Identification of GPAT1 As a Novel Therapeutic Target for Acute Leukemia By Inhibiting Leukemia Specific Metabolism

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.

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The Novel Sulfonamidebenzamide ML291 Activates Apoptotic UPR Signaling in Pediatric Leukemia

Blood ◽

10.1182/blood.v128.22.3523.3523 ◽

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.

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

Blood ◽

10.1182/blood.v122.21.3962.3962 ◽

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.

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

Blood ◽

10.1182/blood.v110.11.4279.4279 ◽

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.

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

Blood ◽

10.1182/blood.v118.21.244.244 ◽

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.

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

Blood ◽

10.1182/blood.v108.11.2605.2605 ◽

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.

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

Blood ◽

10.1182/blood.v122.21.1293.1293 ◽

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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The 3-Drug Combination Treatment ART838, ABT199 Plus Sorafenib Is Effective Against AML Xenografts, Possibly Via Cooperative Targeting of MCL1

Blood ◽

10.1182/blood-2018-99-113040 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4044-4044

Author(s):

Blake S Moses ◽

Jennifer Fox ◽

Xiaochun Chen ◽

Samantha McCullough ◽

Sang Ngoc Tran ◽

...

Keyword(s):

Acute Leukemia ◽

Board Of Directors ◽

Cell Lines ◽

Drug Combination ◽

Research Funding ◽

Leukemia Cell ◽

Advisory Committees ◽

Leukemia Cell Lines ◽

Equity Ownership

Abstract Antimalarial artemisinins have broad antineoplastic activity in vitro, are well tolerated and inexpensive, and can be parenterally or orally administered in humans. Artemisinin-derived trioxane diphenylphosphate dimer 838 (ART838; a potent artemisinin-derivative) inhibited acute leukemia growth in vivo and in vitro, at doses where normal human CD34+ hematopoietic stem-progenitor cell clonogenicity was essentially unaffected (Fox et al, Oncotarget 2016, PMID: 26771236). In our focused drug combination screen for drugs that synergize with ART838, the only BCL2 inhibitors in the screen library of 111 emerging antineoplastic compounds, navitoclax (ABT737) and venetoclax (ABT199; FDA-approved), were identified as 2 of the top 3 candidates. Synergies between ART838 and BCL2 inhibitors were validated in multiple acute leukemia cell lines and primary cases. This ART838-BCL2 inhibitor synergy may be due to reduced levels of MCL1 protein that we and others have observed in multiple acute leukemia cell lines and primary cases treated with artemisinins (Budhraja et al, Clin Cancer Res 2017, PMID: 28974549). Treatment of acute leukemia xenografts with the ART838 plus ABT199 combination reduced leukemia growth rates and prolonged survivals, compared to vehicle or either ART838 or ABT199 alone. To add to the efficacy of this ART838 plus ABT199 treatment regimen, we sought to rationally add a third low-toxicity active antileukemic agent. Sorafenib (SOR; FDA-approved) inhibits multiple kinases which may mediate its antileukemic activity, with the importance of the targets varying from case to case; e.g. FLT3 is an important target in many AMLs. In addition, several reports have found that SOR reduces MCL1 protein stability and translation through inhibition of the ERK and PI3K pathways (Wang et al, Clin Cancer Res 2016, PMID: 26459180; Huber et al, Leukemia 2011, PMID: 21293487). In all acute leukemia cell lines tested, we observed large reductions in MCL1 protein levels with SOR treatment, which may further rationalize the addition of SOR to our ART838 plus ABT199 antileukemic regimen. We had previously observed strong in vitro synergy between ART838 and SOR (PMID: 26771236). Treatment of acute leukemia xenografts with the ART838 plus SOR combination reduced leukemia xenograft growth rates and prolonged survivals, compared to single drugs. Mice bearing luciferase-labelled acute leukemia xenografts were treated (PO daily x5) with single drug or 2-drug or 3-drug combinations of ART838, ABT199, and SOR, each at their individual maximally tolerated doses. Treatment with this 3-drug combination caused rapid regression of luciferase-labelled MV4;11 AML xenografts (Fig 1A). The 5-day treatment cycles were repeated every other week, and mice receiving this 3-drug combination survived >4 times longer than vehicle-treated mice (Fig 1B). Mouse body weights were stable during treatment. Although myelosuppression is the human clinical dose-limiting toxicity of each of these 3 drugs, mouse blood cell counts during 3-drug combination treatment were in the normal range. Treatment of a luciferase-labelled primary AML leukemia xenograft with this 3-drug combination reduced leukemia growth more than the single drugs or 2-drug combinations (Fig 1C). Assessment of efficacy and pharmacokinetics-pharmacodynamics against diverse acute leukemia xenografts will test this combination of ART838, ABT199 plus SOR as a rational low-toxicity drug triad for treatment of acute leukemias and potentially other cancers. Disclosures Fox: Intrexon Corporation: Employment. Tyner:Genentech: Research Funding; Janssen: Research Funding; AstraZeneca: Research Funding; Gilead: Research Funding; Incyte: Research Funding; Constellation: Research Funding; Array: Research Funding; Takeda: Research Funding; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees; Aptose: Research Funding. Civin:ConverGene LLC: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; GPB Scientific LLC: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; 3DBioWorks Inc: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; BD (Becton Dickinson): Honoraria.

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RUNX1/CBFβ Dosage Is Critical for MLL Leukemias Development

Blood ◽

10.1182/blood.v124.21.2187.2187 ◽

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.

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Incorporating Hemoglobin Levels to Map Leukostasis Risk in Acute Leukemia Using Microvasculature-on-Chip Technologies

Blood ◽

10.1182/blood-2020-137804 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 9-10

Author(s):

Jamie Oakley ◽

Evelyn K. Williams ◽

Christina Caruso ◽

Yumiko Sakurai ◽

Reginald Tran ◽

...

Keyword(s):

T Cell ◽

Acute Leukemia ◽

Blood Viscosity ◽

Leukemia Cell ◽

Leukemia Cells ◽

Cell Counts ◽

On Chip ◽

Wbc Count

Hyperleukocytosis, most commonly defined as a white blood cell (WBC) count > 100,000/μL, is an oncologic emergency in acute leukemia that can lead to leukostasis, which occurs when leukemia cells obstruct the microvasculature resulting in significant morbidity and mortality from neurologic (CNS hemorrhage, thrombosis) or pulmonary (respiratory distress, hypoxia) symptoms. The underlying mechanisms are poorly understood but are thought to be related to increased blood viscosity, secondary to high WBC count, leukemia cell aggregation, and the abnormal mechanical properties, size, and cell-cell interactions of leukemia cells. Leukapheresis is a commonly used therapy for rapid cytoreduction in symptomatic patients, but the procedure is not without risks. No existing methods reliably predict leukostasis or guide treatment including the commonly used WBC count, which only loosely correlates with leukostasis and does not accurately describe the blood viscosity at the microvascular level. Importantly, while hematocrit/hemoglobin levels (Hgb) are known to be major contributors to blood viscosity, they have not been systematically assessed in leukostasis risk, and Hgb often decreases as leukemic cell counts rise, complicating the issue. Incorporating Hgb levels may better predict leukostasis and assist physicians balancing the risk of hyperleukocytosis compared to the interventions themselves. To that end, we investigated how the differing presentations of acute leukemia lead to microvessel occlusion, thereby affecting effective blood viscosity at the microvascular level using "microvasculature-on-a-chip" devices that mimic the microvascular geometry (Figure 1) developed by our laboratory. This physiologically relevant microvascular model allows for in vitro investigation as in vivo studies are nearly impossible due to difficulty in visualizing and manipulating the animal microvasculature and cell counts. The devices were microfabricated using polydimethylsiloxane (PDMS). Acute T-cell lymphoblastic (Jurkat) and acute monocytic (THP-1) cell lines were maintained via standard cell culture conditions. Red cells from healthy donors were isolated and mixed with leukemia cells to achieve target Hgb and WBC levels. Various physiologic leukemia "mixtures" were then perfused under physiologic microcirculatory flow conditions through the microvascular device and microchannels occlusion was tracked via videomicroscopy (Figure 2). With T-cell leukemia, Hgb levels affected the risk of "in vitro leukostasis." Specifically, with severe anemia and WBC count less than the hyperleukocytosis range, time to microchannel occlusion was longer, and was more dependent on Hgb rather than WBC count. However, in cases with severe anemia and WBC counts > 100k/μL, WBC count exhibited a stronger effect on occlusion with little dependence on Hgb (Figure 3). At Hgb > 8g/dL, microchannel occlusion was dependent on WBC count regardless of hyperleukocytosis or not. In contrast, our data to date shows that with myeloid leukemia, in vitro leukostasis is not associated with Hgb levels, and is consistent with how myeloid leukemias in vivo cause leukostasis symptoms at lower WBC counts than lymphoid leukemias, not only due to size but also adhesive interactions. These data suggest when determining risk for leukostasis, WBC count should not be the sole determinant. Here we show Hgb levels affect microvascular blood viscosity and propensity for microvascular occlusion, but it appears to have a greater impact with T-cell leukemias versus myeloid leukemias (Figure 4). These studies indicate Hgb is an important clinical parameter for leukostasis risk in acute leukemia and will help inform guidelines for leukapheresis and even phlebotomy, a much simpler and safer procedure, to mitigate hyperviscosity in acute leukemia. These results can also impact decisions regarding the need for red blood cell transfusions, which iatrogenically increase blood viscosity. Studies incorporating patient myeloid and lymphoid leukemia cells and microvasculature-on-chip devices integrating live endothelium to assess leukemia cell adhesion are ongoing. Figure Disclosures Lam: Sanguina, Inc: Current equity holder in private company.

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Marked synergism of cytotoxic agents with either a prenylation inhibitor or M-TOR inhibitor in acute leukemia cell lines: Potential clinical implications

Journal of Clinical Oncology ◽

10.1200/jco.2006.24.18_suppl.13103 ◽

2006 ◽

Vol 24 (18_suppl) ◽

pp. 13103-13103

Author(s):

D. R. Budman ◽

A. Calabro

Keyword(s):

Acute Leukemia ◽

Cell Lines ◽

Single Agent ◽

Leukemia Cell ◽

Cytotoxic Agents ◽

Human Leukemia ◽

Effect Analysis ◽

Leukemia Cell Lines ◽

Classical Combination

13103 Background: The most combinations of anticancer drugs are based upon empiricism. The potential permutations of drugs overwhelm the clinical trials system. Acute leukemia is sensitive to a variety of agents but relapses are common. Targeted agents are attractive new venues of therapy both as single agents and in combination with older agents. Isobologram median effect analysis allows up to three agents to be studied together in vitro to identify interesting combinations. We evaluated a commercially available statin, fluvastatin, to block prenylation which affects a variety of pathways, rapamycin and its experimental analogue RAD001 as M-TOR inhibitors to block downstream of the AKT pathway, and cytotoxic agents. Methods: The human leukemia cell lines AML-193 and KG-1 were obtained from ATTC (Rockville, MD), fluvastatin and RAD001 from Novartis Pharma, and the other agents from Sigma-Aldrich (St. Louis, MO). The IC50 of the single agent was determined by a 72 hr incubation of log growth cells using a MTT assay and the EZ-ED50 program (Perrella Scientific, Conyers, CA). The dosages of all agents were at clinically achievable concentrations. All reported values were the means of at least 3 experiments with each study using 4 wells per point. For isobologram analysis, a minimum of 8 concentrations of drug mixtures were studied above and below the IC50. Median effect CI values less than 1 are synergistic. Results: Doublets of fluvastatin with Ara-C (0.7), daunomycin (0.4), idarubicin (0.7), RAD001 (0.5), or rapamycin (0.3) demonstrated synergy. Doublets of RAD001 with Ara-C (0.3), daunomycin (0.7), or idarubicin (0.5) demonstrated synergy. Triplets of RAD001/daunorubicin/Ara-C, RAD001/daunomycin/fluvastatin, and RAD001/Ara-C/idarubicin all demonstrated marked synergy in both cell lines. Conclusion: A new potential non classical combination for further investigation is RAD001 or rapamycin with an inhibitor of prenylation such as fluvastatin. Additional potential combinations include cytotoxics with either fluvastatin or RAD001, and triplet combinations. No significant financial relationships to disclose.

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