromatin accessibility using ATAC-seq. Over expression of nuclear HK2 enhanced chromatin accessibility, whereas the selective knockdown of nuclear HK2 compacted chromatin. In summary, we discovered that HK2 localizes to nucleus of AML cells and functions independent of its kinase activity to maintain the stem/progenitor state of AML. Thus, we define a new role for mitochondrial enzymes in the regulation of leukemic stemness and differentiation. Disclosures Dick: Bristol-Myers Squibb/Celgene: Research Funding. Schimmer:Takeda: Honoraria, Research Funding; Novartis: Honoraria; Jazz: Honoraria; Otsuka: Honoraria; Medivir AB: Research Funding; AbbVie Pharmaceuticals: Other: owns stock .
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Lahey, Ryan, Jesper Bonde, and Jan A. Nolta. "Uptake of Protamine Sulphate Complexed Fluorescent Nano-Particles Is Defined by Cell Cycle Status in Primary Human CD34+ Cells: Use of a Multi-Color p27 kip1 Based Flow Cytometric Assay." Blood 106, no. 11 (November 16, 2005): 1363. http://dx.doi.org/10.1182/blood.v106.11.1363.1363.
Анотація:
Abstract The use of iron based nano-particles for multi-modal imaging is gaining interest, since it allows high resolution non-invasive in vivo imaging of human hematopoietic homing and engraftment events in xenograft models. The uptake of ferridex nano-particles complexed to cationic protamine sulphate is believed to be non-specific through mechanisms like endocytosis, but this has not been well defined for hematopoietic stem cells (HSC). In defining ex vivo cultivation strategies for manipulation of human HSC, a key factor is the responsiveness of the most primitive cells to the in vitro conditions, with the aim of maintaining viability without inducing terminal differentiation. Here, we present a novel flow cytometry assay which assesses the earliest molecular responses to a defined clinically applicable ex vivo protocol, aimed at facilitating labeling of human stem/progenitor cells using protamine sulphate complexed nano-particles for subsequent in vivo imaging. We used intracellular staining for the cell cycle inhibitor p27kip1, which is present in the highest levels in non-cycling cells, as the primary flow cytometric marker in combination with CD34, CD133 and Alexa 488, 647 and 750 conjugated ferridex nano-particles and the membrane dye PKH26. An assay was developed to simultaneously assess the molecular events occurring in individual human cord blood Lin− or CD34+ cells while they were cultured for up to 72 hours in X-Vivo 15 serum free medium supplemented with Flt3, SCF and TPO on Retronectin (RN) coated plates with or without nano-particles. Co-expression of p27kip1, CD34 or CD133 in the cultured cells slowly decreases from 86.1% CD34+p27kip1 (T=0) to 76.7%+/−12.2% (T=72) and from 89.6% CD133+p27kip1+ (T=0) to 54.1%+/−10.4% (T=72). We suggest that this slow decrease represents cells dividing and potentially differentiating over the time course of the ex vivo cultivation period. Assessing uptake of fluorescent conjugated nano-particles over a 72 hr period showed that the uptake of particles in CD34+ and CD133+ cells declined significantly after the first 24 hrs., from 32.5+/−3.7% nano-positive CD34+ cells to 19.2+/−2.9% at 48 hours ex vivo with a more significant decline to only 8.3+/−3.7% nano positive CD34+ cells in the culture after 72 hours ex vivo. The same decline in uptake over time was observed in cultured human CB cells that were positive for CD133. PKH26 co-staining demonstrated that the majority of cells that undergo cell division within the first 24 hours of ex vivo culture are the most likely to uptake the nano-particles. In summary, using a multi color p27kip1 based flow-cytometry assay, we found that human Lin−, CD133+, and CD34+ cells uptake Fe-Pro in a fashion which is not entirely cell cycle independent as previously suggested. These data indicate that cell cycle or metabolic status may influence the ability of human hematopoietic stem and progenitor subsets to uptake the protamine sulphate-complexed nano-particles. These findings emphasize the need to carefully develop ex vivo conditions for nano-particle labeling of primary human stem cells in order to perform accurate in vivo imaging of the most primitive human hematopoietic stem and progenitor cells.
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Woolthuis, Carolien M., Hendrik JM de Jonge, Annet Z. Vos, Andre B. Mulder, Eva van den Berg, P. M. Kluin, Karen van der Weide, et al. "Gene Expression Profiling In Leukemic Stem Cell-Enriched AML CD34+ Cell Fraction Identifies Target Genes That Predict Prognosis In Normal Karyotype AML." Blood 116, no. 21 (November 19, 2010): 952. http://dx.doi.org/10.1182/blood.v116.21.952.952.
Анотація:
Abstract Abstract 952 Acute myeloid leukemia (AML) is clinically, cytogenetically and molecularly a heterogeneous disease which makes it challenging to classify it properly. In recent years major advances have been achieved in predicting outcome. However, there is still need for more powerful and independent prognostic factors that can guide treatment decisions, especially for the large subgroup of patients presenting with normal karyotype AML. In order to improve the identification of prognostic markers, gene expression studies have been performed. However, most of these studies have analyzed the mononuclear cell fraction. So, very little has been revealed about gene expression programs that drive leukemic transformation in the small population of leukemic stem cells (LSCs). Although considerable heterogeneity appears to exist in the phenotype of LSCs, CD34 is uniformly expressed and LSCs have been found to reside in the CD34+ compartment in the vast majority of leukemias. In the present study AML CD34+-specific gene expression profiles were indentified and used to distinguish prognostically relevant target genes in normal karyotype AML. AML mononuclear cells (n=46) were sorted in CD34+ and CD34- subfractions and genome-wide expression analysis was performed using Illumina BeadChip Arrays. AML CD34+ and CD34- gene expression was compared to a large group of normal CD34+ bone marrow cells (n=31). Unsupervised hierarchical clustering analysis showed that CD34+ AML samples belonged to a distinct cluster compared to normal bone marrow and that in 61% of the cases the AML CD34+ transcriptome did not cluster together with the paired CD34- transcriptome. These data indicate that in the majority of AML cases the leukemic stem cell-enriched CD34+ gene expression profile is quite distinct from the leukemic CD34- compartment. GO analysis revealed that common differences in gene expression between CD34+ and CD34- groups were particularly enriched for genes that were associated with T-cells and erythropoiesis. This association with a more committed phenotype was found in all AML samples and not just in those samples where CD34+ and CD34- transcriptomes did not cluster together. Among the GO-ontologies representing the differentially expressed genes between CD34+ AML versus CD34+ normal bone marrow cells were gene sets related to DNA damage and a number of mitotic and metabolic processes. A top 50 of AML CD34+-specific genes was selected by comparing the AML CD34+ transcriptome with the AML CD34- and CD34+ normal bone marrow transcriptomes. The prognostic relevance of these 50 genes was assessed using univariate cox regression analyses between the continuous transcript levels of these 50 genes and overall survival (OS) in a large series of normal karyotype AML (n=163) (Metzeler et al. Blood 2008). The findings were validated in another independent cohort of 218 normal karyotype AML patients (Valk et al. NEJM 2004). Interestingly, higher transcript levels of three CD34+ AML specific genes, i.e. ankyrin repeat domain 28 (ANKRD28), guanine nucleotide binding protein, alpha 15 (GNA15) and UDP-glucose pyrophosphorylase 2 (UGP2) were associated with a significant poorer OS in both cohorts (p<0.01). For the cohort of 218 normal karyotype AML patients also event free survival (EFS) data were available for further analyses. A significant association between the continuous transcript levels of ANKRD28, GNA15 and UGP2 with poor EFS was evident. Also for the sum of expression of ANKRD28, GNA15 and UGP2 higher transcript levels were strongly associated with poorer OS (p=0.007) and EFS (p=0.006) in the cohort of 218 normal karyotype AML. Similar results were obtained in the cohort of 163 normal karyotype AML patients for OS (p<0.001). Importantly, the prognostic value of the continuous transcript levels of these three genes was independent from the well known risk factors FLT3-ITD, NPM1c+ and CEBPA mutation status as determined by a multivariate analysis in the cohort of 218 normal karyotype AML patients. In conclusion, by microarray analysis of the leukemic stem cell-enriched AML CD34+ cell fraction novel insight was obtained in gene expression programs that are potentially associated with leukemic stem cell self-renewal and transformation. Moreover, the identified new gene expression profiles were shown to have prognostic relevance in normal karyotype AML independent of well known risk factors. Disclosures: No relevant conflicts of interest to declare.
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Wang, DaQuan, Bo Qiu, Qianwen Liu, Liangping Xia, Liu Songran, ChaoJie Zheng, Hui Liu, et al. "Value of Patlak-Ki from ultra-high sensitivity dynamic total body [18F]FDG PET/CT for evaluation of treatment response to induction immuno-chemotherapy in locally advanced non-small cell lung cancer (LA-NSCLC) patients." Journal of Clinical Oncology 41, no. 16_suppl (June 1, 2023): e20508-e20508. http://dx.doi.org/10.1200/jco.2023.41.16_suppl.e20508.
Анотація:
e20508 Background: This study aimed to explore the value of metabolic features in predicting the response to induction immuno-chemotherapy in locally advanced NSCLC, using dynamic total body [18F]FDG PET/CT. Methods: The LA-NSCLC patients who received two cycles of induction immuno-chemotherapy were analyzed in the study. The 60-minute dynamic total body [18F]FDG PET/CT scan was administered before treatment. The primary tumors (PTs) were manually delineated. The metabolic features including the Patlak-Ki, Patlak-Intercept, the SUVmax, metabolic tumor volume (MTV) and total lesion glycolysis (TLG) of PTs were evaluated. Using the Patlak graphical analysis, the Patlak-Ki of PTs was calculated from the 20-60 minutes frames. The Laplacian feature importance scores was used to select the best feature and an unsupervised K-Means method was applied to cluster patients. ROC curve was used to examine the effect of selected metabolic feature in predicting tumor response to treatment. The targeted next generation sequencing on 1021 genes was conducted. The expressions of CD68, CD86, CD163, CD206, CD33, CD34, Ki67 and VEGFA were assayed by immunohistochemistry. The independent samples t test and the Mann-Whitney U test were applied in the intergroup comparison. Results: Thirty-seven LA-NSCLC patients were analyzed between September 2020 and November 2021. All patients received two cycles of induction chemotherapy combined with Nivolumab/ Camrelizumab. The Laplacian scores showed that the Patlak-Ki of PTs had the highest importance for patient clustering, and the unsupervised K-Means derived decision boundary of Patlak-Ki was 2.779 ml/min/100g. Then Patients were divided into a high FDG Patlak-Ki (H-FDG-Ki, Patlak-Ki > 2.779 ml/min/100g) group (n = 23) and a low FDG Patlak-Ki (L-FDG-Ki, Patlak-Ki ≤ 2.779 ml/min/100g) group (n = 14). The ORR to induction immuno-chemotherapy was 67.6% (25/37) in the whole cohort, with 87% (20/23) in H-FDG-Ki group and 35.7% (5/14) in the L-FDG-Ki group (P = 0.001). The sensitivity and specificity of Patlak-Ki in predicting the treatment response were 80% and 75%, respectively [AUC = 0.775 (95%CI 0.605-0.945)]. The expression of CD3+/CD8+ T cells and CD86+/CD163+/CD206+ macrophages were higher in the H-FDG-Ki group, while Ki67, CD33+ myeloid cells, CD34+ micro-vessel density (MVD) and tumor mutation burden (TMB) were comparable between the two groups. Conclusions: The total body [18F]FDG PET/CT scanner performed a dynamic acquisition of the entire body and clustered LA-NSCLC patients into H-FDG-Ki and L-FDG-Ki groups based on the Patlak-Ki. H-FDG-Ki patients had better response to induction immuno-chemotherapy and higher immune cells infiltrations in the PTs than L-FDG-Ki patients. Further studies in a large patient cohort are warranted.
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Xiang, Wei, Yi Hui Lam, Collin Sng, May Anne Cheong, Hein Than, William YK Hwang, and Charles Chuah. "Mefloquine Effectively Targets Blast Phase Chronic Myeloid Leukemia through Inducing Oxidative Stress and Lysosomal Disruption." Blood 128, no. 22 (December 2, 2016): 5426. http://dx.doi.org/10.1182/blood.v128.22.5426.5426.
Анотація:
Abstract Despite the remarkable clinical responses achieved with BCR-ABL tyrosine kinase inhibitors (TKIs) in the treatment of chronic phase-chronic myeloid leukemia (CML), these TKIs have been less effective as single agents in blast phase (BP) CML. Identification of new therapeutic strategies is needed for the better clinical management of BP-CML. It is well known that the mitochondrial metabolic properties of tumor cells are different from those of normal cells, making this as an attractive target for cancer treatment. Previously, we screened a number of antimicrobial drugs with possible mechanisms of action related to mitochondrial metabolism and identified mefloquine as a potential candidate for CML treatment. Mefloquine is a FDA-approved antimalarial drug and has been reported to have anti-cancer activities. In this work, we investigated the effect of mefloquine and its underlying mechanisms in CML. We show that mefloquine induces apoptosis of CML cells in a dose-dependent manner (Fig. 1A). In addition, mefloquine is also effective in targeting BP-CML CD34+ progenitor cells. It induces apoptosis, inhibits colony formation and self-renewal capacity of CD34+ cells derived from a TKI-resistant BP-CML patient (Fig. 2). Mefloquine significantly enhanced anti-proliferative and pro-apoptotic effects of imatinib and dasatinib in CML cell lines as well as BP-CML CD34 cells, suggesting that mefloquine augments the effects of BCR-ABL TKIs (Fig. 1B, 2A and 2B). Mechanistically, we show that mefloquine significantly induces oxidative stress by increasing levels of mitochondrial superoxidase in K562 cells (Fig, 1C). Consistent with this, mefloquine disrupts lysosomal integrity/function in CML cells as measured by LysoTracker labelling (Fig. 1D). Taken together, we demonstrate that mefloquine is active against BP-CML and enhances the efficacy of BCR-ABL TKIs. Our work also highlights the therapeutic value of targeting oxidative stress and lysosome in the treatment of BP-CML. Figure 1 Mefloquine induces apoptosis, ROS, and lysosomal dysfunction in CML cells. (A)Mefloquine induces apoptosis of K562, LAMA84 and KU812 cells in a dose-dependent manner. (B) Combination of mefloquine and imatinib or dasatinib induces more much apoptosis than single drug alone. Cells were treated with drugs for 72 h. (C) Mefloquine increases levels of mitochondrial superoxidase in K562 cells. (D) Less Lysotracker staining in mefloquine-treated K562 cells compared to control. Cells were treated with mefloquine at 15 µM for 24 h. Figure 1. Mefloquine induces apoptosis, ROS, and lysosomal dysfunction in CML cells. (A)Mefloquine induces apoptosis of K562, LAMA84 and KU812 cells in a dose-dependent manner. (B) Combination of mefloquine and imatinib or dasatinib induces more much apoptosis than single drug alone. Cells were treated with drugs for 72 h. (C) Mefloquine increases levels of mitochondrial superoxidase in K562 cells. (D) Less Lysotracker staining in mefloquine-treated K562 cells compared to control. Cells were treated with mefloquine at 15 µM for 24 h. Figure 2 Mefloquine effectively targets BP-CML CD34 progenitor cells. Mefloquine induces apoptosis (A) and colony formation (B) of BP-CML CD34 cells and combination of mefloquine and dasatinib is superior in inducing apoptosis and decreasing colony formation. (C) Mefloquine inhibits self-renewal capacity of BP-CML CD34 cells. Figure 2. Mefloquine effectively targets BP-CML CD34 progenitor cells. Mefloquine induces apoptosis (A) and colony formation (B) of BP-CML CD34 cells and combination of mefloquine and dasatinib is superior in inducing apoptosis and decreasing colony formation. (C) Mefloquine inhibits self-renewal capacity of BP-CML CD34 cells. Disclosures Hwang: Sanofi: Honoraria, Other: Travel support; Janssen: Honoraria, Other: Travel support; BMS: Honoraria, Other: Travel support; Celgene: Honoraria, Other: Travel support; Roche: Honoraria, Other: Travel support; Pfizer: Honoraria, Other: Travel support; Novartis: Honoraria, Other: Travel support; MSD: Honoraria, Other: Travel support. Chuah:Novartis: Honoraria; Bristol-Myers Squibb: Honoraria; Chiltern: Honoraria.
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Bayraktar, Ulas D., and Maricer Escalon. "An Unusual Presentation of Acute Biphenotypic Leukemia without Bone Marrow Involvement." Blood 112, no. 11 (November 16, 2008): 3995. http://dx.doi.org/10.1182/blood.v112.11.3995.3995.
Анотація:
Abstract Acute biphenotypic leukemia (ABL) represents a minority of acute leukemia cases (around 5%) in which blasts co-express markers of different lineages (myeloid, T-cell or B-cell). ABL without bone marrow involvement is exceedingly rare. Case report: An 18-year-old man presented with slowly enlarging, non-tender cervical lymph nodes. He had no other symptoms. Family history included Hodgkin’s disease in his mother and maternal great aunt, histiocytosis X in his maternal cousin, and non-Hodgkin’s lymphoma in his paternal grandfather. Physical exam revealed multiple small cervical and axillary lymph nodes. Laboratory analyses including complete blood count, peripheral blood smear, complete metabolic profile, mononucleosis screen, and hepatitis and HIV serologies were unremarkable. Histopathological examination of the left cervical lymph node revealed rare germinal centers with an interfollicular proliferation of generally medium size cells with a fine blastic chromatin pattern and scant cytoplasm. CD34 was positive in more than half of the cells. Between 30% and 80% of the cells were positive for TdT (Figure 1), CD3, CD4, and CD7. Myeloperoxidase (Figure 2) and lysozyme were positive in about 20% of the cells. Flow cytometry revealed an immature myeloid cell population coexpressing CD33, CD13, CD7, and CD34. Chromosomal analysis revealed a complex karyotype. Bone marrow exam was unremarkable and no evidence of leukemia was observed. Flow cytometry of the bone marrow aspirate revealed only 4% of the cells to be CD34+. Whole body PET scan showed extensive uptake in bilateral axillae, neck, and inguinal regions without any extranodal involvement. The patient received hyper-CVAD regimen (alternating cycles of cyclophosphamide, vincristine, adriamycin, dexamethasone and high dose methotrexate, cytarabine) for 8 cycles and remains in remission five months after the end of hyper-CVAD protocol. To our knowledge, this is the third case reported in the literature with acute biphenotypic leukemia not involving the bone marrow. However, our case cannot accurately be referred to as leukemia since the bone marrow is not involved; neither can it be named as lymphoblastic lymphoma because of its myeloid differentiation. Therefore, we will refer to it as extramedullary granulocytic sarcoma with biphenotypic features. Figure 1 Figure 1. Figure 2 Figure 2.
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Majidi, Fatemeh, Oumaima Stambouli, Ron-Patrick Cadeddu, Simon Kai Brille, Jasmin Ewert, Ulrich Germing, Robert Zeiser, Bernd Giebel, and Norbert Gattermann. "Effect of the Neddylation Inhibitor Pevonedistat on Normal Hematopoietic Stem Cell Subsets and Immune Cell Composition." Blood 138, Supplement 1 (November 5, 2021): 4787. http://dx.doi.org/10.1182/blood-2021-150095.
Анотація:
Abstract Introduction: Antitumor activity of the neddylation inhibitor pevonedistat has been documented in several hematologic and non-hematologic malignancies. Unexpectedly, Zhou et al (PNAS, 2016) discovered a dose-dependent biphasic effect of pevonedistat in solid tumor cell lines. While micromolar concentrations inhibited tumor cell growth, low nanomolar concentrations significantly increased cell proliferation and tumor stem cell self-renewal both in vitro and in vivo. The effect of low-dose pevonedistat has not yet been explored in the field of hematopoietic stem cell transplantation. Therefore, we evaluated how pevonedistat affects the viabiilty, growth and proportions of CD34 + cell subpopulations. In view of the emerging role of neddylation in the regulation of both innate and adaptive immunity, we also investigated the influence of pevonedistat on T-cell activation to explore a potentially beneficial effect on posttransplant immune complications. Methods and Results: Using the WST-1 assay we confirmed the biphasic effect of pevonedistat on normal mobilized CD34 + cells. Incubation for 72 h with 0.1 µM pevonedistat significantly increased metabolic activity as a surrogate parameter for proliferation, while 1.0 µM pevonedistat showed a cytotoxic effect. We explored the underlying mechanism for the low-dose effect. Since Zhou et al. previously showed that pevonedistat can promote tumor stem cell proliferation by inducing EGFR homodimerization, we used a proximity ligation assay and found that 0.1 µM pevonedistat induced EGFR homodimerization in normal mobilized CD34 + cells, too. In addition to homodimerization, we also looked at phosphorylation at Tyr1068, a marker of EGFR activation. By flow cytometry, we showed that phosphorylation was increased by 0.01 µM and 0.1 µM pevonedistat. Using an ELISA-based transcription assay, we also observed a biphasic effect of pevonedistat on c-Myc expression, which is regarded as a marker of 'stemness'. Incubation with pevonedistat for 72 hrs at 0.01 and 0.1 µM stimulated expression of c-Myc, whereas incubation at 1.0 µM downregulated c-Myc. Fractions of hematopoietic stem and progenitor cell (HSPC) subpopulations were measured in CD34 + cells from cord blood after incubation with 0.01, 0.1 and 1.0 µM pevonedistat. Flow cytometry was performed using antibodies against CD34, CD45RA and CD133, as well as 7-AAD for testing cell viability. Exposure to pevonedistat for 72 hrs at 0.1 µM caused an increase in the number of CD34 + cells compared to vehicle-treated CD34+ cells at 72 h as well as compared to initial number of CD34+ cells, whereas 1.0 µM caused a significant decrease. The absolute number of multipotent progenitors (MPP) (CD34 +CD133 +CD45RA -) remained relatively stable at all concentrations, while lympho-myeloid progenitors (LMPP) (CD34+CD133+CD45RA+) and late progenitors (LP) (CD34+CD133-CD45RA+) increased slightly with 0.1 µM pevonedistat compared with controls. However, a significant decrease in LMPP and LP cell numbers was observed at 1.0 µM. Different concentrations of pevonedistat were tested for their capability to modulate allogeneically stimulated T cell activation in a multi-donor mixed lymphocyte reaction (mdMLR) assay in vitro. Mesenchymal stromal cells (MSCs)-derived extracellular vesicles (MSC-EV) were used as internal immuno-modulatory and non-immuno-modulatory controls in the assay. After 5 days, alterations in the immune cell composition were analyzed by flow cytometry. Pevonedistat was not toxic for MNCs in the mdMLR. However, it decreased the number of activated (CD25high CD54+) CD4+ cells and CD8+ cells. Conclusions: One of the problems in the post-transplant period is a rapid decline in MPP numbers, associated with increased risk of engraftment failure. We showed that low-dose pevonedistat (0.1 µM) is capable of increasing the number of CD34 + cells in vitro while keeping the absolute number of MPPs stable. This finding, together with the observed increase in c-Myc expression, suggests that pevonedistat may help to preserve 'stemness' of CD34+ donor cells, thus supporting engraftment of hematopoietic stem and progenitor cells. Furthermore, the immunosuppressive effects revealed by mdMLR suggest that low-dose pevonedistat may also play a useful immunomodulatory role in the post-transplant setting to potentially reduce the risk of graft-versus-host disease. Figure 1 Figure 1. Disclosures Majidi: Takeda: Research Funding. Germing: Jazz Pharmaceuticals: Honoraria; Bristol-Myers Squibb: Honoraria, Other: advisory activity, Research Funding; Celgene: Honoraria; Novartis: Honoraria, Research Funding; Janssen: Honoraria. Zeiser: Incyte, Mallinckrodt, Novartis: Honoraria, Speakers Bureau. Gattermann: Celgene: Honoraria; Takeda: Research Funding; Novartis: Honoraria.
46
Song, Byung Hoo, Su Young Son, Hyun Kyu Kim, Tae Won Ha, Jeong Suk Im, Aeli Ryu, Hyeji Jeon, et al. "Profiling of Metabolic Differences between Hematopoietic Stem Cells and Acute/Chronic Myeloid Leukemia." Metabolites 10, no. 11 (October 26, 2020): 427. http://dx.doi.org/10.3390/metabo10110427.
Анотація:
Although many studies have been conducted on leukemia, only a few have analyzed the metabolomic profiles of various leukemic cells. In this study, the metabolomes of THP-1, U937, KG-1 (acute myelogenous leukemia, AML), K562 (chronic myelogenous leukemia, CML), and cord blood-derived CD34-positive hematopoietic stem cells (HSC) were analyzed using gas chromatography-mass spectrometry, and specific metabolic alterations were found using multivariate statistical analysis. Compared to HSCs, leukemia cell metabolomes were found to have significant alterations, among which three were related to amino acids, three to sugars, and five to fatty acids. Compared to CML, four metabolomes were observed specifically in AML. Given that overall more metabolites are present in leukemia cells than in HSCs, we observed that the activation of glycolysis and oxidative phosphorylation (OXPHOS) metabolism facilitated the incidence of leukemia and the proliferation of leukemic cells. Analysis of metabolome profiles specifically present in HSCs and leukemia cells greatly increases our basic understanding of cellular metabolic characteristics, which is valuable fundamental knowledge for developing novel anticancer drugs targeting leukemia metabolism.
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Li, Zhenrui, Xi He, Ariel Paulson, Meng Zhao, Pengxu Qian, Fang Tao, Wenxing Ding, Rick Dobrowsky, and Linheng Li. "Metabolic Activity Distinguish Reserve and Primed HSCs." Blood 124, no. 21 (December 6, 2014): 2898. http://dx.doi.org/10.1182/blood.v124.21.2898.2898.
Анотація:
Abstract A population of reserve HSCs has been reported to be in a deep-quiescent (or dormant) state and function as a back-up population of HSCs to support life-time hematopoiesis (Li and Clevers, 2010; Wilson et al., 2008). Currently, characterization of reserve HSCs is mainly based on cell cycle quiescence, however, the metabolic state in reserve HSCs is yet to be defined. Here, we show that reserve HSCs maintain not only a quiescent state but also an overall low metabolic activity whereas the primed HSCs maintain still a quiescent state but primed at metabolic state. First, we used CD49b(Benveniste et al., 2010) to further separate conventional long-term (LT) HSCs (CD34-Flk2-Lineage-Sca-1+c-Kit+)(Yang et al., 2005) into CD49blo and CD49bhi subpopulations and confirmed their enrichment with previously identified dormant (Scl-H2B-GFP label retaining cells, LRCs), thus we termed CD49blo and CD49bhi subpopulations as candidates of reserve and primed HSCs. We then determined the cell cycle frequencies of reserve, primed, and ST-HSCs (Cd34+Flk2-LSK) respectively as once in 3 months, 3-4 weeks, and 3-4 days. Functionally, Reserve HSCs had on average 3.5-fold higher functional capacity compared with primed HSCs. RNA-seq analysis revealed that reserve HSC predominantly expressed a list of imprinting genes that associate with growth-restriction functions; primed HSCs expressed relatively-high number of genes involving in mitochondria fusion, organization, and function, while ST-HSCs expressed genes reflecting an active cycling state. Conducting metabolic assays, we found that reserve HSCs not only maintain quiescence but also maintain overall low metabolic activity in both glycolysis and mitochondrial capacity. In contrast, primed HSCs are in a quiescent state, but with their metabolic state primed as evidenced by an increased glycolytic activity and mitochondrial potential for subsequent active proliferating state in ST-HSCs. Intriguingly, our data suggests that the functionality of reserve HSCs correlates to metabolic state rather than cell cycle. Disclosures No relevant conflicts of interest to declare.
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Goncalves, Kevin A., Shuping Li, Melissa L. Brooks, Sharon L. Hyzy, Anthony E. Boitano, and Michael P. Cooke. "MGTA-456, a First-in-Class Cell Therapy Produced from a Single Cord Blood Unit, Enables a Reduced Intensity Conditioning Regimen and Enhances Speed and Level of Human Microglia Engraftment in the Brains of NSG Mice." Blood 132, Supplement 1 (November 29, 2018): 115. http://dx.doi.org/10.1182/blood-2018-99-118258.
Анотація:
Abstract Background. Allogeneic bone marrow transplant (BMT) is a promising, curative approach for patients with inherited metabolic disorders (IMDs), a class of pediatric diseases characterized by a single enzyme deficiency. The goal of transplant is to provide cells that produce functional enzymes otherwise deficient in these patients, and thereby prevent or ameliorate neurological complications associated with selected IMDs. Donor-derived microglial cells are protective, limiting neurological disease progression. For IMD patients who do not have an HLA matched, non-carrier related donor, cord blood (CB) is the preferred HSPC source given its rapid availability and superior clinical outcomes compared to other graft sources. CB, however, is associated with delayed hematopoietic recovery and relatively poor engraftment due to the limited numbers of hematopoietic stem cells (HSCs) in a CB unit, delaying enzyme/protein reconstitution and cross-correction of non-hematopoietic cells. An aryl hydrocarbon receptor antagonist (AHRa)-based culture has been shown to expand CB CD34+ and CD34+CD90+ cells 330-fold and 100-fold, respectively, leading to rapid hematopoietic recovery after infusion of the clinical product, MGTA-456 (Wagner et al., Cell Stem Cell 2016 and Orchard et al., ASH 2018). As microglia are thought to be derived from HSCs, we hypothesized that MGTA-456 might lead to faster and greater microglial engraftment and potentially enable reduced intensity conditioning. Here, we assessed human hematopoietic and brain engraftment in NSG mice after transplant with MGTA-456 and showed that microglia engrafted faster with MGTA-456, less conditioning was needed, and that, mechanistically, these cells are derived from the CD34+CD90+ cell fraction. Methods. CB CD34+ cells were expanded in growth factor-supplemented media with or without an AHRa for 10 days. NSG mice were transplanted with unmanipulated CB CD34+ cells or the expanded product after 200 cGy total body irradiation or busulfan (BU) dosed at 20 or 40 mg/kg ip. Microglial engraftment was measured by flow cytometry of homogenized brains, quantitating the number of CD45+CD11b+Iba1+ cells, and by immunohistochemistry of brain sections. Results. Relative to naïve, unmanipulated CB CD34+ cells, transplant of MGTA-456 into sublethally irradiated mice led to an 8-fold increase in hematopoietic engraftment and a 10-fold increase in microglial engraftment in the brain (p<0.0001, n=15 mice), with histology consistent with engrafting microglia. As high dose BU enables enhanced microglia engraftment relative to irradiation by crossing the blood brain barrier and clearing host microglia (Capotondo et al., PNAS 2013), we evaluated the effectiveness of MGTA-456 after BU conditioning at 20 or 40 mg/kg. Transplant of MGTA-456 led to a 37-fold increase in engraftment relative to mice transplanted with unmanipulated CB CD34+ cells (p<0.001, n=8). Notably, transplant of MGTA-456 into mice conditioned with low-dose BU (20 mg/kg) led to a 15-fold increase in engraftment relative to high-dose BU (40 mg/kg)-conditioned animals transplanted with unmanipulated CB CD34+ cells (p<0.001, n=8). To evaluate speed of microglial engraftment, we evaluated brains weekly to 16 weeks after transplant. A 28-fold increase in microglial engraftment was demonstrated as early as 2 weeks post-transplant with MGTA-456 (p<0.0001, n=8). Number of engrafting hematopoietic cells in the periphery correlated with number of engrafting microglia in the brain (p<0.0001). Lastly, subpopulations of MGTA-456 were evaluated to determine the source of microglial engraftment. Only CD34+CD90+ cells, but not CD34+CD90- or CD34- cells, led to brain engraftment, consistent with the subpopulation of cells that result in hematopoietic engraftment following transplant of unexpanded cells (Radtke et al., Sci Trans Med 2017 and Goncalves et al., Blood 2017 130:659). Conclusions. These studies demonstrate that microglial engraftment is faster and greater in recipients of MGTA-456 even after lower dose BU conditioning, that microglial engraftment correlates with peripheral blood recovery, and that microglia cells are derived from CD34+CD90+ cells. These results suggest that lower dose BU may improve safety without jeopardizing efficacy in IMD recipients of MGTA-456. A Phase 2 clinical trial is ongoing to evaluate transplant of MGTA-456 in patients with select IMDs. Disclosures Goncalves: Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Li:Magenta Therapeutics: Employment, Equity Ownership. Brooks:Magenta Therapeutics: Employment, Equity Ownership. Hyzy:Magenta Therapeutics: Employment, Equity Ownership. Boitano:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Cooke:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties.
49
Ganan-Gomez, Irene, Kelly S. Chien, Feiyang Ma, Hui Yang, Lin Tan, Philip Lorenzi, Guillermo Garcia-Manero, and Simona Colla. "The Transcriptional and Epigenetic Reprogramming of Aged Hematopoietic Stem Cells Drives Myeloid Rewiring in Clonal Hematopoiesis-Associated Cytopenias." Blood 138, Supplement 1 (November 5, 2021): 3273. http://dx.doi.org/10.1182/blood-2021-150663.
Анотація:
Abstract Patients (pts) with myelodysplastic syndromes (MDS) have few therapy options. Interventions to improve outcomes should consider strategies that arrest MDS in its early phases, when symptoms are minimal and prolonged survival is expected. To develop prevention strategies that arrest MDS before the disease outcomes become irreversibly dismal, we dissected the molecular and biological mechanisms that maintain MDS in one of its premalignant phases, clonal cytopenia of undetermined significance (CCUS). Recognizing that CCUS is an aging-related disease, we first sought to determine, at the single-cell level, how CCUS affects the transcriptional and epigenetic profile of the aging hematopoietic stem and progenitor cell (HSPC) compartment and overcomes aging-induced degenerative phenotypes. We performed single-cell RNA sequencing (scRNA-seq) analysis of Lin -CD34 + HSPCs isolated from the bone marrow (BM) of 3 young healthy donors (yHDs), 4 elderly HDs (eHDs), and 3 elderly pts with CCUS carrying mutations in common MDS driver genes. We found that the frequencies of hematopoietic stem cells (HSCs) and megakaryocytic (Mk)/erythroid (Er) progenitors were increased at the expense of myeloid (My) progenitors in eHDs as compared with yHDs (Fig. a). In contrast, CCUS pts had a predominant My-biased HSPC distribution (Fig. a). However, immunophenotypic quantification in large cohorts of eHDs and CCUS pts revealed that CCUS pts' BM had significantly fewer CD34 +CD38 - HSC populations and CD34 +CD38 + My progenitors, suggesting that My bias in CCUS results from the aberrant My differentiation of HSCs rather than My cell expansion. Further differential expression analysis among the scRNA-seq clusters showed that, compared with yHD HSCs, eHD HSCs were characterized by a significant upregulation of genes involved in the TNFα-induced activation of NF-κB (e.g., MCL1; Fig. b), which is consistent with previous findings that aged HSCs undergo transcriptional reprogramming to maintain their survival in response to changes in the systemic environment (He et al. Blood 2020). In contrast, CCUS HSCs, compared with eHD HSCs, overexpressed regulators of translation, respiratory electron transport, and mitochondrial translation initiation (Fig. c), which underscores these cells' state of proliferation and metabolic activation and their ability to overcome aging-induced phenotypic alterations. To evaluate whether the aberrant lineage differentiation in eHD and CCUS HSPCs arose from the altered fate determination of HSCs, we performed single-cell assays for transposase-accessible chromatin sequencing to profile chromatin accessibility in sorted HSCs or Lin -CD34 + HSPCs from yHDs, eHDs, and CCUS pts. Consistent with our transcriptomic data, compared with yHD HSCs, eHD HSCs were mostly poised for Mk differentiation, whereas CCUS HSPCs were poised for lymphoid/My differentiation. Indeed, eHD HSCs had an increased activity of transcriptional factors belonging to the NF-κB family and open peaks at the distal elements of genes involved in hemostasis (Fig. d). In contrast, CCUS HSCs were poised to downregulate the expression of genes involved in NF-κB signaling and Mk/Er differentiation (Fig. e). These results suggested that CCUS HSCs are highly metabolically active to maintain My differentiation. Indeed, metabolomic analyses confirmed that intermediates of oxidative phosphorylation were significantly upregulated in CCUS CD34 + cells as compared with eHD CD34 + cells (Fig. f). Further, scRNA-seq analysis of mononuclear cells isolated from the BM of 3 CCUS and 3 eHD samples revealed the widespread upregulation of genes involved in protein processing and mitochondrial metabolism. This analysis also revealed impaired terminal My differentiation despite the HSPC My bias, with decreased frequencies of monocytic cells, and an intriguing expansion of cytotoxic cell subsets in the BM of CCUS pts. In conclusion, our results demonstrate that CCUS HSCs carrying MDS driver mutations evade aging-induced phenotypic degeneration, become metabolically active, and have aberrant My skewing. Our study clarifies the molecular mechanisms underlying MDS initiation and offers an opportunity for early therapeutic intervention. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.
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Yu, Jiong, Xiaoru Su, Chengxing Zhu, Qiaoling Pan, Jinfeng Yang, Jing Ma, Leyao Shen, Hongcui Cao, and Lanjuan Li. "GFP Labeling and Hepatic Differentiation Potential of Human Placenta-Derived Mesenchymal Stem Cells." Cellular Physiology and Biochemistry 35, no. 6 (2015): 2299–308. http://dx.doi.org/10.1159/000374033.
Анотація:
Background: Stem cell-based therapy in liver diseases has received increasing interest over the past decade, but direct evidence of the homing and implantation of transplanted cells is conflicting. Reliable labeling and tracking techniques are essential but lacking. The purpose of this study was to establish human placenta-derived mesenchymal stem cells (hPMSCs) expressing green fluorescent protein (GFP) and to assay their hepatic functional differentiation in vitro. Methods: The GFP gene was transduced into hPMSCs using a lentivirus to establish GFP+ hPMSCs. GFP+ hPMSCs were analyzed for their phenotypic profile, viability and adipogenic, osteogenic and hepatic differentiation. The derived GFP+ hepatocyte-like cells were evaluated for their metabolic, synthetic and secretory functions, respectively. Results: GFP+ hPMSCs expressed high levels of HLA I, CD13, CD105, CD73, CD90, CD44 and CD29, but were negative for HLA II, CD45, CD31, CD34, CD133, CD271 and CD79. They possessed adipogenic, osteogenic and hepatic differentiation potential. Hepatocyte-like cells derived from GFP+ hPMSCs showed typical hepatic phenotypes. Conclusions: GFP gene transduction has no adverse influences on the cellular or biochemical properties of hPMSCs or markers. GFP gene transduction using lentiviral vectors is a reliable labeling and tracking method. GFP+ hPMSCs can therefore serve as a tool to investigate the mechanisms of MSC-based therapy, including hepatic disease therapy.