Pro-apoptotic BH3-only Protein Bim Research Articles

Proteasome inhibition upregulates Bim and induces caspase-3-dependent apoptosis in human mast cells expressing the Kit D816V mutation

The majority of patients with systemic mastocytosis exhibit a D816V mutation in the activating loop of the Kit receptor expressed on mast cells. The Kit ligand regulates mast cell survival by transcriptional repression of the proapoptotic BH3-only protein Bim and by promoting Bim phosphorylation that makes it vulnerable for proteasomal-dependent degradation. We investigated here whether prevention of Bim degradation by a proteasomal inhibitor, MG132, would induce apoptosis in mast cells with the D816V mutation. Human umbilical cord blood-derived mast cells (CBMCs) with wild-type (wt) Kit and two different subclones of the human mast cell line-1 (HMC-1) were used for the study: HMC-1.1 with the V560G mutation in the juxtamembrane domain and HMC-1.2 carrying the V560G mutation together with the D816V mutation. MG132 at 1 μM induced apoptosis in all cell types, an effect accompanied by increased BH3-only proapoptotic protein Bim. The raise of Bim was accompanied by caspase-3 activation, and a caspase-3 inhibitor reduced MG132-induced apoptosis. Further, MG132 caused a reduction of activated Erk, a negative regulator of Bim expression, and thus Bim upregulation. We conclude that decreased phosphorylation and increased levels of Bim overcome the prosurvival effect of the D816V mutation and that the results warrant further investigations of the clinical effects of proteasomal inhibition in systemic mastocytosis.

BIM doubles its responsibilities

The pro-apoptotic BH3-only protein BIM is inactivated by binding to the dynein light chain protein LC8. However, under conditions of stress BIM is phosphorylated by Jun N-terminal kinase (JNK), which leads to its dissociation from LC8 and localization at mitochondria, where it can induce apoptosis. This study finds that the interaction of BIM with LC8 also regulates autophagy. Under steady-state conditions, LC8 mediates binding of apoptosis-inactive BIM to BECLIN1, leading to the mislocalization of the latter from the endoplasmic reticulum to microtubules, where it cannot promote autophagosome formation and maturation. However, under starvation conditions BIM is phosphorylated by JNK, thereby releasing BECLIN1 and allowing autophagy induction. Consistent with this, loss of BIM leads to enhanced autophagosome induction. Thus, similarly to anti-apoptotic BH3-only proteins, BIM has a key role in inhibiting autophagy under steady-state conditions.

Effect of auranofin on oxidative and endoplasmic reticulum stress as well as anti-CLL activity with proteasome inhibitor.

e13568 Background: The gold-containing compound auranofin (AF) (Ridaura), is a treatment of rheumatoid arthritis. Recently, AF was shown to inhibit thioredoxin reductase (TRR), increase reactive oxygen species (ROS) and induce apoptosis in cancer cells. Methods: We determined the ability of AF (250 to 1000 nM) to induce oxidative, proteotoxic and lethal endoplasmic reticulum (ER) stress in CD19+ primary CLL cells, including those with deletion of chromosome 17p. Results: Treatment with AF induced 20 to 40% increase in ROS levels, decreased TRR activity (mean of 45%), but not its protein expression, in CLL cells. AF-mediated oxidative stress induced NRF2 activity with increase in hemeoxygenase-1 (HO-1) and glutamate cysteine ligase modifier (GCLM) levels. AF induced ER stress, associated with increase in GRP78 and the pro-apoptotic transcription factor CHOP protein levels. AF also induced the pro-apoptotic BH3-only domain protein BIM. Exposure to AF increased the intracellular levels of misfolded polyubiquitylated proteins. This disrupted the binding of heat shock protein (hsp) 90 with histone deacetylase 6 (HDAC6), resulting in hyperacetylation and inhibition of the chaperone function of hsp90. This led to proteasomal degradation of CLL-relevant hsp90 client proteins, including ZAP70, c-Raf and AKT. Nuclear STAT3 levels also declined. Exposure to AF induced significantly more apoptosis (range 40 to 60%) in primary CLL cells, as compared to CD19+ normal B cells and CD34+ human cord blood and bone marrow progenitor cells (< 15% apoptosis) (p < 0.01). AF treatment also induced apoptosis in CD19+ cells from patients with poor prognosis CLL with deletion of 17p or with deletion of 13q. Co-treatment with AF and the proteasome inhibitor carfilzomib or the GCLM antagonist buthionine sulfoximine (BSO) synergistically induced apoptosis in primary CLL cells. A FDA IND-supported phase II clinical trial of AF (6 to 9 mg PO/day) with correlative biomarker analysis (as above) is underway and three patients are enrolled. Conclusions: These findings support the full evaluation of clinical activity and predictive biomarkers of response to the re-purposed AF in patients with CLL.

Abstract 4881: Induction of Bim by the BRAF inhibitor PLX4720 sensitizes human melanoma cells to the BH3 mimetic ABT-737

Abstract Once melanoma has metastasized to distant sites it has proved difficult to treat by a variety of chemotherapeutic and biological agents. Significant advances have been made in identifying subgroups of patients with activating mutations in signalling pathway proteins. In particular, the finding that approximately 50% of melanoma have V600E mutations in the BRAF serine/threonine kinase has resulted in the generation of a number of new drugs which target the mutated active form of BRAF. One of these drugs, Vemurafenib (PLX4032/RG704) has shown significant promise in prolonging survival compared to standard chemotherapy, however reports of patient relapse has prompted investigation into means of overcoming acquired resistance to the agent. We report here that efficacy of the PLX4032 analogue, PLX4720, is greatly enhanced by co-treatment with the BH3 mimetic, ABT-737. Treatment with either agent is able to induce apoptosis in melanoma cells, however the kinetics are typically slow, with cell death occurring 48 or 72 hours post treatment. In this study, while either agent was largely ineffective as a single treatment 24 hours following treatment, addition of the combination of drugs rapidly induced significant killing. We show that apoptosis was caspase-dependent, in that inhibition of caspase activation almost totally blocked induction of cell death by the combination of agents. Apoptosis was also associated with activation of the intrinsic apoptotic pathway, with reduction of mitochondrial membrane potential and activation of caspase-9. Exposure of melanoma cells to PLX4720 rapidly decreased phosphorylation of the pro-apoptotic BH3-only protein Bim, resulting in strong upregulation as early as 6 hours after addition of the compound. Strong upregulation of Bim was shown by RNAi knockdown to play an important role in sensitizing cells to co-treatment with ABT-737, by assisting in the inhibition of anti-apoptotic Bcl-2 family proteins. Further, the combination of agents induced significant levels of apoptosis in melanoma cells derived from patients harbouring the BRAFV600E mutation. Collectively, these results show that the BRAF inhibitor PLX4720 sensitizes melanoma cells to apoptosis by the BH3 mimetic ABT-737 in a Bim- and caspase-dependent manner. Additionally, these studies identify a potential clinical application for BH3 mimetics in overcoming resistance of melanoma to treatment with BRAF inhibitors, increasing efficacy of this class of agent and improving patient outcomes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4881. doi:1538-7445.AM2012-4881

Defective cell death signalling along the Bcl-2 regulated apoptosis pathway compromises Treg cell development and limits their functionality in mice

The Bcl-2 regulated apoptosis pathway is critical for the elimination of autoreactive lymphocytes, thereby precluding autoimmunity. T cells escaping this process can be kept in check by regulatory T (Treg) cells expressing the transcription and lineage commitment factor Foxp3. Despite the well-established role of Bcl-2 family proteins in shaping the immune system and their frequent deregulation in autoimmune pathologies, it is poorly understood how these proteins affect Treg cell development and function. Here we compared the relative expression of a panel of 40 apoptosis-associated genes in Treg vs. conventional CD4+ T cells. Physiological significance of key-changes was validated using gene-modified mice lacking or overexpressing pro- or anti-apoptotic Bcl-2 family members. We define a key role for the Bim/Bcl-2 axis in Treg cell development, homeostasis and function but exclude a role for apoptosis induction in responder T cells as relevant suppression mechanism. Notably, only lack of the pro-apoptotic BH3-only protein Bim or Bcl-2 overexpression led to accumulation of Treg cells while loss of pro-apoptotic Bad, Bmf, Puma or Noxa had no effect. Remarkably, apoptosis resistant Treg cells showed reduced suppressive capacity in a model of T cell-driven colitis, posing a caveat for the use of such long-lived cells in possible therapeutic settings.

Alternative splicing of Bim and Erk-mediated BimEL phosphorylation are dispensable for hematopoietic homeostasis in vivo

The pro-apoptotic BH3-only protein Bim has a major role in hematopoietic homeostasis, particularly in the lymphocyte compartment, where it strongly affects immune function. The three major Bim isoforms (Bim(EL), Bim(L) and Bim(S)) are generated by alternative splicing. Bim(EL), the most abundant isoform, contains a unique sequence that has been reported to be the target of phosphorylation by several MAP kinases. In particular, Erk1/2 has been shown to interact with Bim(EL) through the DEF2 domain of Bim(EL) and specifically phosphorylate this isoform, thereby targeting it for ubiquitination and proteasomal degradation. To examine the physiological importance of this mechanism of regulation and of the alternative splicing of Bim, we have generated several Bim knock-in mouse strains and analyzed their hematopoietic system. Although mutation in the DEF2 domain reduces Bim(EL) degradation in some circumstances, this mutation did not significantly increase Bim's pro-apoptotic activity in vivo nor impact on the homeostasis of the hematopoietic system. We also show that Bim(EL) and Bim(L) are interchangeable, and that Bim(S) is dispensable for the function of Bim. Hence, we conclude that physiological regulation of Bim relies on mechanisms independent of its alternative splicing or the Erk-dependent phosphorylation of Bim(EL).

The proapoptotic protein Bim is not differentially regulated in CD8+ T cells from resolved or chronic HCV infection

Background: CD8 T cells play an important role in the antiviral immune response against HCV. Nevertheless, these cells fail to control the viral replication in patients with chronic infection either due to mutational escape of the virus in targeted epitopes or a dysfunctional phenotype of virus-specific CD8 T cells. Moreover, deletion of virus-specific CD8 T cells due to premature apoptosis upon their activation in the liver has been suggested. The pro-apoptotic BH3-only protein Bim has been found to be upregulated in CD8+ T cells activated by hepatocytes in mouse models as well as in patients chronically infected with HBV. Here, we addressed whether this molecule also plays a role in in HCV infection. Methods: PBMC were collected from anti-HCV positive injection drug users with chronic or spontaneously resolved HCV infection. From this cohort 13 “resolvers” and 13 “chronics” with detectable CD8 T cells by staining with HLA/peptide-multimers ex vivo were used for this study. The expression of Bim and the activation marker CD38 was analysed on HCV-, CMV- and Flu-specific CD8 T cells ex vivo and after 7 days of antigen-specific expansion via flow cytometry. Results: No significant difference in the expression level of Bim or CD38 could be observed in HCV-specific CD8+ T cells from patients with resolved infection compared to those with chronic infection ex vivo. After 7 days of antigen-specific expansion, both Bim and CD38 were upregulated on antigen-specific CD8 T cells, irrespective of the disease status. Both molecules were also upregulated in bulk CD8 T cells after unspecific activation with anti-CD3. There was also no difference between the Bim expression in HCV-specific CD8 T cells and Flu- or CMV-specific CD8 T cells. Conclusions: The pro-apoptotic molecule Bim is upregulated in CD8 T cells upon their activation, however, we find no evidence for differential regulation in HCV-specific cells from patients with chronic or spontaneously resolved HCV-infection.

Bim Is Required to Sensitize Mantle Cell Lymphoma Cells for Killing by Bortezomib, but Not Ara-C, by Selective Inhibition of CDK4/CDK6

Abstract 1655Mantle cell lymphoma (MCL) is characterized by aberrant cyclin D1 expression due to the t (11: 14) translocation. In conjunction with elevation of CDK4/CDK6, this promotes cell cycle progression through G1 and unrestrained cell proliferation. As MCL remains incurable despite initial response to therapy, mechanism- and genome-based therapies that both control the cell cycle and enhance cytotoxic killing are urgently needed. We have recently developed such a regimen by inhibition of CDK4/CDK6 with PD 0332991 (PD), a selective inhibitor of CDK4 and CDK6 that is also potent, reversible and orally bioavailable. We demonstrate that 1) inhibition of CDK4/CDK6 with PD leads to early G1 arrest; 2) upon release of the G1 block, synchronous cell cycle progression to S phase occurs; and 3) S phase synchronization following prolonged early G1 arrest (pG1-S) sensitizes MCL cells to killing by diverse clinically relevant agents at reduced doses, including proteasome inhibitors bortezomib and carfilzomib, and the nucleoside analog Ara-C (cytarabine), both in vitro and in a mouse model of MCL. These findings implicate a unified mechanism for cell cycle sensitization of cytotoxic killing.To elucidate the underpinning mechanism, we show that sensitization to cytotoxic killing by CDK4/CDK6 inhibition requires an intact Rb, the substrate of CDK4/CDK6, but is independent of p53. Gene expression profiling and quantitative RNA and protein analyses further demonstrate that prolonged inhibition of CDK4/CDK6 with PD halts the gene expression program in early G1 and depletes the expression of genes programmed for other phases of the cell cycle, such as cyclin A (G1/S), thymidine kinase (S), CDK1 and cyclin B (G2/M) and selective metabolic genes. Removal of PD restores the CDK4/CDK6 activities and the expression of scheduled cell cycle genes but leaves many others in the pG1 state. This leads to S phase synchronization with impaired metabolism. Accordingly, the magnitude of bortezomib and Ara-C killing in pG1-S greatly exceeds the enrichment of S phase cells. Selective inhibition of CDK4/CDK6, therefore, sensitizes MCL cells for cytotoxic killing in S phase synchronization through induction of a persistent metabolic imbalance in prior pG1.pG1 alone induces caspase activation moderately in MCL cells, but markedly augments apoptosis induced by either bortezomib or Ara-C in pG1-S. This enhancement of apoptosis is apparently mediated by an alteration of the ratios of pro-apoptotic BH3-only proteins (Bim, Noxa and Puma) to anti-apoptotic proteins (Mcl-1, Bcl-2 and Bcl-xL), which lowers the threshold for caspase-9 activation. Importantly, Bim is selectively required to sensitize MCL cells for killing by bortezomib, but not Ara-C, at low doses as indicated in studies of Bim-deficient MCL cell lines. Corroborating these findings, loss of one allele of Bim attenuates the enhancement of bortezomib killing in pG1-S in untransformed primary mouse B cells after activation by BCR and CD40 signaling. Thus, the synergistic actions of PD-bortezomib and PD-AraC in MCL therapy are distinguishable by the requirement for Bim.Furthermore, we found that the three Bim isoforms are expressed at variable levels but undetected in 30% of primary MCL tumor cells, consistent with the reported mutations and bi-allelic deletion of Bim (BCL2L11) in MCL. RNA-Seq analysis of samples from patients enrolled in a phase I study of PD in combination with bortezomib in MCL further reveals that the mutation burden in BCL2L11 is ∼3-fold higher in a clinically non-responder compared with a responder. Collectively, our data demonstrate that by halting scheduled gene expression in prolonged early G1 arrest, selective and reversible inhibition of CDK4/CDK6 provides a mechanism-based strategy to sensitize MCL cells for cytotoxic killing by bortezomib, Ara-C, and potentially other emerging agents. By lowering the threshold for caspase activation, Bim is selectively required for sensitization to killing by low dose bortezomib, but not Ara-C, and may serve as a biomarker for genome-based selection of cytotoxic partners in therapeutic targeting of CDK4/CDK6 in MCL. Disclosures:Martin:Millennium Pharmaceuticals, Inc.: Research Funding, Speakers Bureau. Smith:Pfizer: Research Funding; Millenium: Research Funding. Leonard:Pfizer, Inc.: Consultancy; Millenium: Consultancy; Johnson and Johnson: Consultancy; Onyx: Consultancy. Chen-Kiang:Pfizer, Inc.: Research Funding.

Bone Marrow Stromal Cells Enforce MCL-1 Dependence In Multiple Myeloma Through the Secretion of A Soluble Factor,

Abstract 3950It is well established that the bone marrow microenvironment contributes to the growth and survival of multiple myeloma plasma cells, however the molecular mechanisms that control these changes within the myeloma cell have not been completely elucidated. We have recently demonstrated that myeloma cell lines can be dependent on the anti-apoptotic protein MCL-1 or can be co-dependent on MCL-1 and Bcl-2/xL. The primary distinction between MCL-1 dependence and co-dependence is the distribution of the pro-apoptotic BH3-only protein Bim. In MCL-1-dependent lines, Bim is primarily associated with MCL-1. In contrast in co-dependent lines Bim is either predominantly associated with Bcl-2/xL or when it is released from Bcl-2/xL it can not bind to MCL-1 because of the presence of the MCL-1 inhibitor, Noxa. This renders these cells sensitive to the Bcl-2/xL inhibitor ABT-737. We have confirmed these findings in freshly isolated myeloma cells and demonstrated that among the 4 patient samples tested, Bim was associated with both MCL-1 and Bcl-xL and the cells were sensitive to ABT-737. This suggests that co-dependence on MCL-1 and Bcl-xL may be a common phenomena in myeloma. We now demonstrate that in about 50% of patient samples tested, CD138 column-purified MM cells are more sensitive to ABT-737 than cells that were not purified following ficoll separation of the bone marrow aspirate. This suggests that stromal components in the marrow alter the dependence on Bcl-2 proteins resulting in a cell that is more MCL-1 dependent when in the presence of stroma. To directly test this possibility we determined the effect of co-culturing the co-dependent line, MM.1s with the human stromal line Hs-5 on ABT-737 (0.6 mM) sensitivity and Bim binding. MM1.s cells were incubated with Hs-5 cells for 30 min prior to addition of drug or were cultured in medium containing 50% conditioned medium (48 h growth) from Hs-5 cells to determine if any effects observed were due to a soluble factor. Following a 24 h culture, cells were subjected to CD138 column purification and apoptosis analyzed by Annexin V-FITC/PI flow cytometry. While mono-cultured MM1.s were very sensitive to ABT-737 (88.5% +/− 3.6% control apoptosis, mean +/− SE), the addition of Hs-5 cells or conditioned medium had a profound effect on cell survival resulting in significant resistance to Bcl-2/xL inhibition (23% +/− 4.4% and 24.5% +/− 2.3% control apoptosis respectively). To determine the molecular basis in this shift from co-dependence to MCL-1 dependence, western blot and co-immunoprecipitation analyses were performed. While neither co-culture nor conditioned medium had any significant effect on the expression of levels of Bim, MCL-1 or Bcl-xL, both conditions resulted in a shift of Bim binding from being equally split amongst MCL-1 and Bcl-xL to a binding pattern where Bim was preferentially bound to MCL-1. This is consistent with loss of sensitivity to ABT-737 and a shift to MCL-1 dependence. We next tested the effects of Hs5 co-culture and conditioned medium on other inducers of cell death. We focused on bortezomib (5 nM) and arsenic trioxide (2 mM) as both can induce the MCL-1 inhibitor Noxa, therefore sensitivity would not be expected to be significantly influenced by a change from co-dependence to MCL-1 dependence. Consistent with this possibility we found that bortezomib induced cell death was only modestly affected by co-culture (79% +/− 2.3% vs. 66.5% +/− 1.9% control apoptosis) and no protection was observed with Hs5 conditioned medium. Similar results were observed with arsenic trioxide (monoculture – 31% +/− 2.3%, Hs5 co-culture −15% +/− 1.0%, conditioned medium – 30% +/− 4.2% control apoptosis). Taken together these data demonstrate that a soluble factor produced by stromal support can alter Bim binding to anti-apoptotic Bcl-2 family members, rendering myeloma cells primarily dependent on MCL-1 and protecting them from cell death signals that function through inhibition of Bcl-2/xL. Moreover these data in part explain why bortezomib can overcome survival signaling provided by stromal support, as proteasome inhibition is capable of inducing the MCL-1 inhibitor Noxa. Disclosures:Kaufman:Millenium: Consultancy; Onyx Pharmaceuticals: Consultancy; Novartis: Consultancy; Keryx: Consultancy; Merck: Research Funding; Celgene: Research Funding. Boise:University of Chicago: Patents & Royalties.

The MEK-ERK pathway negatively regulates bim expression through the 3' UTR in sympathetic neurons

BackgroundApoptosis plays a critical role during neuronal development and disease. Developing sympathetic neurons depend on nerve growth factor (NGF) for survival during the late embryonic and early postnatal period and die by apoptosis in its absence. The proapoptotic BH3-only protein Bim increases in level after NGF withdrawal and is required for NGF withdrawal-induced death. The regulation of Bim expression in neurons is complex and this study describes a new mechanism by which an NGF-activated signalling pathway regulates bim gene expression in sympathetic neurons.ResultsWe report that U0126, an inhibitor of the prosurvival MEK-ERK pathway, increases bim mRNA levels in sympathetic neurons in the presence of NGF. We find that this effect is independent of PI3-K-Akt and JNK-c-Jun signalling and is not mediated by the promoter, first exon or first intron of the bim gene. By performing 3' RACE and microinjection experiments with a new bim-LUC+3'UTR reporter construct, we show that U0126 increases bim expression via the bim 3' UTR. We demonstrate that this effect does not involve a change in bim mRNA stability and by using PD184352, a specific MEK1/2-ERK1/2 inhibitor, we show that this mechanism involves the MEK1/2-ERK1/2 pathway. Finally, we demonstrate that inhibition of MEK/ERK signalling independently reduces cell survival in NGF-treated sympathetic neurons.ConclusionsThese results suggest that in sympathetic neurons, MEK-ERK signalling negatively regulates bim expression via the 3' UTR and that this regulation is likely to be at the level of transcription. This data provides further insight into the different mechanisms by which survival signalling pathways regulate bim expression in neurons.

Abstract 2151: Characterization of a novel anti-apoptotic signaling pathway downstream of FGFR4 in rhabdomyosarcoma

Abstract FGFR4 is highly overexpressed in Rhabdomyosarcoma tumors and contains activating mutations in eight percent of the cases. These data strongly suggest that FGFR4 plays an important role in the tumorigenesis of this cancer type. However, the molecular mechanisms behind the benefit of active FGFR4 for Rhabdomyosarcoma cells are largely unknown. Here, we describe a survival pathway activated by FGFR4 in Rhabdomyosarcoma cells. Active FGFR4 is able to block apoptosis induced by inhibition of other central signaling pathways with small molecule inhibitors, such as the IGF1R pathway with AEW541 and PI3K-controlled pathways with BEZ235. Using different biochemical and genetic approaches, we could locate the apoptotic block upstream of the pro-apoptotic BH3-only proteins bmf and bim. To further elucidate the mechanism of this prosurvival signaling, we then performed proteomic studies to define the complete interactome of FGFR4 in Rhabdomyosarcoma cells. These analyses revealed a strong interaction of FGFR4 with PLCγ, linking FGFR signaling to PKCs. Indeed, activation of PKCs strongly inhibits AEW541/BEZ253-induced apoptosis, suggesting that PKCs are central for FGFR4-activated survival signaling. In contrast, the classical survival pathways via Akt and Erk are not or only marginally involved in the rescue mechanism. Treatment approaches targeting the IGF1R/PI3K axis in Rhabdomyosarcoma are under intensive investigation. Our data suggest that rescue mechanisms via FGFR4 and PKCs should be taken into account when applying such treatment strategies. A combination of IGF1R- and FGFR4/PKC-inhibitors therefore might be superior compared to single treatments. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2151. doi:10.1158/1538-7445.AM2011-2151

Egr-1 TransactivatesBimGene Expression to Promote Neuronal Apoptosis

The proapoptotic BH3-only protein Bim is a crucial regulator of neuronal apoptosis. Previous studies have indicated the involvement of the c-Jun, FOXO1/3a, and B/C-Myb transcription factors in the regulation of Bim during neuronal apoptosis. However, the mechanism underlying the transcriptional regulation of Bim in activity deprivation-induced neuronal apoptosis has remained unclear. The present study demonstrates that early growth response 1 (Egr-1), rather than c-Jun, FOXO1/3a, or B/C-Myb, directly transactivates Bim gene expression to mediate apoptosis of rat cerebellar granule neurons. We showed that Egr-1 was sufficient and necessary for neuronal apoptosis. Suppression of Egr-1 activity using dominant-negative mutant or knockdown of Egr-1 using small interfering RNAs led to a decrease in Bim expression, whereas overexpression of Egr-1 resulted in induction of Bim. Deletion and site-directed mutagenesis of the Bim promoter revealed that Bim transcriptional activation depends primarily on a putative Egr-binding sequence between nucleotides -56 and -47 upstream of the start site. We also showed that Egr-1 binding to this sequence increased in response to activity deprivation in vitro and in vivo. Moreover, inhibition of Egr-1 binding to the Bim promoter, by mithramycin A and chromomycin A3, reduced the activity deprivation-induced increases in Bim promoter activity and mRNA and protein levels and protected neurons from apoptosis, further supporting the Egr-1-mediated transactivation of Bim. Additionally, Bim overcame the Egr-1 knockdown-mediated inhibition of apoptosis, whereas Bim knockdown impaired the increase in apoptosis induced by Egr-1. These findings establish Bim as an Egr-1 target gene in neurons, uncovering a novel Egr-1/Bim pathway by which activity deprivation induces neuronal apoptosis.

Structural Basis for Apoptosis Inhibition by Epstein-Barr Virus BHRF1

Epstein-Barr virus (EBV) is associated with human malignancies, especially those affecting the B cell compartment such as Burkitt lymphoma. The virally encoded homolog of the mammalian pro-survival protein Bcl-2, BHRF1 contributes to viral infectivity and lymphomagenesis. In addition to the pro-apoptotic BH3-only protein Bim, its key target in lymphoid cells, BHRF1 also binds a selective sub-set of pro-apoptotic proteins (Bid, Puma, Bak) expressed by host cells. A consequence of BHRF1 expression is marked resistance to a range of cytotoxic agents and in particular, we show that its expression renders a mouse model of Burkitt lymphoma untreatable. As current small organic antagonists of Bcl-2 do not target BHRF1, the structures of it in complex with Bim or Bak shown here will be useful to guide efforts to target BHRF1 in EBV-associated malignancies, which are usually associated with poor clinical outcomes.

Modulation of Bcl-2-related protein expression in pancreatic beta cells by pro-inflammatory cytokines and its dependence on the antioxidative defense status

Pro-inflammatory cytokines are key mediators in the selective and progressive destruction of insulin-producing beta cells during type 1 diabetes development. However, the mechanisms of cytokine-induced beta cell apoptosis are not fully understood.This study demonstrates that pro-inflammatory cytokines strongly modified the expression of the anti-apoptotic protein Bcl-2 and the pro-apoptotic BH3-only proteins Bad, Bim, and Bid in primary rat islets and insulin-producing RINm5F cells. Overexpression of mitochondrially located catalase (MitoCatalase) specifically increased basal Bcl-2 and decreased basal Bax expression, suppressed cytokine-mediated reduction of Bcl-2, and thereby prevented the release of cytochrome c, Smac/DIABLO and the activation of caspase-9 and -3. Thus, cytokine-mediated decrease of Bcl-2 expression and the sequentially changed Bax/Bcl-2 ratio are responsible for the release of pro-apoptotic mitochondrial factors, activation of caspase-9, and ultimately caspase-3.These results indicate that activation of the intrinsic/mitochondrial apoptosis pathway is essential for cytokine-induced beta cell death and the mitochondrial generation of reactive oxygen species, in particular mitochondrial hydrogen peroxide, differentially regulates the Bax/Bcl-2 ratio.

Adenovirus 5 E1A enhances histone deacetylase inhibitors-induced apoptosis through Egr-1-mediated Bim upregulation

Histone deacetylase inhibitors (HDACi) are potent anti-cancer agents for variety of cancer types. Suberoylanilide hydroxamic acid (SAHA) has been approved as a drug to treat cutaneous T cell lymphoma, and the combination of HDACi and other agents have been actively tested in many clinical trials. Adenovirus 5 early region 1A (E1A) has been shown to exhibit high tumor suppressor activity, and gene therapy using E1A has been tested in clinical trials. Here, we showed that proapoptotic activity of HDACi was robustly enhanced by E1A in multiple cancer cells, but not in normal cells. Moreover, we showed that combination of E1A gene therapy and SAHA showed high therapeutic efficacy with low toxicity in vivo ovarian and breast xenograft models. SAHA downregulated Bcl-XL and upregulated proapoptotic BH3-only protein Bim, whose expression was further enhanced by E1A in cancer cells. These alterations of Bcl-2 family proteins were critical for apoptosis induced by the combination in cancer cells. SAHA enhanced acetylation of histone H3 in Bim promoter region, while E1A upregulated Egr-1, which was directly involved in Bim transactivation. Together, our results provide not only a novel insight into the mechanisms underlying anti-tumor activity of E1A, but also a rationale for the combined HDACi and E1A gene therapy in future clinical trials.

Sphingosine kinase regulates oxidized low density lipoprotein-mediated calcium oscillations and macrophage survival

We recently reported that oxidized LDL (oxLDL) induces an oscillatory increase in intracellular calcium ([Ca(2+)](i)) levels in macrophages. Furthermore, we have shown that these [Ca(2+)](i) oscillations mediate oxLDL's ability to inhibit macrophage apoptosis in response to growth factor deprivation. However, the signal transduction pathways by which oxLDL induces [Ca(2+)](i) oscillations have not been elucidated. In this study, we show that these oscillations are mediated in part by intracellular mechanisms, as depleting extracellular Ca(2+) did not completely abolish the effect. Inhibiting sarco-endoplasmic reticulum ATPase (SERCA) completely blocked [Ca(2+)](i) oscillations, suggesting a role for Ca(2+) reuptake by the ER. The addition of oxLDL resulted in an almost immediate activation of sphingosine kinase (SK), which can increase sphingosine-1-phosphate (S1P) levels by phosphorylating sphingosine. Moreover, S1P was shown to be as effective as oxLDL in blocking macrophage apoptosis and producing [Ca(2+)](i) oscillations. This suggests that the mechanism in which oxLDL generates [Ca(2+)](i) oscillations may be 1) activation of SK, 2) SK-mediated increase in S1P levels, 3) S1P-mediated Ca(2+) release from intracellular stores, and 4) SERCA-mediated Ca(2+) reuptake back into the ER.

Abstract 2623: Induction of Bmf and Bim in Vorinostat-induced apoptosis in mantle cell lymphoma

Abstract OBJECTIVES: Mantle cell lymphoma (MCL) is an incurable B-cell neoplasm harboring the t(11;14)(q13;q32) which leads to the overexpression of cyclin D1, with the consequent cell cycle deregulation. Usually, MCL is characterized by bad prognosis and an aggressive course of the disease. Current therapies have shown limited efficacy. Recently, Histone Deacetylase Inhibitors (HDACis) have been successfully introduced for the treatment of several cancers, including hematological malignancies. Accordingly, tumor suppressor genes are frequently epigenetically silenced in these entities, due to histone deacetylation in their promoters. Therefore, our purposes were to evaluate the antitumoral properties of the HDACi suberoylanilide hydroxamic acid (Vorinostat; Merck & Co) in MCL, and to describe the molecular mechanisms involved in HDACi signaling in this disorder. METHODS: MCL cell lines and primary MCL cells were used. HDAC activity was measured using a colorimetric HDAC Assay Kit. Flow cytometry was used to determine sensitivity to Vorinostat through measurement of AnnexinV binding, and to analyze apoptosis features. Vorinostat anti-tumoral signaling was evaluated by RQ-PCR determination of gene transcription, western blot analysis and acetyl Histone H4 ChIP assays. RESULTS: Vorinostat exhibited a heterogeneous cytotoxic effect among MCL cell lines, with a median LD50 of 6.6 μM after 24-hour incubation. Nevertheless, cytotoxicity increased notably after 48h of exposure to the drug with LD50 ranging from 0.4 to 5.3 μM. Interestingly, 7 out of the 10 MCL primary samples tested were extremely sensitive to the compound (with a median LD50 of 2.2 μM after 24-hour incubation). Vorinostat increases the acetylation of H3 and H4 histones, as well as inhibits global HDAC activity in just 1 hour of incubation. The drug notably decreases cyclin D1 protein levels while induces upregulation of the proapoptotic BH3-only proteins Bmf and Bim, triggering the mitochondria-dependent cell death and activation of the caspases cascade. Acetyl Histone H4 ChIP assays showed that Vorinostat increases acetylation of BMF and BIM gene promoters, consequently up regulating Bim and Bmf mRNA levels. CONCLUSIONS: This study suggests that Vorinostat could define a new and attractive therapeutic approach for the treatment of MCL. We identify BMF and BIM as possible crucial target genes of HDAC inhibitors in MCL cells, promoting the induction of mitochondria-mediated apoptosis. GRANTS: This work is supported by Ministerio de Ciencia e Innovación SAF 06-8850; S. X-T is a fellow from Ministerio de Ciencia e Innovación (FPU predoctoral fellowship). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2623.

The proapoptotic BH3-only protein Bim is downregulated in a subset of colorectal cancers and is repressed by antiapoptotic COX-2/PGE2 signalling in colorectal adenoma cells

Overexpression of cyclooxygenase-2 (COX-2) and elevated levels of its enzymatic product prostaglandin E2 (PGE2) occur in the majority of colorectal cancers and play important roles in colorectal tumorigenesis. However, despite the established prosurvival role of PGE2 in cancer, the underlying mechanisms are not fully understood. Here, we have shown that PGE2 suppresses apoptosis via repression of the proapoptotic BH3-only protein Bim in human colorectal adenoma cells. Repression of Bim expression was dependent upon PGE2-mediated activation of the Raf-MEK-ERK1/2 pathway which promoted Bim phosphorylation and proteasomal degradation. Reduction of Bim expression using RNA interference reduced spontaneous apoptosis in adenoma cells and abrogated PGE2-dependent apoptosis suppression. Treatment of COX-2-expressing colorectal carcinoma cells with COX-2-selective NSAIDs induced Bim expression, suggesting that Bim repression via PGE2 signalling may be opposed by COX-2 inhibition. Examination of Bim expression in two established in vitro models of the adenoma-carcinoma sequence revealed that downregulation of Bim expression was associated with tumour progression towards an anchorage-independent phenotype. Finally, immunohistochemical analyses revealed that Bim expression is markedly reduced in approximately 40% of human colorectal carcinomas in vivo. These observations highlight the COX-2/PGE2 pathway as an important negative regulator of Bim expression in colorectal tumours and suggest that Bim repression may be an important step during colorectal cancer tumorigenesis.

IGF-1 suppresses Bim expression in multiple myeloma via epigenetic and posttranslational mechanisms

AbstractInsulin-like growth factor-1 (IGF-1) is an important growth and survival factor in multiple myeloma (MM). Here, we demonstrate that IGF-1 induces significant down-regulation of the proapoptotic BH3-only protein Bim in MM cells. Reduced Bim levels by RNA interference (RNAi) protected cells from drug-induced cell death. The IGF-1–mediated down-regulation of Bim was the result of (1) reduced transcription by activation of the Akt pathway and inactivation of the transcription factor FoxO3a, (2) increased proteasome-mediated degradation of the Bim extra-long protein by activation of the mitogen-activated protein kinase pathway, and (3) epigenetic regulation of both the Bim and the FoxO3a promoter. Treatment of cells with the histone deacetylase inhibitor LBH589 resulted in a clear up-regulation in the expression of Bim. Furthermore, the methylation inhibitor 5-aza-2′deoxycytidine (decitabine) significantly increased the effects of LBH589. On IGF-1 treatment, the Bim promoter region was found to be unmethylated, whereas chromatin immunoprecipitation analysis of the IGF-1–treated cells showed both a reduced histone H3 tail Lys9 (H3K9) acetylation and an increased H3K9 dimethylation, which contributed actively to its silencing. These data identify a new mechanism in the IGF-1–dependent survival of MM cells and emphasize the need for IGF-1–targeted drug therapy.

The pro-apoptotic BH3-only protein Bim regulates cell cycle progression of hematopoietic progenitors during megakaryopoiesis.

The pro-apoptotic BH3-only protein Bim is recognized as a pivotal regulator of apoptosis induced by the depletion of cytokines. In the present study, we examined the role of Bim in megakaryopoiesis. Megakaryocyte (MK) progenitors obtained from bim knockout (KO) mice were analyzed in vitro for liability to apoptosis after the depletion of cytokines, ability to differentiate into MKs and proliferation/cell cycle progression in response to thrombopoietin (TPO). The production of platelets in vitro was evaluated by assaying the formation of proplatelets in MKs. Megakaryopoiesis in vivo was observed in a mouse model of thrombocytopenia induced by injecting fluorouracil (5-FU). Bim-deficient CD34-/c-kit+/Sca-1+/Lineage- stem cells and MKs were highly resistant to apoptosis induced by cytokine depletion, suggesting that Bim is involved in the apoptotic process in both stem cells and MKs. As bim KO mice exhibited splenomegaly and thrombocytopenia, splenectomized mice were used for experiments in vivo. Platelet recovery after 5-FU-induced thrombocytopenia was significantly delayed in bim KO mice. Corresponding with this, numbers of MKs in the recovery phase bone marrow were significantly reduced in bim KO mice. Culture of c-kit+/Lineage- progenitors with TPO revealed that Bim-deficient cells poorly proliferate and differentiate into CD41+ cells in comparison with wild-type (WT) cells. However, once differentiated into MKs, these cells matured normally. Furthermore, cell cycle analyses demonstrated that transition from the G1 to the S phase was delayed in Bim-deficient stem cells. In the present study, we demonstrated that Bim plays a pivotal role in the regulation of cell cycle progression in hepatopoietic progenitors during megakaryopiesis.