Quantifying the inhibitory effect of Bcl-xl on the action of Mff using live-cell fluorescence imaging.

Mitochondrial fission regulates mitochondrial function and morphology, and has been linked to apoptosis. The mitochondrial fission factor (Mff), a tail-anchored membrane protein, induces excessive mitochondrial fission, contributing to mitochondrial dysfunction and apoptosis. Here, we evaluated the inhibitory effect of Bcl-xl, an antiapoptotic protein, on the action of Mff by using live-cell fluorescence imaging. Microscopic imaging analysis showed that overexpression of Mff induced mitochondrial fragmentation and apoptosis, which were reversed by coexpression of Bcl-xl. Microscopic imaging and live-cell fluorescence resonance energy transfer analysis demonstrated that Bcl-xl reconstructs the Mff network from punctate distribution of higher-order oligomers to filamentous distribution of lower-order oligomers. Live-cell fluorescence resonance energy transfer two-hybrid assay showed that Bcl-xl interacted with Mff to form heterogenous oligomers with 1 : 2 stoichiometry in cytoplasm and 1 : 1 stoichiometry on mitochondria, indicating that two Bcl-xl molecules primarily interact with four Mff molecules in cytoplasm, but with two Mff molecules on mitochondria.

Infection with flaviviruses requires BCLXL for cell survival.

BCL2 family proteins including pro-survival proteins, BH3-only proteins and BAX/BAK proteins control mitochondria-mediated apoptosis to maintain cell homeostasis via the removal of damaged cells and pathogen-infected cells. In this study, we examined the roles of BCL2 proteins in the induction of apoptosis in cells upon infection with flaviviruses, such as Japanese encephalitis virus, Dengue virus and Zika virus. We showed that survival of the infected cells depends on BCLXL, a pro-survival BCL2 protein due to suppression of the expression of another pro-survival protein, MCL1. Treatment with BCLXL inhibitors, as well as deficient BCLXL gene expression, induced BAX/BAK-dependent apoptosis upon infection with flaviviruses. Flavivirus infection attenuates cellular protein synthesis, which confers reduction of short-half-life proteins like MCL1. Inhibition of BCLXL increased phagocytosis of virus-infected cells by macrophages, thereby suppressing viral dissemination and chemokine production. Furthermore, we examined the roles of BCLXL in the death of JEV-infected cells during in vivo infection. Haploinsufficiency of the BCLXL gene, as well as administration of BH3 mimetic compounds, increased survival rate after challenge of JEV infection and suppressed inflammation. These results suggest that BCLXL plays a crucial role in the survival of cells infected with flaviviruses, and that BCLXL may provide a novel antiviral target to suppress propagation of the family of Flaviviridae viruses.

BCL-xL-selective BH3 mimetic sensitizes rhabdomyosarcoma cells to chemotherapeutics by activation of the mitochondrial pathway of apoptosis.

BH3 mimetics are a promising new class of anticancer agents that inhibit antiapoptotic BCL-2 proteins. Here, we report that BH3 mimetics selectively targeting BCL-xL, BCL-2 or MCL-1 (i.e. A-1331852, ABT-199, A-1210477) act in concert with multiple chemotherapeutic agents (i.e. vincristine (VCR), etoposide (ETO), doxorubicin, actinomycin D and cyclophosphamide) to induce apoptosis in rhabdomyosarcoma (RMS) cells. Similarly, genetic knockdown of BCL-xL primes RMS cells to VCR- or ETO-induced cell death, highlighting the importance of BCL-xL in mediating chemotherapy resistance in RMS. A-1331852 and VCR or ETO cooperate to stimulate caspase activation and caspase-dependent apoptosis, since the broad-range caspase inhibitor zVAD.fmk rescues cells from cell death. Molecular studies reveal that VCR/A-1331852 co-treatment causes profound mitotic arrest, which initiates phosphorylation of BCL-2, thereby promoting its inactivation. Also, A-1331852 and VCR or ETO act together to trigger BAX and BAK activation, followed by loss of mitochondrial membrane potential (MMP). Consistently, overexpression of BCL-2 or MCL-1 markedly reduces VCR/A-1331852- or ETO/A-1331852-mediated apoptosis, underscoring that mitochondrial apoptosis represents a key event in synergistic drug interaction. In conclusion, our findings provide a rationale for the combination of BH3 mimetics with conventional chemotherapeutic agents to increase the chemosensitivity of RMS.

Transient apoptosis inhibition in donor stem cells improves hematopoietic stem cell transplantation.

During hematopoietic stem cell transplantation, a substantial number of donor cells are lost because of apoptotic cell death. Transplantation-associated apoptosis is mediated mainly by the proapoptotic BCL-2 family proteins BIM and BMF, and their proapoptotic function is conserved between mouse and human stem and progenitor cells. Permanent inhibition of apoptosis in donor cells caused by the loss of these BH3-only proteins improves transplantation outcome, but recipients might be exposed to increased risk of lymphomagenesis or autoimmunity. Here, we address whether transient inhibition of apoptosis can serve as a safe but efficient alternative to improve the outcome of stem cell transplantation. We show that transient apoptosis inhibition by short-term overexpression of prosurvival BCL-XL, known to block BIM and BMF, is not only sufficient to increase the viability of hematopoietic stem and progenitor cells during engraftment but also improves transplantation outcome without signs of adverse pathologies. Hence, this strategy represents a promising and novel therapeutic approach, particularly under conditions of limited donor stem cell availability.

Inhibition of MAPKinase pathway sensitizes thyroid cancer cells to ABT-737 induced apoptosis.

Bcl2 family proteins play an important role in the resistance of thyroid cancer cells to apoptosis induced by chemotherapeutic drugs and targeted therapies. BH3-profiling of seven fresh primary papillary thyroid cancer (PTC) tumors showed dependence for survival on Bcl-xL (2/7), Bcl2 (2/7), and Mcl-1 (2/7), while the majority of thyroid cell lines were mainly dependent on Bcl-xL. Targeting Bcl2 family proteins with the BH3 mimetic, ABT-737, while simultaneously inhibiting ERK pathway proteins with PLX4720 and PD325901 was shown to induce significantly high apoptosis in the majority of cell lines (8505c, SW1736, HTh7, BCPAP) and moderate apoptosis in the TPC-1 cell line. In orthotopic thyroid cancer mouse models of 8505c and BCPAP, treatment with the triple drug combination reduced the size of the tumors and showed significantly higher numbers of cells undergoing apoptosis. This treatment increased the expression of pro-apoptotic protein Bim, while decreasing anti-apoptotic protein Mcl-1. Our results suggest that analyzing the results of BH3-profiling along with the mutational status of tumor can reveal an effective therapy for targeted, personalized treatment of aggressive thyroid cancer.

Non-canonical roles of Bcl-2 and Bcl-xL proteins: relevance of BH4 domain.

Bcl-2 protein family is constituted by multidomain members originally identified as modulators of programmed cell death and whose expression is frequently misbalanced in cancer cells. The lead member Bcl-2 and its homologue Bcl-xL proteins are characterized by the presence of all four conserved BH domain and exert their antiapoptotic role mainly through the involvement of BH1, BH2 and BH3 homology domains, that mediate the interaction with the proapoptotic members of the same Bcl-2 family. The N-terminal BH4 domain of Bcl-2 and Bcl-xL is responsible for the interaction with other proteins that do not belong to Bcl-2 protein family. Beyond a classical role in inhibiting apoptosis, BH4 domain has been characterized as a crucial regulator of other important cellular functions attributed to Bcl-2 and Bcl-xL, including proliferation, autophagy, differentiation, DNA repair, cell migration, tumor progression and angiogenesis. During the last two decades a strong effort has been made to dissect the molecular pathways involved the capability of BH4 domain to regulate the canonical antiapoptotic and the non-canonical activities of Bcl-2 and Bcl-xL, creating the basis for the development of novel anticancer agents targeting this domain. Indeed, recent evidences obtained on in vitro and in vivo model of different cancer histotypes are confirming the promising therapeutic potential of BH4 domain inhibitors supporting their future employment as a novel anticancer strategy.

Bcl-xL Affects Group A Streptococcus-Induced Autophagy Directly, by Inhibiting Fusion between Autophagosomes and Lysosomes, and Indirectly, by Inhibiting Bacterial Internalization via Interaction with Beclin 1-UVRAG.

Anti-apoptotic Bcl-2 and Bcl-xL are proposed to regulate starvation-induced autophagy by directly interacting with Beclin 1. Beclin 1 is also thought to be involved in multiple vesicle trafficking pathways such as endocytosis by binding to Atg14L and UVRAG. However, how the interaction of Bcl-2 family proteins and Beclin 1 regulates anti-bacterial autophagy (xenophagy) is still unclear. In this study, we analyzed these interactions using Group A Streptococcus (GAS; Streptococcus pyogenes) infection as a model. GAS is internalized into epithelial cells through endocytosis, while the intracellular fate of GAS is degradation by autophagy. Here, we found that Bcl-xL but not Bcl-2 regulates GAS-induced autophagy. Autophagosome-lysosome fusion and the internalization process during GAS infection were promoted in Bcl-xL knockout cells. In addition, knockout of Beclin 1 phenocopied the internalization defect of GAS. Furthermore, UVRAG interacts not only with Beclin 1 but also with Bcl-xL, and overexpression of UVRAG partially rescued the internalization defect of Beclin 1 knockout cells during GAS infection. Thus, our results indicate that Bcl-xL inhibits GAS-induced autophagy directly by suppressing autophagosome-lysosome fusion and indirectly by suppressing GAS internalization via interaction with Beclin 1-UVRAG.

Finally, An Apoptosis-Targeting Therapeutic for Cancer.

Resistance to cell death represents one of the hallmarks of cancer. Various genetic and epigenetic changes in malignant cells afford cytoprotection in the face of genomic instability, oncogene activation, microenvironment stress, chemotherapy, targeted anticancer drugs, and even immunotherapy. Central among the regulators of cell life and death are Bcl-2 family proteins, with the founding member of the family (B-cell lymphoma/leukemia-2) discovered via its involvement in chromosomal translocations in lymphomas. The quest for therapeutics that target cell survival protein Bcl-2 represents a long road traveled, with many dead-ends, disappointments, and delays. Finally, a Bcl-2-targeting medicine has gained approval as a new class of anticancer agent. Cancer Res; 76(20); 5914-20. ©2016 AACR.

BCL-B (BCL2L10) is overexpressed in patients suffering from multiple myeloma (MM) and drives an MM-like disease in transgenic mice.

Multiple myeloma (MM) evolves from a premalignant condition known as monoclonal gammopathy of undetermined significance (MGUS). However, the factors underlying the malignant transformation of plasmocytes in MM are not fully characterized. We report here that Eµ-directed expression of the antiapoptotic Bcl-B protein in mice drives an MM phenotype that reproduces accurately the human disease. Indeed, with age, Eµ-bcl-b transgenic mice develop the characteristic features of human MM, including bone malignant plasma cell infiltration, a monoclonal immunoglobulin peak, immunoglobulin deposit in renal tubules, and highly characteristic bone lytic lesions. In addition, the tumors are serially transplantable in irradiated wild-type mice, underlying the tumoral origin of the disease. Eµ-bcl-b plasmocytes show increased expression of a panel of genes known to be dysregulated in human MM pathogenesis. Treatment of Eµ-bcl-b mice with drugs currently used to treat patients such as melphalan and VELCADE efficiently kills malignant plasmocytes in vivo. Finally, we find that Bcl-B is overexpressed in plasmocytes from MM patients but neither in MGUS patients nor in healthy individuals, suggesting that Bcl-B may drive MM. These findings suggest that Bcl-B could be an important factor in MM disease and pinpoint Eµ-bcl-b mice as a pertinent model to validate new therapies in MM.

Inhibition of Bcl-2 family members sensitises soft tissue leiomyosarcomas to chemotherapy.

BACKGROUND: Leiomyosarcoma is an aggressive soft tissue sarcoma with a 5-year survival rate of 15 to 60%. Treatment options for inoperable or metastatic patients are limited owing to frequent resistance of tumours to chemotherapy and radiation. In this study, we hypothesised that antiapoptotic Bcl-2 family proteins might contribute to leiomyosarcoma chemoresistance and therefore inhibition of Bcl-2 family proteins might sensitise leiomyosarcomas to conventional chemotherapy. METHODS: Expression of the Bcl-2 family proteins Bcl-xL, Bcl-w and Bcl-2 was investigated using immunohistochemistry on a tissue microarray containing 43 leiomyosarcomas. Furthermore, we investigated whether ABT-737, a potent BH3 mimetic, sensitises leiomyosarcoma cells to doxorubicin treatment in vitro. RESULTS: Seventy-seven per cent, 84% and 42% of leiomyosarcomas demonstrated high expression of Bcl-2, Bcl-xL and Bcl-w, respectively. Single-agent treatment with ABT-737 resulted in a minor reduction of cell viability. However, combination treatment of ABT-737 and doxorubicin revealed synergism in all four cell lines, by inducing apoptosis. CONCLUSIONS: In conclusion, Bcl-2 family proteins contribute to soft tissue leiomyosarcoma chemoresistance. Antiapoptotic proteins are highly expressed in leiomyosarcoma of soft tissue, and inhibition of these proteins using a BH3 mimetic increases leiomyosarcoma sensitivity to doxorubicin.

The non-apoptotic action of Bcl-xL: regulating Ca(2+) signaling and bioenergetics at the ER-mitochondrion interface.

Bcl-2 family proteins are known to competitively regulate Ca(2+); however, the specific inter-organelle signaling pathways and related cellular functions are not fully elucidated. In this study, a portion of Bcl-xL was detected at the ER-mitochondrion interface or MAM (mitochondria-associated ER membrane) in association with type 3 inositol 1,4,5-trisphosphate receptors (IP3R3); an association facilitated by the BH4 and transmembrane domains of Bcl-xL. Moreover, increasing Bcl-xL expression enhanced transient mitochondrial Ca(2+) levels upon ER Ca(2+) depletion induced by short-term, non-apoptotic incubation with thapsigargin (Tg), while concomitantly reducing cytosolic Ca(2+) release. These mitochondrial changes appear to be IP3R3-dependent and resulted in decreased NAD/NADH ratios and higher electron transport chain oxidase activity. Interestingly, extended Tg exposure stimulated ER stress, but not apoptosis, and further enhanced TCA cycling. Indeed, confocal analysis indicated that Bcl-xL translocated to the MAM and increased its interaction with IP3R3 following extended Tg treatment. Thus, the MAM is a critical cell-signaling junction whereby Bcl-xL dynamically interacts with IP3R3 to coordinate mitochondrial Ca(2+) transfer and alters cellular metabolism in order to increase the cells' bioenergetic capacity, particularly during periods of stress.

Generation of iPS Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors.

Peripheral blood is the easy-to-access, minimally invasive, and the most abundant cell source to use for cell reprogramming. The episomal vector is among the best approaches for generating integration-free induced pluripotent stem (iPS) cells due to its simplicity and affordability. Here we describe the detailed protocol for the efficient generation of integration-free iPS cells from peripheral blood mononuclear cells. With this optimized protocol, one can readily generate hundreds of iPS cell colonies from 1 ml of peripheral blood.

Myeloid leukemia factor 1 interfered with Bcl-XL to promote apoptosis and its function was regulated by 14-3-3

Myeloid leukemia factor 1 (MLF1) was involved in t(3;5) chromosomal rearrangement and aberrantly expressed in myelodysplastic syndromes/acute myeloid leukemia patients. Ex vivo experiments showed that the lymphocytes from the Mlf1-deficient mice were more resistant to apoptotic stimulations than the wild-type cells. Furthermore, the ectopically expressed MLF1 induced apoptosis in the cell models. These findings revealed that MLF1 was required for the cells to respond to the apoptotic stimulations. Ex vivo experiments also demonstrated that cytokine withdrawal significantly up-regulated Mlf1’s expression and promoted its association with B cell lymphoma-extra large (Bcl-XL) in the lymphocytes, at the same time reduced the association of Bax with Bcl-XL The same effects were also observed in the cells that over-expressed MLF1. However, these effects were observed in Mlf1 null lymphocytes as well as the cells over-expressing Bcl-XL. In addition, MLF1’s proapoptosis could be completely prevented by co-expression of Bcl-XL and significantly attenuated in Bax/Bak double null cells. These data, taken together, strongly suggested that in response to the stresses, up-regulated Mlf1 promoted its association with Bcl-XL and reduced the available Bcl-XL for associating with Bax, which resulted in releasing Bax from the Bcl-XL and apoptosis in turn. Lastly, we showed that MLF1 was negatively regulated by 14-3-3 and revealed that 14-3-3 bound to MLF1 and physically blocked MLF1’s Bcl-2 homology domain 3 (BH3) as well as Bcl-XL from associating with MLF1. Our findings suggested that ectopically expressed MLF1 could be responsible for the pathological apoptosis in early myelodysplastic syndrome (MDS) patients

The BH4 domain of anti-apoptotic Bcl-XL, but not that of the related Bcl-2, limits the voltage-dependent anion channel 1 (VDAC1)-mediated transfer of pro-apoptotic Ca2+ signals to mitochondria.

Excessive Ca(2+) fluxes from the endoplasmic reticulum to the mitochondria result in apoptotic cell death. Bcl-2 and Bcl-XL proteins exert part of their anti-apoptotic function by directly targeting Ca(2+)-transport systems, like the endoplasmic reticulum-localized inositol 1,4,5-trisphosphate receptors (IP3Rs) and the voltage-dependent anion channel 1 (VDAC1) at the outer mitochondrial membranes. We previously demonstrated that the Bcl-2 homology 4 (BH4) domain of Bcl-2 protects against Ca(2+)-dependent apoptosis by binding and inhibiting IP3Rs, although the BH4 domain of Bcl-XL was protective independently of binding IP3Rs. Here, we report that in contrast to the BH4 domain of Bcl-2, the BH4 domain of Bcl-XL binds and inhibits VDAC1. In intact cells, delivery of the BH4-Bcl-XL peptide via electroporation limits agonist-induced mitochondrial Ca(2+) uptake and protects against staurosporine-induced apoptosis, in line with the results obtained with VDAC1(-/-) cells. Moreover, the delivery of the N-terminal domain of VDAC1 as a synthetic peptide (VDAC1-NP) abolishes the ability of BH4-Bcl-XL to suppress mitochondrial Ca(2+) uptake and to protect against apoptosis. Importantly, VDAC1-NP did not affect the ability of BH4-Bcl-2 to suppress agonist-induced Ca(2+) release in the cytosol or to prevent apoptosis, as done instead by an IP3R-derived peptide. In conclusion, our data indicate that the BH4 domain of Bcl-XL, but not that of Bcl-2, selectively targets VDAC1 and inhibits apoptosis by decreasing VDAC1-mediated Ca(2+) uptake into the mitochondria.

The functional differences between pro-survival and pro-apoptotic B cell lymphoma 2 (Bcl-2) proteins depend on structural differences in their Bcl-2 homology 3 (BH3) domains.

Bcl-2 homology 3 (BH3) domains are short sequence motifs that mediate nearly all protein-protein interactions between B cell lymphoma 2 (Bcl-2) family proteins in the intrinsic apoptotic cell death pathway. These sequences are found on both pro-survival and pro-apoptotic members, although their primary function is believed to be associated with induction of cell death. Here, we identify critical features of the BH3 domains of pro-survival proteins that distinguish them functionally from their pro-apoptotic counterparts. Biochemical and x-ray crystallographic studies demonstrate that these differences reduce the capacity of most pro-survival proteins to form high affinity "BH3-in-groove" complexes that are critical for cell death induction. Switching these residues for the corresponding residues in Bcl-2 homologous antagonist/killer (Bak) increases the binding affinity of isolated BH3 domains for pro-survival proteins; however, their exchange in the context of the parental protein causes rapid proteasomal degradation due to protein destabilization. This is supported by further x-ray crystallographic studies that capture elements of this destabilization in one pro-survival protein, Bcl-w. In pro-apoptotic Bak, we demonstrate that the corresponding distinguishing residues are important for its cell-killing capacity and antagonism by pro-survival proteins.

MCL-1 but not BCL-XL is critical for the development and sustained expansion of thymic lymphoma in p53-deficient mice.

Apoptosis plays a role in normal lymphopoiesis and lymphoid malignancies. Pro-survival MCL-1 is essential for survival of T-cell progenitors, BCL-XL for immature thymocytes, and BCL-2 for mature T cells. Conversely, little is known about the regulators that are required for the survival of T-cell lymphomas. We used constitutive and conditionally gene-targeted mice to investigate which pro-survival BCL-2 family member is required for the sustained survival of thymic lymphomas initiated by loss of p53. Constitutive loss of a single Mcl-1 allele delayed tumor onset. In contrast, lymphomas emerging in p53(-/-) mice in which Mcl-1 could be conditionally deleted had been selected for retention of MCL-1 expression. In contrast, complete loss of BCL-XL had no impact on lymphoma development in p53(-/-) mice. These results demonstrate that thymic lymphomas elicited by loss of p53 must arise from cancer-initiating cells that require MCL-1 for their survival. Acute deletion of both Mcl-1 alleles abrogated the expansion of p53(-/-) lymphomas in mice, whereas inducible loss of BCL-XL had little impact. This reveals that MCL-1 is essential for the sustained survival of these malignant cells and suggests that targeting MCL-1 may be an attractive strategy for the treatment of T-cell lymphoma.

PI3K/Akt regulates survival during differentiation of human macrophages by maintaining NF-κB-dependent expression of antiapoptotic Bcl-xL.

Resistance to apoptosis is an important characteristic that human macrophages acquire during differentiation from monocytes. However, the intracellular mechanisms that mediate the development of resistance are not well understood. We have used M-CSF-stimulated primary human monocytes and PMA-treated THP1 cells to study apoptosis resistance during differentiation of human macrophages. Our results indicate that PI3K/Akt distinctively regulates survival of macrophages during and after differentiation. More specifically, a signaling pathway consisting of PI3K/Akt-NF-κB-Bcl-xL regulates cell survival during the differentiation process. PI3K/Akt-mediated activation of NF-κB plays a key role in survival of differentiating macrophages by specifically sustaining antiapoptotic Bcl-xL expression. With the use of pharmacological inhibitors and siRNA for Akt and Bcl-xL, we show that in the absence of Akt-dependent Bcl-xL expression during differentiation, cells undergo caspase-mediated apoptosis. In contrast, in differentiated macrophages, Bcl-xL expression is independent of PI3K/Akt activation. Taken together, these results suggest that survival of macrophages is distinctly regulated during and after differentiation. Our results also suggest new, potential therapeutic targets to modulate differentiation and survival of this cell type.

B cell lymphoma-2 (BCL-2) homology domain 3 (BH3) mimetics demonstrate differential activities dependent upon the functional repertoire of pro- and anti-apoptotic BCL-2 family proteins.

The B cell lymphoma-2 (BCL-2) family is the key mediator of cellular sensitivity to apoptosis during pharmacological interventions for numerous human pathologies, including cancer. There is tremendous interest to understand how the proapoptotic BCL-2 effector members (e.g. BCL-2-associated X protein, BAX) cooperate with the BCL-2 homology domain only (BH3-only) subclass (e.g. BCL-2 interacting mediator of death, BIM; BCL-2 interacting-domain death agonist, BID) to induce mitochondrial outer membrane permeabilization (MOMP) and apoptosis and whether these mechanisms may be pharmacologically exploited to enhance the killing of cancer cells. Indeed, small molecule inhibitors of the anti-apoptotic BCL-2 family members have been designed rationally. However, the success of these "BH3 mimetics" in the clinic has been limited, likely due to an incomplete understanding of how these drugs function in the presence of multiple BCL-2 family members. To increase our mechanistic understanding of how BH3 mimetics cooperate with multiple BCL-2 family members in vitro, we directly compared the activity of several BH3-mimetic compounds (i.e. ABT-263, ABT-737, GX15-070, HA14.1, TW-37) in biochemically defined large unilamellar vesicle model systems that faithfully recapitulate BAX-dependent mitochondrial outer membrane permeabilization. Our investigations revealed that the presence of BAX, BID, and BIM differentially regulated the ability of BH3 mimetics to derepress proapoptotic molecules from anti-apoptotic proteins. Using mitochondria loaded with fluorescent BH3 peptides and cells treated with inducers of cell death, these differences were supported. Together, these data suggest that although the presence of anti-apoptotic BCL-2 proteins primarily dictates cellular sensitivity to BH3 mimetics, additional specificity is conferred by proapoptotic BCL-2 proteins.

Bcl-xL protein protects from C/EBP homologous protein (CHOP)-dependent apoptosis during plasma cell differentiation.

Although it is known that the unfolded protein response (UPR) plays a significant role in the process of plasma cell differentiation, the contribution of the individual sensors of the UPR to this process remains unclear. In this study we examine the death signals and compensatory survival signals activated during B cell activation and the first stages of plasma cell differentiation. During in vitro differentiation of both primary murine B cells and the Bcl1 cell line, we demonstrate that in addition to activation of the physiological UPR, changes in the expression of several Bcl-2 proteins occur, which are consistent with a lowering of the apoptotic threshold of the cell. Specifically, we observed decreased expression of Bcl-2 and Mcl-1 and increased expression of the proapoptotic protein Bim. However, these changes were countered by Bcl-xL induction, which is necessary to protect differentiating cells both from ER stress-induced death by tunicamycin and from the death signals inherent in differentiation. Consistent with differentiating cells becoming dependent on Bcl-xL for survival, the addition of ABT-737 resulted in apoptosis in differentiating cells through the inhibition of sequestration of Bim. Confirming this result, differentiation in the context of RNAi-mediated Bcl-xL knockdown also induced apoptosis. This cell death is C/EBP homologous protein (CHOP)-dependent, connecting these events to the UPR. Thus plasma cell differentiation proceeds through a Bcl-xL-dependent intermediate.