Flavaglines Target Primitive Leukemia Cells and Enhance Anti-Leukemia Drug Activity
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Leukemia (2014) 28, 1960–1968 & 2014 Macmillan Publishers Limited All rights reserved 0887-6924/14 www.nature.com/leu ORIGINAL ARTICLE Flavaglines target primitive leukemia cells and enhance anti-leukemia drug activity KP Callahan1, M Minhajuddin2, C Corbett1, ED Lagadinou1, RM Rossi1, V Grose1, MM Balys1,LPan3, S Jacob4, A Frontier4, MR Grever5, DM Lucas5, AD Kinghorn3, JL Liesveld1, MW Becker1 and CT Jordan2 Identification of agents that target human leukemia stem cells is an important consideration for the development of new therapies. The present study demonstrates that rocaglamide and silvestrol, closely related natural products from the flavagline class of compounds, are able to preferentially kill functionally defined leukemia stem cells, while sparing normal stem and progenitor cells. In addition to efficacy as single agents, flavaglines sensitize leukemia cells to several anticancer compounds, including front-line chemotherapeutic drugs used to treat leukemia patients. Mechanistic studies indicate that flavaglines strongly inhibit protein synthesis, leading to the reduction of short-lived antiapoptotic proteins. Notably though, treatment with flavaglines, alone or in combination with other drugs, yields a much stronger cytotoxic activity toward leukemia cells than the translational inhibitor temsirolimus. These results indicate that the underlying cell death mechanism of flavaglines is more complex than simply inhibiting general protein translation. Global gene expression profiling and cell biological assays identified Myc inhibition and the disruption of mitochondrial integrity to be features of flavaglines, which we propose contribute to their efficacy in targeting leukemia cells. Taken together, these findings indicate that rocaglamide and silvestrol are distinct from clinically available translational inhibitors and represent promising candidates for the treatment of leukemia. Leukemia (2014) 28, 1960–1968; doi:10.1038/leu.2014.93 INTRODUCTION cell lymphoma, at doses that caused no discernable toxicity. In the past decade, a relatively new focus in drug development In these studies, the activity of silvestrol was due at least in has been the concept of a cancer stem cell, where the diversity part to loss of the antiapoptotic protein Mcl-1, with within tumors arises at least in part as a consequence of subsequent mitochondrial depolarization and caspase-dependent 7,10 developmental programs that mirror normal tissue systems.1 apoptosis. In addition to leukemia, silvestrol has shown activity Perhaps, the best characterized cancer stem cells are those found toward lung, breast and prostate cancer cells, and thus the utility in the blood cancer acute myelogenous leukemia (AML). Several of these compounds may extend beyond hematologic 11,12 studies have identified malignant stem cells, termed leukemia malignancies. Studies have shown that silvestrol promotes stem cells (LSCs), that are central to the initiation, growth and an aberrant interaction between capped mRNA and eIF4A, relapse of AML.2,3 The high rates of relapse in AML patients thereby interfering with the assembly of the eIF4F translation suggest that current chemotherapeutic strategies do not complex and blocking translation initiation.13,14 Consistent with adequately target LSCs. Thus, there is great interest in the these observations, recent work has identified eIF4A as one of the development/identification of compounds that target LSCs, but primary targets of rocaglamide and silvestrol.15 Hence, the activity still maintain an optimal therapeutic index. of these compounds appears to be related to their ability to inhibit Nearly half of the agents used in cancer therapy today are either translation. Extensive in vitro evidence now points to the natural products or derivatives of natural products.4 A group of translational machinery as a powerful therapeutic target in flavagline compounds from the plant genus Aglaia have attracted cancer, including hematologic malignancies.16,17 The translation attention owing to their insecticidal activities and inhibition of initiation complex constitutes a major node of convergence for tumor growth.5 Two members of this family, rocaglamide and numerous signaling pathways; however, few agents impact this silvestrol, have shown toxicity toward leukemia cells.6–9 The machinery directly, leaving this avenue largely unexplored in vivo. Li-Weber group has shown that rocaglamide induces apoptosis in Thus, flavaglines are a unique set of compounds that represent malignant but not normal proliferating lymphocytes, possibly the first direct inhibitor of translation initiation with clinical attributed to its ability to suppress selectively mitogen-activated potential, as evidenced by their preclinical activity on an array of protein kinase/extracellular signal-regulated kinase survival tumor types in the nanomolar range. activity in the cancer.6,8 Silvestrol has shown efficacy in vitro Here we show that rocaglamide and silvestrol preferentially kill and in mouse models of the B-cell malignancies, chronic phenotypically and functionally defined LSCs, while sparing lymphocytic leukemia, acute lymphoblastic leukemia and mantle normal stem and progenitor cells. Importantly, these compounds 1Department of Medicine, University of Rochester School of Medicine, Rochester, NY, USA; 2Division of Hematology, University of Colorado, Denver, Aurora, CO, USA; 3College of Pharmacy, The Ohio State University, Columbus, OH, USA; 4Department of Chemistry, University of Rochester, Rochester, NY, USA and 5Department of Internal Medicine, The Ohio State University, Columbus, OH, USA. Correspondence: Dr CT Jordan, Division of Hematology, University of Colorado, Denver, 12700 E 19th Avenue, Room 10016, Aurora, CO 80045, USA. E-mail: [email protected] Received 21 August 2013; revised 19 February 2014; accepted 21 February 2014; accepted article preview online 28 February 2014; advance online publication, 1 April 2014 Flavaglines target LSCs KP Callahan et al 1961 are significantly more toxic to leukemia cells as single agents or in shRNA-mediated p53 knockdown and Myc overexpression combination with other anticancer drugs than clinically available The coding sequences of p53 (NM_001126114.1) targeted by short hairpin translational inhibitors. This difference in cytotoxicity, however, is RNAs (shRNAs) are: sh-p53 A, 50-GTCCAGATGAAGCTCCCAGAA-30; sh-p53 E, not attributable to the respective differences after inhibition of 50-GCATCTTATCCGAGTGGAAGG-30; sh-P53 H, 50GGTCTTTGAACCCTTG CTTGC30. The detailed methods for cloning shRNA, generating lentiviral global protein synthesis; rather, it appears that they more 20 efficiently decrease levels of Myc protein and also alter particles and infecting AML cells, are as described previously. Myc mitochondrial integrity via p53 activation. overexpression construct was created as follows: the c-MYC gene was excised from MSCVh c-MYC IRES GFP (addgene plasmid 18119) with EcoRI restriction enzyme and ligated into pLVX-mcherry (Clontech, Mountain View, CA, USA). MATERIALS AND METHODS Primary AML and normal hematopoietic cells Normal and leukemic human bone marrow samples were obtained after Cell cycle analysis informed consent from volunteer donors at the University of Rochester For evaluation of the cell cycle profile, cells were fixed by the BD Fixation/ Medical Center. Total bone marrow mononuclear cells were isolated by Permeabilization Kit (554714; BD Biosciences) according to the manufac- standard Ficoll procedures (GE Healthcare, Niskayuna, NY, USA) and turer’s instructions, stained with anti-Ki67-Alexa Fluor 647 (BD Biosciences) cryopreserved in freezing medium consisting of Cryostor CS10 (BioLife for 45 min at 4 1C and resuspended overnight at 4 1C in 0.5 ml of 5 mg/ml Solutions, Bothell, WA, USA). The viability of leukemic cells after thawing 7-AAD staining solution. Cells were analyzed using BD LSRII flow cytometer was 50–90%. Normal bone marrow total mononuclear cells were further (BD Biosciences). enriched for CD34-positive cells using MACS CD34 Enrichment Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Mitochondrial depolarization Mitochondrial membrane depolarization was assessed using tetramethylr- Cell death assays hodamine, ethyl ester, percholate (TMRE) purchased from Life Technologies. For in vitro cell death assays, normal and leukemic cells were cultured in Following treatment with drugs, cells were incubated with TMRE for 30 min serum-free media for 24 or 48 h in the presence of drug and analyzed with at 37 1C, and then washed and stained with AnnexinV/7-AAD. All analyses AnnexinV/7-aminoactinomycin D (7-AAD) staining using the LSRII flow show the percentage of viable cells with depolarized mitochondrial cytometer (BD Biosciences, San Jose, CA, USA). For ex vivo toxicity assays, membranes. cells were treated in vitro with rocaglamide (Enzo Life Sciences, Farmingdale, NY, USA) for 48 h, and then harvested and injected in Measurement of endogenous reactive oxygen species levels irradiated NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice. For AML and Endogenous reactive oxygen species levels of M9-ENL cells were detected by normal bone marrow specimens, engraftment of human cells was 6 evaluated after 6–8 weeks by flow cytometry. labeling 1 Â 10 cells for 15 min at 37 1C with the redox-sensitive probes CM-H2DCFDA (5 mM), or MitoSox (5 mM) (Life Technologies). Labeled cells were resuspended in 0.2 ml of 10 mM 40,6-diamidino-2-phenylindole Colony-forming assay (Invitrogen, Carlsbad, CA, USA) solution and analyzed using BD