title: Smac mimetic and demethylating agents synergistically trigger cell death in acute myeloid leukemia cells and overcome apoptosis resistance by inducing necroptosis.
abstract: Evasion of apoptosis, for example, by inhibitor of apoptosis (IAP) proteins, contributes to treatment resistance and poor outcome in acute myeloid leukemia (AML). Here we identify a novel synergistic interaction between the small-molecule second mitochondria-derived activator of caspases (Smac) mimetic BV6, which antagonizes X-linked IAP, cellular IAP (cIAP)1 and cIAP2, and the demethylating agents 5-azacytidine or 5-aza-2'-deoxycytidine (DAC) to induce cell death in AML cells, including apoptosis-resistant cells. Calculation of combination index (CI) confirms that this drug combination is highly synergistic (CI 0.02-0.4). In contrast, BV6 and DAC at equimolar concentrations do not cause synergistic toxicity against normal peripheral blood lymphocytes, pointing to some tumor cell selectivity. Molecular studies reveal that BV6 and DAC cooperate to trigger the activation of caspases, mitochondrial perturbations and DNA fragmentation, consistent with apoptotic cell death. However, the broad-range caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD.fmk) fails to protect against BV6/DAC-induced cell death and even significantly increases the percentage of Annexin-V/propidium iodide double-positive cells. Importantly, BV6/DAC-induced cell death in the presence of zVAD.fmk is significantly reduced by pharmacological inhibition of key components of necroptosis signaling, that is, receptor-interacting protein (RIP) 1 using necrostatin-1 or mixed lineage kinase domain-like protein (MLKL) using necrosulfonamide. This indicates a switch from BV6/DAC-induced cell death from apoptosis to necroptosis upon caspase inhibition. Thus, BV6 cooperates with demethylating agents to induce cell death in AML cells and circumvents apoptosis resistance via a switch to necroptosis as an alternative mode of cell death. The identification of a novel synergism of BV6 and demethylating agents has important implications for the development of new treatment strategies for AML.
authors.affiliation: Institute for Experimental Cancer Research in Pediatrics, Goethe-University, Frankfurt, Germany.
authors.forename: L
authors.initials: L
authors.lastname: Steinhart
authors.forename: K
authors.initials: K
authors.lastname: Belz
authors.forename: S
authors.initials: S
authors.lastname: Fulda
mesh_terms: D017209:Apoptosis / Q000187:drug effects*; D019898:Autocrine Communication / Q000187:drug effects; D001374:Azacitidine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D020169:Caspases / Q000378:metabolism; D045744:Cell Line, Tumor; D053938:DNA Fragmentation / Q000187:drug effects; D019175:DNA Methylation / Q000187:drug effects; D000077209:Decitabine; D019008:Drug Resistance, Neoplasm / Q000187:drug effects*; D004357:Drug Synergism; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D015470:Leukemia, Myeloid, Acute / Q000201:enzymology / Q000473:pathology*; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000378:metabolism*; D009336:Necrosis; D009842:Oligopeptides / Q000494:pharmacology*; D019899:Paracrine Communication / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
chemical_list: C582180:BV6 peptide; D024101:Mitochondrial Proteins; D009842:Oligopeptides; D014409:Tumor Necrosis Factor-alpha; D000077209:Decitabine; D020169:Caspases; D001374:Azacitidine
references.citation: Oncogene. 2001 Mar 1;20(9):1063-75
references.pmid: 11314043
references.citation: Leukemia. 2011 Nov;25(11):1728-38
references.pmid: 21701487
references.citation: Cell. 2007 Nov 16;131(4):682-93
references.pmid: 18022363
references.citation: Leukemia. 2012 Jun;26(6):1155-65
references.pmid: 22230799
references.citation: J Biol Chem. 2008 Sep 5;283(36):24295-9
references.pmid: 18621737
references.citation: J Clin Oncol. 2007 Sep 1;25(25):3884-91
references.pmid: 17679729
references.citation: Nat Rev Mol Cell Biol. 2010 Oct;11(10):700-14
references.pmid: 20823910
references.citation: Physiol Rev. 2007 Jan;87(1):99-163
references.pmid: 17237344
references.citation: Mol Cancer Ther. 2007 Jul;6(7):1951-61
references.pmid: 17620426
references.citation: Cytokine Growth Factor Rev. 2008 Jun-Aug;19(3-4):325-31
references.pmid: 18495520
references.citation: Cell. 2011 Mar 4;144(5):646-74
references.pmid: 21376230
references.citation: Nat Rev Drug Discov. 2010 Jun;9(6):447-64
references.pmid: 20467424
references.citation: Invest New Drugs. 2011 Dec;29(6):1264-75
references.pmid: 20614162
references.citation: Cell. 2007 Nov 16;131(4):669-81
references.pmid: 18022362
references.citation: Oncogene. 2006 Aug 7;25(34):4798-811
references.pmid: 16892092
references.citation: Apoptosis. 2009 Apr;14(4):409-23
references.pmid: 19130230
references.citation: Cell Death Differ. 2012 Jan;19(1):107-20
references.pmid: 21760595
references.citation: Cell Cycle. 2008 Jul 15;7(14):2139-45
references.pmid: 18641459
references.citation: Clin Cancer Res. 2000 May;6(5):1796-803
references.pmid: 10815900
references.citation: Best Pract Res Clin Haematol. 2008 Mar;21(1):5-11
references.pmid: 18342807
references.citation: PLoS One. 2008;3(12):e3909
references.pmid: 19079542
references.citation: Lancet. 2006 Nov 25;368(9550):1894-907
references.pmid: 17126723
references.citation: Leukemia. 2012 May;26(5):1020-9
references.pmid: 22173242
references.citation: J Clin Pharmacol. 2005 May;45(5):597-602
references.pmid: 15831784
references.citation: Cell. 2008 May 16;133(4):693-703
references.pmid: 18485876
references.citation: Hematol J. 2004;5(6):489-95
references.pmid: 15570290
references.citation: Cell Death Dis. 2013 Mar 28;4:e564
references.pmid: 23538445
references.citation: Blood. 2005 May 15;105(10):4043-50
references.pmid: 15687241
references.citation: Oncogene. 2006 Aug 24;25(37):5125-33
references.pmid: 16607283
references.citation: Leukemia. 2010 Dec;24(12):2100-9
references.pmid: 20844561
references.citation: Blood. 2009 Feb 19;113(8):1710-22
references.pmid: 19036706
references.citation: Nature. 2011 Mar 17;471(7338):363-7
references.pmid: 21368763
references.citation: Mol Cell. 2008 Jun 20;30(6):689-700
references.pmid: 18570872
references.citation: Ann Pharmacother. 2012 Nov;46(11):1511-7
references.pmid: 23115225
references.citation: Leuk Res. 2010 Mar;34(3):364-72
references.pmid: 19732952
references.citation: Cell. 2012 Jan 20;148(1-2):213-27
references.pmid: 22265413
references.citation: Int J Cancer. 2010 May 1;126(9):2216-28
references.pmid: 19676052
references.citation: Blood. 2007 Aug 15;110(4):1291-300
references.pmid: 17485551
references.citation: Cell Death Differ. 2011 Apr;18(4):656-65
references.pmid: 21052097
references.citation: Blood. 2000 Jan 1;95(1):301-8
references.pmid: 10607716
references.citation: EMBO Rep. 2006 Oct;7(10):988-94
references.pmid: 17016456
references.citation: Cancer Cell. 2007 Nov;12(5):445-56
references.pmid: 17996648
references.citation: Nat Rev Drug Discov. 2012 Feb 01;11(2):109-24
references.pmid: 22293567
references.citation: Cancer Chemother Pharmacol. 2008 Apr;61(5):759-66
references.pmid: 17564707
references.citation: Neoplasia. 2011 Oct;13(10):971-9
references.pmid: 22028622
references.citation: J Clin Oncol. 2007 Apr 1;25(10):1209-15
references.pmid: 17401010