Intrinsic Death Pathway Research Articles

Low Probability Activation of Bax/Bak Can Induce Selective Killing of Cancer Cells by Generating Heterogeneity in Apoptosis

Biomimetic pro-apoptotic agents (e.g., BH3 mimetics) have been shown to activate the intrinsic death pathway (Type 2 apoptosis) selectively in cancer cells, a mechanism that can be key to developing successful anti-cancer therapy. This work reports mathematical modeling and computer simulations to explore the mechanisms for cancer cell apoptosis. The results indicate that a combination of low probability Bid-Bax type reaction along with overexpressed reactant molecules allows specific killing of cancer cells. Low-probability activation of Bax also emerges as a basis for inherent cell-to-cell variability in apoptotic activation. Variations in Bcl-2 to Bax ratio within a cancer cell population can further affect intrinsic fluctuations generated due to the stochastic Bid-Bax reaction. Such heterogeneity in apoptosis resistance can also provide a mechanism for the origin of cells with higher tumorigenic potential (cancer stem-like cells). The implications of our results for cancer therapy, such as in minimizing stochastic fluctuations in cancer cell death, are discussed.

Overexpression of cyclin B1 antagonizes chemotherapeutic-induced apoptosis through PTEN/Akt pathway in human esophageal squamous cell carcinoma cells

The role of cyclin B1 in the clinical therapeutic sensitivity of human esophageal squamous cell carcinoma (ESCC) remains to be defined. In this study, we found that elevated cyclin B1 expression attenuated the apoptosis induced by cisplatin or paclitaxel, while knockdown of cyclin B1 enhanced cisplatin or paclitaxel sensitivity in ESCC cells. Cyclin B1-mediated apoptosis may rely on the Bcl-2-dependent mitochondria-regulated intrinsic death pathway, and the antagonizing effect of cyclin B1 on chemotherapeutic agent-induced apoptosis was through PTEN/Akt pathway. Therefore, cyclin B1 might be a therapeutic target for the development of specific and efficient approaches in the treatment of ESCC.

Acetaminophen Induces Human Neuroblastoma Cell Death through NFKB Activation

Neuroblastoma resistance to apoptosis may contribute to the aggressive behavior of this tumor. Therefore, it would be relevant to activate endogenous cellular death mechanisms as a way to improve neuroblastoma therapy. We used the neuroblastoma SH-SY5Y cell line as a model to study the mechanisms involved in acetaminophen (AAP)-mediated toxicity by measuring CYP2E1 enzymatic activity, NFkB p65 subunit activation and translocation to the nucleus, Bax accumulation into the mitochondria, cytochrome c release and caspase activation. AAP activates the intrinsic death pathway in the SH-SY5Y human neuroblastoma cell line. AAP metabolism is partially responsible for this activation, because blockade of the cytochrome CYP2E1 significantly reduced but did not totally prevent, AAP-induced SH-SY5Y cell death. AAP also induced NFkB p65 activation by phosphorylation and its translocation to the nucleus, where NFkB p65 increased IL-1β production. This increase contributed to neuroblastoma cell death through a mechanism involving Bax accumulation into the mitochondria, cytochrome c release and caspase3 activation. Blockade of NFkB translocation to the nucleus by the peptide SN50 prevented AAP-mediated cell death and IL-1β production. Moreover, overexpression of the antiapoptotic protein Bcl-xL did not decrease AAP-mediated IL-1β production, but prevented both AAP and IL-1β-mediated cell death. We also confirmed the AAP toxic actions on SK-N-MC neuroepithelioma and U87MG glioblastoma cell lines. The results presented here suggest that AAP activates the intrinsic death pathway in neuroblastoma cells through a mechanism involving NFkB and IL-1β.

GdCl3 Induced Hep G2 Cell Death through Mitochondrial and External Death Pathways without Significant Elevation of ROS Generation

Gadolinium (Gd) compounds have important applications as MRI contrast and potential anticancer agents. The present study investigated the mechanisms of the proapoptotic effect of gadolinium chloride (GdCl(3)) on hepatoblastoma cell line (Hep G2) tumor cells. The experimental results indicated that GdCl(3) induced apoptosis of Hep G2 at high concentration and with long time incubation; however, unlike the actions on normal cell lines, GdCl(3) did not cause any oxidative stress on tumor cells. Cytochrome c (Cyt c) and apoptosis inducing factor release, Bax translocation, collapse of mitochondria membrane potential, caspase 3 and 8 activation, and Bid cleavage were observed along with a sustained activation of extracellular signal-regulated kinase (ERK) and c-Jun NH2 terminal kinase (JNK). Addition of ERK and JNK inhibitor attenuated the effect of GdCl(3) induced apoptosis and Cyt c release. All the results suggested a novel mechanism that GdCl(3) induced Hep G2 cell death through intrinsic and external death pathways without significant elevation of reactive oxygen species generation. The present work provided new insight to understand the mechanisms of the biological effects of GdCl(3) and implications for the development of anticancer Gd agents.

Mitoptosis, a Novel Mitochondrial Death Mechanism Leading Predominantly to Activation of Autophagy

Mitoptosis, a Novel Mitochondrial Death Mechanism Leading Predominantly to Activation of Autophagy

AIF depletion provides neuroprotection through a preconditioning effect

Previous studies established a major role for apoptosis inducing factor (AIF) in neuronal cell death after acute brain injury. For example, AIF translocation from mitochondria to the nucleus determined delayed neuronal death, whereas reduced AIF expression provided neuroprotective effects in models of cerebral ischemia or brain trauma. The question remains, however, why reduced AIF levels are sufficient to mediate neuroprotection, since only very little AIF translocation to the nucleus is required for induction of cell death. Thus, the present study addresses the question, whether AIF gene silencing affects intrinsic death pathways upstream of nuclear translocation at the level of the mitochondria. Using MTT assays and real-time cell impedance measurements we confirmed the protective effect of AIF siRNA against glutamate toxicity in immortalized mouse hippocampal HT-22 neurons. Further, AIF siRNA prevented glutamate-induced mitochondrial fragmentation and loss of mitochondrial membrane potential. The protection of mitochondrial integrity was associated with preserved ATP levels, attenuated increases in lipid peroxidation and reduced complex I expression levels. Notably, low concentrations of the complex I inhibitor rotenone (20 nM), provided similar protective effects against glutamate toxicity at the mitochondrial level. These results expose a preconditioning effect as a mechanism for neuroprotection mediated by AIF depletion. In particular, they point out an association between mitochondrial complex I and AIF, which regulate each other's stability in mitochondria. Overall, these findings postulate that AIF depletion mediates a preconditioning effect protecting neuronal cells from subsequent glutamate toxicity through reduced levels of complex I protein.

Pulling the plug on a cancer cell by eliminating XIAP with AEG35156

XIAP, the X-linked inhibitor of apoptosis, is the best example of an endogenous cellular suppressor of apoptosis. XIAP is effective because it directly limits the activity of several critical death-inducing caspases, notably caspase-3, -7 and -9, either by direct enzyme inhibition or through ubiquitin-mediated proteasomal degradation. Furthermore, XIAP acts simultaneously at several nodes in the apoptotic cascade, blocking both the intrinsic and extrinsic death pathways, and thereby preventing feed-forward amplification loops that would otherwise lead to cell death. XIAP over-expression, or increased activity, is associated with cancer progression, resistance to therapy and poor prognosis. Targeting XIAP gene expression by antisense oligonucleotides, or other approaches, demonstrates anti-cancer effects with XIAP down-regulation. These early preclinical studies led to the development of a clinical candidate mixed-backbone antisense oligonucleotide, AEG35156, against XIAP for the treatment of cancer. Published clinical results for the first-in-class and first-in-human trials of AEG35156 are summarized herein, including single agent and combination chemotherapy phase-I or -II trials for solid tumors, lymphoma, and acute myeloid leukemia. These trials demonstrate the safety of AEG35156, as well as some initial promising signs of anti-cancer activity.

Sensitization of cancer cells to TRAIL-induced apoptosis by drugs targeting mitochondrial ROS and endoplasmic reticulum

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is promising for cancer treatment because of its selective cytotoxicity toward tumor cells. However, some cancer cell types including malignant melanoma are resistant to the TRAIL-mediated cytotoxicity. Therefore, drugs that potentiate TRAIL effectiveness are urgently required. A variety of agents such as inhibitors of proteases, histone deacetylases, and tyrosine kinases modulate different processes in the extrinsic or intrinsic (mitochondrial) death pathways and therefore give potential strategies to re-sensitize tumor cells to TRAIL. A growing body of evidence suggests that the endoplasmic reticulum (ER) is the key player in regulation of apoptosis and that ER stress is the major cause of a variety of pro-apoptotic effects. We recently discovered that divergent agents, including ATP-sensitive potassium (KATP) channel inhibitors, hydrogen peroxide (H2O2), the garlic organosulfur compound diallyl trisulfide and the mitochondrial fission inhibitor Mdivi-1, commonly potentiated the TRAIL-induced apoptosis in human leukemia, melanoma, and/or non-small cell lung cancer cells. These agents also potentiated apoptosis induced by agonistic monoclonal antibodies against death receptor 4 and 5. At higher concentrations, these agents alone had substantial apoptosis-inducing activities. Importantly, these agents spared normal cells such as melanocytes and fibroblasts, indicating that their effects are tumor-selective. Our data suggest that (i) these agents commonly induce mitochondrial reactive oxygen species (ROS) generation. (ii) The mitochondrial ROS trigger an ER-mediated death pathway involving X-box-binding protein-1 and caspase-12, which is important for re-sensitizing tumor cells to TRAIL. (iii) Normal cells are more resistant than tumor cells to the TRAIL-induced mitochondrial ROS generation. Based on our findings and others, the new strategies to overcome TRAIL resistance by agents targeting the mitochondria-ER dynamics will be discussed.

Rational Development of a Cytotoxic Peptide To Trigger Cell Death

Defects in the apoptotic machinery can contribute to tumor formation and resistance to treatment, creating a need to identify new agents that kill cancer cells by alternative mechanisms. To this end, we examined the cytotoxic properties of a novel peptide, CT20p, derived from the C-terminal, alpha-9 helix of Bax, an amphipathic domain with putative membrane binding properties. Like many antimicrobial peptides, CT20p contains clusters of hydrophobic and cationic residues that could enable the peptide to associate with lipid membranes. CT20p caused the release of calcein from mitochondrial-like lipid vesicles without disrupting vesicle integrity and, when expressed as a fusion protein in cells, localized to mitochondria. The amphipathic nature of CT20p allowed it to be encapsulated in polymeric nanoparticles (NPs) that have the capacity to harbor targeting molecules, dyes or drugs. The resulting CT20p-NPs proved an effective killer, in vitro, of colon and breast cancer cells, and in vivo, using a murine breast cancer tumor model. By introducing CT20p to Bax deficient cells, we demonstrated that the peptide's lethal activity was independent of endogenous Bax. CT20p also caused an increase in the mitochondrial membrane potential that was followed by plasma membrane rupture and cell death, without the characteristic membrane asymmetry associated with apoptosis. We determined that cell death triggered by the CT20p-NPs was minimally dependent on effector caspases and resistant to Bcl-2 overexpression, suggesting that it acts independently of the intrinsic apoptotic death pathway. Furthermore, use of CT20p with the apoptosis-inducing drug, cisplatin, resulted in additive toxicity. These results reveal the novel features of CT20p that allow nanoparticle-mediated delivery to tumors and the potential application in combination therapies to activate multiple death pathways in cancer cells.

The Apoptosis Related BIM Is An HSP70‐Specific Client Protein

Heat shock protein chaperones have important roles in regulating the function of cells under pathogenic stress, including cancer. Among them, heat inducible Hsp70 and constitutive Hsc70 are of especial interest because they act upstream of the major onco‐protein regulator chaperone, Hsp90, and additionally, they have potent anti‐apoptotic activities by inhibiting both intrinsic and extrinsic death pathways. Here we use a novel Hsp70/Hsc70 inhibitor, YK5, to study a mechanism of Hsp70‐dependent apoptosis in breast cancer cells. We show that the Hsp70 inhibitor activates caspase 8 – depended apoptotic pathways and induces cleavage of the apoptotic protein Bim. We show through chemical precipitation that Bim physically associates with the Hsp70 protein, demonstrating that it is an Hsp70 client protein. Following Hsp70 inhibitor treatment, we identify the formation of a cleaved Bim product, a Bim‐species previously demonstrated to be more efficient at inducing apoptosis than the full‐length protein. Proteasome and serine proteases are involved in Hsp70 – depended Bim degradation and cleavage, and inhibition of serine proteases correlates with a decrease in the Hsp70 inhibitor – induces apoptosis. The data suggest Bim to be an important player in Hsp70 ‐ regulated apoptosis inhibition in cancer cells, and indicate Hsp70 as a potential inhibitor of the formation of the hyperactive cleaved Bim species.

Hypoxia-inducible Factor 1 regulated ARC expression mediated hypoxia induced inactivation of the intrinsic death pathway in p53 deficient human colon cancer cells

Apoptosis repressor with caspase recruitment domain (ARC), an anti-apoptotic protein, plays an important role in the regulation of apoptosis by blocking both the extrinsic and intrinsic death pathways. However, its regulatory mechanism remains largely undefined. Here, we reported that hypoxia up-regulated the expression of ARC in p53 deficient human colon cancer cells. Moreover, ARC is a direct target of the hypoxia-inducible factor 1 (HIF-1), a key transcriptional factor for the cellular response to hypoxia. Silencing the expression of HIF-1α in SW480 colon cancer cells by RNA interference abolished hypoxia induced ARC expression. Using luciferase reporter and ChIP assay, we showed that HIF-1α directly bound to hypoxia-responsive element located at −419 to −414 of ARC gene, which is essential for HIF-1-induced expression. As a result of the increased ARC expression, TRAIL-induced apoptosis was reduced by hypoxia. These discoveries would shed novel insights on the mechanisms for ARC expression regulation and hypoxia induced inactivation of the intrinsic death pathway.

Insulin-like Growth Factor Receptor-1 and Nuclear Factor κB Are Crucial Survival Signals That Regulate Caspase-3-mediated Lens Epithelial Cell Differentiation Initiation

It is now known that the function of the caspase family of proteases is not restricted to effectors of programmed cell death. For example, there is a significant non-apoptotic role for caspase-3 in cell differentiation. Our own studies in the developing lens show that caspase-3 is activated downstream of the canonical mitochondrial death pathway to act as a molecular switch in signaling lens cell differentiation. Importantly, for this function, caspase-3 is activated at levels far below those that induce apoptosis. We now have provided evidence that regulation of caspase-3 for its role in differentiation induction is dependent on the insulin-like growth factor-1 receptor (IGF-1R) survival-signaling pathway. IGF-1R executed this regulation of caspase-3 by controlling the expression of molecules in the Bcl-2 and inhibitor of apoptosis protein (IAP) families. This effect of IGF-1R was mediated through NFκB, demonstrated here to function as a crucial downstream effector of IGF-1R. Inhibition of expression or activation of NFκB blocked expression of survival proteins in the Bcl-2 and IAP families and removed controls on the activation state of caspase-3. The high level of caspase-3 activation that resulted from inhibiting this IGF-1R/NFκB signaling pathway redirected cell fate from differentiation toward apoptosis. These results provided the first evidence that the IGF-1R/NFκB cell survival signal is a crucial regulator of the level of caspase-3 activation for its non-apoptotic function in signaling cell differentiation.

Murine regulatory T cells contain hyperproliferative and death-prone subsets with differential ICOS expression.

Regulatory T cells (Treg) are crucial for self-tolerance. It has been an enigma that Treg exhibit an anergic phenotype reflected by hypoproliferation in vitro after TCR stimulation but undergo vigorous proliferation in vivo. We report in this study that murine Treg are prone to death but hyperproliferative in vitro and in vivo, which is different from conventional CD4(+)Foxp3(-) T cells (Tcon). During in vitro culture, most Treg die with or without TCR stimulation, correlated with constitutive activation of the intrinsic death pathway. However, a small portion of the Treg population is more sensitive to TCR stimulation, particularly weak stimulation, proliferates more vigorously than CD4(+) Tcon, and is resistant to activation-induced cell death. Treg proliferation is enhanced by IL-2 but is less dependent on CD28-mediated costimulation than that of Tcon. We demonstrate further that the surviving and proliferative Treg are ICOS(+) whereas the death-prone Treg are ICOS(-). Moreover, ICOS(+) Treg contain much stronger suppressive activity than that of ICOS(-) Treg. Our data indicate that massive death contributes to the anergic phenotype of Treg in vitro and suggest modulation of Treg survival as a therapeutic strategy for treatment of autoimmune diseases and cancer.

IJU this issue

This issue consists of two Perspectives in Urology, three Review Articles, five Original Articles, one Case Report, one Short Communication and two other reports. The content of this issue focuses on various interesting hot topics associated with clinical, as well as basic, aspects; therefore, I believe that this issue might help provide useful information for a wide variety of readers of IJU. This issue starts with Perspectives in Urology, a newly introduced category in IJU, which briefly presents a scientific view of several fields of urology by specialists. Two articles published in this issue describe the application of gasless single-port surgery to partial nephrectomy by Kihara (Tokyo, Japan) and controversial issues in the decontamination process for urological endoscopes by Yanaihara et al. (Saitama, Japan). This issue then includes three interesting Review Articles. Takenaka et al. (Tottori, Japan) summarized the recent progress in anatomical findings in relation to each basic step during robotic-assisted laparoscopic radical prostatectomy. They emphasized the important message that surgical anatomy should always be developing and changing with advances in surgical approaches. Matsumoto et al. (Asahikawa, Japan) described the pathophysiological relationship between metabolic syndrome and lower urinary tract symptoms (LUTS) with a special emphasis on bladder blood flow. They also proposed a novel therapeutic strategy for patients with LUTS from the viewpoint of bladder blood flow. Azuma et al. (Takatsuki, Japan) reviewed their experience of the development of novel bladder preservation approach for patients with locally advanced bladder cancer. They carried out combined therapy using balloon-occluded arterial infusion anticancer agents and hemodialysis with concurrent radiation, and presented promising oncological outcomes. Consequently, five excellent Original Articles are provided. Jensen et al. (Aarhus, Denmark) compared the prognostic significance between extended and limited lymph node dissection in patients with bladder cancer who underwent radical cystectomy. Although extended nodal dissection provided more accurate staging than limited dissection, recurrence patterns appeared to be similar between these two groups. Shigemura et al. (Kobe, Japan) compared Wallace direct ureteroileal anastomosis with the Le Duc anti-reflux procedure in modified Studer orthotopic neobladder reconstruction after radical cystectomy. The authors concluded that direct ureteroileal anastomosis using the Wallace method is effective for minimizing the stenosis of ureteroileal anastomosis. Bayraktar et al. (Istanbul, Turkey) assessed the effect of constipation on serum prostate-specific antigen (PSA) levels in men. In this study, serum PSA values in the constipated patients before treatment were shown to be significantly higher than those in the control group; therefore, the authors recommended the re-evaluation of PSA value in the constipated patients before carrying out the prostate biopsy. Ip et al. (Taichung, Taiwan) reported the apoptotic effects of bee venom on human bladder cancer cells and the signaling pathways involved in this event. This article clearly showed the induction of both caspase-dependent and caspase-independent apoptotic cell death in human bladder cancer cells by bee venom through the intracellular Ca2+-modulated intrinsic death pathway. Nakane et al. (Gifu, Japan) analyzed the role of cortactin and silent mating type information regulation 2 homolog 1 (SIRT1) in migration and invasion of human prostate cancer DU145 cells. Because knockdown of cortaction or SIRT1 expression resulted in the inhibition of both migration and invasion of DU145 cells, the authors suggest that this strategy could be a promising treatment for prostate cancer by regulating its metastatic spread. This issue also included an interesting Case Report and Short Communication. Fujisaki et al. (Shimotsuke-city, Japan) reported life-threatening anaphylaxis to leuprorelin acetate depot. Sugihara et al. (Iwata, Japan) reported the low incidence of admissions related to interstitial cystitis in Japan, which could possibly be explained by several factors, including racial difference, examination methods, lack of outpatient data and poor health-care coverage of this disease. None declared.

Bee venom induces apoptosis through intracellular Ca2+‐modulated intrinsic death pathway in human bladder cancer cells

To focus on bee venom-induced apoptosis in human bladder cancer TSGH-8301 cells and to investigate its signaling pathway to ascertain whether intracellular calcium iron (Ca(2+)) is involved in this effect. Bee venom-induced cytotoxic effects, productions of reactive oxygen species and Ca(2+) and the level of mitochondrial membrane potential (ΔΨm) were analyzed by flow cytometry. Apoptosis-associated proteins were examined by Western blot analysis and confocal laser microscopy. Bee venom-induced cell morphological changes and decreased cell viability through the induction of apoptosis in TSGH-8301 cell were found. Bee venom promoted the protein levels of Bax, caspase-9, caspase-3 and endonuclease G. The enhancements of endoplasmic reticulum stress-related protein levels were shown in bee venom-provoked apoptosis of TSGH-8301 cells. Bee venom promoted the activities of caspase-3, caspase-8, and caspase-9, increased Ca(2+) release and decreased the level of ΔΨm. Co-localization of immunofluorescence analysis showed the releases of endonuclease G and apoptosis-inducing factor trafficking to nuclei for bee venom-mediated apoptosis. The images revealed evidence of nuclear condensation and formation of apoptotic bodies by 4',6-diamidino-2-phenylindole staining and DNA gel electrophoresis showed the DNA fragmentation in TSGH-8301 cells. Bee venom treatment induces both caspase-dependent and caspase-independent apoptotic death through intracellular Ca(2+) -modulated intrinsic death pathway in TSGH-8301 cells.

The role of mitochondria-mediated intrinsic death pathway in gingerdione derivative I6-induced neuronal apoptosis

Neuronal death induced by I6 displayed apoptotic characteristics but the precise mechanism has not been fully elucidated. In the present studies, I6 at 24h after intraperitoneal administration significantly decreased the density of surviving neurons and increased caspase-3 activity in frontal cortex, suggesting that peripherally administered I6 may cross BBB to induce CNS toxicity. In rat embryonic primary cortical cells, I6-induced reduction of mitochondrial viability and neuronal apoptosis was inhibited by vitamin E. In addition, I6-induced reactive oxygen species (ROS) caused the disruption of mitochondria membrane potential (MMP), the release of cytochrome c, the activation of caspase-9 and caspase-3, and cleavage of poly(ADP-ribose) polymerase (PARP), resulting in activation of mitochondrial-mediated intrinsic death pathway. Pre-treatment with antioxidant vitamin E or N-acetylcysteine (NAC) completely abolished the I6-induced generation of ROS, loss of MMP, release of cytochrome c, activation of caspase-9 and caspase-3, and cleavage of PARP. Carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP), a mitochondrial uncoupler, significantly reduced I6-induced neuronal death as well as caspase-3 activation and PARP cleavage. These results suggest that I6 induces neuronal death by promoting intracellular ROS production to cause a loss of MMP that result in release of cytochrome c and activation of mitochondria-mediated intrinsic death pathway.

The p90RSK Inhibitor BI-D1870 Induces Apoptosis in CLL Cells

Abstract 2492Chronic Lymphocytic leukaemia (CLL) is the most common leukemia in the western world but is currently incurable using conventional therapies. Therefore novel agents which can kill CLL cells either alone or in combination with conventional agents are being continually sought. Activation of the MAPK pathway has been implicated as one of the protective signaling pathway in CLL and is characterised by expression of phosphorylated ERK. In CLL cells ERK can be activated by phosphorylation after BCR activation with αIgM. However in a proportion of CLL isolates ERK is constitutively active. MEK inhibitors (MEKi) are currently being used in clinical trials to treat relapsed or refractory leukemia including CLL (www.clinicaltrials.gov, NCT00920140). Therefore we hypothesised that inhibiting the ribosomal S6 kinase p90RSK which is immediately downstream of the MAPK pathway, may be of therapeutic value for the treatment of CLL. Since p90RSK regulates the majority of the anti-apoptotic signaling of the ERK pathway, we investigated the effect of inhibiting p90RSK with its specific inhibitor BI-D1870 (BI-D) on apoptosis in CLL.Immunoblotting was carried out to investigate phosphorylation of components of the ERK/p90RSK pathway using phosphorylation state-specific and pan-reactive antibodies. BI-D toxicity was evaluated by Annexin V/PI staining, MTT assay and immunoblotting for cleavage of the caspase 3 substrate poly(ADP ribose) polymerase (PARP) from its 116KDa to its 85KDa form.BI-D treatment of CLL cells decreased S6 phosphorylation, and this down regulation correlated with apoptosis. BI-D killed CLL cells rapidly, with a mean IC50 of 12μM following 18h incubation. Treatment of CLL cells with BI-D induced cytochrome C release indicating inhibition of p90RSK induced apoptosis by the intrinsic death pathway. However its toxicity was not due to inhibition of protein translation since inhibition of mTOR, a parallel pathway which also regulates S6 phosphorylation, was minimally toxic to CLL cells at concentrations as high as 500nM. However apoptosis did coincide with a reduction of the antiapoptotic protein Mcl-1. We further demonstrated that BI-D augmented apoptosis induction by cytotoxic drugs, as shown by PARP cleavage and by annexin V/PI binding assays. Analysis of cytotoxicity data using CalcuSyn software provided evidence for synergistic killing of the majority of CLL isolates by combinations of BI-D with either chlorambucil, fludarabine or nutlin 3.Constitutive or antigen induced activation of p90RSK via the MAPK pathway in CLL cells may contribute to apoptosis resistance and its inhibition may be of value in devising novel therapeutic strategies, either alone or in conjunction with conventional cytotoxic drugs. p90RSK inhibition led to apoptosis independently of protein translation inhibition and may be attributable in part to downregulation of Mcl-1. Given the recent interest in using MEK inhibitors (MEKi) for the treatment of CLL, these data suggests MEKi may induce apoptosis in CLL in part by inhibiting pathways downstream of p90RSK. In addition BI-D is a highly specific inhibitor of p90RSK and therefore may negate the off target effects shown with some MEKi. This is the first report on the consequences of inhibition of p90RSK in CLL cells and suggests that inhibiting p90RSK may be of considerable interest for the treatment of CLL. Disclosures:No relevant conflicts of interest to declare.

2.32 The p90RSK Inhibitor BI-D1870 Induces Apoptosis in CLL Cells

Abstract 2492 Chronic Lymphocytic leukaemia (CLL) is the most common leukemia in the western world but is currently incurable using conventional therapies. Therefore novel agents which can kill CLL cells either alone or in combination with conventional agents are being continually sought. Activation of the MAPK pathway has been implicated as one of the protective signaling pathway in CLL and is characterised by expression of phosphorylated ERK. In CLL cells ERK can be activated by phosphorylation after BCR activation with αIgM. However in a proportion of CLL isolates ERK is constitutively active. MEK inhibitors (MEKi) are currently being used in clinical trials to treat relapsed or refractory leukemia including CLL (www.clinicaltrials.gov, NCT00920140). Therefore we hypothesised that inhibiting the ribosomal S6 kinase p90RSK which is immediately downstream of the MAPK pathway, may be of therapeutic value for the treatment of CLL. Since p90RSK regulates the majority of the anti-apoptotic signaling of the ERK pathway, we investigated the effect of inhibiting p90RSK with its specific inhibitor BI-D1870 (BI-D) on apoptosis in CLL. Immunoblotting was carried out to investigate phosphorylation of components of the ERK/p90RSK pathway using phosphorylation state-specific and pan-reactive antibodies. BI-D toxicity was evaluated by Annexin V/PI staining, MTT assay and immunoblotting for cleavage of the caspase 3 substrate poly(ADP ribose) polymerase (PARP) from its 116KDa to its 85KDa form. BI-D treatment of CLL cells decreased S6 phosphorylation, and this down regulation correlated with apoptosis. BI-D killed CLL cells rapidly, with a mean IC50 of 12μM following 18h incubation. Treatment of CLL cells with BI-D induced cytochrome C release indicating inhibition of p90RSK induced apoptosis by the intrinsic death pathway. However its toxicity was not due to inhibition of protein translation since inhibition of mTOR, a parallel pathway which also regulates S6 phosphorylation, was minimally toxic to CLL cells at concentrations as high as 500nM. However apoptosis did coincide with a reduction of the antiapoptotic protein Mcl-1. We further demonstrated that BI-D augmented apoptosis induction by cytotoxic drugs, as shown by PARP cleavage and by annexin V/PI binding assays. Analysis of cytotoxicity data using CalcuSyn software provided evidence for synergistic killing of the majority of CLL isolates by combinations of BI-D with either chlorambucil, fludarabine or nutlin 3. Constitutive or antigen induced activation of p90RSK via the MAPK pathway in CLL cells may contribute to apoptosis resistance and its inhibition may be of value in devising novel therapeutic strategies, either alone or in conjunction with conventional cytotoxic drugs. p90RSK inhibition led to apoptosis independently of protein translation inhibition and may be attributable in part to downregulation of Mcl-1. Given the recent interest in using MEK inhibitors (MEKi) for the treatment of CLL, these data suggests MEKi may induce apoptosis in CLL in part by inhibiting pathways downstream of p90RSK. In addition BI-D is a highly specific inhibitor of p90RSK and therefore may negate the off target effects shown with some MEKi. This is the first report on the consequences of inhibition of p90RSK in CLL cells and suggests that inhibiting p90RSK may be of considerable interest for the treatment of CLL. Disclosures: No relevant conflicts of interest to declare.

Regulation of T‐cell survival and mitochondrial homeostasis by TSC1

The mammalian target of rapamycin (mTOR) is a key regulator of cell growth and metabolism. It associates with multiple proteins and forms two distinct signaling complexes, mTORC1 and mTORC2. Accumulating evidence has revealed critical roles for intact mTOR signaling during T-cell activation and responses to microbial infection. However, the importance of mTOR regulation in T cells has yet to be explored. The TSC1/TSC2 complex has been shown to inhibit mTORC1 signaling in cell line models. We show here that deletion of TSC1 in the murine T-cell lineage results in a dramatic reduction of the peripheral T-cell pool, correlating with increased cell death. While mTORC1 is constitutively activated, mTORC2 signaling, reflected by Akt phosphorylation and activity, is decreased in TSC1-deficient T cells. Furthermore, TSC1-deficient T cells contain elevated reactive oxygen species (ROS) and exhibit decreased mitochondrial content and membrane potential, which is correlated with the activation of the intrinsic death pathway. Overall, our results demonstrate that TSC1 differentially regulates mTORC1 and mTORC2 activity, promotes T-cell survival, and is critical for normal mitochondrial homeostasis in T cells.

Obatoclax effects on MCL1 addiction in mesothelioma and activity in vivo.

e13539 Background: Resistance to apoptosis is a common characteristic of mesothelioma and contributes to multidrug resistance, a particular problem in the clinical setting upon relapse following conventional chemotherapy. Overexpression of the pro-survival BCL-2 family of proteins which include BCL-2, BCL-XL, and MCL-1 contributes to anti-apoptotic signalling, and are highly expressed in mesothelioma. MCL-1 is one of the most commonly amplified genes at 1q21.2 in human cancer and this locus is also amplified in mesothelioma. Methods: All molecular and cell biology techniques used were carried out as described in Crawford et al 2010 or McTavish et al 2007. Results: Across a panel of mesothelioma cell lines several were demonstrated to be primed for death as silencing of Mcl-1 induced apoptosis. This addiction to Mcl-1 was dependent on the intrinsic mitochondrial death pathway as cells that stably expressed shRNA Bax and Bak rescued the addiction due to Mcl-1. Depletion of Bak but not Bax was sufficient to rescue from MCL1 knockdown. In the two Mcl-1 addicted mesothelioma cell lines studied (Ren and MSTO-211H), knockdown of the Noxa also rescued cells from MCL1 silencing induced apoptosis, however depletion of activator BH3 only proteins Bim or Bid only rescued in Ren cells. Amplification of Mcl-1 locus was not essential for addiction. Obatoclax is an inhibitor of MCL1 that is currently in clinical development. On-target activity of obatoclax was demonstrated by BAK/MCL1 co-immunoprecipitation demonstrating dissociation of Bak from Mcl-1 at early timepoints (6-12 hours). Across the panel of mesothelioma cell lines obatoclax exhibited cytotoxicity associated with reduction in cell viability and reduced clonogenic survival in both Mcl-1 addicted and non addicted cell lines. Activity in vivo was evaluated using REN and MSTO-211 xenografts. Obatoclax treatment (8mg/kg) of tumours exhibits significantly reduced tumour growth compared to control tumours. Conclusions: Obatoclax exhibits on target inhibition indicating its activity as a prosurvival BCL-2 antagonist however its activity is not solely dependent on cells being addicted to Mcl-1 for their survival. It is active in both in vitro and in vivo and clinical evaluation mesothelioma is warranted.