CXCR4 Expression and Biological Activity Is Dependent on Oxygen Partial Pressure in Acute Myeloid Leukemia.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 938-938
Author(s):  
Michael Fiegl ◽  
Ismael J. Samudio ◽  
Karen Clise Dwyer ◽  
Jared Burks ◽  
Herbert Fritsche ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Leukemic Cell ◽  
Cxcr4 Expression ◽  
Leukemic Cell Lines ◽  
Acute Myeloid

Abstract CXCR4, the receptor for bone marrow stroma derived SDF-1, has recently been studied in normal hematopoiesis and hematologic malignancies. Increased expression of CXCR4 by leukemic blasts has been reported by us and others (Konoplev S. et al, Cancer 2007) to be associated with poor prognosis in acute myeloid leukemia (AML). However, all in-vitro studies are usually carried out under unphysiological, i.e. normoxic (21% O2) conditions. We hypothesized that the pO2 in vitro has major impact on the expression of CXCR4, a key receptor for cell migration and intracellular signalling. Thus, pO2 of bone marrow aspirates was measured using the i-STAT Portable Clinical Analyzer and a hypoxic workstation was used providing constant low oxygen content. Surface and total CXCR4 expression was examined in leukemic cell lines and patient samples by flow cytometry, confocal microscopy and Western blotting (WB). In 19 patients, the median pO2 of the bone marrow was determined as 46.1±12.8 mmHg (6.1±1.7%) with no significant difference between patients with AML (n=7, pO2 41.3±11.2 mmHg) and patients in CR (n=12, pO2 48.3±15.9 mmHg). This level of hypoxia significantly increases surface and total expression of CXCR4 in the leukemic cell lines U937 and OCI-AML3 as well as in samples from patients with AML, as compared to normoxic conditions (~2.8fold increase). This increase happened mainly within the first 2–8 hours of hypoxia and was unrelated to increased CXCR4 transcription, as shown by PCR. Re-oxygenation of leukemic cells resulted in a statistical significant degradation of CXCR4 (~3fold decrease) in all examined cell lines and patient samples (n=10). This loss of CXCR4 is very rapid (within 5 minutes of re-oxygenation) and was detected by flow cytometry, confocal microscopy and WB. This phenomenon was independent of proteasome activity and ATP. Detailed analysis of membraneous lipid rafts by sucrose density separation, cholesterol depletion and flow cytometry analysis for GM1 gangliosides showed structural (distinct re-distribution of Lck in lipid rafts) and quantitative changes (loss of cholesterol and CXCR4) during re-oxygenation. Moreover, part of the loss of CXCR4 can be attributed to sequestration of microparticles into the extracellular environment as shown by WB of supernatant of re-oxygenated cells and by a significant increase (~1.5fold) in the amount of microparticles released by cells (cell lines U937 and OCI-AML3 and additional patient samples) during the process of re-oxygenation, as measured by flow cytometry. In summary, this study determined the oxygen content of CR and leukemic bone marrow samples as 6.1±1.7%. This pO2 is associated with an increase in CXCR4 expression on AML cells, while re-oxygenation leads to a rapid decrease of CXCR4, perhaps in part by shedding of CXCR4- containing microparticles. These studies point to the importance of studying leukemic blasts under physiologic, i.e. hypoxic conditions.

scholarly journals Circulating Extracellular Vesicles from Acute Myeloid Leukemia Patients Drive Distinct Metabolic Profile of Leukemic Cells and Reveal Crucial Lipidomic Biomarkers

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3471-3471
Author(s):  
Dorian Forte ◽  
Roberto Maria Pellegrino ◽  
Francesco Fabbri ◽  
Ivan Vannini ◽  
Samantha Bruno ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Board Of Directors ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Leukemic Cell ◽  
Mitochondrial Activity ◽  
Advisory Committees ◽  
Leukemic Cell Lines ◽  
Acute Myeloid

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.

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

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1425-1425 ◽  
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.

Identifying Small Molecules That Overcome HoxA9-Mediated Differentiation Arrest in Acute Myeloid Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3513-3513
Author(s):  
David B. Sykes ◽  
Mark K Haynes ◽  
Nicola Tolliday ◽  
Anna Waller ◽  
Julien M Cobert ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Acute Myeloid Leukemia ◽  
Small Molecules ◽  
Cell Line ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Myeloid Differentiation ◽  
Prostacyclin Receptor ◽  
Acute Myeloid

Abstract Abstract 3513 AML in adults is a devastating disease with a 5-year survival rate of 25%. We lack new treatments for AML, and the chemotherapy standard of care remains unchanged in thirty years. One success story in the treatment of AML has been the discovery of drugs that trigger the differentiation of leukemic blasts in the small subset of patients with acute promyelocytic leukemia. However, differentiation therapy is unfortunately not available for the remaining 90% of non-APL acute myeloid leukemia patients. Understanding and targeting the mechanism of differentiation arrest in AML has been under investigation for more than four decades. There is growing evidence to support the role of the homeobox transcription factors in normal hematopoietic differentiation as well as malignant hematopoiesis. The persistent, and inappropriate, expression of the homeobox gene HoxA9 has been described in the majority of acute myeloid leukemias. This implicates HoxA9 dysregulation as a common pathway of differentiation arrest in myeloid leukemias and suggests that by understanding and targeting this pathway, one might be able to overcome differentiation arrest. In cultures of primary murine bone marrow, constitutive expression of HoxA9 blocks myeloid differentiation and results in the outgrowth of immature myeloid cell lines. The mechanism by which HoxA9 causes differentiation arrest is not known and no compounds exist that inhibit HoxA9. We developed a murine cell line model in which the cells were blocked in differentiation by a conditional version of HoxA9. In this system, an estrogen-dependent ER-HoxA9 protein was generated by fusion with the estrogen receptor hormone-binding domain. When expressed in cultures of primary murine bone marrow, immortalized myeloblast cell lines can grow indefinitely in the presence of stem cell factor and beta-estradiol. Upon removal of beta-estradiol, and inactivation of HoxA9, these cell lines undergo synchronous and terminal myeloid differentiation. We took advantage of an available transgenic mouse model in which GFP was expressed downstream of the lysozyme promoter, a promoter expressed only in mature neutrophils and macrophages. Cell lines derived from the bone marrow of this lysozyme-GFP mouse were GFP-negative at baseline and brightly GFP-positive upon differentiation. In this manner, we generated a cell line with a built-in reporter of differentiation. These cells formed the basis of a high-throughput screen in which cells were incubated with small molecules for a period of four days in 384-well plate format. The cells were assayed by multi-parameter flow cytometry to assess for toxicity and differentiation. Compounds that triggered green fluorescence were scored as “HITS” and their pro-differentiation effects confirmed by analysis of morphology and cell surface markers. Given the availability of cells and the simple and reliable assay, we performed both a pilot screen of small molecules at The Broad Institute as well as an extensive screen of the NIH Molecular Libraries Small Molecule Repository. The screen of more than 350,000 small molecules was carried out in collaboration with the University of New Mexico Center for Molecular Discovery. We have identified one lead class of compounds - prostacyclin agonists – capable of promoting myeloid differentiation in this cell line model of AML. Using a parallel cell line derived from a prostacyclin receptor knock-out mouse, we confirmed that activity was due to signaling through the prostacyclin receptor. The role of prostacyclin signaling in myeloid differentiation has not been previously described. Analysis of gene expression demonstrated that the expression of the prostacyclin receptor is seen in ∼60% of in primary human AML samples. This is a potentially exciting finding as prostacyclin agonists (e.g. treprostinil) are clinically relevant as well as FDA-approved. Their potential role in the treatment of acute myeloid leukemia is unknown. Here we present the details of our high-throughput flow cytometry system and preliminary identification of pro-differentiation agents in AML. If successful, we anticipate that one of these small molecules may offer insight into a mechanism for overcoming differentiation arrest, and may also translate into a novel, clinically relevant treatment for acute myeloid leukemia. Disclosures: Sklar: IntelliCyt: Founder of IntelliCyt, the company that sells the HyperCyt high-throughput flow cytometry system. Other. Zon:Fate Therapeutics: Founder Other.

scholarly journals Stromal CYR61 Confers Resistance to Mitoxantrone Via Spleen Tyrosine Kinase Activation in Human Acute Myeloid Leukemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2228-2228
Author(s):  
Xin Long ◽  
Laszlo Perlaky ◽  
Tsz-Kwong Chris Man ◽  
Michele S. Redell
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Stromal Cells ◽  
Myeloid Leukemia ◽  
Stromal Cell ◽  
Chemotherapy Resistance ◽  
Induced Apoptosis ◽  
Acute Myeloid

Abstract Acute myeloid leukemia (AML) is a life-threatening bone marrow malignancy with a relapse rate near 50% in children, despite aggressive chemotherapy. Accumulating evidence shows that the bone marrow stromal environment protects a subset of leukemia cells and allows them to survive chemotherapy, eventually leading to recurrence. The factors that contribute to stroma-induced chemotherapy resistance are largely undetermined in AML. Our goal is to delineate the mechanisms underlying stroma-mediated chemotherapy resistance in human AML cells. We used two human bone marrow stromal cell lines, HS-5 and HS-27A, to study stroma-induced chemotherapy resistance. Both stromal cell lines are equally effective in protecting AML cell lines and primary samples from apoptosis induced by chemotherapy agents, including mitoxantrone, etoposide, and cytarabine. By gene expression profiling using the Affymetrix U133Plus 2 platform, we previously found that CYR61 was among the genes that were commonly upregulated in AML cells by both stromal cell lines. CYR61 is a secreted matricellular protein that is expressed at relatively low levels by AML cells, and at higher levels by stromal cells. CYR61 binds and activates integrins and enhances growth factor signaling in AML cells, and it has been associated with chemoresistance in other malignancies. Our current data provide functional evidence for a role for this protein in stroma-mediated chemoresistance in AML. First, we added anti-CYR61 neutralizing immunoglobulin (Ig), or control IgG, to AML-stromal co-cultures, treated with chemotherapy for 24 hours, and measured apoptosis with Annexin V staining and flow cytometry. In THP-1+HS-27A co-cultures treated with 50 nM mitoxantrone, the apoptosis rate was 33.0 ± 3.7% with anti-CYR61 Ig v. 16.3 ± 4.2% with control IgG; p=0.0015). Next, we knocked down CYR61 in the HS-5 and HS-27A stromal cell lines by lentiviral transduction of two individual shRNA constructs, and confirmed knockdown (KD) at the gene and protein levels for both cell lines. These CYR61-KD stromal cells provided significantly less protection for co-cultured AML cells treated with mitoxantrone, compared to stromal cells transduced with the non-silencing control. For example, the apoptosis rate for THP-1 cells co-cultured with CYR61-KD HS-27A cells was 10.8 ± 0.8%, compared to 6.8 ± 1.1% for THP-1 cells co-cultured with control HS-27A cells (p=0.02). Similar results were obtained with NB-4 AML cells. These results demonstrate that CYR61 contributes to stroma-mediated chemoresistance. CYR61 binds to integrin αvβ3 (Kireeva, et al, J. Biol. Chem., 1998, 273:3090), and this integrin activates spleen tyrosine kinase (Syk) (Miller, et al, Cancer Cell, 2013, 24:45). Using intracellular flow cytometry, we found that activated Syk (pSyk) increased in THP-1 and NB-4 cell lines, and in primary AML patient samples, upon exposure to control HS-27A cells. In primary samples, the mean fluorescence intensity (MFI) for pSyk averaged 11.7 ± 1.3 in co-culture v. 6.6 ± 0.6 for cells cultured alone (p=0.004, n=10). In contrast, pSyk did not significantly increase in AML cells co-cultured with CYR61-KD HS-27A cells (MFI for primary patient samples: 8.6 ± 0.8). This result implicates Syk as a downstream signaling mediator of CYR61. To determine the role of CYR61-induced Syk signaling in chemotherapy resistance, we treated AML-stromal cell co-cultures with 3 uM R406, a potent Syk inhibitor, or DMSO, then added 300 nM mitoxantrone, and measured apoptosis after 24 hours. In AML cells co-cultured with control HS-27A cells, mitoxantrone-induced apoptosis was significantly increased by Syk inhibition (THP-1 cells: 13.7 ± 0.7% with R406 v. 10.0 ± 0.3% with DMSO, p<0.05), consistent with reduced chemoresistance. Notably, R406 did not further increase mitoxantrone-induced apoptosis in AML cells co-cultured with CYR61-KD HS-27A stromal cells (THP-1 cells: 15.7 ± 0.2% with R406 v. 16.9 ± 0.4% with DMSO). Similar results were seen with NB-4 cells, as well. These results support the notion that CYR61 signals through the integrin-Syk pathway to protect AML cells from chemotherapy. Therefore, the CYR61 - integrin - Syk pathway may be a potential therapeutic target for overcoming stroma-induced chemotherapy resistance in AML. Disclosures No relevant conflicts of interest to declare.

The Dual E-Selectin/CXCR4 Inhibitor, GMI-1359, Enhances Efficacy of Anti-Leukemia Chemotherapy in FLT3-ITD Mutated Acute Myeloid Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3790-3790 ◽  
Author(s):  
Weiguo Zhang ◽  
Nalini Patel ◽  
William E. Fogler ◽  
John L. Magnani ◽  
Michael Andreeff
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Leukemic Cell ◽  
Cxcr4 Expression ◽  
Leukemic Cells ◽  
Leukemia Cells ◽  
Acute Myeloid ◽  
Cells Cultured

Abstract Aberrant activation of the FMS-like tyrosine kinase-3 (FLT3) is driven by internal tandem duplication (ITD) mutations in the FLT3 gene, which are commonly observed in patients with acute myeloid leukemia (AML). Hence, FLT3 represents an attractive therapeutic target in AML (Weisberg et al., 2002). Indeed, several small molecule FLT3 inhibitors including sorafenib have showed encouraging efficacy in reducing leukemia blasts in the peripheral blood in FLT3 mutated AML patients. However, these agents have little effect on leukemic stem cells in the bone marrow (BM) microenvironment (Borthakur et al., 2011; Fathi and Chabner, 2011; Zhang et al., 2008). The BM microenvironment is enriched with cytokines and adhesion molecules, such as CXCR4 and E-selectin, which are believed to provide AML cells protection against chemotherapeutic agents (Horacek et al., 2013; Peled and Tavor, 2013). In fact, treatment with sorafenib markedly upregulated CXCR4 levels in FLT3 -mutated cells. In addition, leukemia cells can activate endothelial cells (EC) that induce adhesion of a sub-set of the leukemia cells through E-selectin. The adherent AML cells are sequestered in a nonproliferative state that further protects them from chemotherapy (Pezeshkian et al., 2013). Therefore, blocking CXCR4 and E-selectin in parallel could theoretically eliminate the protection provided by the interaction of leukemic cells with their BM microenvironment and enhance effectiveness of chemotherapy in FLT3-mutant AML patients. In the present study, we evaluated the effectiveness of a dual CXCR4 and E-selectin antagonist, GMI-1359 (GlycoMimetics, Inc., Rockville, MD), in targeting FLT3-ITD-mutant AML in vitro and in vivo. High levels of CXCR4 expression were observed in several human and murine AML cell lines, which was further increased in hypoxic (i.e., 1% oxygen) conditions that mimic the BM microenvironment. These FLT3 -ITD leukemic cell lines also expressed hypoxia-responsive, functional E-selectin ligands identified by reactivity with an antibody (HECA452) that binds the same carbohydrate epitope required for binding to E-selectin. One such E-selectin ligand CD44 increased in FLT3 -ITD cells cultured in hypoxia compared to those cultured in normoxia (i.e. 21% oxygen). In addition, hypoxia also enhanced CXCR4 expression on mesenchymal stem cells (MSC) and EC such as HUVEC. In hypoxic co-cultures of the FLT3 -ITD-mutant leukemia cells MV4-11 or MOLM14 with MSCs and ECs (i.e., HUVEC or TeloHAEC), the presence of the dual E-selectin/CXCR4 inhibitor GMI-1359 effectively reduced leukemic cell adhesion by ~ 50% to the MSC/EC feeder layer compared to the PBS-treated control (p<0.05), even in the presence of TNFa, which induces E-selectin expression in EC. However, an E-selectin specific inhibitor only reduced adhesion of MV4-11 and MOLM14 by ~ 20%. GMI-1359 markedly abrogated the protection provided by the BM microenvironment (i.e., hypoxia and/or MSC and EC) of Baf3-FLT3 -ITD leukemic cells treated with the FLT3 inhibitor sorafenib. Apoptosis was induced in 36.6%, 35.6% and 48.9% of leukemic cells cultured with sorafenib alone, sorafenib and an E-selectin inhibitor or sorafenib and GMI-1359, respectively. The significance of these in vitro findings were studied in vivo. Female SCID beige mice were injected iv with MV4-11 and followed for survival. Beginning 14 days post tumor injection, cohorts of mice (n=10/group) were treated with saline, GMI-1359 (40 mg/kg), standard chemotherapy cytarabine plus daunorubicin, or a combination of GMI-1359 and chemotherapy. Combined treatment of mice with GMI-1359 (40 mg/kg) and chemotherapy demonstrated a profound survival benefit compared to controls or chemotherapy alone at day 135 after leukemia cell injection (i.e., 67% vs. 11% or 30%, p=0.0011 and 0.0406, respectively). Single agent treatment with GMI-1359 was statistically indistinguishable from saline alone or chemotherapy alone. In a separate cohort of MV4.11-engrafted mice, the single administration of GMI-1359 increased circulating WBC and leukemic MV4-11cells, which persisted for at least 8 hrs. This effect was consistent with GMI-1359 disrupting the protective effects of the tumor microenvironment and mobilizing MV4-11 cells from the BM niche.. These findings provide the pre-clinical basis for the evaluation of GMI-1359 in patients with FLT3 -mutant AML. Figure 1. Figure 1. Disclosures Zhang: Karyopharm: Research Funding. Fogler:GlycoMimetics, Inc.: Employment. Magnani:GlycoMimetics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Polo-like kinase 1 is overexpressed in acute myeloid leukemia and its inhibition preferentially targets the proliferation of leukemic cells

Blood ◽  
2009 ◽  
Vol 114 (3) ◽  
pp. 659-662 ◽  
Author(s):  
Annelies G. Renner ◽  
Cédric Dos Santos ◽  
Christian Recher ◽  
Christian Bailly ◽  
Laurent Créancier ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Leukemic Cell Lines ◽  
Clonogenic Potential ◽  
Established Cell ◽  
Interfering Rna ◽  
Acute Myeloid

Abstract Polo-like kinase 1 (Plk1) is a major mitotic regulator overexpressed in many solid tumors. Its role in hematopoietic malignancies is still poorly characterized. In this study, we demonstrate that Plk1 is highly expressed in leukemic cell lines, and overexpressed in a majority of samples from patients with acute myeloid leukemia compared with normal progenitors. A pharmacologic inhibitor, BI2536, blocks proliferation in established cell lines, and dramatically inhibits the clonogenic potential of leukemic cells from patients. Plk1 knockdown by small interfering RNA also blocked proliferation of leukemic cell lines and the clonogenic potential of primary cells from patients. Interestingly, normal primary hematopoietic progenitors are less sensitive to Plk1 inhibition than leukemic cells, whose proliferation is dramatically decreased by the inhibitor. These results highlight Plk1 as a potentially interesting therapeutic target for the treatment of acute myeloid leukemia.

scholarly journals Anti-Leukemic Activity of Daratumumab in Acute Myeloid Leukemia Cells and Patient-Derived Xenografts

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2312-2312 ◽  
Author(s):  
Cedric Dos Santos ◽  
Shan Xiaochuan ◽  
Zhou Chenghui ◽  
Georges Habineza Ndikuyeze ◽  
Joshua Glover ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Cell Killing ◽  
Nsg Mice ◽  
Cd38 Expression ◽  
Acute Myeloid

Abstract Daratumumab is a human antibody that binds to CD38 on the cell surface and induces cell killing by multiple mechanisms including complement mediated cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP) and apoptosis. In pre-clinical and clinical studies, daratumumab has been shown to effectively kill multiple myeloma (MM) cells and to enhance the potency of other treatments against MM. The purpose of the study was to investigate in vitro and in vivo efficacy of daratumumab against 9 acute myeloid leukemia (AML) cell lines and patient-derived samples. First, we evaluated the expression of CD38, complement inhibitory proteins (CIP) CD46, CD55, CD59, and FcgR1 (CD64) on AML cell lines (n=9), AML patient cells (n=10) and healthy donor bone marrow using flow cytometry. CD38 enumeration showed a substantial variation between cell lines (12,827±19, 320 molecules/cell) and between AML patients (11,560±8, 175 molecules/cell), while CD38 expression was more consistent in bone marrow (BM) from healthy donors (1,176±355 molecules/cell). The daratumumab-induced apoptosis observed in cell lines (MOLM-13, MOLM-16, MV-4-11, NB4) in vitro was not correlated with CD38 expression levels. Daratumumab induced minimal ADCC (5-20%) and low levels of (2-5%) CDC mediated cell killing in 6 AML cell lines tested. We did not observe a direct correlation between CD38 expression and ADCC, CDC, nor between CDC and CIP expression. Interestingly, treatment of two human Acute Promyelocytic Leukemia (M3) cell lines HL-60 and NB-4 with all-trans retinoic acid (ATRA) induced a 10-30-fold increase in CD38 expression, suggesting that ATRA could be used in combination with daratumumab. While we, and others, have shown that pre-incubation of primary AML cells with anti-CD38 antibodies inhibits engraftment in NSG mice, we aimed at evaluating the anti-leukemic activity of daratumumab in a therapeutic xenograft model using 3 different AML patients. NSG mice (10/group/patient) were transplanted with T cell-depleted AML cells and BM aspirates were collected 4-6 weeks later to assess leukemia burden in each mouse prior to treatment. Animals were untreated (Ctrl) or received daratumumab (10 mg/kg), or IgG1 isotype once a week for five weeks. We assessed AML burden (% huCD45+ CD33+) in BM, spleen (SPL) and peripheral blood (PB) within 5 days after the last treatment. First, we evaluated an AML (#3406, FLT3-ITD, see figure) with high expression of CD38 (13,445 molecules/cell) and low CD64 (489/cell) was evaluated. Daratumumab significantly reduced leukemia burden in SPL and PB, but had no effect in BM. The same daratumumab-induced reduction in peripheral blasts and lack of effect in BM was observed in 2 other AML patient xenografts (#7577, M1 IDH mutant/FLT3-ITD with 6,529 CD38 molecules/cell; #8096, M2 with 335 CD38 molecules/cell). Interestingly, we observed that daratumumab treatment led to a drastic reduction in CD38 surface expression in AML blasts including in BM, indicating that daratumumab efficiently targeted CD38 in bone marrow blasts. Our results suggest that the bone marrow microenvironment can impair the anti-leukemic activity of daratumumab observed in other tissues. Ongoing xenograft studies are testing whether induction with chemotherapy (Ara-C+doxorubicin), or with other agents disrupting the bone marrow microenvironment, can enhance the anti-leukemic activity of daratumumab. Figure 1: Effect of daratumumab treatment on AML 3406 leukemia burden: Figure 1:. Effect of daratumumab treatment on AML 3406 leukemia burden: Disclosures Dos Santos: Janssen R&D: Research Funding. Xiaochuan:Janssen R&D: Research Funding. Doshi:Janssen R&D: Employment. Sasser:Janssen R&D: Employment. Danet-Desnoyers:Janssen R&D: Research Funding.

The PARP Inhibitor Olaparib Antagonizes Leukemic Growth Induced By TET1 Overexpression in AML1-ETO Positive Acute Myeloid Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4063-4063 ◽  
Author(s):  
Shiva Bamezai ◽  
Jing He ◽  
Deniz Sahin ◽  
Fabian Mohr ◽  
Fabio Ciccarone ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Cell Growth ◽  
Cell Line ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Parp Inhibitor ◽  
Leukemic Cell ◽  
Aberrant Expression ◽  
Acute Myeloid

Abstract DNA methylation patterns are highly deregulated in human acute myeloid leukemia (AML) cases and stratify AML patient samples into different subgroup. AML1-ETO is the most commonly occurring fusion gene in AML and these AML cases exhibit an aberrant and distinct methylation pattern. So far, the underlying mechanisms for this are only poorly understood. The TET1 dioxygenase has recently emerged as an important epigenetic modifier: by catalyzing the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) TET1 plays an important role in active demethylation, thereby regulating a variety of biological processes. It was linked to tumorigenesis based on the observation that its expression is frequently deregulated in solid cancer. However, the role of TET1 in AML1-ETO+ (AE+)human AML cases is yet unexplored. Using quantitative real time (qRT)- PCR we now show that AE+ AML is characterized by high and aberrant expression of TET1: the gene was significantly higher expressed in the majority of AE+ patients (n=7, p<0.01) compared to other AML subtypes such as inv(16) (n=11), PML-RARα+ (n=31), cytogenetically normal (CN)-AML patients (n=33) and CD34+ normal BM cells (n=4). This observation was consistent with published cDNA microarray data on large patient cohorts (Haferlach et al., JCO 2010, p<0.008 t-test, p<0.01 Anova) and recently published transcriptome data (TCGA) of AML patients. In contrast to TET1, TET2 and TET3 did not show significant higher expression in AE+ patients compared to other AML subtypes. In line with patient data, TET1 was highest expressed in the AE+ AML cell line KASUMI-1 and SKNO-1 compared to other AML cell lines (p<0.05 and n=3). Compared to normal CD34+ and myeloid (CD33+, CD15+ and CD14+) cells (n=3), TET1 was 10-fold and 16-fold higher expressed in AE+ patient samples (n= 7). Aberrant expression of TET1 in AE+ leukemic cells was associated with hypomethylation of its promoter and enrichment for H3K4me3 euchromatic marks at its promoter as determined by LC/MS and ChIP-qPCR respectively. Knockdown (KD) of TET1 mRNA using two short hairpin RNAs (shRNAs) in AE+ AML cell lines impaired their cell growth and clonogenicity by over 50% in vitro (n=3 and p<0.01). shRNA mediated depletion of TET1 did not impact the cell growth and clonogenicity of the TET1 negative cell line RAJI, ruling out off target effects of the shRNAs (n=3). In mice, KD of Tet1 in leukemic bone marrow cells expressing the truncated leukemogenic AML1-ETO9a (AE9a) fusion, dramatically inhibited cell growth (>60% compared to scrambled, n=3, p<0.01), clonogenicity (>50-70% reduction in primary CFCs, p<0.01, n=3) and importantly delayed onset of leukemia in vivo (median survival 35 days for scr vs 80 days for shRNA mice, n=4/arm, p<0.03). Tet1-knock-out c-kit+ hematopoietic stem and progenitor cells (HSPCs) transduced with AE9a showed reduced primary colony formation and impaired serial replanting capacity in vitro compared to AE9a transduced Tet1-wild-type HSPCs (>50% and >70%, respectively; p<0.001, n=3). Global analysis of 5hmC and 5mC levels using hMeDIP/MeDIP-seq performed on TET1 depleted KASUMI-1 cells revealed lower global 5hmC levels and increase in 5mC as compared to cells transduced with scrambled control (n=2). 3324 promoter regions lost 5hmC and gained 5mC upon TET1 depletion (-5kTSS, Fold enrichment cut off <2-fold, q-value<1e5). Recent studies have shown that PARP activity induces TET1 expression by regulating its promoter epigenetically. We could show that aberrant TET1 expression could be antagonized by the PARP inhibitor olaparib in AE+ leukemic cell lines. Furthermore, olaparib treatment decreased 5hmC levels and reduced cell growth and clonogenicity of human AE+ cell lines and of the murine AE9a+ leukemic cell line in vitro (n=3, p<0.01). In conclusion, our data indicates that aberrant TET1 expression contributes to the growth of AE+ AML by maintaining the 5-hydroxymethylome and that the PARP inhibitor olaparib can at least partially antagonize the oncogenic effect of TET in AML. Disclosures Mulaw: NuGEN: Honoraria. Buske:Celltrion, Inc.: Consultancy, Honoraria.

scholarly journals Metabolism of Acute Myeloid Leukemia Cell Lines Alters with Passage in 2D Culture and Remains Stable in 3D

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2787-2787
Author(s):  
Sophia Zemenides ◽  
Joana Dos Santos ◽  
Louise Enfield ◽  
Athanasios Mantalaris ◽  
Nicki Panoskaltsis
Keyword(s):  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
3D Culture ◽  
Leukemic Cell ◽  
Type I ◽  
3D Scaffolds ◽  
Metabolomics Analysis ◽  
Acute Myeloid

Abstract Acute Myeloid Leukemia (AML) is a heterogeneous and complex malignancy of the blood and bone marrow (BM). Current treatment options only cure 40% of patients under the age of 65 years and, over the age of 65 years only 10% will be cured. Hence, there is great urgency to further understand pathogenesis, optimize treatment and identify markers for diagnosis, prognosis and targets for novel therapies. It is increasingly evident that metabolism and BM microenvironment are key factors in AML pathogenesis and progression - entities that cannot be accurately investigated within current in vitro methods of two-dimensional (2D) culture. The metabolome is the final downstream product of gene transcription that also responds dynamically to its environment. Metabolomics is an established technique to identify biomarkers with prognostic relevance, such as 2-hydroxygluterate, an onco-metabolite derived from IDH mutations in AML. Targeting metabolic pathways such as oxidative phosphorylation, glycolysis and specific mutations that affect metabolism such as isocitrate dehydrogenase are currently being assessed in clinical trials. An in vitro system that could more accurately represent the in vivo BM may help optimise these metabolically-targeted regimens. Here we show that a previously established three-dimensional (3D) culture within our laboratory recapitulates elements of BM structure enabling long-term culture of AML cell lines without disrupting metabolism, as is the case with serial passage in 2D culture. Metabolism was assessed using gas chromatography-mass spectrometry (GC-MS) metabolomics analysis which reflects the phenotype of the leukemic cell lines. Porous polyurethane scaffolds were fabricated using dioxin by thermally induced phase separation as previously described; scaffolds have a pore size of 100-250µm and a porosity of 90-95% cut to 0.5cm3, coated with type I collagen and seeded with 0.5x106cells of either K562 (erythroleukemia) or HL-60 (promyelocytic leukemia) cells, after expansion in 2D culture. In parallel, 2D cultures of the same cell lines were maintained using passage every 2 days. K562 and HL-60 cells were cultured in IMDM, 1% penicillin and streptomycin and 20% or 10% foetal bovine serum, respectively. Serial sampling occurred at 4 time points over 21 days from 10 scaffolds of each condition. Cells were extracted from the 3D scaffolds using two techniques - needle extraction or TrypLE express (Thermo Fisher Scientific) to assess whether these physical or chemical extraction methods disrupt the metabolome. Once cells were extracted, they were suspended in methanol at 1x106cells/mL cold methanol for metabolite quenching; metabolites were subsequently extracted with methanol/water and derivatised with Methoxamine and N-Methyl-N-(trimethylsilyl)trifluoroacetamide. Metabolomics analysis was then performed using a Shimadzu QP2010 Ultra GC-MS machine detecting 138 metabolites including those of pertinent pathways: glycolysis, tricarboxylic acid, pentose phosphate, urea, glutaminolysis and amino acids. Bioinformatics analysis was performed with MeV TM4(http://mev.tm4.org). Using unsupervised clustering techniques, including hierarchical clustering and principal component analysis, we identified that the metabolome of TrypLE express and needle-extracted cells from 3D scaffolds have a similar metabolic signature and group closely with each other as well as with the metabolome of the seeded cells (day 0); they do not vary significantly over the 21 days of culture. Conversely, with each passage, the metabolomes of the 2D-cultured cells differ to those of day 0 and vary to each other over the same 21-day period. These results highlight limitations in the use of 2D cultures to address AML biology as metabolic changes with passage reflect change in phenotype. Based on these findings, we conclude that 3D cultures provide a more stable environment for leukemic cell culture and assessment of leukemia biology, irrespective of method of cell extraction. The 3D culture platform is more suited than standard 2D in vitro cultures to investigate metabolism, microenvironment and drug targets in AML. Disclosures No relevant conflicts of interest to declare.

scholarly journals Myeloid cell differentiation arrest by miR-125b-1 in myelodysplasic syndrome and acute myeloid leukemia with the t(2;11)(p21;q23) translocation

2008 ◽  
Vol 205 (11) ◽  
pp. 2499-2506 ◽  
Author(s):  
Marina Bousquet ◽  
Cathy Quelen ◽  
Roberto Rosati ◽  
Véronique Mansat-De Mas ◽  
Roberta La Starza ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Cell Differentiation ◽  
Myeloid Leukemia ◽  
Cell Transformation ◽  
Myeloid Cell ◽  
Leukemic Cell ◽  
Granulocytic Differentiation ◽  
Leukemic Cell Lines ◽  
Acute Myeloid

Most chromosomal translocations in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) involve oncogenes that are either up-regulated or form part of new chimeric genes. The t(2;11)(p21;q23) translocation has been cloned in 19 cases of MDS and AML. In addition to this, we have shown that this translocation is associated with a strong up-regulation of miR-125b (from 6- to 90-fold). In vitro experiments revealed that miR-125b was able to interfere with primary human CD34+ cell differentiation, and also inhibited terminal (monocytic and granulocytic) differentiation in HL60 and NB4 leukemic cell lines. Therefore, miR-125b up-regulation may represent a new mechanism of myeloid cell transformation, and myeloid neoplasms carrying the t(2;11) translocation define a new clinicopathological entity.

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