Suppression Of Bim Research Articles

Homoharringtonine Enhances the Anti-Leukemia Effect of Venetoclax in Acute Myeloid Leukemia with t(8;21)(q22;q22) Via Regulation of c-Myc/Bim Axis

Purpose: Recently, small cases reports showed that acute myeloid leukemia (AML) with translocation (8;21)(q22;q22) [t(8;21) AML] responded poorly to venetoclax (VEN) plus azacitidine (AZA) (VA) regimen. Our previous study revealed homoharringtonine (HHT) enhanced the the killing effect of VA in AML. This study went further to explore the mechanism of VEN-resistance and the synergetic effect of VEN and HHT (VH) in overcoming resistance in t(8;21) AML. Methods: Patients with/without t(8;21) AML, being treated with VA with/without HHT were included for response assessment. Synergetic effect and mechanism of VEN combined with HHT or AZA was studied in cell lines, primary cells and mice. Results: Patients with t(8;21) AML acquired significantly lower CR/CRi rate than those without, with VA regimen as frontline (1/5 vs. 56/82, P=0.027) or salvage treatment (1/10 vs. 60/127, P=0.023), while the CR/CRi rate was comparable between the two cohorts with VA plus HHT (VAH) as salvage treatment (9/13 vs. 102/152, P>0.05). Synergetic effect in anti-leukemia was observed in VH but not VA in t(8;21) AML cell lines, primary cells and cell line-derived xenograft mice. Transcriptome RNA-sequencing and western blot detection found c-Myc and its downstring signialing were significantly decreased with the treatment of VH but not VA as compared to the controls. In addition, VEN induced accumulation of c-Myc in t(8;21) cells. Genetic silencing or pharmacological inhibition of c-Myc restored VEN sensitivity in t(8;21) cells. Co-immunoprecipitation assay found Bim, the crucial BH3-only protein, participated in the course of VH-inducing apoptosis. Direct or indirect inhibition of c-Myc increased expression of Bim, while overexpression of c-Myc significantly inhibited the transcription of Bim in t(8;21) cells. CUT&RUN assay revealed two functional sites of c-Myc binded to the promoter of Bim gene. Conclusion: Abnormal activation of c-Myc might contribute to the resistance of VEN via suppression of Bim in t(8;21) AML. HHT might enhance the killing effect of VEN by regulation of c-Myc/Bim axis. Key words: acute myeloid leukemia, t(8;21), homoharringtonine, venetoclax, c-Myc

PRKCQ inhibition enhances chemosensitivity of triple-negative breast cancer by regulating Bim

BackgroundProtein kinase C theta, (PRKCQ/PKCθ) is a serine/threonine kinase that is highly expressed in a subset of triple-negative breast cancers (TNBC) and promotes their growth, anoikis resistance, epithelial-mesenchymal transition (EMT), and invasion. Here, we show that PRKCQ regulates the sensitivity of TNBC cells to apoptosis triggered by standard-of-care chemotherapy by regulating levels of pro-apoptotic Bim.MethodsTo determine the effects of PRKCQ expression on chemotherapy-induced apoptosis, shRNA and cDNA vectors were used to modulate the PRKCQ expression in MCF-10A breast epithelial cells or triple-negative breast cancer cells (MDA-MB231Luc, HCC1806). A novel PRKCQ small-molecule inhibitor, 17k, was used to inhibit kinase activity. Viability and apoptosis of cells treated with PRKCQ cDNA/shRNA/inhibitor +/-chemotherapy were measured. Expression levels of Bcl2 family members were assessed.ResultsEnhanced expression of PRKCQ is sufficient to suppress apoptosis triggered by paclitaxel or doxorubicin treatment. Downregulation of PRKCQ also enhanced the apoptosis of chemotherapy-treated TNBC cells. Regulation of chemotherapy sensitivity by PRKCQ mechanistically occurs via regulation of levels of Bim, a pro-apoptotic Bcl2 family member; suppression of Bim prevents the enhanced apoptosis observed with combined PRKCQ downregulation and chemotherapy treatment. Regulation of Bim and chemotherapy sensitivity is significantly dependent on PRKCQ kinase activity; overexpression of a catalytically inactive PRKCQ does not suppress Bim or chemotherapy-associated apoptosis. Furthermore, PRKCQ kinase inhibitor treatment suppressed growth, increased anoikis and Bim expression, and enhanced apoptosis of chemotherapy-treated TNBC cells, phenocopying the effects of PRKCQ downregulation.ConclusionsThese studies support PRKCQ inhibition as an attractive therapeutic strategy and complement to chemotherapy to inhibit the growth and survival of TNBC cells.

MiR-30e-5p Mitigates Hypoxia-Induced Apoptosis in Human Stem Cell-Derived Cardiomyocytes by Suppressing Bim.

Coronary microembolization can cause slow or no reflow, which is one of the crucial reasons for reverse of clinical advantage from cardiac reperfusion therapy. miRNAs and apoptosis are dramatically involved in the occurrence and process of cardiovascular diseases. Fortunately, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have emerged as an appealing model for the evaluation of cardiovascular diseases. Therefore, our study was designed to explore the role of miR-30e-5p and apoptosis in a hypoxia-induced hiPSC-CM injury model. Our results showed that the expression levels of miR-30e-5p were overtly downregulated in a time-dependent manner under hypoxic conditions. Expression of miR-30e-5p was significantly downregulated after 24 hours of hypoxia, hypoxia treatment dramatically induced apoptosis. Calcium handling capability significantly decreased after 24 hours of hypoxia treatment. miR-30e-5p overexpression partially mitigated hypoxia-induced apoptosis and rescued hypoxia-induced calcium handling defects in hiPSC-CMs. The luciferase reporter assay showed that miR-30e-5p can directly target the 3'-UTR of Bim, which is an apoptosis activator and autophagy suppressor. The mRNA and protein of Bim remarkably increased after hypoxia treatment and reduced with miR-30e-5p overexpression. Moreover, downregulation of Bim mitigated hypoxia-induced apoptosis and activated autophagy. These results demonstrated that miR-30e-5p mitigated hypoxia-induced apoptosis in hiPSC-CMs at least in part via Bim suppression and subsequent autophagy activation. Our study suggested miR-30e-5p may act as a potential therapeutic target for coronary microembolization.

Abstract 4118: The EMT transcription factor TWIST1 mediates resistance to EGFR inhibitors in EGFR-mutant non-small cell lung cancer

Abstract Recent advances in the treatment of non-small cell lung cancer (NSCLC) stem from the paradigm shift of classifying patients into subtypes based upon the presence of distinct molecular drivers. Subsets of patients, such as those with EGFR mutations and ALK translocations, have dramatic responses in their tumors to tyrosine kinase inhibitors (TKIs) that specifically inhibit these oncogenic drivers. While many patients initially response to TKIs, therapeutic resistance is inevitable. For EGFR-mutant NSCLC, there are multiple described mechanisms of resistance to EGFR TKIs, including epithelial-mesenchymal transition (EMT). Previous studies have implicated the AXL kinase and ZEB1, an EMT transcription factor (EMT-TF), in EMT-mediated EGFR TKI resistance. We have previously demonstrated that the EMT-TF, TWIST1, is required for oncogene-driven NSCLC tumorigenesis, including those tumors with EGFR mutations. In this study, we investigated the role of TWIST1 in EMT-mediated resistance to EGFR TKIs. We have demonstrated that genetic or pharmacologic inhibition of TWIST1 resulted in growth inhibition in a panel of EGFR-mutant NSCLC cell lines and apoptosis in a subset of these lines. Interestingly, TWIST1 overexpression in EGFR-mutant NSCLC cell lines led to EGFR TKI resistance. Conversely, knockdown of TWIST1 in an erlotinib resistant EGFR-mutant NSCLC cell line restored erlotinib sensitivity. We found that TWIST1 mediates resistance to EGFR TKIs through suppression of apoptosis possibly through decreasing the expression of the pro-apoptotic Bcl-2 member, BCL2L11 (BIM). We observed that TWIST1 knockdown increased BIM levels, while TWIST1 overexpression decreased BIM expression. Furthermore, TWIST1-mediated resistance was overcome by treatment with the BCL-2/BCL-XL inhibitor, ABT-737. Knockdown of BIM recapitulated the resistance seen following TWIST1 overexpression, suggesting that TWIST1 suppression of BIM is a mechanism through which TWIST1 leads to EGFR TKI resistance. To explore the role of TWIST1 in modulating EGFR inhibitor sensitivity in vivo, we used an inducible EGFR-mutant transgenic mouse model, CCSP-rtTA/tetO-EGFRL858R (CE), which expresses EGFRL858R in the lung and a EGFR-mutant/Twist1 transgenic model, CCSP-rtTA/tetO-EGFRL858R/ Twist1- tetO7-luc (CET), which expresses both Twist1 and EGFRL858R in the lung. CET mice had a significantly increased tumor burden, decreased apoptosis and a decreased overall survival compared to CE mice following erlotinib treatment. In summary, we found that TWIST1 overexpression leads to EGFR TKI resistance by suppressing EGFR TKI-induced apoptosis through suppressing BIM expression. Future studies aim to establish the mechanisms of TWIST1 suppression of BIM expression and determine if our TWIST1 inhibitor, harmine, is effective in overcoming EMT-mediated resistance. Citation Format: Zachary A. Yochum, Hailun Wang, Jessica A. Cades, Susheel Khetarpal, Eric H. Huang, Phouc T. Tran, Timothy F. Burns. The EMT transcription factor TWIST1 mediates resistance to EGFR inhibitors in EGFR-mutant non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4118. doi:10.1158/1538-7445.AM2017-4118

CRISPR/Cas9 Screens Reveal Epstein-Barr Virus-Transformed B Cell Host Dependency Factors

Epstein-Barr virus (EBV) causes endemic Burkitt lymphoma (BL) and immunosuppression-related lymphomas. These B cell malignancies arise by distinct transformation pathways and have divergent viral and host expression programs. To identify hostdependency factors resulting from these EBV+,B cell-transformed cell states, we performed parallel genome-wide CRISPR/Cas9 loss-of-function screens in BL and lymphoblastoid cell lines (LCLs). These highlighted 57 BL and 87 LCL genes uniquely important for their growth and survival. LCL hits were enriched for EBV-induced genes, including viral super-enhancer targets. Our systematic approach uncovered key mechanisms by which EBV oncoproteins activate the PI3K/AKT pathway and evade tumor suppressor responses. LMP1-induced cFLIP was found to be critical for LCL defense against TNFα-mediated programmed cell death, whereas EBV-induced BATF/IRF4 were critical for BIM suppression and MYC induction in LCLs. Finally, EBV super-enhancer-targeted IRF2 protected LCLs against Blimp1-mediated tumor suppression. Our results identify viral transformation-driven synthetic lethal targets for therapeutic intervention.

Fork Head Box Class O1 (FOXO1) Activates Bim Expression to Mediate Cardiac Apoptosis in Chronic Intermittent Hypoxia-Induced Cardiac Hypertrophy.

BackgroundObstructive sleep apnea syndrome (OSAS) is characterized by chronic intermittent episodes of upper-airway obstruction with hypoxia and is associated with increased risk of cardiovascular diseases, including myocardial hypertrophy. Chronic intermittent hypoxia (CIH) has been shown to induce apoptosis in cardiomyocytes. However, the mechanisms of cardiomyocytes apoptosis under CIH largely remain unclear.Material/MethodsWe used male Sprague-Dawley rats and human cardiomyocyte cell line H9C2, and Annexin V/PI, Western blot analysis, co-immunoprecipitation, RT-PCR, immunohistochemistry, and TUNEL assay were carried out.ResultsWe show that Bim was significantly up-regulated by CIH in cardiomyocytes, and the function of Bim in CIH-induced apoptosis was supported by the genetic suppression of Bim with si-RNA. We also observed that CIH-motivated expression of Bim was directly related to fork head box class O1 (FOXO1), which is increased in CIH. Genetic ablation and pharmacological inhibition of FOXO1 in cardiomyocytes attenuated CIH-induced apoptosis, hypertrophy, and features of perivascular fibrosis in cardiomyocytes in vitro and in vivo.ConclusionsFOXO1 is a key integrator of the apoptosis signal transduction pathway, driving chronic intermittent hypoxia-induced cardiac hypertrophy, and inhibition of FOXO1 provides a potential target for the treatment of OSAS with cardiac hypertrophy.

Suppression of Bim by microRNA-19a may protect cardiomyocytes against hypoxia-induced cell death via autophagy activation.

Microvascular obstruction (MO), one of unfavorable complications of percutaneous coronary intervention (PCI), is responsible for the lost benefit of reperfusion therapy. Determination of microRNA-19a, a member of the miR-17-92 cluster, using quantitative real-time polymerase chain reaction (PCR) revealed notably down-regulated microRNA-19a, in myocardium with MO. Nonetheless, the role of miR-19a in MO and the underlying mechanism remains to be elucidated. To this end, an in vitro microembolization model in cardiomyocytes was used. Our data revealed that hypoxic exposure prompted cardiomyocyte apoptosis in a time-dependent manner accompanied by reduced miR-19a. miR-19a overexpression clearly ameliorated hypoxia-induced cell death (necrosis and apoptosis), at least in part, through switching on autophagy. Further dual-luciferase reporter assay and immunoblotting studies demonstrated that miR-19a-induced cytoprotection might be achieved in part through modulation of the specific target Bcl-2 interacting mediator of cell death, Bim, an apoptotic activator. Bim sufficiently interfered with miR-19a-induced LC3 conversion and increased cardiomyocyte apoptosis under hypoxia. Moreover, cardiomyocytes pretreated with 3-methyladenine conferred resistance to the cytoprotective effect of miR-19a and displayed notably increased TUNEL staining and caspase-3 activity. In conclusion, miR-19a protected cardiomyocytes against hypoxia-induced lethality at least in part via Bim suppression and subsequently autophagy activation.

Modulation of Glucocorticoid Resistance in Pediatric T-cell Acute Lymphoblastic Leukemia by Increasing BIM Expression with the PI3K/mTOR Inhibitor BEZ235.

The aim of our study is to evaluate the preclinical therapeutic activity and mechanism of action of BEZ235, a dual PI3K/mTOR inhibitor, in combination with dexamethasone in acute lymphoblastic leukemia (ALL). The cytotoxic effects of BEZ235 and dexamethasone as single agents and in combination were assessed in a panel of ALL cell lines and xenograft models. The underlying mechanism of BEZ235 and dexamethasone was evaluated using immunoblotting, TaqMan RT-PCR, siRNA, immunohistochemistry, and immunoprecipitation. Inhibition of the PI3K/AKT/mTOR pathway with the dual PI3K/mTOR inhibitor BEZ235 enhanced dexamethasone-induced anti-leukemic activity in in vitro (continuous cell lines and primary ALL cultures) and systemic in vivo models of T-ALL (including a patient-derived xenograft). Through inhibition of AKT1, BEZ235 was able to alleviate AKT1-mediated suppression of dexamethasone-induced apoptotic pathways leading to increased expression of the proapoptotic BCL-2 protein BIM. Downregulation of MCL-1 by BEZ235 further contributed to the modulation of dexamethasone resistance by increasing the amount of BIM available to induce apoptosis, especially in PTEN-null T-ALL where inhibition of AKT only partially overcame AKT-induced BIM suppression. Our data support the further investigation of agents targeting the PI3K/mTOR pathway to modulate glucocorticoid resistance in T-ALL.

Regulation of cell survival by sphingosine-1-phosphate receptor S1P1 via reciprocal ERK-dependent suppression of Bim and PI-3-kinase/protein kinase C-mediated upregulation of Mcl-1.

Although the ability of bioactive lipid sphingosine-1-phosphate (S1P) to positively regulate anti-apoptotic/pro-survival responses by binding to S1P1 is well known, the molecular mechanisms remain unclear. Here we demonstrate that expression of S1P1 renders CCL39 lung fibroblasts resistant to apoptosis following growth factor withdrawal. Resistance to apoptosis was associated with attenuated accumulation of pro-apoptotic BH3-only protein Bim. However, although blockade of extracellular signal-regulated kinase (ERK) activation could reverse S1P1-mediated suppression of Bim accumulation, inhibition of caspase-3 cleavage was unaffected. Instead S1P1-mediated inhibition of caspase-3 cleavage was reversed by inhibition of phosphatidylinositol-3-kinase (PI3K) and protein kinase C (PKC), which had no effect on S1P1 regulation of Bim. However, S1P1 suppression of caspase-3 was associated with increased expression of anti-apoptotic protein Mcl-1, the expression of which was also reduced by inhibition of PI3K and PKC. A role for the induction of Mcl-1 in regulating endogenous S1P receptor-dependent pro-survival responses in human umbilical vein endothelial cells was confirmed using S1P receptor agonist FTY720-phosphate (FTY720P). FTY720P induced a transient accumulation of Mcl-1 that was associated with a delayed onset of caspase-3 cleavage following growth factor withdrawal, whereas Mcl-1 knockdown was sufficient to enhance caspase-3 cleavage even in the presence of FTY720P. Consistent with a pro-survival role of S1P1 in disease, analysis of tissue microarrays from ER+ breast cancer patients revealed a significant correlation between S1P1 expression and tumour cell survival. In these tumours, S1P1 expression and cancer cell survival were correlated with increased activation of ERK, but not the PI3K/PKB pathway. In summary, pro-survival/anti-apoptotic signalling from S1P1 is intimately linked to its ability to promote the accumulation of pro-survival protein Mcl-1 and downregulation of pro-apoptotic BH3-only protein Bim via distinct signalling pathways. However, the functional importance of each pathway is dependent on the specific cellular context.

Abstract 4990: PP2A signaling is overridden by MEK/ERK activity leading to suppression of Bim and protection of melanoma cells from ER stress-induced apoptosis

Abstract Endoplasmic reticulum (ER) stress triggers apoptosis by activating Bim in diverse types of cells. This involves dephosphorylation of Bim by protein phosphatase 2A (PP2A). However, melanoma cells are largely resistant to ER stress-induced apoptosis, indicating that Bim activation is suppressed in melanoma cells undergoing ER stress. We show here that this is due to increased activation of MEK/ERK signaling that overrides the PP2A activity leading to enhanced Bim phosphorylation and subsequent proteosomal degradation. Upon pharmacological ER stress, the Bim protein expression was rapidly downregulated after an initial increase, despite its sustained upregulation at the transcriptional level. Although PP2A exerted the dephosphorylating effect on BimEL, its activity appeared to be superseded by increased activation of MEK/ERK signaling that led to enchanced phosphorylation and accelerated turnover of the protein. Moreover, in contrast to its upregulation by ER stress in MCF-7 and HEK293 cells, PP2A was downregulated in melanoma cells undergoing ER stress. Pharmacological activation or enforced expression of PP2A decreased ERK and Bim phosphorylation, up-regulated Bim expression, and rendered melanoma cells sensitive to ER stress-induced apoptosis. Notably, instead of direct interaction with Bim, PP2A reduced Bim phosphorylation indirectly by dephosphorylating ERK in melanoma cells. Together, these results identify a key resistance mechanism of melanoma cells against apoptosis induced by ER stress. 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 4990. doi:1538-7445.AM2012-4990

Over Expression of MN1 Accelerates Leukemia Onset and Confers Resistance to Chemotherapy by Suppression of p53 and Bim

Abstract 2501Acute myeloid leukemia (AML) is an aggressive malignancy of immature myeloid precursors that leads to progressive marrow failure and death. This disease will affect approximately 12,950 people this year in the United States, causing 9,050 deaths. The most common treatment is combination chemotherapy containing cytarabine (Ara-C) and an anthracycline. Resistance to these therapies is a major problem and most patients diagnosed with AML will ultimately die from resistant disease.AML is a genetically diverse malignancy and karyotype can be used to delineate prognosis. There is a clear link between chromosomal abnormalities and resistance to chemotherapy as complete remission rates are significantly different between groups. Additionally, there are now multiple submicroscopic genetic alterations that have been found to effect prognosis. These alterations can be mutations, over or under expression of a particular gene.MN1 is a transcription co-factor and several studies have demonstrated its over-expression confers a worse prognosis. High MN1 expressers were less likely to achieve a remission and had lower 3 year survival rates. Additionally, over expression of MN1 in murine bone marrow leads to AML in transplanted recipients and predicts for resistance to ATRA in elderly AML patients. The effect of MN1 on response to standard chemotherapy is currently unknown.To determine the effect of MN1 expression on therapy response we infected murine MLL-ENL driven AML cells with retroviral vectors that expressed MN1. When partially infected populations were exposed to a titration of either Ara-C or doxorubicin MN1 expressing cells were significantly enriched compared to untreated controls. When cells were exposed to a titration of Ara-C the MN1 expressing cells were enriched up to1.69 fold and when exposed to doxorubicin were enriched up to 3.80 fold. Both results were highly statistically significant with p values of 0.004 and < 0.0001. Consistent results were obtained with repeated infections and with separately derived MLL-ENL lines. Additionally, MN1 was able to confer therapy resistance to anthracycline resistant Flt3-ITD expressing cells suggesting non-overlapping mechanisms. Purified populations of cells expressing MN1 were resistant to Ara-C when compared to the parental leukemia (IC50 175.6nM vs 67.28nM) and highly resistant to doxorubicin. Consistent with these results human OCI-AML3 cells expressing MN1were enriched by 1.6 fold when exposed to doxorubicin, a highly significant result with a p value of 0.0002. In contrast a control vector without MN1 was not significantly enriched.In vivo when mixed leukemia cells were injected into syngeniec recipients MN1 expressers were significantly enriched in the femoral bone marrow of treated animals compared to controls. Treated animals had 90.58% (+/−0.66) MN1 expressing blasts compared to 55.38% (+/−5.25) in controls. This result was highly statistically significant with a p value of < 0.0001. This observation was reproducible in a separately derived MLL-ENL driven cell line. Additionally, the engraftment of MLL-ENL and Flt3-ITD expressing cells was significantly increased by MN1 expression leading to shorter survival in recipient animals despite the already highly aggressive nature of the parental leukemia.When MN1 expressing cells were exposed to doxorubicin or Ara-C they displayed significantly lower Annexin V positivity consistent with an attenuated apoptotic response (3.65 vs 34.79, p=<0.0001). When we examined BH3 only family member induction following exposure to Ara-C and doxorubicin we found significantly decreased levels of Bim induction by QPCR in cells expressing MN1. Similarly, stabilization of p53 following treatment was blunted in MN1 expressers as was induction of its downstream targets p21 and MDM2. Importantly the amount of DNA damage induced by doxorubicin as assessed by γH2AX foci was not different between MN1 expressing cells and the parental leukemia.These data suggest that over expression of MN1 confers resistance to both Ara-C and doxorubicin in vitro and in vivo by suppression of Bim induction and p53 response. These observations suggest a biological explanation for the clinical observation that it confers a worse prognosis. Disclosures:No relevant conflicts of interest to declare.

Glucocorticoids Inhibit Cell Death Induced by Oncogenic Stress After Imatinib Withdrawal In TKI Resistant p190Bcr/Abl Overexpressing Cells

AbstractAbstract 3150Imatinib resistance is a major problem in treatment of Bcr-Abl positive leukemias, particularly in patients with ALL and advanced CML. One mechanism of this resistance is overexpression of Bcr-Abl. We investigated the effects of Imatinib deprivation in Bcr-Abl overexpressing Imatinib-resistant ALL cell lines. Removal of Imatinib from culture medium led to induction of a non-apoptotic cell death starting approximately 40 hours after Imatinib withdrawal. This cell death was preceded by a rapid and robust induction of Bcr-Abl autophosphorylation and concomitant overstimulation of PI3K-, MAPK-, and JAK/STAT signalling. Over-activation of Bcr-Abl downstream signalling was accompanied by a significant enhanced glucose and amino acid metabolism and an elevated intracellular protein and nucleic acid content. As a result of this enhanced metabolisms we observed a massive increase in cell size, multinucleation, cytoplasmic vacuolization, and reduced intracellular ATP level. The phase-lucent vacuoles did not incorporate the endosome/lysosome tracker, Lyso Tracker Red indicating that the vacuoles were formed by dilation of ER cisternae. To determine if Imatinib deprivation induces an ER stress response concurrent with vacuolization we investigated typical ER stress markers. Upon removal of Imatinib a massive induction of CHOP expression and eIF2 alpha phosphorylation, as well as alternative splicing of XPB could be detected. It has been demonstrated that severe ER stress promotes cell death either by induction of a BIM-dependent apoptitic process or by a TRAF2-RIP1 dependent pathway. Despite of a robust induction of BIM and posttranslational modification of RIP1, siRNA-mediated suppression of BIM and RIP1 had no effect on Imatinib deprivation-induced cell death whereas downmodulation of CHOP partially rescued those cells. Using different small molecule inhibitor libraries, we also identified inhibitors of glycogen synthase kinase-3 (GSK3) and p38-MAPK, as well as glucocorticoids as potent compounds which not only completely prevented metabolic stress and induction of cell death upon removal of Imatinib but also partially repressed induction of CHOP upon Imatinib withdrawal. Importantly, in the presence of glucocorticoids, we found a significant enhanced number of cell clones with Bcr-Abl overexpression in lympoid cell lines upon de novo transfection with Bcr-Abl.In conclusion, acute over-activation of the oncogene Bcr-Abl led to metabolic stress and ER stress followed by a delayed induction of a necrosis-like cell death. These cellular responses to Bcr-Abl-mediated oncogenic stress could be completely blocked by glucocorticoids. Therefore, treatment of glucocorticoid resistant ALL may generate selective pressure towards Bcr-Abl overexpession. Disclosures:No relevant conflicts of interest to declare.

Hypoxia Suppression of Bim and Bmf Blocks Anoikis and Luminal Clearing during Mammary Morphogenesis

Proper adhesion to extracellular matrix is critical for epithelial cell survival. Detachment from matrix signals results in apoptosis, referred to as anoikis. Selective apoptosis of cells that become detached from matrix is associated with the formation of a lumen in three-dimensional mammary epithelial acinar structures in vitro. Because early breast cancer lesions such as carcinoma in situ, characterized by ducts exhibiting lumens filled with cells, are often associated with hypoxic markers, we sought to examine the role of hypoxia in anoikis and lumen formation in mammary epithelial cells. Here, we show that hypoxic conditions inhibit anoikis and block expression of proapoptotic BH3-only family members Bim and Bmf in epithelial cells. Hypoxia-mediated anoikis protection is associated with increased activation of the epidermal growth factor receptor-mitogen-activated protein kinase kinase-extracellular signal-regulated kinase (Erk) kinase pathway and requires the hypoxia-activated transcription factor. Consistent with these data, hypoxic conditions inhibit luminal clearing during morphogenesis in human mammary epithelial acini when grown in three-dimensional cultures and are associated with decreased expression of Bim and Bmf as well as Erk activation. We show that hypoxia regulates specific cell survival pathways that disrupt tissue architecture related to clearing of luminal space during mammary morphogenesis and suggest that hypoxia-mediated anoikis resistance may contribute to cancer progression.

The Polycomb Group Protein Bmi-1 Is Essential for the Growth of Multiple Myeloma Cells

Bmi-1 is a member of the Polycomb group family of proteins that function in the epigenetic silencing of genes governing self-renewal, differentiation, and proliferation. Bmi-1 was first identified through its ability to accelerate c-Myc-induced lymphomagenesis. Subsequent studies have further supported an oncogenic role for Bmi-1 in several cancers including those of the breast, lung, prostate, and brain. Using a stable and inducible shRNA system to silence Bmi-1 gene expression, we show a novel role for Bmi-1 in regulating the growth and clonogenic capacity of multiple myeloma cells both in vitro and in vivo. Moreover, to elucidate novel gene targets controlled by Bmi-1, global transcriptional profiling studies were performed in the setting of induced loss of Bmi-1 function. We found that the expression of the proapoptotic gene Bim is negatively regulated by Bmi-1 and that Bim knockdown functionally rescues the apoptotic phenotype induced upon loss of Bmi-1. Therefore, these studies not only highlight Bmi-1 as a cancer-dependent factor in multiple myeloma, but also elucidate a novel antiapoptotic mechanism for Bmi-1 function involving the suppression of Bim.

Role for the Proapoptotic Factor BIM in Mediating Imatinib-induced Apoptosis in a c-KIT-dependent Gastrointestinal Stromal Tumor Cell Line

The c-KIT receptor tyrosine kinase is constitutively activated and oncogenic in the majority of gastrointestinal stromal tumors. The identification of selective inhibitors of c-KIT, such as imatinib, has provided a novel therapeutic approach in the treatment of this chemotherapy refractory tumor. However, despite the clinical importance of these findings and the potential it provides as a model system for understanding targeted therapy, this approach has not yielded curative outcomes in most patients, and the biochemical pathways connecting c-KIT inhibition to cell death are not completely understood. Here, we show that inhibition of c-KIT with imatinib in gastrointestinal stromal tumors (GISTs) triggered the up-regulation of the proapoptotic protein BIM via both transcriptional and post-translational mechanisms. The inhibition of c-KIT by imatinib increased levels of the dephosphorylated and deubiquitinated form of BIM as well as triggered the accumulation of the transcription factor FOXO3a on the BIM promoter to activate transcription of BIM mRNA. Furthermore, using RNA interference directed against BIM, we demonstrated that BIM knockdown attenuated the effects of imatinib, suggesting that BIM functionally contributes to imatinib-induced apoptosis in GIST. The identification and characterization of the pathways that mediate imatinib-induced cell death in GIST provide for a better understanding of targeted therapy and may facilitate the development of new therapeutic approaches to further exploit these pathways.

Glutathione S‐transferase M1 inhibits dexamethasone‐induced apoptosis in association with the suppression of Bim through dual mechanisms in a lymphoblastic leukemia cell line

Glutathione S-transferase mu (GSTM1) is mainly known as a detoxification enzyme but it has also been shown to be a negative regulator of apoptosis-related signaling cascades. Recently GSTM1 has been reported to be a significant risk factor for hematological relapse in childhood acute lymphoblastic leukemia, although the underlying mechanism remains largely unknown. Glucocorticoids play a crucial role in the treatment of childhood acute lymphoblastic leukemia, therefore we hypothesized that GSTM1 plays important roles in glucocorticoid-induced apoptotic pathways. To clarify the relationship between GSTM1 and drug resistance, GSTM1 was transfected into a T-acute lymphoblastic leukemia cell line, CCRF-CEM (CEM), and we established the GSTM1-expressing cell lines CEM/M1-4 and CEM/M1-9. Transduction of GSTM1 into CEM selectively decreased cellular sensitivity to dexamethasone in a manner that was independent of glutathione conjugation, but was due to apoptosis inhibition. Dexamethasone-induced p38-MAPK and Bim activation were concomitantly suppressed. Interestingly, nuclear factor kappa b (NF-kappaB) p50 activity was upregulated in GSTM1-expressing CEM. Inhibition of NF-kappaB by the pharmacological agent BAY11-7082 greatly enhanced the sensitivity of the GSTM1-expressing CEM to dexamethasone and was accompanied by an increase in Bim expression. Thus, we propose that GSTM1, a novel regulator of dexamethasone-induced apoptosis, causes dexamethasone resistance by suppression of Bim through dual mechanisms of both downregulation of p38-MAPK and upregulation of NF-kappaB p50.

Bim-targeted cancer therapy: A link between drug action and underlying molecular changes

In the past few years, the pro-apoptotic molecule Bim has attracted increasing attention as a plausible target for tumor therapy. A variety of normal and pathological systems regulated by Bim, dependent on cell type, apoptotic stimulation, and chemotherapeutic agents, have been documented. Bim promotes anoikis of many tumor cells, such as lung cancer, breast cancer, osteosarcoma, and melanoma. Various chemotherapeutic agents use Bim as a mediating executioner of cell death. Hence, Bim suppression supports metastasis and chemoresistance. Imatinib, gefitinib, bortezomib, and Bim protein itself are spotlighted as current and future Bim-targeting therapeutic agents. The potential benefits of Bim-targeted therapies are selectivity of treatment for tumor cells and reduction in tumor-associated phenomena such as chemoresistance and metastasis. Thus, Bim-targeting therapies may provide more effective and unique tumor management modalities in future. This review article discusses all these issues.

Mesothelin Promotes Anchorage-Independent Growth and Prevents Anoikis via Extracellular Signal-Regulated Kinase Signaling Pathway in Human Breast Cancer Cells

Mesothelin (MSLN) is a glycoprotein that is overexpressed in various tumors. MSLN is present on the cell surface and is also released into body fluids or culture supernatants from MSLN-positive tumor cells. Despite intensive study of MSLN as a diagnostic marker or target for immunotherapy, its biological function is largely unknown. In the present study, we examined the effects of ectopic expression of MSLN in human breast cancer cell lines (MCF-7, T47D, and MDA-MB-231). We found that overexpression of MSLN promoted anchorage-independent growth in soft agar. In addition, MDA-MB-231 cells expressing high levels of MSLN exhibited resistance to anoikis (a type of apoptosis induced by detachment from substratum), as indicated by decreased DNA fragmentation and down-regulation of the proapoptotic protein Bim. Incubating MSLN-expressing MDA-MB-231 cells in the presence of U0126, an inhibitor of mitogen-activated protein/extracellular-signal-regulated kinase kinase, induced accumulation of Bim and restored susceptibility to anoikis. Western blot analysis also revealed that overexpression of MSLN resulted in sustained activation of extracellular signal-regulated kinase 1/2 and suppression of Bim. The present results constitute novel evidence that MSLN enables cells to survive under anchorage-independent conditions by suppressing Bim induction via the extracellular signal-regulated kinase signaling pathway.

PINCH-1 Regulates the ERK-Bim Pathway and Contributes to Apoptosis Resistance in Cancer Cells

Resistance to apoptosis is a hallmark of cancer cells. We report here that PINCH-1, a cytoplasmic component of cell-extracellular matrix adhesions, is required for protection of multiple types of cancer cells from apoptosis. Furthermore, using HT-1080 fibrosarcoma cells as a model system, we have investigated the signaling pathway through which PINCH-1 contributes to apoptosis resistance. Loss of PINCH-1 markedly increases the level of Bim and promotes Bim translocation to mitochondria, resulting in activation of the intrinsic apoptosis pathway. Depletion of Bim completely blocked apoptosis induced by the loss of PINCH-1. Thus, PINCH-1 contributes to apoptosis resistance through suppression of Bim. Mechanistically, PINCH-1 suppresses Bim not only transcriptionally but also post-transcriptionally. PINCH-1 promotes activating phosphorylation of Src family kinase and ERK1/2. Consistent with this, ERK1/2-mediated Ser(69) phosphorylation of Bim, a key signal for turnover of Bim, is suppressed by the removal of PINCH-1. Our results demonstrate a strong dependence of multiple types of apoptosis-resistant cancer cells on PINCH-1 and provide new insights into the molecular mechanism by which cancer cells are protected from apoptosis.

HOXA9 Represses Bim Expression in MLL Rearranged Leukemia: Implications for Drug Therapy.

MLL translocations can be found in roughly 10% of acute leukemias, including over 70% of infant leukemias. These leukemias are often associated with a poor prognosis, making new therapeutics for this subtype of leukemias a priority. HoxA cluster genes have been shown to be upregulated by MLL fusion proteins, and make up a portion of a self-renewal associated gene expression signature found in murine models of MLL-AF9-expressing leukemia stem cells. The role of HoxA cluster genes in MLL-fusion mediated leukemia is not clearly understood, mainly due to lack of knowledge of HoxA downstream targets. One possible role for HoxA proteins in MLL-fusion induced leukemia is enhancement of growth and survival pathways. Evidence for this possibility can be found in C.elegans, where the pro-apoptotic BH3-only gene egl-1 is regulated by homeobox genes. Therefore we sought to determine whether a HoxA cluster gene could in fact regulate survival/apoptosis machinery. Here we demonstrate that HoxA9 serves as a repressor of the pro-apoptotic BH3-only family member Bim. Knockdown of HoxA9 using shRNA in the MLL-AF9 expressing AML cell line Molm14 leads to upregulation of Bim, and rapid induction of apoptosis. Interestingly, knockdown of Meis1 or Pbx2, known binding partners of HoxA9, showed only an intermediate effect, with slower growth, but without an increase in Bim levels. Other BH3-only family members Noxa and Puma showed no significant change in expression after HoxA9 suppression as determined by quantitative RT-PCR. Using a FLAG-HOXA9 expressing Molm14 cell line, we were able to determine by chromatin immunoprecipitation that HOXA9 is present at a DNA region containing a canonical HOX-PBX binding site (TGATTTAT) roughly 100bp downstream of the first exon-intron junction in the human Bim gene. As histone deacetylases (HDACs) are generally transcription repressors, it is plausible that HOXA9 recruits HDACs to the Bim gene, thus regulating its expression. Since HDAC inhibitors also activate BIM, we assessed BIM activation in Molm14 cells after treatment with an HDAC inhibitor, and noted similar kinetics of BIM activation, suggesting a possible link between HoxA9 mediated suppression of BIM and HDACs. We are now assessing HOXA9/HDAC interactions in MLL rearranged leukemia cells. These data suggest that HOXA9 ensures survival of leukemia cells by directly inhibiting transcription of the pro-apoptotic gene Bim, and that interfering with this mechanism may improve treatment for MLL-fusion bearing leukemias.