scholarly journals A SIX1/EYA2 Inhibitor Impairs CALM-AF10 and Jurkat Leukemia Cell Proliferation

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4331-4331
Author(s):  
Waitman Kurt Aumann ◽  
Catherine P. Lavau ◽  
Dongdong Julie Chen ◽  
Amanda E. Conway ◽  
Heide Ford ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Lines ◽  
Nuclear Export ◽  
Leukemia Cell ◽  
Homeobox Gene ◽  
Jurkat Cells ◽  
Leukemia Cells ◽  
Leukemia Cell Lines ◽  
Gene And Protein Expression ◽  
Hoxa Genes

Abstract Background : The CALM-AF10 translocation is found in 5-10% of T-cell acute lymphoblastic leukemias (T-ALL), and a subset of acute myeloid leukemias (AML). CALM-AF10 leukemias are characterized by elevated expression of proleukemic HOXA genes. Since HOXA genes are difficult to target, we hypothesized that identification of non-HOXA CALM-AF10 effector genes could potentially yield novel therapeutic targets. To discover novel CALM-AF10-regulated genes, we took advantage of our prior observation that the nuclear export factor CRM1/XPO1 tethers CALM-AF10 to HOXA genes by interacting with a nuclear export signal within CALM. Using microarrays, we identified a set of genes that showed decreased expression in response to the CRM1 inhibitor Leptomycin B (LMB), similar to Hoxa genes, in murine CALM-AF10 leukemia cells. Then using RNA-sequencing, we discovered a set of genes increased in murine hematopoietic stem cells transduced with CALM-AF10. There were 11 genes that were both decreased in response to LMB and increased in response to CALM-AF10, which included the Hoxa gene cluster, as well as Six1. We demonstrated that CALM-AF10 increases Six1 expression and localizes to the Six1 locus, as it does the Hoxa genes. SIX1, like the Hoxa genes, is a homeobox gene that is associated with embryogenesis and is quiescent post-embryologically. In addition, SIX1 and its cofactor EYA2 are overexpressed in numerous solid tumors, and an inhibitor of the SIX1/EYA2 complex (Compound 8430) has recently been described. While there is evidence of a role for SIX1 in solid tumors, its role in leukemias has not been explored. Objective : Evaluate the effect of a SIX1/EYA2 complex inhibitor on leukemia cell proliferation. Design/Methods : SIX1 gene and protein expression were assessed in CALM-AF10, Jurkat (T-ALL) and NOMO1 (AML) leukemia cell lines via Western Blot and RT-qPCR. CALM-AF10 leukemias were derived from murine models in our lab, Jurkat and NOMO1 cell lines were obtained from ATCC. The effect of compound 8430 - an inhibitor of the Six1/Eya2 interaction - on cell proliferation was evaluated using Cell-Titer-Glo Assays and liquid culture proliferation assays. In addition, we used the the CRM1 Nuclear Export Inhibitor KPT-330 alone and in combination with 8430 in these cell lines. SynergyFinder2 (https://synergyfinder.fimm.fi/) was used to assess synergy of 8430 and KPT-330. δ-score is a calculated value that indicates synergistic drug interaction, with a higher δ-score indicative of a synergistic effect of the drugs. Results : SIX1 gene and protein expression are increased in CALM-AF10 leukemia cell lines and Jurkat T-ALL cells, but not NOMO1 cells. Compound 8430 decreases cell proliferation in CALM-AF10 leukemias and Jurkat leukemia cell lines, however it did not affect the AML line NOMO1. Correspondingly, liquid cultures showed that 8430 alone slowed the proliferation of CALM-AF10 leukemia and the Jurkat cells, but not NOMO1 cells. The addition of KPT-330 to 8430 was synergistic in CALM-AF10 leukemia cells with a KPT-330 dose of 60 nM and multiple dose levels of 8430 (δ-scores 17-19) while in the Jurkat leukemia cells a dose of 30 nM of KPT-330 was synergistic at multiple dose levels of 8430 (δ-score 6-8) (Figure 1). Conclusions : The SIX1 homeobox gene is highly expressed during development, and its expression is silenced post-embryogenesis. Through an initial unbiased screen, we discovered that Six1 may play a role in CALM-AF10 leukemogenesis. We have determined that Six1 expression is upregulated in the presence of CALM-AF10. A role for Six1 in CALM-AF10 leukemogenesis is further supported by the ability of a SIX1/EYA2 inhibitor to slow the proliferation of CALM-AF10 leukemia cells. Importantly, based on our observation that 8430 slows proliferation of Jurkat cells, SIX1 inhibition may be relevant in other leukemias. Finally, our demonstration that 8430 synergizes with KPT-330, a Nuclear Export Inhibitor, suggests the possibility of a novel therapeutic approach for CALM-AF10 and other leukemias. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Clathrin Assembly Protein CALM Is Necessary for Leukemia Cell Proliferation and Erythroid Development Via Regulating Transferrin Receptor Endocytosis

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 244-244
Author(s):  
Yuichi Ishikawa ◽  
Manami Maeda ◽  
Min Li ◽  
Sung-Uk Lee ◽  
Julie Teruya Feldstein ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Leukemia Cell Lines ◽  
Erythroid Development

Abstract Abstract 244 Clathrin assembly lymphoid myeloid leukemia (CALM) protein is implicated in clathrin dependent endocytosis (CDE) and the CALM gene is the target of the t(10;11)(p13;q14-21) CALM/AF10 translocation, which is observed in multiple types of acute leukemia. Although the translocation generally dictates poor prognosis, the molecular mechanisms by which the fusion protein exerts its oncogenic activity remains elusive. To determine the role of CALM and CDE in normal hematopoiesis and leukemogenesis, we generated and characterized both conventional (Calm+/−) and conditional (CalmF/FMx1Cre+) Calm knockout (KO) mutants. Furthermore, we determined the impact of Calm loss on leukemia cell growth in vitro and in vivo employing a series of leukemia cell lines and leukemia mouse models. Hematopoietic-specific Calm knockout mice (CalmF/FMx1Cre+) exhibited a hypocromatic anemia with increased serum iron levels. We observed significant reduction in mature erythroblasts/erythrocytes (TER119+CD71-) with concomitant increase in immature erythroblasts (TER119+CD71+) in the spleen of CalmF/FMx1Cre+ mice. The frequencies of erythroblasts in S phase were lower and the proportions of apoptotic (cleaved PARP positive) erythroblasts were increased in CalmF/FMx1Cre+ mice. Surface transferrin receptor 1 (Tfr1, CD71) levels were significantly up-regulated in Calm-deficient hematopoietic progenitors, and uptake of Alexa647-conjugated transferrin was abrogated in Calm-deficient erythroblasts, revealed by immunofluorescence analysis. Freez-etch electron microscopy analysis showed a defective clathrin coated vesicle (CCV) formation in Calm-deficient erythroblasts, indicating that Calm is indispensable for iron-bound transferrin internalization by regulating CCV formation, thereby critical for erythroid differentiation and hemoglobinization. CALM was highly expressed in leukemia/lymphoma cell lines and primary acute myeloid leukemia samples, although its expression was limited to erythroblasts in normal hematopoietic lineage cells. Treatment of leukemia cell lines with Desferoxamine (DFO), an iron chelator, led to a significant increase in Calm mRNA levels, suggesting that Calm expression is regulated by intracellular iron levels. Since highly proliferative leukemia cells demand iron as a cofactor for ribonucleotide reductase (RNR), we hypothesized that Calm is required for leukemia cell proliferation by regulating iron-bound transferrin internalization. To determine the effect of Calm inactivation in leukemia cells, we transduced a series of leukemia cell lines with a lentivirus-based ShRNA vector (pLKO-GFP), which allowed shRNA-expressing cells to be traced by green fluorescent protein (GFP). Calm shRNA transduced cells, but not cells transduced with scrambled shRNA, showed a proliferative disadvantage compared to non-transduced cells. To determine the effect of Calm deletion in leukemia cells in vivo, the CALM/AF10 oncogene was retrovirally transduced into either wild type (WT) or CalmF/FMx1Cre+ bone marrow (BM) cells and the cells were subsequently transferred to lethally-irradiated recipient mice. The Calm gene was deleted in donor cells via pIpC injections one month after transplant (before leukemia development) and survival curves generated. The recipients transplanted with the BM cells from CalmF/FMx1Cre+ mice showed a significantly delayed onset of leukemia and longer survivals compared to control (p=0.001), indicating that Calm is necessary for the development of CALM/AF10-induced leukemia. We next assessed whether Calm is required for the “maintenance” of leukemia in vivo. Leukemia cells were harvested from the primary recipients transplanted with the CALM/AF10-transduced CalmF/FMx1Cre+ BM cells (in which the endogenous Calm genes were intact) and transferred to the secondary recipients. The leukemic secondary recipient mice were then injected with pIpC and survival curves generated. Calm inactivation significantly delayed leukemia progression by blocking leukemia cell proliferation. Taken together, our data indicate that Calm is essential for erythroid development and leukemia cell proliferation by regulating TFR1 internalization. Since Calm inactivation significantly blocked the leukemia cell proliferation in vitro and in vivo, our findings may provide new therapeutic strategies for acute myeloid leukemia. Disclosures: Naoe: Kyowa-Hakko Kirin.: Research Funding; Dainipponn-Sumitomo Pharma.: Research Funding; Chugai Pharma.: Research Funding; Novartis Pharma.: Honoraria, Speakers Bureau; Zenyaku-Kogyo: Research Funding; Otsuka Pharma.: Research Funding.

scholarly journals Ampelopsin Inhibits Cell Proliferation and Induces Apoptosis in HL60 and K562 Leukemia Cells by Downregulating AKT and NF-κB Signaling Pathways

2021 ◽  
Vol 22 (8) ◽  
pp. 4265
Author(s):  
Jang Mi Han ◽  
Hong Lae Kim ◽  
Hye Jin Jung
Keyword(s):  
Signaling Pathways ◽  
Cell Lines ◽  
White Blood Cells ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Ros Generation ◽  
Anticancer Activities ◽  
Nuclear Condensation ◽  
Leukemia Cell Lines ◽  
Antileukemic Effect

Leukemia is a type of blood cancer caused by the rapid proliferation of abnormal white blood cells. Currently, several treatment options, including chemotherapy, radiation therapy, and bone marrow transplantation, are used to treat leukemia, but the morbidity and mortality rates of patients with leukemia are still high. Therefore, there is still a need to develop more selective and less toxic drugs for the effective treatment of leukemia. Ampelopsin, also known as dihydromyricetin, is a plant-derived flavonoid that possesses multiple pharmacological functions, including antibacterial, anti-inflammatory, antioxidative, antiangiogenic, and anticancer activities. However, the anticancer effect and mechanism of action of ampelopsin in leukemia remain unclear. In this study, we evaluated the antileukemic effect of ampelopsin against acute promyelocytic HL60 and chronic myelogenous K562 leukemia cells. Ampelopsin significantly inhibited the proliferation of both leukemia cell lines at concentrations that did not affect normal cell viability. Ampelopsin induced cell cycle arrest at the sub-G1 phase in HL60 cells but the S phase in K562 cells. In addition, ampelopsin regulated the expression of cyclins, cyclin-dependent kinases (CDKs), and CDK inhibitors differently in each leukemia cell. Ampelopsin also induced apoptosis in both leukemia cell lines through nuclear condensation, loss of mitochondrial membrane potential, increase in reactive oxygen species (ROS) generation, activation of caspase-9, caspase-3, and poly ADP-ribose polymerase (PARP), and regulation of Bcl-2 family members. Furthermore, the antileukemic effect of ampelopsin was associated with the downregulation of AKT and NF-κB signaling pathways. Moreover, ampelopsin suppressed the expression levels of leukemia stemness markers, such as Oct4, Sox2, CD44, and CD133. Taken together, our findings suggest that ampelopsin may be an attractive chemotherapeutic agent against leukemia.

Aurora-A kinase: a novel target of cellular immunotherapy for leukemia

Blood ◽  
2009 ◽  
Vol 113 (1) ◽  
pp. 66-74 ◽  
Author(s):  
Toshiki Ochi ◽  
Hiroshi Fujiwara ◽  
Koichiro Suemori ◽  
Taichi Azuma ◽  
Yoshihiro Yakushijin ◽  
...  
Keyword(s):  
Cell Lines ◽  
Leukemia Cell ◽  
Myelogenous Leukemia ◽  
Leukemia Cells ◽  
Cellular Immunotherapy ◽  
Aurora A Kinase ◽  
Aurora A ◽  
Hematopoietic Stem ◽  
Specific Ctls ◽  
Leukemia Cell Lines

Abstract Aurora-A kinase (Aur-A) is a member of the serine/threonine kinase family that regulates the cell division process, and has recently been implicated in tumorigenesis. In this study, we identified an antigenic 9–amino-acid epitope (Aur-A207-215: YLILEYAPL) derived from Aur-A capable of generating leukemia-reactive cytotoxic T lymphocytes (CTLs) in the context of HLA-A*0201. The synthetic peptide of this epitope appeared to be capable of binding to HLA-A*2402 as well as HLA-A*0201 molecules. Leukemia cell lines and freshly isolated leukemia cells, particularly chronic myelogenous leukemia (CML) cells, appeared to express Aur-A abundantly. Aur-A–specific CTLs were able to lyse human leukemia cell lines and freshly isolated leukemia cells, but not normal cells, in an HLA-A*0201–restricted manner. Importantly, Aur-A–specific CTLs were able to lyse CD34+ CML progenitor cells but did not show any cytotoxicity against normal CD34+ hematopoietic stem cells. The tetramer assay revealed that the Aur-A207-215 epitope–specific CTL precursors are present in peripheral blood of HLA-A*0201–positive and HLA-A*2402–positive patients with leukemia, but not in healthy individuals. Our results indicate that cellular immunotherapy targeting Aur-A is a promising strategy for treatment of leukemia.

Metabolic Profile Of Leukemia Cells Influences Treatment Efficacy Of L‑Asparaginase

2019 ◽  
Author(s):  
Katerina Hlozkova ◽  
Alena Pecinova ◽  
David Pajuelo Reguera ◽  
Marketa Simcikova ◽  
Lenka Hovorkova ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Cell Lines ◽  
Mitochondrial Membrane ◽  
Metabolic Profile ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Lower Sensitivity ◽  
Leukemia Cell Lines

Abstract Background Effectiveness of L-asparaginase administration in acute lymphoblastic leukemia treatment is mirrored in overall outcome of patients. Generally, leukemia patients differ in their sensitivity to L-asparaginase; however, the mechanism underlying their inter-individual differences is still not fully understood. We have previously shown that L-asparaginase rewires the biosynthetic and bioenergetic pathways of leukemia cells to activate both anti-leukemic and pro-survival processes. Herein, we investigated the relationship between the metabolic profile of leukemia cells and their sensitivity to currently used cytostatic drugs.Methods Altogether, 19 leukemia cell lines and primary leukemia cells from 11 patients were used. Glycolytic function and mitochondrial respiration were measured using Seahorse bioanalyzer. Sensitivity to cytostatics was measured using MTS assay and/or absolute count and flow cytometry. Mitochondrial membrane potential was determined as TMRE fluorescence.Results We characterized the basal metabolic state of the cells derived from different leukemia subtypes using cell lines and primary samples and assessed their sensitivity to cytostatic drugs. We found that leukemia cells cluster into distinct groups according to their metabolic profile, which is mainly driven by their hematopoietic lineage of origin from which they derived. However, majority of lymphoid leukemia cell lines and patients with lower sensitivity to L-asparaginase clustered regardless their hematopoietic phenotype together with myeloid leukemias. Furthermore, we observed a correlation of specific metabolic parameters with sensitivity to L-asparaginase. Greater ATP-linked respiration and lower basal mitochondrial membrane potential in cells significantly correlated with higher sensitivity to L-asparaginase. No such correlation was found in other tested cytostatic drugs.Conclusions These data support the prominent role of the cell metabolism in the treatment effect of L-asparaginase. Based on these findings metabolic profile could identify leukemia patients with lower sensitivity to L-asparaginase with no specific genetic characterization.

Elevated FoxM1 Expression Promotes Cell Cycle Progression by Induction of KIS Expression and Contributes to the Development of Leukemia Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 888-888 ◽  
Author(s):  
Okinaka Keiji ◽  
Satoki Nakamura ◽  
Isao Hirano ◽  
Takaaki Ono ◽  
Shinya Fujisawa ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Lines ◽  
Leukemia Cell ◽  
Aurora Kinase ◽  
Leukemia Cells ◽  
Clinical Samples ◽  
Acute Leukemias ◽  
Aurora Kinase B ◽  
Leukemia Cell Lines ◽  
Foxm1 Expression

Abstract [Background] FoxM1, a member of the Fox transcription factor family, plays an important cell cycle regulator of both the transition from G1 to S phase and progression to mitosis. FoxM1 expression was also found to be up-regulated in some solid tumors (basal cell carcinomas, hepatocellular carcinoma, and primary breast cancer). These results suggested that FoxM1 plays a role in the oncogenesis of malignancies. However, it is unknown whether FoxM1 expression contributes to the development or progression of leukemia cells. Therefore, we investigated how FoxM1 regulated the cell cycle of leukemia cells and the expression analysis of the FoxM1 gene in patients with acute leukemias. [Methods] The cells used in this study were human acute leukemia cell lines, U937 and YRK2 cells. Primary acute myeloblastic (25 AML (4 M1, 11 M2, 6 M4, 4 M5)) cells were obtained from the peripheral blood. Human normal mononuclear cells (MNCs) were isolated from peripheral blood (PB) of healthy volunteers after obtaining informed consents. For analysis of proliferation and mitotic regulatory proteins (p27, p21, Skp2, Cdc25B, Cyclin D1, Survivin, Aurora kinase B, and KIS) in leukemia cells, MTT assays and western blot were performed in all cell lines, which untransfected or transfected with siRNA FoxM1, respectively. For cell cycle analysis, flow cytometory analysis was performed in leukemia cells untransfected or transfected with siRNAFoxM1 by PI staining. For analysis of FoxM1 mRNA, quantitative RT-PCR was performed in all cell lines and clinical samples. [Results] In all leukemia cell lines, the expression of FoxM1B mRNA were significantly higher than normal MNCs. When transfected with the siRNA FoxM1 in leukemia cells, suppression of FoxM1 caused a mean 71% (range 62 to 80%) reduction in S phase cells and a mean 4.4-fold (range 3.2 to 5.6-fold) increase in G2/M phase cells compared to controls. MTT assay demonstrated that the proliferation of the siRNA FoxM1 transfected cells was inhibited compared to the untransfected cells. Moreover, FoxM1 knockdown by siRNA in leukemia cells reduced protein and mRNA expression of Aurora kinase B, Survivin, Cyclin D1, Skp2 and Cdc25B, while increased protein expression of p21and p27. In the clinical samples obtained from patients with acute leukemias, the FoxM1B gene was overexpressed in 22/25 (88%). The relative folds of FoxM1B gene expression were for AML: 2.83 compared to normal MNCs. [Conclusions] In this study, we report in the first time that FoxM1 is overexpressed in myeloid leukemia cells. These results demonstrated that expression of FoxM1 is an essential transcription factor for growth of leukemia cells, and regulate expression of the mitotic regulators. Moreover, we showed that FoxM1 induced the expression of KIS protein. Therefore, FoxM1 might be one of moleculer targets of therapy for acute leukemias.

Dysregulated Expression of HSP70 Isoforms in Acute Myelogenous Leukemia (AML)

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1370-1370
Author(s):  
Krishan K. Sharma ◽  
Juan Felipe Rico ◽  
Duane C Hassane ◽  
Gabriela Chiosis ◽  
Monica L. Guzman
Keyword(s):  
Drug Resistance ◽  
Heat Shock ◽  
Cord Blood ◽  
Cell Lines ◽  
Blood Cells ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Normal Cells ◽  
Leukemia Cell Lines ◽  
Cord Blood Cells

Abstract Abstract 1370 Stress-inducible heat shock protein 70 (HSP70) is a major cytoprotective factor and a molecular chaperone that interacts with HSP90 to form a multi-chaperone complex. Cancer cells are highly dependent on this complex due to their increased demand for protein synthesis. HSP70 overexpression inhibits apoptosis and has been associated with drug resistance. However, the contribution to drug resistance in AML of specific HSP70 isoforms remains unknown. As there is growing interest in therapeutically targeting HSP70, we investigated the expression of 7 different HSP70 isoforms in AML primary cells and leukemia cell lines and their response to a novel HSP70-inhbitor, YK5. A panel of 12 leukemia cell lines and 11 primary samples was used to determine the expression of HSP70 and their response to YK5. We also evaluated the changes to the HSP70 isoforms when exposed to either heat shock or YK5. We found MV4-11, MOLM-13, and U937 sensitive to YK5 (LD50 = 1.18μM, 1.03μM, and 2.31μM at 24 hours, respectively). In contrast, OCI-AML3, TUR and THP-1 were more resistant to the inhibitor. (LD50 = 9.92μM, 9.74μM, and 8.84μM at 24 hours, respectively). Non-tumor cells, however, were significantly less affected by treatment with YK5 (72% viable cord blood mononuclear cells after 24 hour treatment with 5μM YK5). We found that the cell surface expression of HSP70 was higher in both cell lines and primary samples when compared their normal counterparts. Furthermore, quantitative PCR revealed that cell lines with higher levels of HSPA1A and lower levels of HSPA6 demonstrated higher sensitivity to YK5. Interestingly, higher levels of HSPA1A and lower levels of HSPA6 were also found in primary AML samples when compared to CD34+ cord blood cells, consistent with the relative insensitivity of normal cells to YK5. We further discovered, mining publicly available databases, that high levels of HSPA1A were associated poorer prognosis (p = 0.004), suggesting that YK5 would be beneficial to patients presenting high HSP70 expression. We also evaluated the effect of YK5 on the gene expression of the various HSP70 isoforms. Quantitative PCR revealed the ability of YK5 to downregulate HSPA6 and HSC70 (HSPA8) in both cell lines and primary samples. Strikingly, all HSP70 isoforms exhibited similar fold changes upon heat shock in primary samples, CD34+ cord blood cells, and leukemia cell lines, indicating that the cellular stress response is not damaged in AML. However, the specificity of HSP70 inhibition to leukemia cells and not normal cells suggests a dysregulated set of client proteins and increased dependency on HSP70 to maintain leukemic homeostasis. In summary, we have found dysregulated expression of the HSP70 isoforms HSPA1A and HSPA6 in leukemia cells and that the expression levels of these isoforms correlate to the sensitivity of YK5-mediated HSP70 inhibition (HSPA1A: p=0.0012 and r2=0.801, HSPA6: p=0.0011 and r2=0.847). *KKS and JFR contributed equally to this project Disclosures: No relevant conflicts of interest to declare.

MicroRNAs Expression signatures of microvesicles Derived-From Three Different Leukemia Cell Lines

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4649-4649
Author(s):  
Xiaomei Chen ◽  
Wei Xiong ◽  
Dongmei Guo ◽  
Junfeng Wang ◽  
Juanjuan Li ◽  
...  
Keyword(s):  
Cell Lines ◽  
Normal Control ◽  
Target Genes ◽  
Leukemia Cell ◽  
Jurkat Cells ◽  
Differentially Expressed ◽  
Mirna Microarray ◽  
Leukemia Cell Lines ◽  
Differentially Expressed Mirnas ◽  
In Cells

Abstract Abstract 4649 MicroRNAs(miRNAs) are short non-coding RNAs that are involved in post-transcriptional regulation of gene expression in multicellular organisms by affecting both the stability and translation of mRNAs. Recent studies revealed that genetic exchange of miRNAs between cells can be accomplished through microvesicles(MVs),which are small exosomes/vesicles of endocytic origin shed from the surface membranes of activated platelets and normal or malignant cells. Generally, MVs are enriched in various bioactive molecules of their parental cells, such as proteins, DNA, mRNA and miRNAs. So, exploration of changes of the miRNAs profiles of MVs derived from leukemia cells will contribute to a better understanding of the pathogenesis of leukemias and then provide valuable suggestions about how to diagnose, prevent and control leukemia. In the present study,we used Agilent miRNA microarray to analyze the miRNAs of MVs derived from three different leukemia cell lines (K562, Nalm-6 and Jurkat) and their cell counterparts, and normal plasma derived MVs as normal control. The significantly differentially expressed miRNAs between test group and normal control group were determined by t-test(P<0.05 and Fold Change>2).The putative target genes of the differentially expressed miRNAs were predieted by Targetscan. We observed that 125 miRNAs (93 up-regulated and 32 down-regulated) were determined to be differentially expressed in MVs derived from three leukemia cell lines compared with their normal control counterparts. Four miRNAs including miR-1290, miR-1268,miR-1246 and miR-1305 were found to be hundreds fold alteration. In addition, let-7f, miR-26a, miR-26b and miR-223 were significantly under expressed. We next found that MVs derived from lymphocytic leukemia cell lines(Nalm-6 and Jurkat) shared 100 miRNAs of 888 miRNAs, 99 upregulated and only one miRNA downregulated. Meanwhile, 44 miRNAs were just altered in Nalm-6-MVs, 9 miRNAs are just altered in Jurkat-MVs. Moreover, we found 22 miRNAs were only altered in K562-MVs, only one miRNAs(miR-191) altered in lymphocytic-MVs. We also found that MVs miRNA profiles were significantly different, compared the MVs derived from three leukemia cell lines with their cell counterparts. K562 cells and K562 MVs shared 112 miRNAs. 122 miRNAs were only altered in cells and 77 miRNAs were only altered in their MVs. Similarly, Nalm-6 cells and nalm-6 MVs shared 154 miRNAs, and 9 miRNAs were only altered in cells and 102 miRNAs were only altered in their MVs. Jurkat cells and Jurkat MVs shared 111 miRNAs, and 15 miRNAs were only altered in cells and 95 miRNAs were only altered in their MVs. The results of real-time qRT-PCR consisitanted with that observed in the microarray assays. We identified target genes of these differently expressed miRNAs by TargetScan, and retrievaled recent literature on the properties and biogenesis of these aberrant miRNAs and their potential role in cancer progression. Differentially expressed miRNAs include known oncomirs (e.g miR-96) as well as miRNAs that were not previously universally associated with cancer. Specific examples include let-7f,miR-191 and miR-21, which were consistently down regulated and miR-223 which is consistently up regulated in cancer, in the context of our cohort. Furthermore, miR-1246 mediates the functions of p53 family members, and BCL-2,KRAS may be target of miR-1305. In summary,we firstly used the miRNA microarray to seek for miRNAs with differential expression in MVs derived from K562, Nalm-6 and Jurkat cells. Predicting the putative target genes of the miRNAs with bioinformatic softwares may play a foundation for further studies exploring the biomolecular mechanism of oncogensis and development of leukemia and searching for related biomolecular markers of diagnosis and treatment in leukemia. Disclosures: No relevant conflicts of interest to declare.

Preclinical Evaluation of Oncolytic Reovirus in Targeted Therapeutics for High-Risk Pediatric Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3571-3571
Author(s):  
Matthew F. Clarkson ◽  
Aru Narendran ◽  
Randal N. Johnston
Keyword(s):  
Cell Death ◽  
High Risk ◽  
Cell Viability ◽  
Cell Lines ◽  
Leukemia Cell ◽  
Treatment Strategies ◽  
Leukemia Cells ◽  
Western Blots ◽  
Pediatric Leukemia ◽  
Leukemia Cell Lines

Abstract Abstract 3571 Purpose: Leukemia is the most common malignancy in children. Improved treatment strategies in recent decades have yielded substantially enhanced outcomes for children with leukemia, reaching survival rates >80%. However, there remain significant issues with current treatment. Certain subgroups of patients who are resistant to or relapse from current treatments have a dismal prognosis. Furthermore, there are significant late effects of intensive treatments, including secondary cancers, neurocognitive defects, cardiotoxicity, obesity and infertility. For these reasons, novel treatment strategies are urgently needed for high-risk leukemia in children. Reovirus type 3 Dearing is a wild-type double-stranded RNA virus that has shown great promise as a selective oncolytic agent by its ability to replicate in transformed cells but not in normal cells. Although a number of early phase clinical studies have been completed in patients with advanced, refractory solid tumors in adults, systematic evaluation of this agent in the treatment of refractory pediatric leukemia has not been reported. As an initial step towards developing an oncolytics based treatment approach, we report preclinical data with respect to the activity, target validation, target modulation and drug combinability of reovirus in childhood leukemia cells. Experimental Design: A panel of pediatric leukemia cell lines representing high-risk molecular features such as Bcr-Abl, MLL rearranged and mixed lineage was used (n =6). Expression of JAM-A, the cell surface receptor for reovirus, was assessed by flow cytometry. The Ras Activation Assay Kit (EMD Millipore) was used to assess activity of the RAS protein. Western Blots were used to assess the activation (phosphorylation) of the signaling partners downstream of RAS. Cells treated with reovirus, chemotherapy drugs, or both for distinct treatment schedules were assessed for cell viability by the CellTiter-Glo© Luminescent Cell Viability Assay (Promega), and cell death by apoptosis was confirmed by cleavage of PARP. Productive viral infection was assessed by measuring reoviral protein synthesis by Western Blots, and reoviral replication was assessed by virus plaque titration assay. Drug synergies were calculated according to the method of Chou and Talalay. Results: Target validation assays showed the expression of JAM-A, which facilitates effective viral entry into malignant cells, in five of six cell lines. These cell lines also demonstrated differential activation of RAS and downstream kinases, suggesting targeted susceptibility of these cells to reovirus oncolysis. To further test this, we infected cells with reovirus for 1–4 days and assessed cytopathic effects. Using phase contrast microscopy, we observed the virus treated cell lines to demonstrate morphological changes characteristic of cell death following infection. Cell viability assays were used to quantify this effect, and the mechanism of cell death was determined to be apoptotic as evidenced by caspase-dependent cleavage of PARP. Reovirus-induced cell death was correlated with viral protein production and replication. Next, we screened for the ability of reovirus to induce synergistic activity in a panel of conventional and novel targeted therapeutic agents. Our studies showed that, in contrast to the current antileukemic agents, the Bcl-2 inhibitor BH3 mimetic ABT-737 was able to significantly synergize with reovirus in all cell lines tested. Conclusions: In our in vitro studies, oncolytic reovirus as a single agent showed potent oncolytic activity against all pediatric leukemia cell lines tested that express the receptor for reovirus, regardless of the status of the RAS signaling pathway. Further, we found reovirus-induced oncolysis can be enhanced by combination with Bcl-2 inhibition but was unaltered or antagonized by the other drugs indicating a key relationship between the two pathways. As such, our data for the first time, show that pediatric leukemia cells carry the potential to be targeted by reovirus induced oncolysis and the identification of drug synergy and the biomarkers of target modulation provide the basis for further studies to develop this novel therapeutic approach for clinical studies in the near future. Disclosures: No relevant conflicts of interest to declare.

Uncovering Cardioprotective Strategies For Anthracycline Therapy By Analysis Of Redox and Apoptotic Effects

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1293-1293
Author(s):  
Daniela E. Egas Bejar ◽  
Joy M. Fulbright ◽  
Fernando F. Corrales-Medina ◽  
Mary E. Irwin ◽  
Blake Johnson ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Cell Death ◽  
Vitamin E ◽  
Acute Leukemia ◽  
Cell Lines ◽  
Leukemia Cell ◽  
Cell Types ◽  
Leukemia Cells ◽  
Soluble Form ◽  
Leukemia Cell Lines

Abstract Anthracyclines are among the most powerful drugs used for the treatment of leukemia, however their use has been associated with cardiotoxicity. Reactive oxygen species (ROS) are generated in both cancer and normal cells after anthracycline exposure and have been implicated in both early and late onset cardiotoxicity. Counteracting this ROS generation are intracellular antioxidants such as the ubiquitous antioxidant glutathione (GSH), levels of which are depleted upon anthracycline exposure. Basal expression of GSH pathway components and other antioxidants vary greatly between different cell types. Due to this differential expression of cellular antioxidants in cardiomyocytes versus leukemia cells, we posit that anthracyclines exert distinct effects on oxidative stress and consequent apoptosis induction in leukemia cells and nontransformed hematopoietic cells (PBMC) relative to cardiomyocytes. As a result, we expect potentially varied mechanisms of cell death induction in these cell lines after anthracycline treatment. To test this hypothesis, the acute leukemia cell lines Jurkat and ML-1 and the cardiomyocyte line H9C2 were used. Dose responses with the anthracyclines, doxorubicin and daunorubicin, were carried out and trypan blue exclusion and propidium iodide staining followed by flow cytometry were used to assess viability and DNA fragmentation respectively. Cardiomyocytes had a 25-150 fold higher IC50 value than the acute leukemia cell lines, indicating selectivity. To assess whether apoptosis was induced by anthracyclines, caspase 3 activity was measured and found to be increased at 24 hours in Jurkat cells which preceded decreases in viability, supporting an apoptotic mechanism of cell death. GSH levels also decreased markedly after 24 hours of treatment with anthracyclines in this cell line, however, a pan-caspase inhibitor did not block GSH depletion, indicating that these events occur independent of each other. To evaluate whether antioxidants conferred protection against loss of viability in all cell types, cells were pretreated for at least 30 minutes with antioxidants and then treated with doxorubicin and daunorubicin for 24 hours. Antioxidants used were N-acetylcysteine (NAC, a GSH precursor and amino acid source), GSH ethyl ester (cell permeable form of GSH), tiron (free radical scavenger) and trolox (a water soluble form of vitamin E). GSH ethylester did not prevent cytotoxicity of anthracyclines in acute leukemia lines or cardiomyocytes. Therefore boosting GSH levels in leukemia cells does not reverse cytotoxicity. Trolox, however, did block anthracycline induced cell death in ML-1 cells, suggesting that vitamin E supplementation would counteract leukemia cell specific effects of anthracyclines on AML cells. Tiron protected PBMC from doxorubicin cytotoxicity but did not protect leukemia cells or cardiomyocytes, hinting at a protective strategy for normal non-leukemia blood cells. Interestingly, NAC did not interfere with the cytotoxic effects of anthracyclines on acute leukemia cells or PBMC, but protected H9C2 cells from daunorubicin cytotoxicity. Taken together, these data reveal differential protective effects of antioxidants in cardiomyocytes and PBMCs relative to ALL and AML cells. Our work indicates that NAC can protect cardiomyocytes without interfering with anthracycline cytotoxicity in acute leukemia cells. In humans, one randomized control trial tested the addition of NAC to doxorubicin therapy, detecting no evidence of cardioprotective activity by chronic administration of NAC. However, the schedule used for administration of NAC in that study may not have been optimal, and biomarkers for oxidative stress reduction by NAC were not incorporated into the trial. Previously, other antioxidants have been used with very limited clinical success and possible contributing factors include inadequate sample size, choice of agent, dose used, duration of intervention and the lack of biomarker endpoints. Designing a cardioprotective and antioxidant strategy with attention to these factors may prove to be efficacious in protecting cardiac cells without interfering with the antitumoral effect of anthracyclines. To this end, our data suggests that trolox and vitamin E analogues should not be used in acute leukemia as they may interfere with the cytotoxic action of anthracyclines but NAC or cysteine may be used as cardioprotectants. Disclosures: No relevant conflicts of interest to declare.

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