ABT-737 is a BH3-mimetic that has a wide spectrum of single-agent activity against acute lymphoblastic leukemia (ALL) cell lines and xenografts. Previously, we reported that in response to ABT-737, ABT-737-resistant ALL cell lines showed an apparent increase in Mcl-1 (an anti-apoptotic Bcl-2 family protein that is not effectively inhibited by ABT-737) while ABT-737-sensitive ALL cell lines showed decreased Mcl-1 levels. Here we explored the mechanism of Mcl-1 cleavage by ABT-737 and the effect of adjacent phosphorylation sites on Mcl-1 cleavage and apoptosis induced by ABT-737 in a human B-lineage ALL cell line. Caspase cleavage sites in Mcl-1 and the effect of mutation in Mcl-1 phosphorylation sites were determined by transducing Mcl-1 variants tagged with the V5 epitope into human ALL cells. Cytotoxicity was by fluorescence-based DIMSCAN, and changes in protein by immunoblotting. ABT-737 induced a caspase-dependent cleavage of Mcl-1. Of the two Mcl-1 caspase cleavage sites (D127 and D157), D157 was the site of ABT-737-induced cleavage in ALL cells. Cells with exogenously expressed Mcl-1 Δ157 fragment showed greater caspase-3 and caspase-9 activation when they were treated with ABT-737 compared with cells expressing wild-type or D157A mutant Mcl-1. Cells with mutated phosphorylation sites on Mcl-1 (S159A and T163A) were less susceptible to Mcl-1 cleavage and apoptosis induced by ABT-737. Our data showed that Mcl-1 is post-translationally regulated in response to ABT-737 treatment, primarily via a caspase-dependent cleavage that generates a pro-apoptotic Mcl-1 fragment.
KangMHReynoldsCP. Bcl-2 Inhibitors: targeting mitochondrial apoptotic pathways in cancer therapy. Clin Cancer Res2009; 15: 1126–32.
2.
AddeoRCaragliaMBaldiAD'AngeloVCasaleFCrisciSAbbruzzeseAVincenzeBCampioniMDi TullioMTIndolfiP. Prognostic role of bcl-xL and p53 in childhood acute lymphoblastic leukemia (ALL). Cancer Biol Ther2005; 4: 32–8.
3.
Coustan-SmithEKitanakaAPuiCHMcNinchLEvansWERaimondiSCBehmFGAricoMCampanaD. Clinical relevance of BCL-2 overexpression in childhood acute lymphoblastic leukemia. Blood1996; 87: 1140–46.
4.
LauriaFRaspadoriDRondelliDVenturaMAFiacchiniMVisaniGForconiFTuraS. High bcl-2 expression in acute myeloid leukemia cells correlates with CD34 positivity and complete remission rate. Leukemia1997; 11: 2075–8.
5.
SalomonsGSSmetsLAVerwijs-JanssenMHartAAHaarmanEGKaspersGJWeringEVDer Does-Van Den BergAVKampsWA. Bcl-2 family members in childhood acute lymphoblastic leukemia: relationships with features at presentation, in vitro and in vivo drug response and long-term clinical outcome. Leukemia1999; 13: 1574–80.
6.
PeregoPRighettiSSupinoRDeliaDCaseriniCCareniniNBedognéBBroomeEKrajewskiSReedJZuninoF. Role of apoptosis and apoptosis-related proteins in the cisplatin-resistant phenotype of human tumor cell lines. Apoptosis1997; 2: 540–48.
7.
MinnAJRudinCMBoiseLHThompsonCB. Expression of bcl-xL can confer a multidrug resistance phenotype. Blood1995; 86: 1903–10.
8.
ReedJC. Bcl-2-family proteins and hematologic malignancies: history and future prospects. Blood2008; 111: 3322–30.
9.
YoshinoTShiinaHUrakamiSKikunoNYonedaTShigenoKIgawaM. Bcl-2 expression as a predictive marker of hormone-refractory prostate cancer treated with taxane-based chemotherapy. Clin Cancer Res2006; 12: 6116–24.
10.
CampanaDCoustan-SmithEManabeABuschleMRaimondiSCBehmFGAshmunRAricoMBiondiAPuiCH. Prolonged survival of B-lineage acute lymphoblastic leukemia cells is accompanied by overexpression of bcl-2 protein. Blood1993; 81: 1025–31.
11.
CamposLSabidoOSebbanCCharrinCBertheasMFFiereDGuyotatD. Expression of BCL-2 proto-oncogene in adult acute lymphoblastic leukemia. Leukemia1996; 10: 434–8.
12.
Del PoetaGVendittiADel PrincipeMIMaurilloLBuccisanoFTamburiniACoxMCFranchiABrunoAMazzoneCPanettaPSuppoGMasiMAmadoriS. Amount of spontaneous apoptosis detected by Bax/Bcl-2 ratio predicts outcome in acute myeloid leukemia (AML). Blood2003; 101: 2125–31.
13.
OsorioLMDe SantiagoAguilar-SantelisesHaMMellstedtkJondalM. CD6 ligation modulates the Bcl-2/Bax ratio and protects chronic lymphocytic leukemia B cells from apoptosis induced by anti-IgM. Blood1997; 89: 2833–41.
14.
GalaJLVermylenCCornuGFerrantAMichauxJLPhilippeMMartiatP. High expression of bcl-2 is the rule in acute lymphoblastic leukemia, except in Burkitt subtype at presentation, and is not correlated with the prognosis. Ann Hematol1994; 69: 17–24.
15.
OltvaiZNMillimanCLKorsmeyerSJ. Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell1993; 74: 609–19.
16.
ArefSSalamaOAl TonbaryYMansourA. Assessment of bcl-2 expression as modulator of fas mediated apoptosis in acute leukemia. Hematology2004; 9: 113–21.
17.
OltersdorfTElmoreSWShoemakerARArmstrongRCAugeriDJBelliBABrunckoMDeckwerthTLDingesJHajdukPJJosephMKKitadaSKorsmeyerSJKunzerARLetaiALiCMittenMJNettesheimDGNgSNimmerPMO'ConnorJMOleksijewAPetrosAMReedJCShenWTahirSKThompsonCBTomaselliKJWangBWendtMDZhangHFesikSWRosenbergSH. An inhibitor of Bcl-2 family proteins induces regression of solid tumours. Nature2005; 435: 677–81.
18.
KojimaKKonoplevaMSamudioIJSchoberWDBornmannWGAndreeffM. Concomitant inhibition of MDM2 and Bcl-2 protein function synergistically induce mitochondrial apoptosis in AML. Cell Cycle2006; 5: 2778–86.
19.
KonoplevaMContractorRTsaoTSamudioIRuvoloPPKitadaSDengXZhaiDShiYXSneedTVerhaegenMSoengasMRuvoloVRMcQueenTSchoberWDWattJCJiffarTLingXMariniFCHarrisDDietrichMEstrovZMcCubreyJMayWSReedJCAndreeffM. Mechanisms of apoptosis sensitivity and resistance to the BH3 mimetic ABT-737 in acute myeloid leukemia. Cancer Cell2006; 10: 375–88.
20.
ChauhanDVelankarMBrahmandamMHideshimaTPodarKRichardsonPSchlossmanRGhobrialIRajeNMunshiNAndersonKC. A novel Bcl-2/Bcl-X(L)/Bcl-w inhibitor ABT-737 as therapy in multiple myeloma. Oncogene2006; 26: 2374–80.
21.
van DelftMFWeiAHMasonKDVandenbergCJChenLCzabotarPEWillisSNScottCLDayCLCorySAdamsJMRobertsAWHuangDC. The BH3 mimetic ABT-737 targets selective Bcl-2 proteins and efficiently induces apoptosis via Bak/Bax if Mcl-1 is neutralized. Cancer Cell2006; 10: 389–99.
22.
Del Gaizo MooreVBrownJRCertoMLoveTMNovinaCDLetaiA. Chronic lymphocytic leukemia requires BCL2 to sequester prodeath BIM, explaining sensitivity to BCL2 antagonist ABT-737. J Clin Invest2007; 117: 112–21.
23.
TahirSKYangXAndersonMGMorgan-LappeSESarthyAVChenJWarnerRBNgSCFesikSWElmoreSWRosenbergSHTseC. Influence of Bcl-2 family members on the cellular response of small-cell lung cancer cell lines to ABT-737. Cancer Res2007; 67: 1176–83.
24.
ShoemakerAROleksijewABauchJBelliBABorreTBrunckoMDeckwirthTFrostDJJarvisKJosephMKMarshKMcClellanWNellansHNgSNimmerPO'ConnorJMOltersdorfTQingWShenWStavropoulosJTahirSKWangBWarnerRZhangHFesikSWRosenbergSHElmoreSW. A small-molecule inhibitor of Bcl-XL potentiates the activity of cytotoxic drugs in vitro and in vivo. Cancer Res2006; 66: 8731–9.
25.
KangMHKangYHSzymanskaBWilczynska-KalakUSheardMAHarnedTMLockRBReynoldsCP. Activity of vincristine, L-ASP, and dexamethasone against acute lymphoblastic leukemia is enhanced by the BH3-mimetic ABT-737 in vitro and in vivo. Blood2007; 110: 2057–66.
26.
GoldsmithKCGrossMPeirceSLuyindulaDLiuXVuASliozbergMGuoRZhaoHReynoldsCPHogartyMD. Mitochondrial Bcl-2 family dynamics define therapy response and resistance in neuroblastoma. Cancer Res2012; 72: 2565–77.
27.
FangHHarnedTMKalousOMaldonadoVDeClerckYAReynoldsCP. Synergistic activity of fenretinide and the Bcl-2 family protein inhibitor ABT-737 against human neuroblastoma. Clin Cancer Res2011; 17: 7093–104.
28.
GoldsmithKCGrossMPeirceSLuyindulaDLiuXVuASliozbergMGuoRZhaoHReynoldsCPHogartyMD. Mitochondrial Bcl-2 family dynamics define therapy response and resistance in neuroblastoma. Cancer Res2012; 72: 2565–77.
29.
ChenSDaiYHaradaHDentPGrantS. Mcl-1 down-regulation potentiates ABT-737 lethality by cooperatively inducing Bak activation and Bax translocation. Cancer Res2007; 67: 782–91.
30.
LinXMorgan-LappeSHuangXLiLZakulaDMVernettiLAFesikSWShenY. ‘Seed’ analysis of off-target siRNAs reveals an essential role of Mcl-1 in resistance to the small-molecule Bcl-2/Bcl-X(L) inhibitor ABT-737. Oncogene2007; 26: 3972–9.
31.
GandhiLCamidgeDRRibeiro de OliveiraMBonomiPGandaraDKhairaDHannCLMcKeeganEMLitvinovichEHemkenPMDiveCEnschedeSHNolanCChiuYLBusmanTXiongHKrivoshikAPHumerickhouseRShapiroGIRudinCM. Phase I study of Navitoclax (ABT-263), a novel Bcl-2 family inhibitor, in patients with small-cell lung cancer and other solid tumors. J Clin Oncol2011; 29: 909–16.
32.
WilsonWHO'ConnorOACzuczmanMSLaCasceASGerecitanoJFLeonardJPTulpuleADunleavyKXiongHChiuYLCuiYBusmanTElmoreSWRosenbergSHKrivoshikAPEnschedeSHHumerickhouseRA. Navitoclax, a targeted high-affinity inhibitor of BCL-2, in lymphoid malignancies: a phase 1 dose-escalation study of safety pharmacokinetics, pharmacodynamics, and antitumour activity. Lancet Oncol2010; 11: 1149–59.
33.
KangMHWanZKangYSpostoRReynoldsCP. Mechanism of synergy of N-(4-hydroxyphenyl)retinamide and ABT-737 in acute lymphoblastic leukemia cell lines: Mcl-1 inactivation. J Natl Cancer Inst2008; 100: 580–95.
34.
SheardMAKangMHCabralDLeeJCastroLKhankaldyyanVWuSQReynoldsCP. Bone marrow-level oxygen tension enables enhanced and sustained growth of 3 new pediatric acute lymphoblastic leukemia cell lines [abstract]. Proc Am Assoc Cancer Res2007; 48: 1808.
35.
MastersJRThomsonJAly-BurnsBReidYADirksWGPackerPTojiLHOhnoTTanabeHArlettCFKellandLRHarrisonMVirmaniAWardTHAyresKLDebenhamPG. Short tandem repeat profiling provides an international reference standard for human cell lines. PNAS2001; 98: 8012–7.
36.
FrgalaTKalousOProffittRTReynoldsCP. A fluorescence microplate cytotoxicity assay with a 4-log dynamic range that identifies synergistic drug combinations. Mol Cancer Ther2007; 6: 886–97.
37.
KeshelavaNFrgalaTKrejsaJKalousOReynoldsCP. DIMSCAN: a microcomputer fluorescence-based cytotoxicity assay for preclinical testing of combination chemotherapy. Methods Mol Med2005; 110: 139–53.
NijhawanDFangMTraerEZhongQGaoWDuFWangX. Elimination of Mcl-1 is required for the initiation of apoptosis following ultraviolet irradiation. Genes Dev2003; 17: 1475–86.
40.
DerouetMThomasLCrossAMootsRJEdwardsSW. Granulocyte macrophage colony-stimulating factor signaling and proteasome inhibition delay neutrophil apoptosis by increasing the stability of Mcl-1. J Biol Chem2004; 279: 26915–21.
41.
WengCLiYXuDShiYTangH. Specific cleavage of Mcl-1 by caspase-3 in tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in Jurkat leukemia T cells. J Biol Chem2005; 280: 10491–500.
42.
ClohessyJGZhuangJBradyHJ. Characterisation of Mcl-1 cleavage during apoptosis of haematopoietic cells. Br J Haematol2004; 125: 655–65.
43.
WilsonWHCzuczmanMSLaCasceASGerecitanoJFLeonardJPDunleavyKKrivoshikAPXiongHChiuYO'ConnorOA. A phase 1 study evaluating the safety, pharmacokinetics, and efficacy of ABT-263 in subjects with refractory or relapsed lymphoid malignancies [abstract]. JClin Oncol2008; 26S: 8511.
44.
HerrantMJacquelAMarchettiSBelhaceneNColosettiPLucianoFAubergerP. Cleavage of Mcl-1 by caspases impaired its ability to counteract Bim-induced apoptosis. Oncogene2004; 23: 7863–73.
45.
HanJGoldsteinLAGastmanBRRabinovitzARabinowichH. Disruption of Mcl-1.Bim complex in granzyme B-mediated mitochondrial apoptosis. J Biol Chem2005; 280: 16383–92.
46.
DayCLChenLRichardsonSJHarrisonPJHuangDCHindsMG. Solution structure of prosurvival Mcl-1 and characterization of its binding by proapoptotic BH3-only ligands. J Biol Chem2005; 280: 4738–44.
47.
ThomasLWLamCEdwardsSW. Mcl-1; the molecular regulation of protein function. FEBS Lett2010; 584: 2981–9.
48.
RahmaniMAustMMAttkissonEWilliamsDCJrFerreira-GonzalezAGrantS. Dual inhibition of Bcl-2 and Bcl-xL strikingly enhances PI3K inhibition-induced apoptosis in human myeloid leukemia cells through a. Cancer Res2013; 73: 1340–51.
49.
MaurerUCharvetCWagmanASDejardinEGreenDR. Glycogen synthase kinase-3 regulates mitochondrial outer membrane permeabilization and apoptosis by destabilization of MCL-1. Mol Cell2006; 21: 749–60. .
50.
WengCLiYXuDShiYTangH. Specific cleavage of Mcl-1 by Caspase-3 in tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in Jurkat leukemia T cells. J Biol Chem2005; 280: 10491–500.
51.
KobayashiSLeeSHMengXWMottJLBronkSFWerneburgNWCraigRWKaufmannSHGoresGJ. Serine 64 phosphorylation enhances the antiapoptotic function of Mcl-1. J Biol Chem2007; 282: 18407–17.
52.
WarrMRMillsJRNguyenMLemaire-EwingSBaardsnesJSunKLWMalinaAYoungJCJeyarajuDVO'Connor-McCourtMPellegriniLPelletierJShoreGC. Mitochondrion-dependent N-terminal processing of outer membrane Mcl-1 protein removes an essential Mule/Lasu1 protein-binding site. J Biol Chem2011; 286: 25098–107.
53.
HerrantMJacquelAMarchettiSBelhaceneNColosettiPLucianoFAubergerP. Cleavage of Mcl-1 by caspases impaired its ability to counteract Bim-induced apoptosis. Oncogene2004; 23: 7863–73.
54.
MorelCCarlsonSMWhiteFMDavisRJ. Mcl-1 integrates the opposing actions of signaling pathways that mediate survival and apoptosis. Mol Cell Biol2009; 29: 3845–52.
55.
DobleBWWoodgettJR. GSK-3: tricks of the trade for a multi-tasking kinase. J Cell Sci2003; 116: 1175–86.
56.
DingQHeXHsuJMXiaWChenCTLiLYLeeDFLiuJCZhongQWangXHungMC. Degradation of Mcl-1 by beta-TrCP mediates glycogen synthase kinase 3-induced tumor suppression and chemosensitization. Mol Cell Biol2007; 27: 4006–17.
57.
TseCTahirSKAndersonMG. The balance between Bcl-2 family members governs the cellular sensitivity of small cell lung cancer to Bcl-2/Bcl-xl antagonist ABT-737 [abstract]. Clin Cancer Res2005Suppl 24B34.
58.
YeciesDCarlsonNEDengJLetaiA. Acquired resistance to ABT-737 in lymphoma cells that up-regulate MCL-1 and BFL-1. Blood2010; 115: 3304–13.
