Enhancing Ceramide Cytotoxicity in Acute Myelogenous Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4905-4905
Author(s):  
Timothy J Brown ◽  
Brian Barth ◽  
David F. Claxton
Keyword(s):  
Cell Death ◽  
Drug Discovery ◽  
Cell Lines ◽  
Leukemia Cell ◽  
Sphingosine Kinase ◽  
Myelogenous Leukemia ◽  
Sphingosine Kinase 1 ◽  
Liposomal Formulations ◽  
Acute Myelogenous ◽  
Complete Cell

Abstract Abstract 4905 Background: While understanding of Acute Myelogenous Leukemia (AML) pathogenesis has advanced greatly in recent years, drug discovery and development have added little to therapy. Ceramide and other sphingolipids are of therapeutic interest for many neoplasms (Jiang Y, DiVittore NA, Kaiser JM, et al. Combinatorial therapies improve the therapeutic efficacy of nanoliposomal ceramide for pancreatic cancer. Cancer Biol Ther. Oct 1 2011;12(7):574–585). Cellular ceramide accumulation favors a pro-apoptotic state, while accumulation of sphingosine-1-phosphate promotes survival. We have targeted the ceramide balance of AML cells in vitro with varying concentrations of liposomal formulations of C6 ceramide (Lip-C6), the sphingosine kinase-1 inhibitor safingol (Lip-Saf), and tamoxifen (Lip-Tam) to determine potential synergistic anti-leukemic efficacy. Safingol is a sphingosine kinase inhibitor currently in phase 1 trials. In addition, Tamoxifen can reverse drug resistance of many cancer cell types (Chapman JV, Gouaze-Andersson V, Messner MC, et al. Metabolism of short-chain ceramide by human cancer cells–implications for therapeutic approaches. Biochem Pharmacol. Aug 1 2010;80(3):308–315.). This has been shown to be due to the ability of Tamoxifen to block the activities of glucosylceramide synthase (GCS) and p-glycoprotein (P-GP), which coordinate to detoxify ceramide at the Golgi membrane. In the present study, liposomal drug formulations were chosen to enhance drug delivery and prevent premature drug metabolism. Methods: The cell lines C1498, HL-60, HL-60/VCR, GFPp210, Wehi-3B, K562, U937, and KG-1 were used in this study. Drugs were synthesized into liposomal formulations by the Penn State Hershey Drug Discovery and Delivery Core laboratory. Cellular viability was measured after treatment with Lip-C6, Lip-Saf, Lip-Tam, or a combination for 48 hours. Synergy and dose-response curves were modeled using CalcuSyn software. Apoptosis and cell proliferation were assessed using flow cytometry after treatment of drug for 24 hours at the calculated IC50 from MTS assays. Autophagy was also measured in C1498 cells to confirm an established safingol cell-death mechanism. Primary human AML collected by our lab from consenting, patients was assessed in methylcellulose for blast clonogenicity. Results and Conclusions: Several cell lines showed a favorable change in the IC50 of the drugs when used in combination, indicating a possible synergistic anti-leukemic mechanism of action. When Lip-C6 was combined with Lip-Saf in a varying ratios, synergistic growth inhibition was observed in the human AML cell lines HL-60, HL-60/VCR, and KG-1 (Figure 1). Interestingly, Lip-Tam caused complete cell population death at concentrations less than 15 μM in the Wehi-3B, K562, GFPp210, and C1498 lines. When cells were treated with Lip-C6 and Lip-Tam in a 1:1 combination, complete cell population killing was noted at concentrations of less than 10 μM in every cell line tested. Additionally, flow cytometric data confirmed findings of other investigators suggesting that Lip-Saf caused enhanced autophagy. Therefore, the observed synergistic leukemia cell death is likely due in part to the novel combination of an autophagy-inducer with an apoptosis-inducer. Clonogenic data has shown that combination of Lip-C6and Lip-Saf cooperate to inhibit formation of blast colonies from human AML, indicating a potential use in lessening leukemia burden (Figure 1). In conclusion, novel ceramide-centered drug combinations promote improved cell death of leukemia cell lines via accumulation of ceramide and inhibition of ceramide metabolic pathways. This study acts as a persuasive proof-of-concept of the effect of inhibiting a single ceramide degradation pathway within cells. By inhibiting sphingosine kinase-1 or the GCS activity of P-GP in these cell lines, it becomes apparent that the cellular ceramide balance shifts to favor a pro-apoptotic state. 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.

TNF-related apoptosis-inducing ligand (TRAIL) frequently induces apoptosis in Philadelphia chromosome–positive leukemia cells

Blood ◽  
2003 ◽  
Vol 101 (9) ◽  
pp. 3658-3667 ◽  
Author(s):  
Kanako Uno ◽  
Takeshi Inukai ◽  
Nobuhiko Kayagaki ◽  
Kumiko Goi ◽  
Hiroki Sato ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Lines ◽  
Philadelphia Chromosome ◽  
Leukemia Cell ◽  
Negative Regulator ◽  
Myelogenous Leukemia ◽  
Leukemia Cells ◽  
Caspase 8 ◽  
Death Domain ◽  
Leukemia Cell Lines

Tumor necrosis factor (TNF)–related apoptosis-inducing ligand (TRAIL) and Fas ligand (FasL) have been implicated in antitumor immunity and therapy. In the present study, we investigated the sensitivity of Philadelphia chromosome (Ph1)–positive leukemia cell lines to TRAIL- or FasL-induced cell death to explore the possible contribution of these molecules to immunotherapy against Ph1-positive leukemias. TRAIL, but not FasL, effectively induced apoptotic cell death in most of 5 chronic myelogenous leukemia–derived and 7 acute leukemia–derived Ph1-positive cell lines. The sensitivity to TRAIL was correlated with cell-surface expression of death-inducing receptors DR4 and/or DR5. The TRAIL-induced cell death was caspase-dependent and enhanced by nuclear factor κB inhibitors. Moreover, primary leukemia cells from Ph1-positive acute lymphoblastic leukemia patients were also sensitive to TRAIL, but not to FasL, depending on DR4/DR5 expression. Fas-associated death domain protein (FADD) and caspase-8, components of death-inducing signaling complex (DISC), as well as FLIP (FLICE [Fas-associating protein with death domain–like interleukin-1–converting enzyme]/caspase-8 inhibitory protein), a negative regulator of caspase-8, were expressed ubiquitously in Ph1-positive leukemia cell lines irrespective of their differential sensitivities to TRAIL and FasL. Notably, TRAIL could induce cell death in the Ph1-positive leukemia cell lines that were refractory to a BCR-ABL–specific tyrosine kinase inhibitor imatinib mesylate (STI571; Novartis Pharma, Basel, Switzerland). These results suggested the potential utility of recombinant TRAIL as a novel therapeutic agent and the possible contribution of endogenously expressed TRAIL to immunotherapy against Ph1-positive leukemias.

Regulation and Functional Role of Beta2-Adrenergic Receptor in Acute Myelogenous Leukemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2563-2563
Author(s):  
Ningfei An ◽  
Yeong-Bin Im ◽  
Amr Abdallah Moh'D Qudeimat ◽  
Luciano J Costa ◽  
Robert K Stuart ◽  
...  
Keyword(s):  
Cell Lines ◽  
Acute Myelogenous Leukemia ◽  
Leukemia Cell ◽  
Hematopoietic Cell ◽  
Myelogenous Leukemia ◽  
Pim Kinases ◽  
Leukemia Cell Lines ◽  
Acute Myelogenous ◽  
Hematopoietic Cell Lines

Abstract Abstract 2563 Acute myelogenous leukemia (AML) occurs with an incidence of 2.7 per 100,000 population in the year of 2009, and is associated with significant mortality and morbidity. Despite recent advances in molecular and cytogenetic analytic technologies, initial treatment for AML patients has remained essentially the same over the last 30 years; and the treatment outcome is dreadful with a 5-year survival rate of ∼25%. In an effort to gain a better understanding of AML cell biology and to develop more effective treatments for AML, we have been exploring the roles of the b2-adrenergic receptor (B2-AR) in AML. B2-AR is a G-protein- coupled catecholamine receptor and was recently found to play a direct role in the engraftment, proliferation and migration of hematopoietic stem cells [Spiegel, A., et al. Nat Immunol8, 1123–1131 (2007)]. However, very little is known about the roles of B2-AR in AML. We first screened a number of leukemia/hematopoietic cell lines, including K562, NBAL3, Jurkat, RPMI8226, U937, HEL, HL60, NB4, THP-1, and MV4;11, for B2-AR expression using flow cytometry. We found that B2-AR expression was not uniform in the leukemia/hematopoietic cell lines we tested. B2-AR was absent in acute non-myelogenous leukemia/hematopoietic cell lines such as K562, NBAL3, Jurkat or RPMI8226. In contrast, B2-AR was expressed on all acute myelogenous leukemia cell lines tested, with the highest expression on 2 myelomonocytic leukemia cell lines (THP-1 and MV4;11). We next examined the roles of B2-AR in leukemia cell survival, in leukemia cell responses to chemotherapy, and in leukemia cell migration in response to stromal derived factor-1 (SDF-1). We found that treatment with a B2-AR antagonist (ICI115881) modestly inhibited leukemia cell growth. Interestingly, treatment with a B2-AR agonist (i.e., isoproteronol or clenbuterol) inhibited leukemia cell migration to SDF-1. Additionally, combined treatment of MV4-11 cells with Isoproterenol and SDF-1 increased downstream ERK phosphorylation synergistically, suggesting a potential interaction or reciprocal regulation between B2-AR and CXCR4 receptor. To further understand the regulation and functional role of B2-AR in AML cells, we have been focusing on two biphenotypic leukemia cell lines, that is, MV4;11 and RS4;11 cells. Both MV4;11 and RS4;11 cells carry the t(4;11)(q21;q23) chromosomal translocation. While RS4;11 cells bear only the wild-type version of the Flt3 gene, MV4;11 cells express exclusively the mutated Flt3-ITD gene. Interestingly, we found that in contrast to MV4;11 leukemia cells, RS4;11 cells did not express surface B2-AR (panel A), although the total amount of B2-AR in the whole cell lysate was comparable between these two cell lines (panel B). This significantly different B2-AR expression pattern between these 2 cell lines may be related to the difference in the expression level of proviral insertion in murine lymphoma (Pim) kinases (panel B); RS4;11 has reduced/absent expression of Pim kinases compared to MV4;11 cells. Consistent with the potential regulation of B2-AR expression by Pim kinases, we found that Pim 2 and 3 double knockout mice had reduced B2-AR surface expression in peripheral blood mononucleated cells. Additional experiments are currently ongoing to further dissect the interaction between B2-AR and Pim kinases. Taken together, our current studies demonstrated a novel role of B2-AR in AML and a potential functional interaction between B2-AR, CXCR4, Pim kinase, and Flt3 gene. This work is supported by MUSC Hollings Cancer Center Startup Fund, Hollings Cancer Center ACS IRG, and ASCO Conquer Cancer Foundation Career Development Award Disclosures: No relevant conflicts of interest to declare.

scholarly journals Reduced sphingosine kinase 1 activity in dengue virus type-2 infected cells can be mediated by the 3′ untranslated region of dengue virus type-2 RNA

2013 ◽  
Vol 94 (11) ◽  
pp. 2437-2448 ◽  
Author(s):  
J. M. Carr ◽  
T. Kua ◽  
J. N. Clarke ◽  
J. K Calvert ◽  
J. R. Zebol ◽  
...  
Keyword(s):  
Cell Death ◽  
Dengue Virus ◽  
Virus Type ◽  
Elongation Factor ◽  
Sphingosine Kinase ◽  
Sphingosine Kinase 1 ◽  
Diarrhea Virus ◽  
Infected Cells ◽  
Dengue Virus Type 2

Sphingosine kinase 1 (SphK1) is a lipid kinase with important roles including regulation of cell survival. We have previously shown reduced SphK1 activity in cells with an established dengue virus type-2 (DENV-2) infection. In this study, we examined the effect of alterations in SphK1 activity on DENV-2 replication and cell death and determined the mechanisms of the reduction in SphK1 activity. Chemical inhibition or overexpression of SphK1 after established DENV-2 infection had no effect on infectious DENV-2 production, although inhibition of SphK1 resulted in enhanced DENV-2-induced cell death. Reduced SphK1 activity was observed in multiple cell types, regardless of the ability of DENV-2 infection to be cytopathic, and was mediated by a post-translational mechanism. Unlike bovine viral diarrhea virus, where SphK1 activity is decreased by the NS3 protein, SphK1 activity was not affected by DENV-2 NS3 but, instead, was reduced by expression of the terminal 396 bases of the 3′ UTR of DENV-2 RNA. We have previously shown that eukaryotic elongation factor 1A (eEF1A) is a direct activator of SphK1 and here DENV-2 RNA co-localized and co-precipitated with eEF1A from infected cells. We propose that the reduction in SphK1 activity late in DENV-2-infected cells is a consequence of DENV-2 out-competing SphK1 for eEF1A binding and hijacking cellular eEF1A for its own replication strategy, rather than a specific host or virus-induced change in SphK1 to modulate viral replication. Nonetheless, reduced SphK1 activity may have important consequences for survival or death of the infected cell.

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