scholarly journals A Novel WNT5A-Mimicking Peptide Affects Leukemia Cell Survival in the Bone Marrow Microenvironment

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2949-2949
Author(s):  
Fernanda Marconi Roversi ◽  
Maura Lima Pereira Bueno ◽  
Rafael Gonçalves Barbosa Gomes ◽  
Guilherme Rossi Assis-Mendonça ◽  
Paulo Latuf Filho ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Mrna Expression ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Actin Polymerization ◽  
Leukemia Cell ◽  
Ros Production ◽  
Protein Levels ◽  
Cd34 Cells

Abstract Background: The crosstalk between hematopoietic cells and bone marrow (BM) microenvironment in hematological malignancies is related to disease initiation, maintenance and relapse. BM niche sustains a protective response against currently available treatments that have shown unwanted adverse effects and high levels of toxicity for patients. WNT5a is a glycoprotein secreted by mesenchymal stromal cells (MSC) that activates the WNT non-canonical pathway in hematopoietic cells, modulating important biological processes related to neoplasia development. Aims: To investigate WNT5a mRNA expression, protein levels and methylation pattern in Myelodysplastic Syndrome (MDS) and de novo Acute Myeloid Leukemia (AML) patients and their impact on clinical outcomes, and to analyze effects of Foxy-5 (WNTResearch), a new WNT5a-mimicking compound. Methods: WNT5a mRNA expression was analyzed in mononuclear cells (MC) from 371 AML patients (212 male, median age 61 years [range: 2-87]) (Ohsu, Nature 2018). BMMSC and BM biopsies from 5 healthy donors (HD), 6 MDS and 13 AML patients at diagnosis were submitted to analysis of WNT5a mRNA expression and methylation after Azacytidine (1μM) treatment by real-time PCR, and WNT5a protein levels by immunohistochemistry and immunofluorescence (IF). A panel of myeloid leukemia cell lines (U937, HL60, THP1, KG1a, K562) were treated with Foxy-5 (1 to 100μM) for 72h in monoculture, coculture and 3D-coculture (with MSC) and evaluated for ROS production (DCFDA dye), cell proliferation (Ki-67 stain), autophagy (acridine orange dye), chemotaxis (Transwell), actin polymerization (phalloidin stain), cell cycle (PI/RNAse stain), cell viability (MTT assay), apoptosis (Annexin-V stain) and protein expression (Western blot, WB). MC and CD34+ cells from HD were submitted to cytotoxic assays. Statistical analyzes were performed using ANOVA or Mann-Whitney tests, as appropriate. Results: WNT5a gene expression was reduced in MC from AML patients with adverse cytogenetic risk compared to favorable and intermediate cytogenetic risk (fold-decrease [FD]: 42.9; 18.8, respectively) (P<.05) and in BMMSC from AML compared to HD (FD: 53.3) (P<.05). Accordingly, WNT5a gene expression in MDS and AML BMMSC treated with Azacytidine was restored (fold-increase [FI]: 3.99; 1.50, respectively). WNT5a protein expression were diminished in BMMSC from MDS and AML patients compared to HD onto a 3D-coculture (IF)(P<.05) and immunohistochemically detected in all BM hematopoietic lineages. Foxy-5 reduced ROS production in U937 (FD of mean fluorescence intensity [MFI]: 48.2; 46.6), HL60 (FD: 47.1; 115.0), KG1a (FD: 34.9; 20.7) and K562 (FD: 19.0; 24.3) at 100μM in monoculture and coculture, respectively (P<.05). Foxy-5 also significantly decreased proliferation in U937 (FD: 41.0), HL60 (FD: 18.0), THP1 (FD: 36.0) and K562 (FD: 68.0) at 100μM (P<.05), confirmed by a 3D-coculture containing these cell lines and MSC. Foxy-5 reduced monocyte differentiation and inhibited CD11b expression in U937 (FD: 16.4) and THP1 (FD: 14.4). Cell cycle progression was blocked in sub G0/G1 phase in all cell lines (P<.05) after Foxy-5 treatment, probably mediated by the reduction of cyclin D1 protein levels, as verified by WB. Further, Foxy-5 reduced AKT1/2/3 and ERK1/2 phosphorylation levels, possibly by beta-catenin inhibition, with disruption of actin polymerization (U937 (FD: 65.3), HL60 (FD: 35.9), THP1 (FD: 58.5), K562 (FD: 15.0)) at 100μM (P<.05) and consequent impairment of CXCL12-induced chemotaxis (U937 (FD: 27.9), HL60 (FD: 42.5), THP1 (FD: 82.4), K562 (FD: 45.1)) at 100μM (P<.05). In coculture, cell autophagy was reduced in U937 (FD: 27.8), HL60 (FD: 35.9), KG1a (FD: 16.4) and K562 (FD: 35.8) when treated with Foxy-5 at 100μM (P<.05). Finally, Foxy-5 treatment did not affect cytotoxicity in MC and CD34+ cells from HD. Conclusion: WNT5a downregulation in MDS and AML patients occurs probably by methylation and contributes to poor prognosis. Foxy-5, by restoring WNT5a levels, could represent a strategy to counterbalance several oncogenic processes present in leukemia by reducing ROS production and, consequently, inhibiting cell growth and differentiation, downregulating PI3K and MAPK pathways, disrupting actin polymerization and decreasing autophagy. Thus, Foxy-5 treatment may be an important approach to impair leukemia growth and maintenance and arises as a promising therapeutic target. Disclosures No relevant conflicts of interest to declare.

Bromodomain Inhibition By OTX015 Regulates c-MYC and HEXIM1 in a Panel of Human Acute Leukemia Cell Lines

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5957-5957
Author(s):  
Marie-Magdelaine Coudé ◽  
Thorsten Braun ◽  
Jeannig Berrou ◽  
Mélanie Dupont ◽  
Raphael Itzykson ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Expression ◽  
Mrna Expression ◽  
Rna Polymerase Ii ◽  
Cell Lines ◽  
Leukemia Cell ◽  
K562 Cells ◽  
Antileukemic Activity ◽  
Protein Levels ◽  
Gene And Protein Expression

Abstract Background: The bromodomain-containing protein 4 (BRD4) activates the transcription elongation factor b (P-TEFb) which regulates RNA polymerase II. Conversely, hexamethylene bisacetamide (HMBA) inducible protein 1 (HEXIM1) inactivates P-TEFb. BRD4/HEXIM1 interplay influences cell cycle progression and tumorigenesis. It has been widely demonstrated that BRD4 knockdown or inhibition by JQ1 is associated with c-MYC downregulation and antileukemic activity. We recently reported that the small molecule BRD2/3/4 inhibitor OTX015 (Oncoethix, Lausanne, Switzerland), currently in clinical development, mimics the effects of JQ1 (Braun et al, ASH 2013). We evaluated the effect of OTX015 on c-MYC, BRD2/3/4, and HEXIM1 in human in vitro leukemic models. Methods: c-MYC, BRD2/3/4 and HEXIM1 expression was assessed in six acute myeloid leukemia (AML; K562, HL-60, NB4, NOMO-1, KG1, OCI-AML3) and two acute lymphoid leukemia (ALL; JURKAT and RS4-11) cell lines after exposure to 500 nM OTX015. Quantitative RT-PCR and Western blotting were performed at different time points (24-72h). A heatmap was computed with R-software. Results: c-MYC RNA levels were ubiquitously downregulated in all AML and ALL cell lines after 24h exposure to OTX015 (Figure 1). c-MYC protein levels decreased to a variable extent at 24-72h in all cell lines evaluated other than KG1. BRD2, BRD3 and BRD4 mRNA expression was significantly decreased in K562 cells (known to be OTX015-resistant) after 48h exposure to OTX015 but was increased in HL60 and NOMO-1 cells, while minimal to no increases were observed in other cell lines. OTX015 induced a decrease in BRD2 protein expression in most cell lines, but not in K562 cells. In contrast, decreased BRD4 protein expression was only seen in the OCI-AML3, NB4 and K562 cell lines. BRD3 protein levels were not modified after OTX015 exposure in all cell lines evaluated other than KG1. HEXIM1 mRNA expression increased after 24h exposure to 500 nM OTX015 in all cell lines except OTX015-resistant K562 cells in which the increase was considered insignificant (less than two-fold). Increases in HEXIM1 protein levels were observed in OCI-AML3, JURKAT and RS4-11 cell lines at 24-72h but not in K562 cells. Conclusion: Taken together, these results show that BRD inhibition by OTX015 modulates HEXIM1 gene and protein expression, in addition to c-MYC decrease and BRD variations. HEXIM1 upregulation seems to be restricted to OTX015-sensitive cell lines and was not significantly affected in OTX015-resistant K562 cells. Further studies are needed to clarify the role of HEXIM1 in antileukemic activity of BRD inhibitors. Figure 1: Heatmap of gene expression after exposure to 500 nM OTX015 for 24 or 48h in AML and ALL cell lines. Repression in blue. Overexpression in red. Figure 1:. Heatmap of gene expression after exposure to 500 nM OTX015 for 24 or 48h in AML and ALL cell lines. Repression in blue. Overexpression in red. Disclosures Riveiro: OTD: Employment. Herait:OncoEthix: Employment. Dombret:OncoEthix: Research Funding.

scholarly journals Analysis of ROS-Responsive Genes in Mutant RAS Expressing Hematopoietic Progenitors Identifies the Glycolytic Pathway As a Major Target Promoting Both Proliferation and Survival

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2210-2210
Author(s):  
Goitseone Lucy Hopkins ◽  
Andrew J Robinson ◽  
Paul S Hole ◽  
Richard L. Darley ◽  
Alex Tonks
Keyword(s):  
Gene Expression ◽  
Gene Expression Profile ◽  
Cell Lines ◽  
Expression Profile ◽  
Myeloid Leukemia ◽  
Lactate Production ◽  
Glycolytic Pathway ◽  
Ros Production ◽  
Cd34 Cells ◽  
Major Target

Abstract Acute myeloid leukemia (AML) is a heterogeneous clonal disorder with a generally poor clinical outcome. Previously we have shown that over-production of reactive oxygen species (ROS) occurs in >60% of AML patients due to NOX oxidase activation and that this promotes growth factor independent proliferation of AML blasts (Hole et al., 2013). Normal CD34+ cells overexpressing mutant NRAS serve as a model for this since these cells also overproduce ROS through NOX activation, which in turn promotes their proliferation (Hole et al., 2010). We used this model to investigate the mechanism by which ROS promote proliferation by examining the effect of ROS on gene expression. CD34+ expressing NRASG12D showed an 8-fold (p<0.05) increase in ROS production compared to empty vector controls. DPI, a NOX inhibitor, virtually ablated ROS production (>90%; p<0.05) and also selectively inhibited proliferation of CD34+-NRAS cells (>60%; p<0.05). We next compared the gene expression profile (GEP) of NRAS and control cells ±DPI to determine the ROS-specific gene expression profile (Affymetrix Human Exon 1.0ST). NRAS changed the expression of 342 genes (>1.2 fold; p<0.05) of which 24 were specifically attributed to ROS production (Table 1). Most of these were associated with metabolic change; particularly glycolysis (p<0.0001 n=4). Consistent with this we found a doubling in the level of extracellular lactate production (indicating increased glycolysis) from NRAS cells compared to controls (n=4). Extracellular ROS (generated from GOX) also directly promoted a 1.3 fold increase in lactate production (n=4). These data suggest that ROS directly promotes glycolysis in hematopoietic cells. To examine this in AML we analysed a GEP database of 139 AML patients; this showed that those with high ROS (defined by high NOX2 oxidase expression; Hole et al., 2013) had a distinct profile of glycolytic enzyme overexpression, particularly ALDOC (r=0.4; p=2x10-25), GPI (r=0.4; p=2x10-8) and FBP1(r=0.7; p=5x10-8). These are amongst the most significant ROS-responsive genes in Table 1 and suggest that promotion of glycolysis through extracellular ROS production is also seen in AML blasts. To establish the functional significance of upregulated expression of glycolytic enzymes, we focused on the aldolase enzyme, ALDOC, since it showed the biggest induction with ROS and because overexpression of this enzyme has been recently associated with elevated glycolysis and poor prognosis in primary AML (Chen et al., 2014). We found that ALDOC was directly induced (2 fold) by physiological levels (150 nM/hour) of ROS in both normal CD34+ cells and in AML cell lines. We next examined the effect of stable ALDOC knockdown in 3 myeloid leukemia cell lines: Mv4;11 (1.5 fold reduction at the protein level), K562 (3.5 fold) and THP-1 (1.5 fold). Knockdown was associated with a reduction in proliferation in Mv4;11 and THP-1 cells (2- and 5-fold respectively; p<0.05 n=3) and also reduction in survival of THP-1 (1.7 fold; p<0.05, n=3). These data have identified ROS-responsive genes in CD34+ hematopoietic cells and show for the first time that a major target of ROS are enzymes of the glycolytic pathway. We also show evidence that ROS promotes glycolysis in both cell lines and in AML patients and that myeloid leukemia cells show dependency on ALDOC, for their growth and survival. Given the frequent overexpression of ROS in primary AML, these data provide a plausible mechanism for the enhanced glycolysis seen in AML (Chen et al., 2014) and suggest that agents restoring the redox environment could be used to correct metabolic imbalances which contribute to treatment resistance in this disease. Table 1. Effect of ROS on gene expression Gene Gene expression (fold and direction of change) p-value Protein validation (fold and direction of change where available) Process ALDOC +4.3 1×10-6 +2 Glycolysis ENO2 +2.6 1×10-4 +1.5 FBP1 +1.8 2×10-7 GPI +1.5 2×10-6 PFK-1 +1.4 9×10-6 GATM +2.8 3×10-5 +2 Metabolism SULF2 +2.1 2×10-5 CKB +2.1 7×10-5 ASPH +1.4 1×10-4 PTPRD +2.2 2×10-5 +2 Signal transduction KIT -2.1 5×10-5 0 CD32 +1.5 7×10-5 0 TNS1 +1.7 8×10-6 REC8 +1.4 6×10-5 STARD8 +1.4 3×10-5 CMTM8 -1.2 1×10-4 CNR2 -3.6 2×10-6 0 CD34 +1.7 4×10-5 0 Other CITED1 +1.7 6×10-5 +1.5 CYTL1 -1.9 6×10-6 CACNB1 +1.3 1×10-4 SLC6A8 +1.7 1×10-5 JAKMIP2 +1.2 3×10-5 WDR54 +1.8 5×10-6 Disclosures No relevant conflicts of interest to declare.

In Vitro and In Vivo Monitoring of Valproic Acid Effects on Gene Expression Signatures in Adult Acute Myeloid Leukemia.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2605-2605
Author(s):  
Lars Bullinger ◽  
Konstanze Dohner ◽  
Richard F. Schlenk ◽  
Frank G. Rucker ◽  
Jonathan R. Pollack ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Leukemia Cell ◽  
Serum Levels ◽  
Leukemia Cell Lines ◽  
Acute Myeloid ◽  
Myeloid Leukemia Cell

Abstract Inhibitors of histone deacetylases (HDACIs) like valproic acid (VPA) display activity in murine leukemia models, and induce tumor-selective cytoxicity against blasts from patients with acute myeloid leukemia (AML). However, despite of the existing knowledge of the potential function of HDACIs, there remain many unsolved questions especially regarding the factors that determine whether a cancer cell undergoes cell cycle arrest, differentiation, or death in response to HDACIs. Furthermore, there is still limited data on HDACIs effects in vivo, as well as HDACIs function in combination with standard induction chemotherapy, as most studies evaluated HDACIs as single agent in vitro. Thus, our first goal was to determine a VPA response signature in different myeloid leukemia cell lines in vitro, followed by an in vivo analysis of VPA effects in blasts from adult de novo AML patients entered within two randomized multicenter treatment trials of the German-Austrian AML Study Group. To define an VPA in vitro “response signature” we profiled gene expression in myeloid leukemia cell lines (HL-60, NB-4, HEL-1, CMK and K-562) following 48 hours of VPA treatment by using DNA Microarray technology. In accordance with previous studies in vitro VPA treatment of myeloid cell lines induced the expression of the cyclin-dependent kinase inhibitors CDKN1A and CDKN2D coding for p21 and p19, respectively. Supervised analyses revealed many genes known to be associated with a G1 arrest. In all cell lines except for CMK we examined an up-regulation of TNFSF10 coding for TRAIL, as well as differential regulation of other genes involved in apoptosis. Furthermore, gene set enrichment analyses showed a significant down-regulation of genes involved in DNA metabolism and DNA repair. Next, we evaluated the VPA effects on gene expression in AML samples collected within the AMLSG 07-04 trial for younger (age<60yrs) and within the AMLSG 06-04 trial for older adults (age>60yrs), in which patients are randomized to receive standard induction chemotherapy (idarubicine, cytarabine, and etoposide = ICE) with or without concomitant VPA. We profiled gene expression in diagnostic AML blasts and following 48 hours of treatment with ICE or ICE/VPA. First results from our ongoing analysis of in vivo VPA treated samples are in accordance with our cell line experiments as e.g. we also see an induction of CDKN1A expression. However, the picture observed is less homogenous as concomitant administration of ICE, as well as other factors, like e.g. VPA serum levels, might substantially influence the in vivo VPA response. Nevertheless, our data are likely to provide new insights into the VPA effect in vivo, and this study may proof to be useful to predict AML patients likely to benefit from VPA treatment. To achieve this goal, we are currently analyzing additional samples, and we are planning to correlate gene expression findings with histone acetylation status, VPA serum levels, cytogenetic, and molecular genetic data.

Molecular Characterization of the Leukemic Niche in Chronic Myeloid Leukemia (CML) and Evaluation of a Leukemia / Niche Cross-Talk

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 908-908
Author(s):  
Djamel Aggoune ◽  
Nathalie Sorel ◽  
Sanaa El Marsafy ◽  
Marie Laure Bonnet ◽  
Denis Clay ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Bone Marrow ◽  
Mrna Expression ◽  
Expression Pattern ◽  
Myeloid Leukemia ◽  
Gene Expression Pattern ◽  
Fold Increase ◽  
Leukemic Cells ◽  
Cd34 Cells

Abstract Abstract 908 There is growing evidence that the bone marrow microenvironment could participate to the progression of chronic myeloid leukemia (CML). Recent data show indeed that placental growth factor (PGF) expression is highly induced in stromal cells from CML patients although they are not part of the leukemic clone as they are Ph1-negative (Schmidt et al, Cancer Cell 2011). It is possible that leukemic cells instruct the niche components via extracellular or contact signals, transforming progressively the “normal niche” into a functionally “abnormal niche” by inducing aberrant gene expression in these cells, similar to the pattern that has been identified in cancer-associated fibroblasts (CAF). In an effort to identify the differential gene expression pattern in the CML niche, we have undertaken two strategies of gene expression profiling using a Taqman Low Density Arrays (TLDA) protocol designed for 93 genes involved in antioxidant pathways (GPX, PRDX, SOD families), stromal cell biology (Collagen, clusterin, FGF, DHH), stem cell self-renewal (Bmi1, MITF, Sox2) and hematopoietic malignancies (c-Kit, hTERT, Dicer, beta-catenin, FOXO3). The first strategy consisted in the analysis of mesenchymal stem cells (MSCs) isolated from the bone marrow of newly diagnosed CP-CML patients (n=11). As a control, we have used MSCs isolated from the bone marrow of age-matched donors (n=3). MSCs were isolated by culturing 6–8.106 bone marrow mononuclear cells in the presence of b-FGF (1 ng/ml). At 2–3 weeks, cells were characterized by the expression of cell surface markers (CD105+, CD90+) and by their potential of differentiation towards osteoblastic, chondrocytic and adipocytic lineages. The second strategy aimed to study the potential instructive influence of leukemic cells in the gene expression program of normal MSC after co-culture with either the UT7 cell line expressing BCR-ABL (3 days) or with CD34+ cells isolated from CP-CML at diagnosis (5 days) as compared to co-culture with cord blood CD34+ cells. After culture, CD45-negative MSC were cell-sorted and analyzed by TLDA. All results were analyzed using the StatMiner software. Results: TLDA analysis of gene expression pattern of MSC from CML patients (n=11) as compared to normal MSCs (n=3) identified 6 genes significantly over-expressed in CML-MSC: PDPN (10-Fold Increase), V-CAM and MITF (∼8 Fold increase), MET, FOXO3 and BMP-1 (∼ 5 Fold increase). To confirm these results we have performed Q-RT-PCR in a cohort of CML-MSC (n= 14, including the 11 patients as analyzed in TLDA) as compared to normal MSC. High levels of PDPN (Podoplanin, ∼8 fold increase), MITF (Microphtalmia Associated Transcription factor, 4-Fold) and VCAM (Vascular Cell Adhesion Protein, 2 fold increase) mRNA were again observed on CML MSCs. Our second strategy (co-culture of normal MSC with BCR-ABL-expressing UT7) revealed an increase of IL-8 and TNFR mRNA expression in co-cultured MSCs (∼5-fold ) whereas there was a major decrease in the expression of DHH (∼ 25-fold) upon contact with BCR-ABL-expressing cells. No modification of the expression of PDPN, MITF or VCAM was noted in normal MSC after this 3-day co-culture strategy using UT7-BCR-ABL cells. Current experiments are underway to determine if primary CD34+ cells from CML patients at diagnosis could induce a specific gene expression pattern in normal MSC after 5 days of co-culture. PDPN is a glycoprotein involved in cell migration and adhesion, acting downstream of SRC. It has been shown to promote tumor formation and progression in solid tumor models and is highly expressed in CAFs. MITF is a bHLH transcription factor involved in the survival of melanocyte stem cells and metastatic melanoma. Finally, high VCAM1 mRNA expression by MSCs from CML patients could be involved in increased angiogenesis known to be present on CML microenvironment. In conclusion, our results demonstrate an abnormal expression pattern of 3 important genes (PDPN, MITF and VCAM1) in MSC isolated in CP-CML patients at diagnosis. The mechanisms leading to an increased mRNA expression (instructive or not instructive by leukemic cells) and their relevance to CML biology are under evaluation. Our results, confirming previous data, suggest strongly the existence of a molecular cross-talk between leukemic cells and the leukemic niche. The elucidation of such aberrant pathways in the microenvironment could lead to the development of “niche-targeted” therapies in CML. Disclosures: Turhan: Novartis, Bristol Myers Squibb: Honoraria, Research Funding.

RASSF2 Functions As a Tumor Suppressor in t(8;21) AML Via Promotion of Apoptosis

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3590-3590
Author(s):  
Samuel A Stoner ◽  
Russell Dekelver ◽  
Miao-Chia Lo ◽  
Dong-Er Zhang
Keyword(s):  
Gene Expression ◽  
Tumor Suppressor ◽  
Cell Lines ◽  
Hematopoietic Cells ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Mrna Levels ◽  
Human Cd34 ◽  
Leukemia Cell Lines ◽  
Cd34 Cells

Abstract The t(8;21) chromosomal translocation is one of the most common chromosomal translocations associated with acute myeloid leukemia (AML), found in approximately 12% of de novo AML cases. The majority of these cases are classified as FAB-subtype M2 AML. The t(8;21) results in the stable fusion of the AML1 (RUNX1) and ETO (RUNX1T1) genes. The AML1-ETO fusion protein is composed of the N-terminal portion of AML1, which includes the DNA-binding Runt-homology domain, and nearly the full-length ETO protein. The primary accepted mechanism by which AML1-ETO promotes leukemia development is through the aberrant recruitment of transcriptional repression/activation complexes to normal AML1 target genes. Therefore, the identification of individual genes or biological pathways that are specifically disrupted in the presence of AML1-ETO will provide further molecular insight into the pathogenesis of t(8;21) AML and lead to the possibility for improved treatment for these patients. We identified RASSF2 as a gene that is specifically downregulated in (2-4 fold) in total bone marrow of t(8;21) patients compared to non-t(8;21) FAB-subtype M2 AML patients by analyzing publicly available gene expression datasets. Similarly, using a mouse model of t(8;21) AML we found Rassf2 mRNA levels to be nearly 30-fold lower in t(8;21) leukemia cells compared to wild-type Lin-Sca-cKit+ (LK) myeloid progenitors. Gene expression analysis by RT-qPCR in leukemia cell lines confirmed that RASSF2 mRNA levels are significantly downregulated (8-10-fold) in both Kasumi-1 and SKNO-1 t(8;21) cell lines as compared to a similar non-t(8;21) HL-60 cell line and to primary human CD34+ control cells. In addition, expression of AML1-ETO in HL-60 or CD34+ cells results in a decrease in RASSF2 mRNA expression, which further suggests that RASSF2 is a target for regulation by AML1-ETO. Assessment of published ChIP-seq data shows that AML1-ETO binds the RASSF2 gene locus at two distinct regions in both primary t(8;21) AML patient samples and in the Kasumi-1 and SKNO-1 cell lines. These regions are similarly bound by several important hematopoietic transcription factors in primary human CD34+ cells, including AML1, ERG, FLI1, and TCF7L2, implicating these two regions as important for the regulation of RASSF2 expression during blood cell differentiation. Overexpression of RASSF2 in human leukemia cell lines using an MSCV-IRES-GFP (MIG) construct revealed that RASSF2 has a strong negative effect on leukemia cell proliferation and viability. The overall percentage of GFP-positive cells in MIG-RASSF2 transduced cells markedly decreased compared to MIG-control transduced cells over a period of 14 days. This effect was primarily due to significantly increased apoptosis in the RASSF2 expressing cell populations. Similarly, we found that expression of RASSF2 significantly inhibits the long-term self-renewal capability of hematopoietic cells transduced with AML1-ETO in a serial replating/colony formation assay. AML1-ETO transduced hematopoietic cells were normally capable of serial replating for more than 6 weeks. However, AML1-ETO transduced cells co-expressing RASSF2 consistently had reduced colony number and lost their ability to replate after 3-4 weeks. This was due to a dramatically increased rate of apoptosis in RASSF2 expressing cells. RASSF2 is reported to be a tumor suppressor that is frequently downregulated at the transcriptional level by hypermethylation in primary tumor samples, but not healthy controls. Here we have identified RASSF2 as a target for repression, and demonstrated its tumor suppressive function in t(8;21) leukemia cells. Further insights into the molecular mechanisms of RASSF2 function in AML will continue to be explored. Disclosures No relevant conflicts of interest to declare.

Preclinical Evaluation of the BET-Bromodomain (BET-BRD) Inhibitor OTX015 in Leukemia Cell Lines Harboring the JAK2 V617F Mutation

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 873-873
Author(s):  
Maria Eugenia Riveiro ◽  
Lucile Astorgues-Xerri ◽  
Charlotte Canet-jourdan ◽  
Mohamed Bekradda ◽  
Esteban Cvitkovic ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Line ◽  
Cell Lines ◽  
Research Funding ◽  
Leukemia Cell ◽  
Jak2 V617f ◽  
Jak2 V617f Mutation ◽  
Mrna Levels ◽  
Protein Levels ◽  
V617f Mutation

Abstract Background: Exposure of cancer cells to BET-BRD protein inhibitors has been associated with a significant downregulation of C-MYC expression, leading to suppression of the transcriptional program linked to proliferation and survival. C-MYC mRNA expression, mediated by STAT5 activation, is induced by the JAK2 (V617F) mutation (JAK2mu) in transfected BA/F3 cells (Funakoshi-Tago, et al. 2013). We selected JAK2mu leukemia-derived cell lines for preclinical evaluation of OTX015 (Oncoethix, Switzerland), a selective orally-bioavailable inhibitor of BET-BRD proteins with promising early results in an ongoing phase I study in hematologic malignancies (Herait et al, AACR 2014, NCT01713582). Material and Methods: Antiproliferative effects of OTX015 and JQ1 were evaluated in three established JAK2mu human myeloid leukemia cell lines (SET2, MUTZ8, HEL 92.1.7). GI50 (OTX015 concentration inducing 50% growth inhibition) and Emax (% cell proliferation at 6 µM OTX015) values were determined by MTT assay after 72h exposure. Protein levels were analyzed by Western blot, and RT-PCR was performed with Fast SYBR Green Master Mix on a StepOnePlus Real-Time PCR System. For cell cycle analysis, cells were stained with propidium iodide and analyzed with a FACScan flow cytometer. Induction of apoptosis was evaluated by Annexin-V. Simultaneous schedules of OTX015 combined with ruxolitinib, a JAK2 inhibitor, were evaluated. Combination index (CI) was determined using the Chou & Talalay method; CI<1 reflects synergy, CI=1 additivity and CI>1 antagonism. Results: After 72h exposure, SET2 was the most sensitive cell line (GI50=0.12 µM and Emax=15%), and HEL92.1.7 cells had a GI50=1.9 µM with an Emax=23%. MUTZ8 was the most resistant cell line with an Emax=61%. Similar GI50 and Emax values are observed with JQ1. A significant increase in the fraction of apoptotic cells was observed in SET2 cells after 72h 500 nM OTX015 exposure. Non-significant increases in Annexin-positive cells were seen in HEL92.1.7 and MUTZ8 cells. Cell cycle analysis revealed a significant increase in the percentage of SET2 cells in subG0/G1 after 24, 48, and 72h 500 nM OTX015, correlating with the increase in apoptosis. Conversely, an increase in the percent cells in the G1 phase was observed in HEL 92.1.7 cells. After 4h 500 nM OTX015, BRD2 mRNA levels were significantly increased in all three cell lines, whereas BRD3 levels were not modified. BRD4 mRNA levels increased significantly after 48h in SET2 cells. OTX015 treatment induced a transitory reduction of C-MYC mRNA levels after 4h with an increase at 24h in all cell lines. At the protein level, C-MYC decreased substantially in SET2 cells after 4h, with complete disappearance after 48h without recovery, while in the less sensitive MUTZ8 cell line, the decrease in C-MYC protein levels was transitory. Conversely, this proto-oncogene was not modified in HEL92.1.7 cells. In addition, p-STAT5 protein was downregulated by OTX015 in SET2 cells, but was increased in MUTZ8 cells after longer exposure time. Furthermore, BCL2 mRNA and protein levels decreased in SET2 cells, correlating with the apoptosis induction seen with OTX015 treatment. In HEL92.1.7 cells, P21 mRNA levels and cyclin D1 protein levels increased after 4h and 48h OTX015 treatment, respectively. Moreover, concomitant combination of OTX015 with ruxolitinib showed a highly antagonist effect (CI>7) in SET2 cells, the most sensitive cell line to both agents. On the other hand, very strong synergy was observed in HEL92.1.7 (CI=0.19) and MUTZ8 (CI=0.41), despite their low sensitivity to single agent OTX015. Conclusions. Our findings demonstrate that OTX015 exhibits potent activity against cultured leukemic cells expressing the JAK2 V617F mutation, inducing apoptosis or cell cycle arrest at submicromolar concentrations. This activity correlates with modulation of C-MYC, p-STAT5, BCL2, P21 and cyclin D1 mRNA and protein levels following OTX015 treatment. Our study highlights the novel and synergistic activity of the combination of a BRD antagonist and a JAK inhibitor in human leukemic cells harboring the JAK2 V617 F mutation, supporting the rationale for in vivo testing of OTX015 in combination with JAK inhibitors in leukemic JAK2mu models. Disclosures Riveiro: Oncoethix SA: Research Funding. Astorgues-Xerri:Oncoethix SA: Research Funding. Canet-jourdan:Oncoethix SA: Research Funding. Bekradda:Oncoethix SA: Research Funding. Cvitkovic:Oncoethix SA: Membership on an entity's Board of Directors or advisory committees, Shareholder and CSO Other. Herait:Oncoethix SA: CMO and Shareholder Other. Raymond:Oncoethix SA: Membership on an entity's Board of Directors or advisory committees, Research Funding.

scholarly journals JQ1 Inhibits Proliferation and Induces Apoptosis of Leukemia Cells Through BCL-2 Regulated Pathway

2021 ◽  
Author(s):  
Yifan Zeng ◽  
Xing-Hua Liang ◽  
Yong Xia ◽  
Wen-Yin He
Keyword(s):  
Cell Cycle ◽  
Tumor Cells ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
High Concentration ◽  
Apoptosis Pathway ◽  
Leukemia Cell Lines ◽  
Myeloid Leukemia Cell

Abstract Objective To explore the mechanism of JQ1 on leukemia cells. Methods This study takes two myeloid leukemia cell lines as a research model. Cells treated with high concentration of JQ1 were collected for quantitative real-time PCR, immunoblot and flow cytometry to verify the effects of JQ1 on myeloid leukemia tumor cells. Combined with mRNA sequencing of cell lines to identify the differences in mRNA expression of different cell lines. Results Two cell lines changed cell morphology under JQ1 treatment. The cell membrane appeared in varying degrees of wrinkled internal subsidence. K562 cell lines can maintain stable proliferation after being induced by a specific concentration of JQ1. However, JQ1 cannot induce the death of the K562 cells. Although the MYC and BCL2 gene expression decreased, JQ1 did not affect the c-Myc targeted genes to affect the cell cycle, nor did it trigger the BCL2-mediated apoptosis pathway. On the contrary, after JQ1 induced the MV-4-11 cells, the MYC-mediated cell cycle significantly slowed down and arrested at the G0/G1 phase. The death of MV-4-11 tumor cells through the apoptosis pathway regulated by BCL-2 family. Conclusion JQ1 has different pharmacological effects on two myeloid leukemia cell lines. For MV-4-11, JQ1 mainly inhibited cell cycle by regulating MYC pathway and induced BCL-2-mediated apoptosis to kill myeloid leukemia tumor cells and thus perform anti-tumor effects. K-562 cells showed drug resistance to JQ1 which confirmed that the K-562 cell line has a feedback mechanism that prevents JQ1-induced apoptosis.

scholarly journals 19-nor Vitamin-D Analogs: A New Class of Potent Inhibitors of Proliferation and Inducers of Differentiation of Human Myeloid Leukemia Cell Lines

Blood ◽  
1998 ◽  
Vol 92 (7) ◽  
pp. 2441-2449 ◽  
Author(s):  
Hiroya Asou ◽  
Michiaki Koike ◽  
Elena Elstner ◽  
Moray Cambell ◽  
Jennifer Le ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Kinase Inhibitor ◽  
Leukemia Cell ◽  
Cyclin Dependent Kinase ◽  
Cyclin Dependent Kinase Inhibitor ◽  
Clonal Proliferation ◽  
Leukemia Cell Lines ◽  
Myeloid Leukemia Cell

Abstract We have studied the in vitro biological activities and mechanisms of action of 1,25-dihydroxyvitamin D3 (1,25D3) and nine potent 1,25D3 analogs on proliferation and differentiation of myeloid leukemia cell lines (HL-60, retinoic acid-resistant HL-60 [RA-res HL-60], NB4 and Kasumi-1). The common novel structural motiff for almost all the analogs included removal of C-19 (19-nor); each also had unsaturation of the side chain. All the compounds were potent; for example, the concentration of analogs producing a 50% clonal inhibition (ED50) ranged between 1 × 10−9 to 4 × 10−11 mol/L when using the HL-60 cell line. The most active compound [1,25(OH)2-16,23E-diene-26-trifluoro-19-nor-cholecalciferol (Ro 25-9716)] had an ED50 of 4 × 10−11mol/L; in contrast, the 1,25D3 produced an ED50of 10−9 mol/L with the HL-60 target cells. Ro 25-9716 (10−9 mol/L, 3 days) was a strong inducer of myeloid differentiation because it caused 92% of the HL-60 cells to express CD11b and 75% of these cells to reduce nitroblue tetrazolium (NBT). This compound (10−8 mol/L, 4 days) also caused HL-60 cells to arrest in the G1 phase of the cell cycle (88% cells in G1v 48% of the untreated control cells). The p27kip-1, a cyclin-dependent kinase inhibitor which is important in blocking the cell cycle, was induced more quickly and potently by Ro 25-9716 (10−7 mol/L, 0 to 5 days) than by 1,25D3, suggesting a possible mechanism by which these analogs inhibit proliferation of leukemic growth. The NB4 promyelocytic leukemia cells cultured with the Ro 25-9716 were also inhibited in their clonal proliferation (ED50, 5 × 10−11mol/L) and their expression of CD11b was enhanced (80% positive [10−9 mol/L, 4 days] v 27% untreated NB4 cells). Moreover, the combination of Ro 25-9716 (10−9mol/L) and all-trans retinoic acid (ATRA, 10−7 mol/L) induced 92% of the NB4 cells to reduce NBT, whereas only 26% of the cells became NBT positive after a similar exposure to the combination of 1,25D3 and ATRA. Surprisingly, Ro 25-9716 also inhibited the clonal growth of poorly differentiated leukemia cell lines (RA-res HL-60 [ED50, 4 × 10−9 mol/L] and Kasumi-1 [ED50, 5 × 10−10 mol/L]). For HL-60 cells, Ro 25-9716 markedly decreased the percent of the cells in S phase of the cell cycle and increased the expression of the cyclin-dependent kinase inhibitor, p27kip-1. In summary, 19-nor vitamin D3 compounds strongly induced differentiation and inhibited clonal proliferation of various myeloid leukemia cell lines, suggesting a therapeutic niche for their use in myeloid leukemia.

scholarly journals Transcriptomic Profiles of MV4-11 and Kasumi 1 Acute Myeloid Leukemia Cell Lines Modulated by Epigenetic Modifiers Trichostatin A and 5-Azacytidine

2020 ◽  
Author(s):  
Mat Jusoh Siti Asmaa ◽  
Hamid Ali Al-Jamal ◽  
Abdul Rahim Hussein ◽  
Badrul Hisham Yahaya ◽  
Azlan Husin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Trichostatin A ◽  
Leukemia Cell ◽  
Leukemic Cell ◽  
Enrichment Score ◽  
Acute Leukemias ◽  
Acute Myeloid

Background: Acute myeloid leukemia (AML) is the most common form of acute leukemias in adults which is clinically and molecularly heterogeneous. Several risk and genetic factors have been widely investigated to characterize AML. However, the concomitant epigenetic factors in controlling the gene expression lead to AML transformation was not fully understood. This study was aimed to identify epigenetically regulated genes in AML cell lines induced by epigenetic modulating agents, Trichostatin A (TSA) and 5-Azacytidine (5-Aza). Materials and Methods: MV4-11 and Kasumi 1 were treated with TSA and/or 5-Aza at IC50 concentration. Gene expression profiling by microarray was utilized using SurePrint G3 Human Gene Expression v3. Gene ontology and KEGG pathway annotations were analyzed by DAVID bioinformatics software using EASE enrichment score. mRNA expression of the differentially expressed genes were verified by quantitative real time PCR. Results: Gene expression analysis revealed a significant changes in the expression of 24,822, 15,720, 15,654 genes in MV4-11 and 12,598, 8828, 18,026 genes in Kasumi 1, in response to TSA, 5-Aza and combination treatments, respectively, compared to non-treated (p<0.05). 7 genes (SOCS3, TUBA1C, CCNA1, MAP3K6, PTPRC, STAT6 and RUNX1) and 4 genes (ANGPTL4, TUBB2A, ADAM12 and PTPN6) shown to be predominantly expressed in MV4-11 and Kasumi 1, respectively (EASE<0.1). The analysis also revealed phagosome pathway commonly activated in both cell lines. Conclusion: Our data showed a distinct optimal biological characteristic and pathway in different types of leukemic cell lines. These finding may help in the identification of cell-specific epigenetic biomarker in the pathogenesis of AML.  

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