BH3-only Bcl-2 Family Member Bim Research Articles

Abstract 602: High-Density Lipoprotein Protects Macrophages Against Apoptosis Through BH3-only Bcl-2 Family Member Bim

Macrophage apoptosis contributes to the formation of necrotic cores within atherosclerotic lesions, increasing the susceptibility of atherosclerotic plaques to rupture and the risk of cardiovascular events. In advanced atherosclerotic plaques, lipid-laden foam cells are exposed to a diverse number of apoptosis inducers, including prolonged endoplasmic reticulum (ER) stress. The pro-apoptotic BH3-only protein Bim has been reported to mediate apoptosis in response to ER stress induction in a variety of cells including macrophages. Treatment of macrophages with high-density lipoprotein (HDL) protects them against apoptosis induced by a variety of stressors. We hypothesize that HDL mediated protection against macrophage apoptosis occurs via pathways that regulate Bim activity. To test this, thioglycollate-elicted peritoneal macrophages were treated in lipoprotein deficient culture with different apoptosis inducers in the absence or presence of HDL. Cell apoptosis was assessed by cleaved caspase 3 staining and terminal deoxynucleotidal transferase dUTP nick end labeling. Treatment of mouse peritoneal macrophages in culture with 50μg/ml of human HDL protected against apoptosis induced by tunicamycin (P<0.05) without affecting ER stress markers, Grp78, Grp94 and CHOP. Peritoneal macrophages from mice deficient in Bim were less susceptible to apoptosis induced by treatment with either tunicamycin or 7-ketocholesterol. Furthermore, treatment with HDL provided no additional protection to apoptosis in Bim KO macrophages. Our results suggest that protection of macrophage apoptosis by HDL is regulated through pathways involving the activity of BH3-only Bcl-2 family member Bim.

PI3Kδ Inhibition Elicits Anti-Leukemic Effects through Bim-Dependent Apoptosis

Introduction: Phosphatidylinositol-3-kinase δ (PI3Kδ)-signaling provides key maintenance, proliferation, and survival cues during both normal and malignant B-lymphocyte development. Consequently, isoform-selective PI3Kδ inhibitors (PI3Kδi) have generated huge interest as a potential treatment for lymphoid malignancies. In particular, PI3Kδi demonstrate impressive clinical efficacy in combination with anti-CD20 monoclonal antibodies (mAbs) for relapsed chronic lymphocytic leukemia (CLL). However, these combinations function primarily to delay disease progression, but are not curative. With the ever-rising number of new targeted therapeutics, the challenge is to identify combinations that will ultimately deliver curative regimes. In order to guide these selections, a detailed mechanistic understanding is required. To date, only limited data are available regarding the exact in vivo therapeutic mechanism of PI3Kδi. Currently, studies identify immunomodulation, inhibition of BCR-, chemokine/cytokine-signaling, and induction of apoptosis as putative therapeutic mechanisms. Here we characterize the molecular mechanisms responsible for PI3Kδi-induced cytotoxicity and determine the relative contribution toward in vivo therapeutic responses utilizing the Eµ-TCL1-Tg mouse model of CLL alongside human CLL samples.Methods: The molecular mechanisms of PI3Kδi alone or in combination with anti-CD20 mAbs were assessed using the Eµ-TCL1-Tg mouse model, an in vivo model system of CLL. To inhibit PI3Kδ, the δ isoform-selective inhibitor GS-9820 was chosen, as it is highly structurally related to idelalisib, and critically demonstrates improved pharmacokinetic properties in the mouse in comparison to idelalisib. In vitro GS-9820 IC50 are as follows: PI3Kδ 27 nM; PI3Kα 83,424 nM; PI3Kβ 14,899 nM; and PI3Kγ 15,606 nM. Assays to measure its effects on BCR-mediated kinase activation, chemokine signaling/chemotaxis, and cytokine- and cell-mediated support were performed. GS-9820 was administered in vivo at 10 mg/kg per os BID (formulated in 0.5% methylcellulose, 0.05% tween-80), once leukemias were detected, and maintained throughout the treatment period (GS-9820 Cmax 3114 nM, Ctrough 48.6 nM).Results: GS-9820 induced substantive in vitro cell death and disruptedBCR-mediated kinase activation, chemokine signaling/chemotaxis, and inhibited both cytokine- and cell-mediated support in murine (Eµ-TCL1) and human CLL cells. In vivo administration of GS-9820 imparted significant therapeutic responses in Eµ-TCL1-bearing animals, reducing leukemic burden by 75% and splenic tumor deposits by 66% 4 weeks posttreatment. GS-9820 appeared well tolerated in recipient animals with no obvious toxicity apparent (e.g. weight loss or behavioral symptoms). When in combination with anti-CD20 mAbs, GS-9820 extended leukemia depletion by several weeks. GS-9820 enhanced overall survival by 66% in comparison with vehicle control-recipient animals and enhanced the survival benefit of anti-CD20 mAb therapy. These therapeutic responses were associated with a 2-fold increase in expression of the pro-apoptotic BH3-only Bcl-2 family member Bim and a 3-fold increase in the extent of Bim/Bcl-2 interaction. Accordingly, Bim-/- Eµ-TCL1-Tg leukaemias exhibited profound resistance to PI3Kδi-induced cytotoxicity, were refractory to PI3Kδi in vivo, and failed to display combination efficacy with anti-CD20 mAbs. These findings informed the rational design of a GS-9820 + ABT199 (Venetoclax) complementary drug combination strategy. Combinations of GS-9820 and ABT199 were well tolerated with an absence of weight loss or altered behavioral symptoms. The GS-9820 + ABT199 combination effectively halted leukemia progression in vivo with increased efficacy compared to monotherapy regimes, resulting in a 90% reduction in leukemic burden at the end of the treatment period.Conclusions: Bim-dependent apoptosis represents the key in vivo effector mechanism for PI3Kδi in the Eµ-TCL1-Tg mouse model, both alone and in combination with anti-CD20 mAbs. As such, combinations of PI3Kδ and Bcl-2 inhibitors may represent an efficacious drug combination strategy. DisclosuresTannheimer:Gilead Sciences: Employment. Packham:Karus Therapeutics: Other: Share Holder & Founder; Aquinox Pharmaceuticals: Research Funding. Cragg:Baxalta: Consultancy; Roche: Consultancy, Research Funding; Bioinvent International: Consultancy, Research Funding; Gilead Sciences: Research Funding; GSK: Research Funding.

Quantitative Measurement of BIM Protein in Lung Cancer: Diagnosis/Staging

A proportion of EGFR-mutant and ALK-rearranged lung cancers do not respond to tyrosine kinase inhibition for as-yet unknown reasons. The BH3-only Bcl-2 family member BIM has been implicated as a potential biomarker of clinical benefit from kinase inhibitors. Quantifying BIM expression in tumor samples of patients with lung cancer may help to predict outcome, and potentially to guide therapy.

Impact of conditional deletion of the pro-apoptotic BCL-2 family member BIM in mice.

The pro-apoptotic BH3-only BCL-2 family member BIM is a critical determinant of hematopoietic cell development and homeostasis. It has been argued that the striking hematopoietic abnormalities of BIM-deficient mice (accumulation of lymphocytes and granulocytes) may be the result of the loss of the protein throughout the whole animal rather than a consequence intrinsic to the loss of BIM in hematopoietic cells. To address this issue and allow the deletion of BIM in specific cell types in future studies, we have developed a mouse strain with a conditional Bim allele as well as a new Cre transgenic strain, Vav-CreER, in which the tamoxifen-inducible CreER recombinase (fusion protein) is predominantly expressed in the hematopoietic system. We show that acute loss of BIM in the adult mouse rapidly results in the hematopoietic phenotypes previously observed in mice lacking BIM in all tissues. This includes changes in thymocyte subpopulations, increased white blood cell counts and resistance of lymphocytes to BIM-dependent apoptotic stimuli, such as cytokine deprivation. We have validated this novel conditional Bim knockout mouse model using established and newly developed CreER strains (Rosa26-CreER and Vav-CreER) and will make these exciting new tools for studies on cell death and cancer available.

Role of p53 in cAMP/PKA pathway mediated apoptosis

Deregulated β-adrenoceptor/cAMP-PKA pathway is implicated in a range of human diseases, such as neuronal loss during aging, cardiomyopathy and septic shock. The molecular mechanism of this process is, however, only poorly understood. We recently had demonstrated that the β-adrenoceptor/cAMP-PKA pathway triggers apoptosis through the transcriptional induction of the pro-apoptotic BH3-only Bcl-2 family member BIM in tissues, such as the thymus and the heart. Induction of BIM is driven by the transcriptional co-activator CBP (CREB Binding Protein) together with the proto-oncogene c-Myc. Association of CBP with c-Myc leads to altered histone acetylation and methylation pattern at the BIM promoter site [Lee et al., Cell Death Difference 20(7):941-952 (2013)]. However since CBP is a co-factor for multiple transcription factors, BH-3 only proteins other than Bim could also contribute to this apoptosis pathway. Here we provide evidence for the involvement of p53-CBP axis in apoptosis through Puma/Noxa induction, in response to β-adrenoceptor activation. Our findings highlight the molecular complexity of pathophysiology associated with a deregulated neuro-endocrine system and for developing novel therapeutic strategies for these diseases.

CREB-binding protein (CBP) regulates β-adrenoceptor (β-AR)−mediated apoptosis

Catecholamines regulate the β-adrenoceptor/cyclic AMP-regulated protein kinase A (cAMP/PKA) pathway. Deregulation of this pathway can cause apoptotic cell death and is implicated in a range of human diseases, such as neuronal loss during aging, cardiomyopathy and septic shock. The molecular mechanism of this process is, however, only poorly understood. Here we demonstrate that the β-adrenoceptor/cAMP/PKA pathway triggers apoptosis through the transcriptional induction of the pro-apoptotic BH3-only Bcl-2 family member Bim in tissues such as the thymus and the heart. In these cell types, the catecholamine-mediated apoptosis is abrogated by loss of Bim. Induction of Bim is driven by the transcriptional co-activator CBP (CREB-binding protein) together with the proto-oncogene c-Myc. Association of CBP with c-Myc leads to altered histone acetylation and methylation pattern at the Bim promoter site. Our findings have implications for understanding pathophysiology associated with a deregulated neuroendocrine system and for developing novel therapeutic strategies for these diseases.

Insulin-like growth factor 1 attenuates antiestrogen- and antiprogestin-induced apoptosis in ER+ breast cancer cells by MEK1 regulation of the BH3-only pro-apoptotic protein Bim

IntroductionIn this pre-clinical in vitro study conducted in estrogen receptor positive (ER+) breast cancer cells, we have characterized the effects of insulin-like growth factor I (IGF-1) on the cytostatic and cytotoxic action of antiestrogen treatment when used as a single agent or in combination with the antiprogestin mifepristone (MIF). Our goal was to identify new molecular targets to improve the efficacy of hormonal therapy in breast cancer patients that have a poor response to hormonal therapy, in part, due to high circulating levels of unbound insulinIGF-1.MethodsIGF-1-mediated effects on cytostasis and apoptotic cell death were determined with cell counts conducted in the presence and absence of trypan blue; enzyme-linked immunosorbent assays to determine the intracellular levels of cleaved cytokeratin 18, a marker of epithelial cancer cell apoptosis; and immunoblot analysis to determine the levels of cleaved poly-ADP ribose polymerase (PARP) and lamin A that result from caspase-dependent apoptosis. Cytotoxicity was further characterized by determination of the levels of reactive oxygen species (ROS) and the percent of mitochondrial membrane depolarization in cell populations treated with the different hormones in the presence and absence of IGF-1. Small molecule inhibitors of the dual-specificity protein kinase MEK1, MEK1 siRNA, Bim siRNA, and vectors overexpressing MEK1 wild type and mutant, dominant negative cDNA were used to identify key IGF-1 downstream prosurvival effectors.ResultsIGF-1, at physiologically relevant levels, blocked the cytotoxic action(s) of the antiestrogens 4-hydroxytamoxifen (4-OHT) and tamoxifen (TAM) when used as single agents or in combination with the antiprogestin MIF. The antiapoptotic action of IGF-1 was mediated primarily through the action of MEK1. MEK1 expression reduced the levels of ROS and mitochondrial membrane depolarization induced by the hormonal treatments via a mechanism that involved the phosphorylation and proteasomal turnover of the proapoptotic BH3-only Bcl-2 family member Bim. Importantly, small-molecule inhibitors of MEK1 circumvented the prosurvival action of IGF-1 by restoring Bim to levels that more effectively mediated apoptosis in ER+ breast cancer cells.Conclusionhis study provides strong support for the use of MEK1 inhibitors in combination with hormonal therapy to effectively affect cytostasis and activate a Bim-dependent apoptotic pathway in ER+ breast cancer cells. We discuss that MEK1 blockade may be a particularly effective treatment for women with high circulating levels of IGF-1, which have been correlated to a poor prognosis.

MicroRNA-10b is a Prognostic Indicator in Colorectal Cancer and Confers Resistance to the Chemotherapeutic Agent 5-Fluorouracil in Colorectal Cancer Cells

Recent evidence has shown that altered patterns of microRNA (miRNA) expression correlate with various human cancers. We investigated the clinical significance of miR-10b and its involvement in chemotherapeutic resistance to 5-fluorouracil (5-FU), which is a key component of common chemotherapy regimens in colorectal cancer. Quantitative RT-PCR was used to evaluate the clinicopathologic significance of miR-10b expression in 88 colorectal cancer cases. We also investigated the chemotherapeutic sensitivity to 5-FU in miR-10b-overexpressing colorectal cancer cells. To explore the mechanism of chemoresistance in miR-10b transfected cells, we examined whether miR-10b inhibits the pro-apoptotic BH3-only Bcl-2 family member BIM(BCL2L11), a key mediator of chemotherapy-induced cell death. High level miR-10b expression was found to be significantly associated with high incidence of lymphatic invasion (P = 0.0257) and poor prognosis (P = 0.0057). Multivariate analysis indicated that high miR-10b expression is an independent prognostic factor for survival. In vitro studies revealed that miR-10b directly inhibits pro-apoptotic BIM, and the overexpression of miR-10b confers chemoresistance in colorectal cancer cells to 5-FU. MiR-10b is a novel prognostic marker in colorectal cancer. Moreover, the expression of miR-10b is a potential indicator of chemosensitivity to the common 5-FU-based chemotherapy regimen.

Caspase-8 inactivation in T cells increases necroptosis and suppresses autoimmunity in Bim−/− mice

Dysregulation of either the extrinsic or intrinsic apoptotic pathway can lead to various diseases including immune disorders and cancer. In addition to its role in the extrinsic apoptotic pathway, caspase-8 plays nonapoptotic functions and is essential for T cell homeostasis. The pro-apoptotic BH3-only Bcl-2 family member Bim is important for the intrinsic apoptotic pathway and its inactivation leads to autoimmunity that is further exacerbated by loss of function of the death receptor Fas. We report that inactivation of caspase-8 in T cells of Bim(-/-) mice restrained their autoimmunity and extended their life span. We show that, similar to caspase-8(-/-) T cells, Bim(-/-) T cells that also lack caspase-8 displayed elevated levels of necroptosis and that inhibition of this cell death process fully rescued the survival and proliferation of these cells. Collectively, our data demonstrate that inactivation of caspase-8 suppresses the survival and proliferative capacity of Bim(-/-) T cells and restrains autoimmunity in Bim(-/-) mice.

BIM is a prognostic biomarker for early prednisolone response in pediatric acute lymphoblastic leukemia

Glucocorticoids such as prednisolone (PRED) are widely used in the treatment of pediatric acute lymphoblastic leukemia. In PRED-induced apoptosis, Bcl-2 family members play important regulatory roles. However, the exact members involved remain unknown. In this study, the roles of Bcl-2 family members in PRED-induced apoptosis and their prognostic value to day 8 PRED response are evaluated. Four clinically important acute lymphoblastic leukemia cell lines, three PRED-sensitive (697, Sup-B15, and RS4;11) and one PRED-resistant (REH) were studied. Thirty paired patient bone marrow samples were obtained at diagnosis (day 0) and after 7 days (day 8) of PRED monotherapy. Twenty-five patients had PRED good response and five PRED poor response. Differential expressions of Bcl-2 members were observed in those samples and BIM was further investigated using gene silencing technology in representative cell line Sup-B15. The proapoptotic BH3-only Bcl-2 family member BIM was upregulated only in PRED-sensitive cells. Receiver operating characteristic curve analysis showed that BIM expression was highly predictive of PRED response (area under the curve = 0.81; p = 0.032) in paired patient bone marrow samples and is, most excitingly, independent of molecular subtype. Patients whose BIM protein expression levels fail to upregulate at day 8 compared to day 0 (D8/D0 ratio <0.93) have significantly poorer event-free survival (60%) than those patients whose BIM protein expression levels did upregulate (92%). By silencing BIM in PRED-sensitive cells, PRED-induced apoptosis was inhibited. Upregulation of BIM by PRED in acute lymphoblastic leukemia cells regardless of molecular subtype is significantly prognostic of outcomes, confirming BIM's essential regulatory role in the PRED-induced apoptosis.

Bim Nuclear Translocation and Inactivation by Viral Interferon Regulatory Factor

Viral replication efficiency is in large part governed by the ability of viruses to counteract pro-apoptotic signals induced by infection of the host cell. Human herpesvirus 8 (HHV-8) uses several strategies to block the host's innate antiviral defenses via interference with interferon and apoptotic signaling. Contributors include the four viral interferon regulatory factors (vIRFs 1–4), which function in dominant negative fashion to block cellular IRF activities in addition to targeting IRF signaling-induced proteins such as p53 and inhibiting other inducers of apoptosis such as TGFβ receptor-activated Smad transcription factors. Here we identify direct targeting by vIRF-1 of BH3-only pro-apoptotic Bcl-2 family member Bim, a key negative regulator of HHV-8 replication, to effect its inactivation via nuclear translocation. vIRF-1-mediated relocalization of Bim was identified in transfected cells, by both immunofluorescence assay and western analysis of fractionated cell extracts. Also, co-localization of vIRF-1 and Bim was detected in nuclei of lytically infected endothelial cells. In vitro co-precipitation assays using purified vIRF-1 and Bim revealed direct interaction between the proteins, and Bim-binding residues of vIRF-1 were mapped by deletion and point mutagenesis. Generation and experimental utilization of Bim-refractory vIRF-1 variants revealed the importance of vIRF-1:Bim interaction, specifically, in pro-replication and anti-apoptotic activity of vIRF-1. Furthermore, blocking of the interaction with cell-permeable peptide corresponding to the Bim-binding region of vIRF-1 confirmed the relevance of vIRF-1:Bim association to vIRF-1 pro-replication activity. To our knowledge, this is the first report of an IRF protein that interacts with a Bcl-2 family member and of nuclear sequestration of Bim or any other member of the family as a means of inactivation. The data presented reveal a novel mechanism utilized by a virus to control replication-induced apoptosis and suggest that inhibitory targeting of vIRF-1:Bim interaction may provide an effective antiviral strategy.

Abstract 1033: Prednisolone induces BIM expression in pediatric acute lymphoblastic leukemia and synergizes with BH3-mimetics GX15-070 and ABT-737

Abstract Chemotherapeutic regimens for pediatric acute lymphoblastic leukemia (ALL) treatment entail the use of glucocorticoids such as prednisolone (PRED). Around 5% of patients fail to attain remission due to drug resistance. We have previously shown that the pro-apoptotic BH3-only BCL-2 family member BIM was up-regulated in only in PRED-sensitive cells. After the specific silencing of BIM by siRNA, inhibition of apoptosis was observed and validated by levels of cleaved PARP and caspase activity, providing a clear indication of BIM's significance in PRED induced apoptosis. In this study, protein expression levels of BIM were further evaluated using patient bone marrow. We also examined the ability of two BH3 mimetics, GX15-070 (obatoclax) and ABT-737, to induce apoptosis in ALL cell lines and the effect of drug combinations. Patient bone marrow samples were obtained at diagnosis (Day 0) and after 7 days (Day 8) of PRED monotherapy. Response to PRED was defined as Good (PGR, peripheral blast count &amp;lt; 1000 /µl), and Poor (PPR, peripheral blast count ≥1000 /µl). Twenty-five PGR and five PPR patients were included in this study. Using western immunoblotting, the protein expression of BIM was reproducibly observed in patient samples. The value of ODBim/ODActin at Day 8 was significantly higher than that on Day 0 (p-Value=0.0006) for PGR patients. In contrast, this value did not differ greatly in PPR patients. At Day 0, BIM expression levels of the entire patient cohort was comparable, but levels were significantly different at Day 8 (p-Value=0.01). These results indicate that the upregulation of BIM was induced by PRED in ALL patients and further confirms the essential role of BIM in this apoptotic pathway. Treatment with GX15-070 and ABT-737 as single agents yielded an increase in cell death in a dose dependent manner both in PRED-sensitive and -resistant cell lines. Apoptosis induced by both GX15-070 and ABT-737 could be inhibited by caspase-9 inhibitor (Z-LEHD-FMK) and caspase-3 inhibitor (Z-DEVD-FMK). Simultaneous exposure of ALL cells to PRED and GX15-070 or ABT-737 resulted in synergistic levels of cell death in SUP-B15 and RS4;11 cell lines with combination index (CI) values that are significantly less than one (as calculated by CalcuSyn software version 2.1). This highlights the possibility of enhancing cell death in ALL cells even with a reduction in drug dosage. In conclusion, BH3-only BCL-2 family member BIM, is vital for PRED-induced apoptosis in ALL. BH3 mimetics GX15-070 and ABT-737 can induce apoptosis in ALL cells regardless of PRED response. The synergism between PRED and BH3-mimetics is likely to minimize severe side effects, and more importantly, provide alternative therapies especially for patients that are resistant to conventional chemotherapeutic drugs in the clinic. 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 1033.

CAML regulates Bim-dependent thymocyte death

Appropriate control of apoptosis during T lymphocyte differentiation is critical for destruction of T cells bearing potentially autoreactive or useless immuno-receptors, and for survival of those T cells bearing antigen receptors that may recognize foreign proteins. Despite the well-established importance of thymocyte survival, the exact signals regulating thymocyte apoptosis have not been fully elucidated. Here, we show that thymocytes lacking the endoplasmic reticulum (ER) protein CAML failed to undergo normal T cell development and exhibited dramatically increased rates of apoptosis. In vitro, CAML-deficient thymocytes accumulated high levels of reactive oxygen species (ROS) and underwent abnormally accelerated death in response to several cytotoxic stimuli, including treatment with etoposide, cytokine deprivation or Fas ligation. Although neither p53 deletion nor loss of Fas rescued the survival and continued development of CAML-deficient thymocytes, removal of the pro-apoptotic BH3-only Bcl-2 family member Bim significantly restored their survival. This work reveals CAML to be a critically important regulator of ROS- and Bim-dependent thymocyte death.

Epigenetic Silencing of the Pro-Apoptotic Bim Gene in Glucocorticoid Poor-Responsive Pediatric Acute Lymphoblastic Leukemia, and Its Reversal by Histone Deacetylase Inhibition.

Abstract 939 Introduction:The glucocorticoids dexamethasone and prednisolone are critical components of combination chemotherapy regimens used to treat pediatric acute lymphoblastic leukemia (ALL). While approximately 80% of patients are cured, poor response to upfront prednisolone monotherapy in 5-10% of patients is a strong predictor of adverse treatment outcome. A greater understanding of the mechanisms responsible for glucocorticoid resistance in pediatric ALL is likely to result in the design of novel strategies to overcome resistance and improve outcome for patients with refractory disease. The pro-apoptotic BH3-only BCL-2 family member BIM (BCL-2L11) has previously been identified as a critical component of glucocorticoid-induced apoptosis in normal and malignant lymphocytes. The purpose of this study was to elucidate clinically relevant mechanisms of glucocorticoid resistance in pediatric ALL, and design and test resistance reversal strategies. Patients and Methods:The study included biopsy specimens obtained at diagnosis from a cohort of patients who received 7 days of prednisolone monotherapy (60 mg/m⋀2/day) and a single age-related dose of intrathecal methotrexate, and whose prednisolone response was determined on Day 8 to be Good (PGR, peripheral blast count < 1 × 10⋀9/L, n=11) or Poor (PPR, peripheral blast count >= 1 × 10⋀9/L, n=11). The study also included biopsies from a cohort of patients who experienced early relapse (within 2 years of diagnosis, n=12). Experimental models of the disease included xenografts (n=12) established in immune-deficient (NOD/SCID) mice using direct explants of patient biopsies. Methods of analysis included: real-time quantitative RT-PCR and immunoblotting of glucocorticoid-induced mRNA and proteins following dexamethasone treatment of ex vivo cultured xenograft cells; DNA methylation analysis of the BIM 5' Untranslated Region (5'UTR) by methylated DNA immunoprecipitation (MeDIP), bisulfite sequencing, and SEQUENOM MassArray Epityper analysis; real time and array based chromatin immunoprecipitation (ChIP) analysis of histone-H3K9 acetylation, H3K4 and H3K27 tri-methylation across the entire BIM locus; as well as assessment of the histone deacetylase inhibitor SAHA (Vorinostat) to reverse dexamethasone resistance both in vitro and in vivo. Results:Dexamethasone resistance in pediatric ALL xenografts was consistently associated with failure to up-regulate BIM mRNA and protein in response to dexamethasone treatment, despite verification that other known glucocorticoid-responsive genes (GILZ, FKBP5) were highly induced in all xenografts. These results indicate specific silencing of BIM in dexamethasone-resistant xenografts rather than a dysfunctional glucocorticoid receptor, leading us to focus on epigenetic regulation of BIM transcription. DNA methylation of the BIM 5'UTR was variable between xenografts, and showed no clear association with dexamethasone resistance. In contrast, the extent of H3K9 acetylation at the BIM locus significantly correlated with the ability of dexamethasone to up-regulate BIM expression in ALL xenografts (R=0.90; P<0.001; n=12): relatively deacetylated H3K9 was associated with BIM repression, indicating that the BIM locus was in a “closed” and transcriptionally inaccessible conformation in dexamethasone-resistant xenografts. These results were in direct contrast to the other BH3-only genes examined, NOXA and PUMA, where H3K9 deacetylation was not associated with dexamethasone resistance. Moreover, H3K9 acetylation at the BIM promoter was significantly decreased in patients classified as PPRs compared with PGRs (P=0.013), while the decrease in BIM H3K9 acetylation in patients at early relapse approached a significant difference from PGRs (P=0.096). These results confirm that the results obtained with xenografts were not an artifact of engraftment. Treatment of a dexamethasone-resistant xenograft with SAHA increased BIM H3K9 acetylation, up-regulated BIM, and caused synergistic anti-leukemic efficacy with dexamethasone both in ex vivo cultures and in an in vivo model of systemic disease. Conclusions:Poor response to glucocorticoid therapy in pediatric ALL is significantly associated with epigenetic silencing of the BIM gene locus, and strategies aimed at improving glucocorticoid sensitivity and clinical outcome should consider incorporating epigenetic modifiers. Disclosures:No relevant conflicts of interest to declare.

Loss of the pro-apoptotic BH3-only Bcl-2 family member Bim sustains B lymphopoiesis in the absence of IL-7

IL-7 is pivotal for B cell development. Proteins of the Bcl-2 family are essential regulators of lymphocyte survival. Particularly, the pro-apoptotic BH3-only members Bim and Puma mediate lymphocyte apoptosis provoked by cytokine deprivation. Herein, we addressed whether the absence of Bim or Puma within the hematopoietic compartment could bypass the requirement for IL-7-driven B cell development in adult mice. We found that deficiency of Bim, but not Puma, partially rescued B cell development in the absence of IL-7. The numbers of both sIgM(-) and sIgM(+) B cells were markedly increased in the bone marrow of recipients lacking IL-7 upon reconstitution with Bim-deficient hematopoietic progenitors, compared with their control or Puma-deficient counterparts. The augmentation of B cell lymphopoiesis in the absence of Bim was reflected in the mature peripheral compartment by an increase in both the number of immature and mature B cells in the spleen and in the circulating IgM levels. Bim-deficient B cells were also increased in IL-7-sufficient recipients suggesting that peripheral B cells homeostasis is governed by a Bim-dependent and IL-7-independent mechanism. Our data highlight the role of Bim as a key regulator of cell survival during B lymphocyte development in vivo.

ON-III inhibits erbB-2 tyrosine kinase receptor signal pathway and triggers apoptosis through induction of Bim in breast cancer cells

ErbB-2 gene encodes tyrosine kinase receptor p185neu. Overexpression of erbB-2 plays a key role in tumorigenesis or progression such as breast cancer and ovarian cancer. Our previous study showed that ON-III (2’,4’-dihydroxy-6’-methoxy-3’,5’- dimethylchalcone) extracted from Traditional Chinese Medicine Cleistocaly xoperculatus dry flower could inhibit KDR tyrosine kinase phosphorylation and tumor growth in vivo. In this study, we reported that ON-III repressed tyrosine phosphorylation of erbB-2 without reduced erbB-2 receptor expression in MDA-MB- 453 cells. Activation of mitogen-activated protein kinase (MAPK) and AKT, downstream molecules of erbB-2-mediated signal transduction pathway, was inhibited following exposure to ON-III. ON-III induced apoptosis in breast cancer cells as determined by caspase-3 and PARP cleavage. Also, ON-III up-regulated the expression of proapoptotic BH3-only Bcl2 family member Bim. Bim siRNA could inhibit ON-III- mediated apoptosis in MDA-MB-453 cells. It concludes that ON-III inhibits erbB-2 tyrosine kinase phosphorylation, shuts down its downstream pathway and triggered apoptosis via induction of Bim. These results suggest that ON-III is a potential novel anti-cancer agent for erbB-2-overexpressing cancer.

Synergy between phosphatidylinositol 3-kinase/Akt pathway and Bcl-xL in the control of apoptosis in adenocarcinoma cells of the lung

Adenocarcinomas of the lung commonly show an increase in the activity of phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway, yet many are resistant to apoptosis induced by the inhibition of PI3K. We hypothesized that Bcl-xL would have a synergistic effect on the apoptotic response induced by inhibition of the PI3K/Akt pathway in lung adenocarcinoma. To test this, we examined the effect of the PI3K inhibitor (LY294002) on lung adenocarcinoma cell lines expressing varying levels of Bcl-xL. We found that cells that overexpress Bcl-xL are resistant to LY294002-induced apoptosis, whereas cells that express little Bcl-xL readily are not. Restoring Bcl-xL expression in cells that express low level of Bcl-xL conferred resistance to apoptosis in response to LY294002. The simultaneous inhibition of the PI3K/Akt pathway by LY294002 or Akt1 small interfering RNA and Bcl-xL function by ABT-737 or Bcl-xL small interfering RNA greatly enhanced the apoptotic response. Moreover, this response was associated with the induction of proapoptotic BH3-only Bcl-2 family member Bim. Our data suggest that PI3K/Akt and Bcl-xL pathways control cell death in lung adenocarcinoma cells in a synergistic manner. Modulation of Bcl-xL expression may represent one important strategy to optimize the efficacy of therapeutic agents targeting the PI3K/Akt pathway in adenocarcinoma of the lung.

Induction of BIM Is Essential for Apoptosis Triggered by EGFR Kinase Inhibitors in Mutant EGFR-Dependent Lung Adenocarcinomas

BackgroundMutations in the epidermal growth factor receptor (EGFR) gene are associated with increased sensitivity of lung cancers to kinase inhibitors like erlotinib. Mechanisms of cell death that occur after kinase inhibition in these oncogene-dependent tumors have not been well delineated. We sought to improve understanding of this process in order to provide insight into mechanisms of sensitivity and/or resistance to tyrosine kinase inhibitors and to uncover new targets for therapy.Methods and FindingsUsing a panel of human lung cancer cell lines that harbor EGFR mutations and a variety of biochemical, molecular, and cellular techniques, we show that EGFR kinase inhibition in drug-sensitive cells provokes apoptosis via the intrinsic pathway of caspase activation. The process requires induction of the proapoptotic BH3-only BCL2 family member BIM (i.e., BCL2-like 11, or BCL2L11); erlotinib dramatically induces BIM levels in sensitive but not in resistant cell lines, and knockdown of BIM expression by RNA interference virtually eliminates drug-induced cell killing in vitro. BIM status is regulated at both transcriptional and posttranscriptional levels and is influenced by the extracellular signal-regulated kinase (ERK) signaling cascade downstream of EGFR. Consistent with these findings, lung tumors and xenografts from mice bearing mutant EGFR-dependent lung adenocarcinomas display increased concentrations of Bim after erlotinib treatment. Moreover, an inhibitor of antiapoptotic proteins, ABT-737, enhances erlotinib-induced cell death in vitro.ConclusionsIn drug-sensitive EGFR mutant lung cancer cells, induction of BIM is essential for apoptosis triggered by EGFR kinase inhibitors. This finding implies that the intrinsic pathway of caspase activation may influence sensitivity and/or resistance of EGFR mutant lung tumor cells to EGFR kinase inhibition. Manipulation of the intrinsic pathway could be a therapeutic strategy to enhance further the clinical outcomes of patients with EGFR mutant lung tumors.

ER Stress Triggers Apoptosis by Activating BH3-Only Protein Bim

Endoplasmic reticulum (ER) stress caused by misfolded proteins or cytotoxic drugs can kill cells and although activation of this pathway has been implicated in the etiology of certain degenerative disorders its mechanism remains unresolved. Bim, a proapoptotic BH3-only member of the Bcl-2 family is required for initiation of apoptosis induced by cytokine deprivation or certain stress stimuli. Its proapoptotic activity can be regulated by several transcriptional or posttranslational mechanisms, such as ERK-mediated phosphorylation, promoting its ubiquitination and proteasomal degradation. We found that Bim is essential for ER stress-induced apoptosis in a diverse range of cell types both in culture and within the whole animal. ER stress activates Bim through two novel pathways, involving protein phosphatase 2A-mediated dephosphorylation, which prevents its ubiquitination and proteasomal degradation and CHOP-C/EBPalpha-mediated direct transcriptional induction. These results define the molecular mechanisms of ER stress-induced apoptosis and identify targets for therapeutic intervention in ER stress-related diseases.

Growth Factor Withdrawal and Apoptosis: The Middle Game

In this issue of Molecular Cell, detail molecular events that trigger apoptosis following growth factor withdrawal, linking irreversible mitochondrial permeabilization to activation of GSK-3 and subsequent phosphorylation, ubiquitinylation, and degradation of the antiapoptotic BCL-2 family member MCL-1.