The G Protein-Coupled Receptor cysLT1 Is Expressed in Acute Myeloid Leukemia Mediating Cell Migration and Survival.

Abstract Previous studies have shown that the G protein-coupled receptor (GPCR) cysLT1, which recognizes inflammatory lipid mediators (cysteinyl-leukotrienes), is expressed in immature hematopoietic and tumor (eg. colon cancer) cells. In the present study, we analyzed leukemic blasts from 19 patients with newly diagnosed acute myeloid leukemia for analysis of expression and function of cysLT1. By RT-PCR, cysLT1 mRNA was found in all primary AML cells. Quantitative TaqMan PCR demonstrated high levels particularly in acute myeloblastic leukemia without maturation (FAB M1). There was also a positive correlation between the cysLT1 mRNA level and the expression of CD34 and CD-117 (c-kit), indicating that high cysLT1 expression corresponds to an AML phenotype resembling normal hematopoietic progenitor cells. CysLT1 was functionally active in AML blasts, as demonstrated by intracellular calcium fluxes and actin polymerization induced by the ligand LTD4. Similar to mRNA expression, strongest responses were seen in AML FAB M1. As LTD4 can be produced in the bone marrow by stromal cells and may contribute to bone marrow infiltration of AML, chemotaxis was analyzed. Surprisingly, already low concentrations (10nM) induced significant chemotaxis of AML blasts, while higher concentrations (up to 1 uM) were less effective in a dose-dependent manner. Incubation of myeloid leukemic cell lines (eg. KG1a) and primary AML blasts with the specific cysLT1 antagonist MK571 resulted in significantly reduced viability after 48 h in a dose-dependent manner (10nM-10uM), suggesting also an autocrine function of cysLT1 ligands. To explore signal transduction pathways involved in leukemic cell proliferation and chemotaxis, we found that in AML cell lines, LTD4 induced phosphorylation of Erk/MAP kinase, wich is related to proliferation, and Pyk2, which represents a focal adhesion kinase-like signaling molecule that links GPCRs with cell migration, while the Akt pathway was not involved. We conclude that cysLT1 is consistently expressed in acute myeloid leukemia, and mediates both chemotactic and proliferative responses. Therefore, cysLT1 antagonists, which are already used in the treatment of allergy, may improve the effectiveness of antileukemic therapy.

Download Full-text

Acute myeloid leukemia–induced remodeling of the human bone marrow niche predicts clinical outcome

Blood Advances ◽

10.1182/bloodadvances.2020001808 ◽

2020 ◽

Vol 4 (20) ◽

pp. 5257-5268

Author(s):

Yiyang Chen ◽

Lina Marie Hoffmeister ◽

Yasmin Zaun ◽

Lucas Arnold ◽

Kurt Werner Schmid ◽

...

Keyword(s):

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Survival Rate ◽

Clinical Outcome ◽

Myeloid Leukemia ◽

Bone Marrow Niche ◽

Dependent Manner ◽

Hematopoietic Stem ◽

Acute Myeloid ◽

First Diagnosis

Abstract Murine models of myeloid neoplasia show how leukemia infiltration alters the hematopoietic stem cell (HSC) niche to reinforce malignancy at the expense of healthy hematopoiesis. However, little is known about the bone marrow architecture in humans and its impact on clinical outcome. Here, we dissect the bone marrow niche in patients with acute myeloid leukemia (AML) at first diagnosis. We combined immunohistochemical stainings with global gene expression analyses from these AML patients and correlated them with clinical features. Mesenchymal stem and progenitor cells (MSPCs) lost quiescence and significantly expanded in the bone marrow of AML patients. Strikingly, their HSC- and niche-regulating capacities were impaired with significant inhibition of osteogenesis and bone formation in a cell contact–dependent manner through inhibition of cytoplasmic β-catenin. Assessment of bone metabolism by quantifying peripheral blood osteocalcin levels revealed 30% lower expression in AML patients at first diagnosis than in non-leukemic donors. Furthermore, patients with osteocalcin levels ≤11 ng/mL showed inferior overall survival with a 1-year survival rate of 38.7% whereas patients with higher osteocalcin levels reached a survival rate of 66.8%. These novel insights into the human AML bone marrow microenvironment help translate findings from preclinical models and detect new targets which might pave the way for niche-targeted therapies in AML patients.

Download Full-text

Acute myeloid leukemia M4 with bone marrow eosinophilia (M4Eo) and inv(16)(p13q22) exhibits a specific immunophenotype with CD2 expression

Blood ◽

10.1182/blood.v81.11.3043.3043 ◽

1993 ◽

Vol 81 (11) ◽

pp. 3043-3051 ◽

Cited By ~ 84

Author(s):

HJ Adriaansen ◽

PA te Boekhorst ◽

AM Hagemeijer ◽

CE van der Schoot ◽

HR Delwel ◽

...

Keyword(s):

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Low Frequency ◽

Leukemic Cell ◽

Leukemic Cells ◽

Terminal Deoxynucleotidyl Transferase ◽

Cell Populations ◽

Spontaneous Proliferation ◽

Acute Myeloid

Abstract Extensive immunologic marker analysis was performed to characterize the various leukemic cell populations in eight patients with inv(16)(p13q22) in association with acute myeloid leukemia with abnormal bone marrow eosinophilia (AML-M4Eo). The eight AML cases consisted of heterogeneous cell populations; mainly due to the presence of multiple subpopulations, which varied in size between the patients. However, the immunophenotype of these subpopulations was comparable, independent of their relative sizes. Virtually all AML-M4Eo cells were positive for the pan-myeloid marker CD13. In addition, the AML were partly positive for CD2, CD11b, CD11c, CD14, CD33, CD34, CD36, CDw65, terminal deoxynucleotidyl transferase (TdT), and HLA-DR. Double immunofluorescence stainings demonstrated coexpression of the CD2 antigen and myeloid markers and allowed the recognition of multiple AML subpopulations. The CD2 antigen was expressed by immature AML cells (CD34+, CD14-) and more mature monocytic AML cells (CD34-, CD14+), whereas TdT expression was exclusively found in the CD34+, CD14- cell population. The eight AML-M4Eo cases not only expressed the CD2 antigen, but also its ligand CD58 (leukocyte function antigen-3). Culturing of AML-M4Eo cell samples showed a high spontaneous proliferation in all three patients tested. Addition of a mixture of CD2 antibodies against the T11.1, T11.2, and T11.3 epitopes diminished cell proliferation in two patients with high CD2 expression, but no inhibitory effects were found in the third patient with low frequency and low density of CD2 expression. These results suggest that high expression of the CD2 molecule in AML-M4Eo stimulates proliferation of the leukemic cells, which might explain the high white blood cell count often found in this type of AML.

Download Full-text

The pan-Bcl-2 inhibitor obatoclax promotes differentiation and apoptosis of acute myeloid leukemia cells

Investigational New Drugs ◽

10.1007/s10637-020-00931-4 ◽

2020 ◽

Vol 38 (6) ◽

pp. 1664-1676

Author(s):

Małgorzata Opydo-Chanek ◽

Iwona Cichoń ◽

Agnieszka Rak ◽

Elżbieta Kołaczkowska ◽

Lidia Mazur

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Treatment Strategies ◽

Leukemic Cell ◽

Leukemic Cells ◽

Dependent Manner ◽

Apoptotic Pathway ◽

Acute Myeloid Leukemia Cells ◽

Induced Apoptosis ◽

Acute Myeloid

Summary One of the key features of acute myeloid leukemia (AML) is the arrest of differentiation at the early progenitor stage of myelopoiesis. Therefore, the identification of new agents that could overcome this differentiation block and force leukemic cells to enter the apoptotic pathway is essential for the development of new treatment strategies in AML. Regarding this, herein we report the pro-differentiation activity of the pan-Bcl-2 inhibitor, obatoclax. Obatoclax promoted differentiation of human AML HL-60 cells and triggered their apoptosis in a dose- and time-dependent manner. Importantly, obatoclax-induced apoptosis was associated with leukemic cell differentiation. Moreover, decreased expression of Bcl-2 protein was observed in obatoclax-treated HL-60 cells. Furthermore, differentiation of these cells was accompanied by the loss of their proliferative capacity, as shown by G0/G1 cell cycle arrest. Taken together, these findings indicate that the anti-AML effects of obatoclax involve not only the induction of apoptosis but also differentiation of leukemic cells. Therefore, obatoclax represents a promising treatment for AML that warrants further exploration.

Download Full-text

Anti-VEGFC Treatment Reduces the Leukemic Outgrowth of Primary CD34+ Pediatric Acute Myeloid Leukemia Cells

Blood ◽

10.1182/blood.v118.21.1425.1425 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1425-1425 ◽

Cited By ~ 1

Author(s):

Kim R Kampen ◽

Arja ter Elst ◽

André B Mulder ◽

Megan E Baldwin ◽

Klupacs Robert ◽

...

Keyword(s):

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Leukemic Cell ◽

Suspension Cells ◽

Pediatric Aml ◽

Limiting Dilution ◽

Acute Myeloid

Abstract Abstract 1425 Previously, it was demonstrated that exogenous addition of vascular endothelial growth factor C (VEGFC) increased the leukemic cell viability, reduced apoptosis via activation of Bcl-2, and decreased chemotherapy induced apoptosis via its receptor FLT-4 (Further revert to as VEGFR3) (Dias et al. Blood 2002). Furthermore, it was shown that VEGFC promotes angiogenesis by induction of COX-2 through VEGFR3 activation in THP-1 cells (Chien et al. Carcinogenesis 2005). We have previously found that endogenous VEGFC expression is associated with decreased drug responsiveness in childhood acute myeloid leukemia (AML), both in vitro as well as in vivo (de Jonge et al. Clinical Cancer Research 2008). In addition, high VEGFC mRNA expression is strongly associated with reduced complete remission and overall survival in adult as well as pediatric AML (de Jonge et al. Blood 2010). It was thought that the leukemic blast population is organized as a hierarchy, whereby leukemia initiating cells (LICs) reside at the top of this hierarchy, and it is only these cells that have the capacity to engraft in non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice. The LIC is thought to be enriched in the CD34+ leukemic cell fraction and is shown to expand in vitro using a myeloid cytokine mix of IL-3, TPO, and G-CSF in colony forming cell (CFC) assays and long-term culture-initiating cell (LTC-IC) assays (Guan et al. Exp. Hematol. 2002, van Gosliga et al. Exp. Hematol. 2007). Moreover, LTC-IC assays performed in limiting dilution detect the in vitro outgrowth potential of stem-like cells that reside underneath the stromal cell layer. In this study, we set out to investigate the potential of anti-VEGFC treatment as an inhibitor of the outgrowth of LICs within the CD34+ fraction of primary AML samples. First, we determined the possibility of an autocrine loop for VEGFC in AML. Pediatric AML cell (n=7) derived VEGFC levels were found to be 1.4-fold increased (P =.008) compared to secreted VEGFC levels from normal bone marrow (NBM) cells (n=4). Pediatric AML blast cells showed KDR (further revert to as VEGFR2) membrane expression in 44 out of 50 patient samples (varying 8–99% of the total blast population), whereas on NBM cells VEGFR2 expression was below 5%. VEGFR3 expression was below 5% on both leukemic blasts and NBM cells. We evaluated the effect of anti-VEGFC (VGX-100, kindly provided by Vegenics, used at a concentration of 30 μg/ml) treatment on the CD34+ isolated compartment of pediatric AML bone marrow samples. Anti-VEGFC treatment reduced the outgrowth potential of AML derived CD34+ cells (n=2) with >25% in CFC assays. Interestingly, morphological analysis revealed a 3-fold enhanced formation of macrophages. LTC-IC assays demonstrated a (15% to 50%) decrease in the long-term growth of CD34+ isolated AML cells in 3 out of 4 patient samples. Morphological characterization of the suspension cells suggested a shift in development along the myelomonocytic lineage after two weeks of anti-VEGFC treatment. With FACS analysis, these cells showed a higher number of cells stained positive for CD11b, and CD14, and lower numbers where positive for CD34. Anti-VEGFC treated LTC-IC assays in limiting dilution demonstrated a (44% and 74%) reduction in the outgrowth potential of long-term cultured CD34+ isolated AML cells and blocked the erythroid colony formation in 2 out of 3 patient samples. Anti-VEGFC treatment did not have an effect on the outgrowth of CD34+ sorted NBM cells in the various assays (n=2). In conclusion, anti-VEGFC treatment of the CD34+ isolated fraction from primary pediatric AML samples showed a reduction of AML outgrowth. Differentiating cells are skewed to the myelomonocytic lineage upon anti-VEGFC treatment. We hypothesize that deprivation of VEGFC in primary CD34+ AML cell cultures results in enhanced leukemic cell death and abates an important proliferation signal for AML cells. Yet, further investigations are warranted.Figure 1.Skewing of LTC-IC assay suspension cells towards the myelomonocytic lineage upon anti-VEGFC treatment. MGG stained cytospins of suspension cells of the LTC-IC co-culture obtained during demi-depopulation at week 2.Figure 1. Skewing of LTC-IC assay suspension cells towards the myelomonocytic lineage upon anti-VEGFC treatment. MGG stained cytospins of suspension cells of the LTC-IC co-culture obtained during demi-depopulation at week 2. Disclosures: Baldwin: Circadian Technologies Limited: Employment. Robert:Circadian Technologies Limited: Employment, Membership on an entity's Board of Directors or advisory committees.

Download Full-text

A Novel Non-Genetic Photostable Approach to Labelling Acute Myeloid Leukemia and Bone Marrow Cells with Quantum Dots

Blood ◽

10.1182/blood.v120.21.4818.4818 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4818-4818

Author(s):

Yanwen Zheng ◽

Zhengwei Mao ◽

Bin Yin

Keyword(s):

Bone Marrow ◽

Quantum Dots ◽

Acute Myeloid Leukemia ◽

Blood Cells ◽

Myeloid Leukemia ◽

Hematopoietic Cells ◽

Leukemia Cell ◽

Leukemia Cells ◽

Dependent Manner ◽

Acute Myeloid

Abstract Abstract 4818 Acute myeloid leukemia (AML) is a detrimental disease with difficult diagnosis and treatment. Understanding the biology of AML at the molecular and cellular levels would be essential to successful management of the disease. However, the notoriously known difficulty in manipulation of leukemia cells has long hindered the dissection of AML pathogenesis. The advent of CdSe/ZnS quantum dots (QDs) represents an important advancement in the research field of nanotechnology, which have recently also been applied for imaging of live cells. Here, we have introduced a non-genetic approach of marking blood cells, by taking advantage of QD technology. We compared QDs complexed with different vehicles, including a peptide Tat (QDs-Tat), cationic polymer Turbofect (QDs-Tf) and liposome Lipofectamine 2000 (QDs-Lip), in their abilities to mark cells. QDs-Tat showed the highest efficiency in delivery into hematopoietic cells, among the three vehicles. We then examined QDs-Tat labelling of leukemia cell lines, and found that QDs-Tat could label 293T, bone marrow (BM) cells, THP-1, MEG-01 and HL-60 with a decreasing efficiency. The efficiency of QDs-Tat delivery was dependent on the concentration of QDs-Tat applied, but not the length of incubation time. In addition, more uniform intracellular distributions of QDs in 293T and leukemia cells were obtained with QDs-Tat, compared with the granule-like formation obtained with QDs-Lip. Clearly, QD fluorescence was sharp and tolerant to repetitive photo excitations, and could be detected in 293T for up to one week following labelling. In summary, our results suggest that QDs have provided a photostable, non-genetic and transient approach that labels normal and malignant hematopoietic cells in a cell type-, vehicle-, and QD concentration-dependent manner. We expect for potentially wide applications of QDs as an easy and fast tool assisting investigations of various types of blood cells in the near future. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

2559 Role of tissue non-specific alkaline phosphatase (TNAP) in promoting the survival of acute myeloid leukemia (AML) cells within the bone marrow microenvironment

Journal of Clinical and Translational Science ◽

10.1017/cts.2018.119 ◽

2018 ◽

Vol 2 (S1) ◽

pp. 26-27

Author(s):

Bradley Bowles ◽

Rosalie M. Sterner ◽

Kimberly N. Kremer ◽

Amel Dudakovic ◽

Jennifer J. Westendorf ◽

...

Keyword(s):

Bone Marrow ◽

Alkaline Phosphatase ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Histone Deacetylase Inhibitors ◽

Leukemic Cell ◽

Bone Marrow Microenvironment ◽

Matrix Mineralization ◽

Altered Expression ◽

Acute Myeloid

OBJECTIVES/SPECIFIC AIMS: Treatment of acute myeloid leukemia (AML) is challenging, as apoptosis-resistant AML cells often persist within the bone marrow microenvironment despite chemotherapy. The overall goal of our laboratory is to identify and ultimately target the bone marrow factors that protect AML cells. METHODS/STUDY POPULATION: Using cell cultures, we previously reported that SDF-1 (CXCL12), an abundant bone marrow chemokine, induces apoptosis of isolated CXCR4+ AML cells, including freshly isolated bone marrow-derived AML cells from approximately one-third of AML patients. However, co-culture of AML cells with differentiating osteoblasts protected AML cells from apoptosis. RESULTS/ANTICIPATED RESULTS: Histone deacetylase inhibitors (HDACi) abrogated the ability of osteoblasts to protect AML cells and altered expression of matrix mineralization genes including tissue nonspecific alkaline phosphatase (TNAP). A different drug, cyclosporine A (CSA), similarly inhibited osteoblast-mediated protection of AML cells and reduced TNAP expression. Specifically targeting osteoblast TNAP via siRNA was sufficient to prevent osteoblasts from protecting AML cells in co-cultures. In addition, we are targeting TNAP enzymatically. DISCUSSION/SIGNIFICANCE OF IMPACT: Our results indicate that targeting TNAP may be useful in AML treatment to render the bone marrow microenvironment more hostile to leukemic cell survival.

Download Full-text

High Expression of ENPP2/Autotaxin in Patients with FLT3-ITD Positive Acute Myeloid Leukemia.

Blood ◽

10.1182/blood.v108.11.1911.1911 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1911-1911

Author(s):

Claudia Ortlepp ◽

Christine Steudel ◽

Sina Koch ◽

Martin Ryser ◽

Gerhard Ehninger ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Cell Migration ◽

Tyrosine Kinase ◽

Myeloid Leukemia ◽

Leukemic Cell ◽

High Expression ◽

Functional Assay ◽

Close Link ◽

Lysophospholipase D ◽

Acute Myeloid

Abstract The Fms-like tyrosine kinase 3 (FLT-3) receptor tyrosine kinase acts as an important player in controlling the growth and differentiation of haematopoietic progenitor cells. It was shown that activating mutations of FLT-3 are involved in the pathogenesis of acute myeloid leukemia (AML). The most frequent aberration is an internal tandem duplication (ITD) of the juxtamembrane domain coding sequence, which is present in up to 27% of the patients with AML and has been associated with poorer prognosis. To further understand the factors involved, we performed microarray analysis to identify genes specifically upregulated in cases with FLT3-ITD mutations. One gene we identified was ENPP2/Autotaxin (ENPP2/ATX). The ENPP2/ATX protein acts as a lysophospholipase D through generating lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P). These two bioactive small lipids have crucial functions in cell migration and proliferation and act via G-protein coupled receptors. ENPP2/ATX has been associated with tumor cell migration in several solid tumors, expression has not been reported in myeloid cells before. The aim of the current study was to investigate the expression pattern of ENPP2/ATX in normal haematopoiesis and leukemia as well as its role in the pathogenesis of AML. Real-Time PCR analysis of 215 primary AML samples from all major cytogenetic risk groups confirmed a significant upregulation in FLT3-ITD patients. High expression was also found in cases with complex karyotype and −5 and −7, but not in cases with good risk cytogenetics. To explore the function of ENPP2/ATX in the haematopoietic system, we stably overexpressed two different splice variants of ENPP2/ATX in several leukemic cell lines. The expression was detected at both, mRNA and protein level by RT-PCR and Western blot analyses, respectively. The lysophospholipase D activity of the protein could be measured by a functional assay using an artificial substrate for ENPP2/ATX. Transwell migration assays showed an increased migration capacity of cells with high expression of ENPP2, which indicates that the overexpressed protein is functional. Based on our results, we suggest that ENPP2/ATX is involved in leukemic cell migration. Our findings point to a close link between ENPP2/ATX and FLT3 in the pathogenesis of AML. Current work focuses on its role in cell survival and prognosis.

Download Full-text

Targeting FLT3 Phosphorylation and Signaling in Acute Myeloid Leukemia.

Blood ◽

10.1182/blood.v110.11.912.912 ◽

2007 ◽

Vol 110 (11) ◽

pp. 912-912

Author(s):

Alan K. Ikeda ◽

Dejah Judelson ◽

Junling Li ◽

Ru Qi Wei ◽

Paul Tapang ◽

...

Keyword(s):

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Bone Marrow Transplantation ◽

Marrow Transplantation ◽

Myeloid Leukemia ◽

Scid Mice ◽

Parp Cleavage ◽

Dependent Manner ◽

Acute Myeloid

Abstract Children with acute myeloid leukemia (AML) have 50% overall survival despite aggressive chemotherapy and bone marrow transplantation. Similarly, only one third of adults diagnosed with AML will be cured. AML blast cells from approximately 30% of patients express a constitutively active receptor tyrosine kinase, FLT3-ITD, which contains internal tandem duplications in the juxtamembrane domain. Patients with FLT3-ITD have a worse prognosis. ABT-869 is a multi-targeted small molecule inhibitor of receptor tyrosine kinases and is a potent inhibitor of FLT3, c-Kit, and members of the VEGF and PDGF receptor families. We previously demonstrated that ABT-869 in vitro induces apoptosis of AML cell lines harboring the FLT3-ITD and primary AML cells, and in vivo in tumors from MV-411 xenograft models. Phosphorylation of FLT3 and activation of downstream signaling molecules, STAT5 and ERK, were inhibited by ABT-869 in a concentration-dependent manner. Cells were also stained with Annexin V-FITC and propidium iodide, and analyzed using FACS. ABT-869 induced apoptosis, caspase-3 activation, and PARP cleavage after 48 hours. Toxic effects were not observed on normal hematopoietic progenitor cells in methylcellulose-based colony assays at concentrations that were effective in AML cells. To examine the effects of ABT-869 in vivo, we treated SCID mice injected with MV-411 with oral preparations of ABT-869. Complete regression of MV4-11 tumors was observed in mice treated with ABT-869 at 20 and 40 mg/kg/day. No adverse effects were detected in the peripheral blood counts, bone marrow, spleen or liver. Tumors from mice treated with ABT-869 showed decreased proliferation by Ki67 and increased apoptosis by TUNEL staining. We also observed that the mice treated with ABT-869 the day after injection of AML cells remained tumor-free for over 3 months in contrast to the mice receiving the vehicle alone. Inhibition of FLT3 phosphorylation was demonstrated in the tumors from mice treated with ABT-869. ABT-869 also suppresses the growth of Molm-13 (human AML cell line that expresses both FLT3-ITD and wt FLT3) at an IC50 between 1 and 10nM. To examine the effects of ABT-869 in vivo, we employed a murine bone marrow transplantation model. After chemical ablation of the bone marrow, SCID mice were injected with Molm-13 cells through the tail vein to allow engraftment. We observed that mice treated with an oral preparation of ABT-869 at 40 mg/kg/day prevented the engraftment of Molm-13 cells. The SCID mice that were not administered ABT-869 demonstrated clinical engraftment with hind leg paralysis and chloroma formation. Chloroma formation was confirmed by immunohistochemical staining with CD33 and CD45. NOD-SCID mouse models are currently being used to analyze the effects of ABT-869 on primary AML cells in vivo. We will also determine if there is any difference in efficacy in relation to the FLT3 status of each primary AML sample. Our preclinical studies demonstrate that ABT-869 is effective and nontoxic at the doses studied, and provide rationale for the treatment and prevention of relapse in AML patients.

Download Full-text

Treatment With G-CSF Reduces Acute Myeloid Leukemia (AML) Blasts Viability In Presence Of Bone Marrow Stroma

Blood ◽

10.1182/blood.v122.21.1422.1422 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1422-1422

Author(s):

Meritxell Nomdedeu ◽

Marta Pratcorona ◽

Marina Díaz-Beyá ◽

Xavier Calvo ◽

Mari Carmen Lara-Castillo ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Cell Viability ◽

Clinical Benefit ◽

Myeloid Leukemia ◽

Conflicts Of Interest ◽

Leukemic Cells ◽

Cell Counting ◽

Dependent Manner ◽

Dose Dependent ◽

Acute Myeloid

Abstract Background The simultaneous administration of G-CSF and chemotherapy as a priming strategy has resulted in a clinical benefit in determined subsets of patients diagnosed with acute myeloid leukemia (AML) (Löwenberg et al, NEJM 2003; Pabst T, et al, Blood 2012). However, the mechanism responsible for this anti-leukemic effect is not fully characterized. We hypothesize that the clinical benefit may occur at least partially by the effect of G-CSF on leukemic stem cells (LSC). Objective The main goal of this project was to determine the effect of G-CSF on primary AML samples in vitro, especially on LSCs. Methods and patients Peripheral blood mononuclear cells (PBMC) from 10 AML patients were treated with G-CSF at increasing doses, alone or in co-culture with HS-5 stroma cells. Cell viability (7-AAD -eBioscience- cell death exclusion and volumetric cell counting) and surface phenotype was determined by flow cytometry (FACSVerse, BD) 72 hours after treatment. Data were analyzed using the FlowJo (Trastar) software. For clonogenicity assays, AML primary samples were treated for 18 hours with G-CSF at increasing concentrations and cultured in H4034 Optimum MethoCult (StemCell Technologies) for 14 days. Colonies were counted based on cellularity and morphology criteria. Results G-CSF treatment showed no effect on cell viability of the bulk leukemic population or on the CD34 + immature subpopulation. A dose-dependent increase in CXCR4 surface expression was observed, reaching a 1.4-fold of change at the highest concentration of G-CSF (100 μg/mL). In contrast, treatment of leukemia cells with G-CSF in the presence of stroma cells reduced the overall cell viability. Thus, a 32% decrease of cell viability was measured at the highest concentration used (p = 0.0006), while no significant changes in the frequency of each leukemic subpopulations were observed. Clonogenic capacity was significantly reduced in a dose-dependent manner upon treatment with G-CSF, achieving a 41% reduction at the highest G-CSF concentration (100 μg/mL). Conclusions G-CSF reduces the viability of leukemic cells when these cells are in co-culture with the HS-5 stroma cell line, suggesting that the presence of stroma cells is required for the cytotoxical effect of G-CSF on the blast population. Interestingly, G-CSF treatment decreased the clonogenic capacity of AML samples, therefore suggesting that G-CSF exerts its effect at least partially on LSCs. Our findings support the design of studies to explore new strategies of chemotherapy priming in AML patients. Disclosures: No relevant conflicts of interest to declare.

Download Full-text