title: BECN1 and BIM interactions with MCL-1 determine fludarabine resistance in leukemic B cells.
abstract: The purine analog fludarabine (Fd) is an essential therapeutic for chronic lymphocytic leukemia (CLL). Innate or acquired resistance to Fd is a significant clinical problem and is largely mediated by increased expression of BCL-2 family members. The antiapoptotic BCL-2 family proteins inhibit both apoptosis and autophagy, therefore, downregulation of antiapoptotic BCL-2 family proteins and enhanced autophagy must coexist in cells dying in response to an apoptosis inducing therapeutic. However, in the drug-resistant cells that have an increased dependence on antiapoptotic proteins, whether autophagy is also inhibited remains unclear. Here, we examined the role of the BCL-2 family in regulating cell death and autophagy in leukemic cell lines and their derivative isogenic Fd-resistant (FdR) cells. MCL-1 degradation following Fd treatment freed the proapoptotic effectors BIM and BECN1, thus leading to cell death-associated autophagy in Fd-sensitive cells. However, in FdR cells, low BIM expression and BECN1 sequestration by MCL-1 prevented cell death. Consistently, in sensitive cells inhibition of apoptosis using siBIM and of both the early-phase autophagy nucleation steps by siBECN1, shATG7 or 3-methyladenine and the late-phase autophagy by shLAMP2, significantly reduced Fd-induced cell death. Paradoxically, FdR cells were addicted to basal autophagy, which was dependent on AMP-activated protein kinase (AMPK) but not BECN1. Moreover, in FdR cells, inhibition of autophagy by shLAMP2, but not siBECN1, enhanced cell death. The BH3-mimetic obatoclax released BIM and BECN1 from MCL-1 in Fd-sensitive and BECN1 from MCL-1 in FdR cells, and was effective at killing both Fd-sensitive and - resistant leukemic cells, including primary CLL cells. Therefore, a differential regulation of autophagy through BECN1 and AMPK signaling in Fd-sensitive and - resistant cells determines the different possible outcomes of autophagy inhibition. These findings suggest effective means to overcome Fd resistance by induction of BIM-dependent apoptosis and activation of BECN1-dependent autophagy.
authors.affiliation: Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195, USA.
authors.forename: A
authors.initials: A
authors.lastname: Sharma
authors.forename: K
authors.initials: K
authors.lastname: Singh
authors.forename: S
authors.initials: S
authors.lastname: Mazumder
authors.forename: B T
authors.initials: BT
authors.lastname: Hill
authors.forename: M
authors.initials: M
authors.lastname: Kalaycio
authors.forename: A
authors.initials: A
authors.lastname: Almasan
mesh_terms: D055372:AMP-Activated Protein Kinases / Q000378:metabolism; D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000494:pharmacology* / Q000627:therapeutic use; D017209:Apoptosis / Q000187:drug effects; D051017:Apoptosis Regulatory Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D001343:Autophagy / Q000187:drug effects*; D000071193:Autophagy-Related Protein 7; D000072224:Bcl-2-Like Protein 11; D000071186:Beclin-1; D045744:Cell Line, Tumor; D019008:Drug Resistance, Neoplasm; D006801:Humans; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology; D052119:Lysosomal-Associated Membrane Protein 2 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D011485:Protein Binding; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D044764:Ubiquitin-Activating Enzymes / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D014740:Vidarabine / Q000031:analogs & derivatives* / Q000494:pharmacology / Q000627:therapeutic use
chemical_list: D000970:Antineoplastic Agents; D051017:Apoptosis Regulatory Proteins; C000606775:BCL2L11 protein, human; C491997:BECN1 protein, human; D000072224:Bcl-2-Like Protein 11; D000071186:Beclin-1; D052119:Lysosomal-Associated Membrane Protein 2; D008565:Membrane Proteins; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D034741:RNA, Small Interfering; C025946:3-methyladenine; D055372:AMP-Activated Protein Kinases; C509349:ATG7 protein, human; D000071193:Autophagy-Related Protein 7; D044764:Ubiquitin-Activating Enzymes; D014740:Vidarabine; D000225:Adenine; C024352:fludarabine