However, in the drug-resistant cells that have an increased dependence on antiapoptotic proteins, whether autophagy is also inhibited remains unclear. 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 AGI-6780 apoptosis using siBIM and of both the early-phase autophagy nucleation actions 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 CCND2 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 main 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. Upon activation, the effector’ proteins BAX and BAK oligomerize and form pores around the outer mitochondrial membrane to release cytochrome and subsequently lead to caspase activation and apoptosis.8, 9 Activation of effector’ proteins requires conversation with the direct activators’, BIM and BID. Sensitizerssuch as PUMA and NOXA interact with and prevent antiapoptotic proteins from interacting with BIM and BID. 10 The functionally diverse BCL-2 family proteins,11 in addition to inhibition of apoptosis, also regulate autophagy,12, 13 a catabolic process maintaining cellular turnover in both normal and malignancy cells. A double membrane vesicle autophagosome’ in the beginning forms around the target AGI-6780 substrate and later fuses with lysosomes to form autolysosomeswhere degradation takes place.14, 15 The nucleation of the autophagosomal membrane is regulated by beclin1 (BECN1, ATG6), a BH3-domain name containing protein, which forms the core class III phosphatidylinositol-3 kinase (PI3K)Ccomplex, BECN1/Vps34/Vps15, that recruits essential autophagic proteins to a preautophagosomal membrane.12, 14 The BCL-2 family proteins, BCL-2, BCL-XL and MCL-1 block autophagy by direct conversation and inhibition of BECN1.13, 16, 17 As autophagy can cause both cell death and survival,18, 19, 20 we investigated the molecular alterations of autophagy and BCL-2 family proteins in response to acquired chemoresistance. By comparing Fd-sensitive and – resistant (FdR) cells that were generated by chronic exposure to Fd, we delineate how the drug-resistant cells adapt to chemotherapy by their ability to evade apoptosis by activating autophagy. Targeting alternate cell survival or cell death pathways could provide attractive treatment strategies. Results Fd induces autophagy and enhances autophagic flux To study the regulation of Fd-induced cell death or acquired resistance by autophagy, we first examined Fd-induced autophagy using LC3 (also known as ATG8) processing as a marker of autophagy. As you will find no Fd-sensitive CLL cell lines available, we selected pre-B leukemic cell lines as a Fd-sensitive model (IC50 10?degradation of LC3-I/II.14 AGI-6780 To determine autophagic flux, chloroquine (CQ) was used to inhibit degradation through autophagy by blocking lysosomal acidification.14 CQ pretreatment enhanced LC3 processing in both Nalm-6 and Reh (Determine 1d) and LC3 puncta in 4-h-Fd treated Nalm-6 cells, siControl-expressing Nalm-6 (drug resistance in CLL.6, 35 Importantly, the BIM-MCL-1Ccomplex is known to be critical for apoptosis AGI-6780 modulation in CLL.36 We show that endogenous MCL-1 sequestered BIM in untreated Fd-sensitive cells to inhibit apoptosis. Fd treatment reduced MCL-1 levels and released BIM to initiate apoptosis. Interestingly, FdR cells experienced amazingly low-BIM levels, which at.