Hemangioblastic Activity of Infant Leukemia with t(4;11).

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4279-4279
Author(s):  
Zhongbo Hu ◽  
Xiaomiao Li ◽  
William B. Slayton
Keyword(s):  
Bone Marrow ◽  
Blood Vessels ◽  
Cell Lines ◽  
Leukemia Cell ◽  
Scid Mice ◽  
Leukemia Cells ◽  
Leukemia Cell Lines ◽  
Infant Leukemia

Abstract Background: Infant leukemia patients with t(4;11) have an extremely high risk for treatment failure. Hemangioblasts are cells that were initially described in the embryonic yolk sac, where they make both blood and blood vessels when these two systems are forming. It was demonstrated that hemangioblasts are present in the adult bone marrow and CML patients. Recently, subgroups of infant ALL patients with t(4;11) were found to have gene expression profiles similar to hemangioblasts by microarray. However, whether infant leukemia cells behave like hemangioblasts and produce their own blood vessels remains unknown. Objective: We sought to determine whether infant leukemia cells with t(4;11) are derived from malignant hemangioblasts and can produce their own blood vessels. Design/Methods: Three childhood leukemia cell lines with t(4;11): MV4-11, SEM-K2 and RS4-11, were used to analyze the expression pattern of key angiogenic receptors by flow cytometry and angiogenesis related proteins by protein array in comparison with benign endothelial cells. These cell lines were also cultured in vitro using Matrilgel, an in vitro angiogenesis assay system, in order to test their ability to produce vascular tubes. These cell lines were injected into the immune deficient NOD/SCID mice after sublethal irradiation to establish leukemia in vivo. Some of primary cells from MLL patients were obtained and subcutaneously injected into NOD/SCID mice mixed with BD Matrigel to observe their vessel development in vivo together with these three cell lines. The bone marrow, liver, spleen and tumor tissues together with Matrigel were collected to look for the evidence of t(4;11) in endothelial cells by immunohistochemical staining and fluorescent in situ hybridization (FISH). Results: The leukemia cell lines expressed many angiogenetic cytokines, such as VEGF, VEGF-D, RANTES, PIGF, PDGF-BB, MCP-1, IGF-1, ENA-78, and angiogenin, at the same levels as HUVEC cells, a human umbilical vessel endothelial cell line. Different cell lines expressed some angiogenesis-related receptors, such as CD31, Tie-2, PDGFRalpha, CD141, CD146, and KDR. None of the cell lines formed tubes in Matrigel. When these three cell lines generated leukemia in NOD/SCID mice, the microvessel density level increased in the tumor areas. However, immunostaining for human and murine endothelial markers demonstrated that all the vessels came from the mouse, not from the human leukemia cells. Conclusions: We conclude that infant leukemia cell lines with t(4;11) have proangiogenesic activity. However, these cells do not function as hemangioblasts, as they do not produce blood vessels in culture or in vivo in NOD/SCID mice. We plan to look further by examing bone marrow biopsies from patients with t(4;11) leukemia to determine whether the translocation is present in their blood vessels.

The Novel Sulfonamidebenzamide ML291 Activates Apoptotic UPR Signaling in Pediatric Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3523-3523
Author(s):  
Danielle Garshott ◽  
Nicole Melong ◽  
Tania T. Sarker ◽  
Yue Xi ◽  
Amy Brownell ◽  
...  
Keyword(s):  
Cell Lines ◽  
Leukemia Cell ◽  
Leukemic Cell ◽  
K562 Cells ◽  
Leukemia Cells ◽  
Wild Type ◽  
Leukemia Cell Lines ◽  
Proliferation Assays

Abstract Background: Acute leukemias are the most common cancers in childhood. Despite multi-agent chemotherapy protocols and the introduction of novel molecularly targeted therapies which have resulted in improved survival over the last few decades, relapsed acute lymphoblastic leukemia remains the second most common pediatric cancer diagnosis. In addition, morbidities from current chemotherapy regimens are unacceptably high. Abundant evidence point to a major role for mediators of the unfolded protein response (UPR) in normal and leukemic white blood cell biology. We have demonstrated that activation of the UPR is a productive approach to inhibit the proliferation of solid tumor cell lines in vitro and to reducing xenograft burden in vivo. The UPR consists of genetically distinct mechanisms that serve to clear misfolded proteins from the endoplasmic reticulum (ER) and enhance protein folding, or induce apoptosis if the initiating stress is prolonged or robust. ML291 is a novel UPR-inducing sulfonamidebenzamide, identified through cell-based high throughput screening and iterative SAR-guided chemical synthesis, that overwhelms the adaptive capacity of the UPR and induces apoptosis in a variety of solid cancer models. Objective: To determine the ability of ML291 to activate the UPR and induce apoptosis in a panel of leukemia cell lines, and to use CHOP-null K562 cells to elucidate the relative contribution of the UPR. We hypothesized that ML291 might activate the PERK/eIF2a/CHOP (apoptotic) arm of the UPR and reduce leukemic cell burden in vitro and in vivo. Methods: MTT and luciferase-based proliferation assays, flow cytometry and RT-qPCR were used to evaluate cell growth, UPR activation and apoptosis in a panel of leukemia cell lines that included AML, ALL and CML in cells exposed to ML291. CRISPR-Cas9 genome editing was used to delete CHOP in K562 (human myeloid leukemia) cells. Deletion was validated by immunoblot analysis and these cells were subjected to the same proliferation and gene analyses described above. The in vivo response to ML291 therapy was evaluated in an established zebrafish xenograft assay (Corkery et al. BJH 2011) in which embryos were xenotransplanted with wild type or CHOP knockdown K562 cells and embryos bathed in ML291. Results: Immunoblot and RT-qPCR analysis revealed an accumulation of proteins and increased gene expression for downstream UPR genes, including CHOP, GRP78/BiP, GADD34 and XBP1 in leukemia cells following ML291 treatment, indicating the activation of the UPR. Increased expression of the apoptotic genes, NOXA, PUMA and DR5 was also observed post-treatment with ML291; and dose response proliferation assays performed after 24 hours revealed IC50 concentrations of 1 - 30µM across cell lines. CHOP deleted K562 cells were protected from cell death when cultured with increasing concentrations of ML291, and were significantly less able to translocate phosphatidylserine across the cell membrane and activate the caspase cascade. When zebrafish embryos xenotransplanted with K562-wild type or -CHOP-null cells were bathed in water containing 5mM ML291 for three days, there was a significant reduction in leukemia cell burden exclusively in theK562 wild type xenografts. Conclusion: Collectively these data indicate that intact PERK/eIF2a/CHOP signaling is required for efficient leukemic cell apoptosis in response to ML291 in vitro and in vivo, and support the hypothesis that small molecule enforcement of the UPR might be a productive therapeutic approach in leukemia. Disclosures No relevant conflicts of interest to declare.

A-Type Proanthocyanidins Prevent Engraftment Of Primary Acute Myelogenous Leukemia Cells In Mice and Exhibit Potentially Novel Anti-Leukemia Mechanisms

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3962-3962
Author(s):  
Laura M Bystrom ◽  
Hongliang Zong ◽  
Hsiao-Ting Hsu ◽  
Neng Yang ◽  
Noa Greenberg ◽  
...  
Keyword(s):  
Cell Lines ◽  
Iron Metabolism ◽  
Leukemia Cell ◽  
Myelogenous Leukemia ◽  
Leukemia Cells ◽  
Survival Pathways ◽  
Leukemia Cell Lines ◽  
Acute Myelogenous

Abstract Acute myelogenous leukemia (AML) is often a fatal disease where after strong induction therapy most patients relapse and die. AML originates and is maintained by leukemia stem cells (LSCs). Failure to eliminate LSCs by chemotherapy is likely to result in disease relapse. Therefore, it is a priority to identify new therapies that eliminate blasts while ablating LSCs and preventing a relapse. We have found that a unique class of compounds in cranberries (Vaccinium macrocarponAit.), known as A-type proanthocyanidins (A-PACs), were effective against several leukemia cell lines and primary AML samples in vitro. A-PACs consist of monomeric epicatechin units attached to one another by a carbon-carbon bond and a distinctive ether bond that differentiates these compounds from other proanthocyanidins found in nature. Moreover, A-PACs possess ortho-hydroxyl phenolic groups that have the potential to bind to iron and alter redox status. Preliminary work showed that pre-treatment with antioxidants or holo-transferrin (iron-saturated transferrin) partially protected AML cells from A-PAC induced cell death (p<0.01). A-PACs were also found to selectively ablate leukemia stem and progenitor cells, with minimal effects on normal hematopoetic stem cells. Furthermore, AML engraftment of cells treated ex vivo with 62.5 µg/ml A-PACs was decreased (90.6%, n=3, p<0.001), while normal CD34+ cells retained engraftment capability in immunodeficient mice. It was also found that a fraction of A-PACs of up to 7 degree of polymerization was more effective than individual A-PACs. This information prompted us to investigate the in vivo anti-leukemia effects of A-PACs in xenotransplanted mice with primary AML samples, and to further investigate the mechanisms associated with these compounds. Primary AML cells were injected in sub-lethally irradiated NOD/SCID mice. Four weeks after injections, when human leukemia cells have engrafted, intraperitoneal injections of cytarabine (AraC) at 60 mg/kg were given to the mice for 1 week everyday or A-PACs (100 mg/kg dose every 3 days for A-PACs) and vehicle control (1% DMSO in PBS every 3 days) were injected for 2.5 weeks. Mice were sacrificed and leukemia engraftment evaluated using anti-human CD45 and CD33. Moreover, primary cells treated with A-PACs were assessed for effects on iron metabolism, ROS, and survival pathways either by gene expression analysis, flow cytometry or mass spectrometry. Administration of A-PACs to NOD-SCID mice bearing AML tumors reduced tumor burden. Mice that were treated with the vehicle control had engraftment of AML primary cells equivalent to 16.1% (95% CI: -6.0, 38.37; n=4), whereas the mice treated with the A-PACs and AraC showed a level of engraftment of 4.9% (95% CI: 2, 8; n=5) and 5.8% (95% CI: -1.1, 12.7; n=5), respectively. No significant changes in hemoglobin or weight were found between the different treatment groups. Moreover, qPCR analysis of sensitive leukemia cell lines treated with A-PACs showed changes in gene expression of several iron metabolism genes in sensitive leukemia cell lines (up-regulation of ferritin and transferrin receptors 1 and down-regulation of ferroportin) and several ROS-relevant genes (down-regulation of nuclear factor erythroid-2-related factor 2 and glutamate-cysteine ligase regulatory subunit). Mass spectrometry also confirmed that A-PACs bind iron. The results indicate that A-PACs not only target primary AML cells in vitro but are also effective in vivo. Secondary transplants are also being performed to determine the effects on LSC activity. Some of the anti-leukemia mechanisms under investigation include effects related to iron metabolism, ROS or inhibition of survival pathways. Understanding the unique structure and biological effects of A-PACs may provide novel information about pathways involved in the survival of LSCs and provide crucial information in preparation for clinical trials and/or optimal combination drug therapies. Disclosures: Rivella: Novartis: Consultancy; Bayer: Consultancy; Isis: Consultancy, Research Funding; Merganser: Equity Ownership, Research Funding; Biomarin: Consultancy; Alexion: Consultancy; Imago: Consultancy.

Repression of Mir-495, a Microrna Associated with Favorable Outcome of Acute Myeloid Leukemia Patients, Is Required for the MLL-Associated Leukemogenesis,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3462-3462
Author(s):  
Xi Jiang ◽  
Hao Huang ◽  
Zejuan Li ◽  
Yuanyuan Li ◽  
Ping Chen ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Leukemia Cell ◽  
Mouse Bone Marrow ◽  
Human Leukemia ◽  
Leukemia Cell Lines ◽  
Acute Myeloid

Abstract Abstract 3462 Acute myeloid leukemia (AML) bearing MLL (mixed lineage leukemia) translocations are associated with poor survival, and only fewer than 50% of the patients survive longer than 5 years. Thus, an improved strategy leading to a higher cure rate is urgently needed to treat MLL-associated AML. MicroRNAs (miRNAs), a class of small non-coding RNAs, have been postulated to be important gene expression regulators in all biology including human leukemia. Through large-scale, genome-wide miRNA expression profiling assays, we determined that miR-495 is significantly down-regulated in the majority of human AML samples, particularly, in those with MLL rearrangements. More interestingly, through correlating the expression signature of miR-495 with clinical outcome of AML patients, we revealed that a low expression level of miR-495 is a predictor of poor prognosis in most AML patients. Our further qPCR assays confirmed that the expression of miR-495 is even more significantly downregulated in MLL-rearranged AML primary patient samples and cell lines. Through in vitro colony-forming/replating assays and in vivo bone marrow transplantation studies, we found that forced expression of miR-495 significantly inhibits the capacity of the MLL-AF9 fusion gene to support colony formation in mouse bone marrow progenitor cells in vitro and to induce leukemia in vivo. In leukemia cell lines, overexpression of miR-495 greatly inhibits the viability of the cells, while increasing apoptosis. Furthermore, by using 3 algorithms for miR-495 3'UTR binding sites, we identified several well-known MLL leukemia-related genes, e. g. BMI1, MEF2C, BID and MEIS1, as potential targets of miR-495. Results of qPCR revealed that forced expression of miR-495 significantly inhibits the expression levels of these genes in leukemia cell lines, mouse bone marrow progenitor cells, as well as mouse peripheral blood cells with MLL fusion genes. Therefore we hypothesize that miR-495 may function as a tumor suppressor in AML with MLL rearrangements by targeting essential tumor-related genes. Further studies will focus on: 1) effects of miR-495 on the functions of target genes studied in vitro and in vivo; 2) the epigenetic mechanisms and the signaling pathways involved in regulating the expression level of miR-495 in human leukemia. 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 Identification of GPAT1 As a Novel Therapeutic Target for Acute Leukemia By Inhibiting Leukemia Specific Metabolism

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1384-1384
Author(s):  
Hidetoshi Irifune ◽  
Yu Kochi ◽  
Masayasu Hayashi ◽  
Yoshikane Kikushige ◽  
Toshihiro Miyamoto ◽  
...  
Keyword(s):  
Acute Leukemia ◽  
Cell Lines ◽  
Mass Spectrometer ◽  
Mitochondrial Function ◽  
Therapeutic Target ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Leukemia Cell Lines

With the development of mass spectrometer technology, recent studies revealed the critical roles of cancer-specific metabolism for tumor propagation in several types of cancers. In leukemia, many studies have been conducted to elucidate a leukemia-specific metabolism, and several effective treatments such as IDH1/2 inhibitors targeting acute myeloid leukemia (AML) with IDH1/2 mutation have been developed. To identify the new metabolic pathways on which acute leukemia cells depend, we purified water-soluble metabolites from CD34+ hematopoietic stem and progenitor cells (HSPCs) of healthy donors, AML and acute lymphoblastic leukemia (ALL) patients, and we comprehensively measured 116 metabolites using mass spectrometer analysis. From this experiment, we found that the cellular content of glycerol 3-phosphate (G3P) in CD34+ AML and ALL cells was lower than that of normal CD34+ HSPCs. G3P is an intermediate metabolite in the glycolysis metabolic pathway and is utilized as a substrate for phospholipids synthesis. The initial and rate-limiting step of phospholipids synthesis is the synthesis of lysophosphatidic acid (LPA) from G3P and acyl-CoA mediated by glycerol 3-phosphate acyltransferases (GPATs). Since CD34+ acute leukemia cells contained significantly lower level of G3P, we hypothesized that leukemia cells actively consumed G3P and synthesized LPA by GPATs. GPATs are classified into four isoforms based on intracellular localization and substrate preference. GPAT1 and GPAT2 are mitochondrial GPATs that are localized to the mitochondrial outer membrane, but on the other hand, GPAT3 and GPAT4 are microsomal GPATs that are localized to the endoplasmic reticulum membrane, each encoded by independent genes. GPAT1 is identified as an essential gene for the growth of leukemia cells by RNAi screen analysis in the public database (DepMap). We found that CD34+ immature AML cells exhibited higher GPAT1 expression as compared to CD34- more differentiated AML cells and normal T cells. GPAT1 knockdown inhibited the proliferation of several acute leukemia cell lines including THP-1 and Kasumi-1 in vitro and in vivo. Moreover, a mitochondrial GPATs specific inhibitor (FSG67), which was originally developed as a drug to treat obesity and diabetes, suppressed the growth of the leukemia cell lines through the induction of G1 cell cycle arrest. Growth inhibition was rescued by exogenous administration of LPA, suggesting that the synthetic activity mediated by mitochondrial GPATs should be required for acute leukemia growth. Furthermore, FSG67 induced the apoptosis of leukemia cells derived from AML and ALL patients without affecting normal CD34+ HSPCs at least in vitro. We also confirmed that the injection of FSG67 resulted in the suppression of AML and ALL propagation in vivo using patient-derived xenograft models (see figure). GPAT1 regulates the mitochondrial function by producing LPA which is an essential metabolite for maintaining mitochondrial fusion. Actually, the amount of LPA was decreased in GPAT1 knockdown acute leukemia cells. We next examined mitochondrial energy production by extracellular flux assay, and found that GPAT1 knockdown as well as FSG67 significantly suppressed oxygen consumption rate of acute leukemia cells. Consistent with the impaired mitochondrial function, FSG67 suppressed the mitochondrial membrane potential, indicating that GPAT1 should play a pivotal role in maintaining leukemia-specific mitochondrial function. These results collectively suggest that the synthesis of LPA from G3P catalyzed by GPAT1 has a critical role in propagation of acute leukemia cells irrespective of their lineage origin. Thus, GPAT1 is a novel and common therapeutic target for human acute leukemia through suppressing leukemia-specific mitochondrial function. Figure Disclosures Akashi: Celgene, Kyowa Kirin, Astellas, Shionogi, Asahi Kasei, Chugai, Bristol-Myers Squibb: Research Funding; Sumitomo Dainippon, Kyowa Kirin: Consultancy.

The Multi-Targeted Receptor Tyrosine Kinase Inhibitor, ABT-869, Induces Apoptosis of AML Cells Both In Vitro and In Vivo.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 616-616 ◽  
Author(s):  
Deepa B. Shankar ◽  
Jenny C. Chang ◽  
Bertrand Parcells ◽  
Salemiz Sandoval ◽  
Junling Li ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Tyrosine Kinase ◽  
Cell Line ◽  
Progenitor Cells ◽  
Cell Lines ◽  
Receptor Tyrosine Kinase ◽  
Scid Mice ◽  
Parp Cleavage

Abstract Children with acute myeloid leukemia (AML) have less than 60% overall survival despite aggressive chemotherapy and bone marrow transplantation. Only one third of the adult patients diagnosed with AML will be cured. AML blast cells from up to 30% of patients express a constitutively active receptor tyrosine kinase, FLT3-ITD, which contains an internal tandem duplication in the juxtamembrane domain. Patients with FLT3-ITD have a worse prognosis. ABT-869 is a novel multi-targeted small molecule inhibitor of receptor tyrosine kinases and is a potent inhibitor of FLT3, c-Kit, and all members of the VEGF and PDGF receptor families. To determine the effects of ABT-896 on AML cells, we treated AML cell lines, primary cells, and tumors in xenograft models with varying concentrations of the drug. In vitro viability assays showed that ABT-869 inhibited the growth of two different cell lines, MV-4-11 (human AML cell line that expresses FLT3-ITD) and BAF3-ITD (murine B-cell line stably transfected with the FLT3-ITD) at an IC50 of 10nM. ABT-869 was also effective against another mutation of FLT3, D835V, but at higher concentrations (IC50 of 100nM). Phosphorylation of FLT3 and activation of downstream signaling molecules, STAT5 and ERK, were inhibited by ABT-869 in a concentration-dependent manner. Cells were also stained with Annexin V-FITC and Propidium Iodide, and analyzed using FACS. ABT-869 induced apoptosis, caspase-3 activation, and PARP cleavage after 48 hours. To examine the in vitro effects of ABT-869 on normal hematopoietic progenitor cells, we performed methylcellulose-based colony assays with human bone marrow. No significant difference was observed in the number and type of colonies formed using BM cells treated with ABT-869 or control, up to a concentration of 1 micromolar. These results suggest that ABT-869 is not toxic to normal bone marrow progenitor cells at concentrations that are effective against AML cells. To examine the effects of ABT-869 in vivo, we treated SCID mice injected with MV-4-11, Baf3-ITD, Baf3-D835V, or Baf3-WT cells, with oral preparations of ABT-869. Complete regression of MV-4-11 tumors was observed in mice treated with ABT-869 at 20 and 40 mg/kg/day. No adverse effects were detected in the peripheral blood counts, bone marrow, spleen or liver. Histology of the tumors from the control-treated group showed a high degree of proliferation by Ki-67 staining, increased mitotic figures, and a well-defined tumor mass. In contrast, the tumors from mice treated with ABT-869 showed a number of apoptotic bodies by TUNEL staining and the presence of reactive, inflammatory cells. Interestingly, we also observed that mice that received ABT-869 the day after injection of AML cells remained tumor-free for over 2 months in contrast to the mice receiving the vehicle alone. Inhibition of FLT3 phosphorylation was demonstrated in the tumors from mice treated with ABT-869. We are evaluating the activity of ABT-869 treatment of SCID mice injected with Baf3-ITD, Baf3-D835V, or Baf3-WT cells. NOD-SCID mouse models are currently being used to analyze the effects of ABT-869 on primary AML cells in vivo. Our preclinical studies demonstrate that ABT-869 is effective and nontoxic, and provide rationale for the treatment and prevention of relapse in AML patients.

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.

ARC Is Regulated by MAPK and PI3K and Confers Drug Resistance and Survival Advantage to AML in Vitro and in Vivo

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 893-893
Author(s):  
Po Yee Mak ◽  
Duncan H Mak ◽  
Yuexi Shi ◽  
Vivian Ruvolo ◽  
Rodrigo Jacamo ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Apoptosis Resistance ◽  
Control Mscs ◽  
Extrinsic Apoptosis ◽  
Induced Apoptosis ◽  
And Control

Abstract Abstract 893 ARC (Apoptosis repressor with caspase recruitment domain) is a unique antiapoptotic protein that has been shown to suppress the activation of both intrinsic and extrinsic apoptosis. We previously reported that ARC is one of the most potent adverse prognostic factors in AML and that high ARC protein expression predicted shorter survival and poor clinical outcome in patients with AML (Carter BZ et al., Blood 2011). Here we report how ARC is regulated and its role in inhibition of AML apoptosis and in cell survival. We provide evidence that ARC expression is regulated by MAPK and PI3K signaling. Inhibition of MAPK and PI3K pathways decreased ARC mRNA and protein levels in AML cells. ARC expression in AML cells is upregulated in co-cultures with bone marrow-derived mesenchymal stromal cells (MSCs) and the upregulation is suppressed in the presence of MAPK or PI3K inhibitors. To investigate the role of ARC in apoptosis resistance in AML, we generated stable ARC overexpressing (O/E) KG-1 and stable ARC knock down (K/D) OCI-AML3 and Molm13 cells and treated them with Ara-C and agents selectively inducing intrinsic (ABT-737) or extrinsic (TRAIL) apoptosis. We found that ARC O/E cells are more resistant and ARC K/D cells more sensitive to Ara-C, ABT-737, and TRAIL-induced apoptosis: EC50s of Ara-C, ABT-737, or TRAIL treatment at 48 hours for ARC O/E KG-1 and control cells were 1.5 ± 0.1 μM vs. 83.5 ± 4.6 nM, 2.2 ± 0.2 μM vs. 60.2 ± 3.1 nM, or 0.97 ± 0.03 μg/mL vs. 0.17 ± 0.08 μg/mL, respectively and for ARC K/D OCI-AML3 and control cells were 0.33 ± 0.02 μM vs. 3.4 ± 0.2 μM, 0.24 ± 0.01 μM vs. 1.3 ± 0.1 μM, or 0.13 ± 0.09 μg/mL vs. 0.36 ± 0.03 μg/mL, respectively. Bone marrow microenvironment is known to play critical roles in AML disease progression and in protecting leukemia cells from various therapeutic agent-induced apoptosis. Leukemia cells were co-cultured with MSCs in vitro study to mimic the in vivo condition. ARC was found to be highly expressed in MSCs and stable ARC K/D MSCs were generated. AML cell lines and primary patient samples were co-cultured with ARC K/D or control MSCs and treated with Ara-C, ABT-737, or TRAIL. Interestingly, ARC K/D MSCs lost their protective activity for leukemia cells treated with these agents. EC50s for OCI-AML3 cells co-cultured with ARC K/D or control MSCs for 48 hours treated with Ara-C, ABT-737, or TRAIL were 1.0 ± 0.04 μM vs. 4.5 ± 0.2 μM, 0.15 ± 0.06 μM vs. 0.53 ± 0.02 μM, or 1.4 ± 0.8 μg/mL vs. 8.1 ± 0.3 μg/mL, respectively. In addition, ARC O/E KG-1 cells grew faster and ARC K/D OCI-AML3 and Molm13 cells and ARC K/D MSCs grew slower than their respective controls. We then injected KG-1 cells into mice and found that NOD-SCID mice harboring ARC O/E KG-1 had significantly shorter survival than mice injected with the vector control KG-1 (median 84 vs. 111 days) as shown in the figure. Collectively, results demonstrate that ARC plays critical roles in AML. ARC is regulated by MSCs through various signaling pathways in AML cells, protects leukemia cells from apoptosis induced by chemotherapy and by agents selectively inducing intrinsic and extrinsic apoptosis. ARC regulates leukemia cell growth in vitro and in vivo. The results suggest that ARC is a potential target for AML therapy. In addition, targeting ARC in MSCs suppresses microenvironmental protection of AML cells. Disclosures: No relevant conflicts of interest to declare.

scholarly journals RUNX1/CBFβ Dosage Is Critical for MLL Leukemias Development

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2187-2187
Author(s):  
Xiaomei Yan ◽  
Yoshihiro Hayashi ◽  
Xinghui Zhao ◽  
Aili Chen ◽  
Yue Zhang ◽  
...  
Keyword(s):  
Protein Expression ◽  
Cell Lines ◽  
Research Funding ◽  
Leukemia Cell ◽  
Down Regulation ◽  
Leukemia Cell Lines ◽  
Flanking Regions ◽  
The Impact

Abstract Transcription factors RUNX1/CBFβ play critical roles in hematopoiesis. Both of them are frequently involved in chromosomal translocations, point mutations, or deletions in acute leukemia. The mixed lineage leukemia (MLL) gene is also frequently involved in chromosomal translocations or partial tandem duplication in acute leukemia. We have previously shown that MLL, RUNX1, and CBFβ interact and form a regulatory complex to regulate downstream target genes. However, the functional consequence of MLL fusions on RUNX1/CBFβ activity remains unknown. To determine the impact of MLL fusion protein on RUNX1/CBFβ, we introduced either MLL, MLL-BP (longer N-terminal Flag-tagged MLL construct which contains CXXC domain; 1-1406), or MLL-fusions together with RUNX1, CBFβ, or both RUNX1 and CBFβ into 293T cells. MLL-BP and MLL fusions significantly decreased RUNX1 levels compared with controls (empty vector and MLL). CBFβ protein was mildly decreased by MLL-BP and MLL-fusions when expressed alone. However, when CBFβ was co-expressed with RUNX1, it was significantly decreased compared with controls. The expression levels of RUNX1 and CBFβ proteins in LSK cells from Mll-Af9 knock-in mice were significantly lower than those from wild-type (WT) mice. To confirm these findings in human acute myeloid leukemia (AML), we measured the expression of RUNX1 and CBFβ at both mRNA and protein levels in various leukemia cell lines. The expression levels of RUNX1 and CBFβ proteins were significantly decreased in AML cells with MLL fusion and MLL partial tandem duplication (MLL-PTD) compared with those in AML cells without MLL aberrations. MLL fusions still have CXXC domain. In MLL-PTD, the CXXC domain is duplicated. Our data showed that RUNX1 protein is not only down-regulated by MLL fusion proteins, but also by MLL-BP. Thus, to determine which region is involved in the down-regulation of RUNX1, we introduced a series of MLL deletion mutants into 293T cells and measured RUNX1 protein expression. MLL deletion mutants without CXXC domain had no effect on RUNX1 stability. The construct which contains point mutations in CXXC domain also lacked the ability to reduce RUNX1 expression. Furthermore, overexpression of only CXXC domain and flanking regions could down-regulate RUNX1 protein expression. These results suggest that MLL fusion proteins and the N-terminal MLL portion of MLL fusions down-regulate RUNX1 and CBFβ protein expression via the MLL CXXC domain and flanking regions. To understand the impact of RUNX1/CBFβ down-regulation on hematopoietic stem and progenitor cells (HSPCs), we generated RUNX1+/–/CBFβ+/– mice as a hypomorph model. The percentage of bone marrow (BM) LSK cells from RUNX1+/–/CBFβ+/– mice was significantly increased compared with that from WT mice. Using BM cells from these mice, we performed in vitro CFU assay and in vivo bone marrow transplantation (BMT) assay. BM cells from RUNX1+/–/CBFβ+/– mice provided more colonies in CFU assay compared with those from WT mice. To determine whether restoration of RUNX1 could repress the MLL mediated leukemogenesis, we retrovirally overexpressed WT RUNX1 in BM cells from Mll-Af9 knock-in mice. Using transduced BM cells, we performed in vitro CFU assay and in vivo BMT assay. RUNX1 overexpressed Mll-Af9 (Mll-Af9/RUNX1) cells underwent terminal differentiation after 2 times replating, while control vector transduced Mll-Af9 (Mll-Af9/Control) cells could still be replated more than 4 times. All the recipient mice transplanted with Mll-Af9/Control cells developed AML. In contrast, all the recipient mice transplanted with Mll-Af9/RUNX1 never develop AML. Furthermore, when we treated MLL leukemia cell lines with DOT1L inhibitor (EPZ-5676), RUNX1 protein levels in these MLL leukemia cell lines were significantly increased 48 hours after the treatment in comparing with controls treated with DMSO. However, there was no significant mRNA expression level change of RUNX1within 48 hours. Future studies are needed to fully understand the mechanism of whether this increasing RUNX1 protein level by DOT1L inhibitor is through blocking CXXC domain and flanking regions mediated degradation. In conclusion, MLL aberrations down-regulate RUNX1/CBFβ via their CXXC domain and flanking regions. Down-regulation of RUNX1/CBFβ plays critical role for MLL mediated leukemia development. Targeting RUNX1/CBFβ levels allows us to test novel therapies for MLL leukemias. Disclosures Mulloy: Celgene: Research Funding; Seattle Genetics: Research Funding; Amgen: Research Funding; NovImmune: Research Funding.

A Novel N-Oxide Drug, AQ4N, Has In Vitro Activity in Lymphoma and Leukemia Cell Lines and Selectively Targets Lymphocytes and Lymphatic Tissues in Vivo.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2498-2498
Author(s):  
Jeffrey L. Cleland ◽  
Alvin Wong ◽  
Susan E. Alters ◽  
Peter A. Harris ◽  
Chris R. Dunk ◽  
...  
Keyword(s):  
Cell Lines ◽  
Leukemia Cell ◽  
Lymphoid Tissues ◽  
Human Leukemia ◽  
Repeat Dose ◽  
Cynomolgus Monkeys ◽  
Leukemia Cell Lines ◽  
Human Lymphoma

Abstract An ideal treatment for lymphoma and leukemia is the use of highly selective compounds to eliminate diseased cells with minimal systemic toxicity to normal tissues (cf. imatinib mesylate; Gleevec). AQ4N (1,4 bis[[2-(dimethylamino)ethylamino}-5,8-hydroxyanthracene-9,10-dione bis N-oxide) is designed to have little or no toxicity until selectively activated by bioreduction in hypoxic cells to AQ4 (reduced AQ4N), a highly potent DNA topoisomerase II inhibitor. In a series of studies, AQ4 has been shown to have potent cytotoxicity on lymphoma and leukemia cell lines in vitro and AQ4N has selective activity in lymphatic tissues in vivo. The IC50 of AQ4, was 0.63, 12.0, 90.5 and 150 nM in Namalwa, Daudi, Ramos, and Raji human lymphoma cell lines and 1.0, 6.0, and 20 nM in HL-60, KG1a and K562 human leukemia cell lines. On several of the tumor lines the activity of AQ4 was more potent than doxorubicin (i.e. IC50 for Dox was 20.3 nM on Namalwa). AQ4N also had anti-proliferative activity at μM levels indicating a potential mechanism for activation by these cell lines. In repeat dose toxicology studies of AQ4N in pigmented rats and cynomolgus monkeys, the maximum tolerated doses (MTD; rats: 20 mg/kg/wk x 6; monkeys 6 mg/kg/wk x 6) resulted in lymphoid tissue atrophy. A decrease in lymphocyte levels and atrophy of the spleen, thymus, and mandibular and mesenteric lymph nodes were observed at terminal sacrifice of the animals. In contrast, there was an absence of myelosuppression and only mild neutropenia and minor bone marrow atrophy at the MTD. Administration of radiolabeled AQ4N (14C-benzene) to pigmented rats and cynomolgus monkeys indicated persistence of AQ4N radioactivity in lymphoid tissues for several weeks after a single dose (rats: 20 mg/kg (130–140 μCi/kg); monkeys: 10 mg/kg (135 μCi/kg)). For example, in rats the half-life of radioactive AQ4N in the spleen was 538 hrs with 0.9 μg AQ4N/g tissue (spleen) remaining one week after dosing. Monkeys demonstrated a similar effect with 76.5–86.8 μg AQ4N/g tissue observed in the spleen one week after treatment. Other tissues contained significantly less radioactive AQ4N with the exception of the liver (67.9–78.6 μg AQ4N/g tissue) and adrenal cortex (78.7–86.6 μg AQ4N/g tissue). While some hypertrophy and eosinophila was observed in the adrenal glands, liver toxicity was not observed at the MTD in the repeat dose cynomolgus monkey toxicology study. Overall, these initial findings indicate that AQ4N is active in vitro against human lymphoma and leukemia cell lines and selectively targets lymphoid tissues in vivo suggesting the potential benefit of AQ4N in the treatment of lymphoproliferative diseases.

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