Loss Of Mcl-1 Research Articles

Deletion of MCL-1 causes lethal cardiac failure and mitochondrial dysfunction

MCL-1 is an essential BCL-2 family member that promotes the survival of multiple cellular lineages, but its role in cardiac muscle has remained unclear. Here, we report that cardiac-specific ablation of Mcl-1 results in a rapidly fatal, dilated cardiomyopathy manifested by a loss of cardiac contractility, abnormal mitochondria ultrastructure, and defective mitochondrial respiration. Strikingly, genetic ablation of both proapoptotic effectors (Bax and Bak) could largely rescue the lethality and impaired cardiac function induced by Mcl-1 deletion. However, while the overt consequences of Mcl-1 loss were obviated by combining with the loss of Bax and Bak, mitochondria from the Mcl-1-, Bax-, and Bak-deficient hearts still revealed mitochondrial ultrastructural abnormalities and displayed deficient mitochondrial respiration. Together, these data indicate that merely blocking cell death is insufficient to completely overcome the need for MCL-1 function in cardiomyocytes and suggest that in cardiac muscle, MCL-1 also facilitates normal mitochondrial function. These findings are important, as specific MCL-1-inhibiting therapeutics are being proposed to treat cancer cells and may result in unexpected cardiac toxicity.

The Stress Protein BAG3 Stabilizes Mcl-1 Protein and Promotes Survival of Cancer Cells and Resistance to Antagonist ABT-737

Members of the Bcl-2 family of proteins are important inhibitors of apoptosis in human cancer and are targets for novel anticancer agents such as the Bcl-2 antagonists, ABT-263 (Navitoclax), and its analog ABT-737. Unlike Bcl-2, Mcl-1 is not antagonized by ABT-263 or ABT-737 and is considered to be a major factor in resistance. Also, Mcl-1 exhibits differential regulation when compared with other Bcl-2 family members and is a target for anticancer drug discovery. Here, we demonstrate that BAG3, an Hsp70 co-chaperone, protects Mcl-1 from proteasomal degradation, thereby promoting its antiapoptotic activity. Using neuroblastoma cell lines, with a defined Bcl-2 family dependence, we found that BAG3 expression correlated with Mcl-1 dependence and ABT-737 resistance. RNA silencing of BAG3 led to a marked reduction in Mcl-1 protein levels and overcame ABT-737 resistance in Mcl-1-dependent cells. In ABT-737-resistant cells, Mcl-1 co-immunoprecipitated with BAG3, and loss of Mcl-1 after BAG3 silencing was prevented by proteasome inhibition. BAG3 and Mcl-1 were co-expressed in a panel of diverse cancer cell lines resistant to ABT-737. Silencing BAG3 reduced Mcl-1 protein levels and overcame ABT-737 resistance in several of the cell lines, including triple-negative breast cancer (MDA-MB231) and androgen receptor-negative prostate cancer (PC3) cells. These studies identify BAG3-mediated Mcl-1 stabilization as a potential target for cancer drug discovery.

Abstract 19297: Cardiac Specific Deletion of Mcl-1 Leads to Impaired Autophagy and Rapid Development of Heart Failure

Mcl-1 is an anti-apoptotic Bcl-2 protein which is expressed at high levels in the heart compared to other anti-apoptotic Bcl-2 proteins. However, very little is known with respect to how Mcl-1 regulates cell survival in cardiomyocytes. To investigate the functional role of Mcl-1 in the heart, we used a loxP/α-MHC-Cre-recombinase system to generate inducible, cardiomyocyte-specific Mcl-1 knockout mice. Mcl-1 deletion led to rapid contractile dysfunction, cardiac hypertrophy, and interstitial fibrosis in the absence of stress. Surprisingly, loss of Mcl-1 did not activate apoptosis. Instead, Mcl-1 deficiency led to necrotic cell death characterized by mitochondrial permeability transition pore (mPTP) opening, mitochondrial swelling, and cellular rupture. To clarify how mPTP opening and necrosis contribute to heart failure in Mcl-1-/- mice, we generated Mcl-1/Cyclophilin D (CypD) double knockouts. CypD is an essential component of the mPTP, and CypD deficiency has been shown to reduce cardiac necrosis. CypD deletion significantly delayed development of heart failure in Mcl-1-/- mice, but did not rescue the phenotype. Although autophagy is an important cellular quality control mechanism, electron microscopy revealed no autophagosome formation in Mcl-1-/- cardiomyocytes. Further analysis revealed that loss of Mcl-1 led to impaired basal autophagic flux. Western blot analysis for autophagy proteins revealed reduced LC3II/I ratios and accumulation of p62 in Mcl-1-/- hearts. Chloroquine injection, which inhibits the fusion between autophagosomes and lysosomes, led to accumulation of LC3II and p62 in WT hearts but not in Mcl-1-/- hearts. To investigate whether autophagy could be induced in Mcl-1-/- hearts, we subjected mice to acute exercise. Autophagy was rapidly enhanced in wild type hearts after 80 minutes of swimming, but no change in autophagy occurred in Mcl-1-/- hearts. Conversely, adenoviral overexpression of Mcl-1 in rat neonatal cardiomyocytes led to increased LC3 processing and autophagy. These data suggest that, in addition to its anti-apoptotic role, Mcl-1 is essential in initiating autophagy in cardiomyocytes. Increased understanding of Mcl-1 mechanisms in the myocardium may provide new therapeutic avenues for heart disease.

Bcl-xL Protects From UPR-Associated Apoptosis During Plasma Cell Differentiation

Abstract 3288Understanding factors that control plasma cell survival is important for the development of therapeutic approaches to diseases including multiple myeloma and autoimmune disorders. As part of the program that allows for B cell differentiation to a plasma cell, a required signal includes the activation of an unfolded protein response (UPR). However unlike stress-induced activation of the UPR, induction of apoptosis does not occur, suggesting that compensatory survival signals are also activated during plasma cell differentiation. The compensatory survival pathways are less defined and require further research. Therefore we employed a model of plasma cell differentiation to better define the survival signaling during this process. The murine B cell lymphoma cell line, Bcl1 can be stimulated to secrete immunoglobulin using IL-5 and LPS. To determine the effects of exogenous ER stress on plasma cell differentiation, we treated the cells with the inhibitor of N-linked glycosylation, tunicamycin, for 5 hours prior to the differentiation signal. The 5 hour pulse of tunicamycin was sufficient to induce significant apoptosis in undifferentiated cells or cells treated with IL-5, resulting in 78% and 74% cell death respectively by 24 hours post treatment. However, if LPS was included in the differentiation stimulus the cells were able to differentiate into IgM-secreting plasma cells with similar kinetics as cells differentiated in the absence of tunicamycin pretreatment. Thus LPS-induced differentiation is sufficient to block ER stress-induced cell death. Since these cells also activate a UPR during differentiation, we hypothesized that part of the differentiation program included protection from UPR-associated cell death. To investigate this effect, we first examined the levels of the antiapoptotic proteins Bcl-2, Bcl-xL and Mcl-1 during plasma cell differentiation. We found that differentiation induced Bcl-xL and caused the loss of Mcl-1. From this data we hypothesized that the differentiation of these cells resulted in Bcl-xL dependence during plasma cell differentiation. To test this we used ABT-737, which selectively blocks the binding pocket of Bcl-xL and Bcl-2 but not Mcl-1 and kills cells that are dependent on Bcl-2 or Bcl-xL. Undifferentiated Bcl1 cells were insensitive to ABT-737 with an IC50 > 2μM. However ABT-737 sensitized LPS-treated Bcl1 cells to tunicamycin pretreatment resulting in 89% death in 24 h compared to 23% in untreated cells. These data suggest that the induction of Bcl-xL is responsible for the survival of cells undergoing ER stress. Most importantly, cells treated with LPS and IL-5 for differentiation became sensitive to ABT-737 with 59% cell death versus 26% in untreated cells, thus demonstrating that during plasma cell differentiation, cells switch to a Bcl-xL-dependent state. To determine the molecular basis for these findings we investigated the effects of ABT-737 on the expression levels of Bcl-2 proteins as well as the effects of differentiation on their interactions. ABT-737 did not induce changes in the expression of Bcl-2 family proteins. However, co-immunoprecipitation demonstrated a shift in Bim binding from Mcl-1 in untreated cells to Bcl-xL in differentiating cells. This latter finding is consistent with a shift from Mcl-1 dependence to Bcl-xL during plasma cell differentiation. To validate these data, primary C57BL/6 splenocytes were isolated, depleted of non-B cells and subsequently stimulated with IL-4 and LPS to differentiate into plasmablasts. Realtime qPCR showed an increase in Bcl-xL mRNA and loss of Mcl-1 and Bcl-2 mRNA in both the primary B cells and the Bcl1 cell line. Western blotting of primary B cell lysates also showed an increase in Bcl-xL protein and loss of Bcl-2 and Mcl-1 protein. Together these data indicate that during plasma cell differentiation the cell enters a Bcl-xL-dependent state that protects against differentiation-induced apoptosis. Disclosures:No relevant conflicts of interest to declare.

Combination treatments with ABT‐263 and an immunotoxin produce synergistic killing of ABT‐263‐resistant small cell lung cancer cell lines

Synergistic killing was achieved when Small Cell Lung Cancer (SCLC) cell lines were incubated with ABT-263 and an immunotoxin directed to the transferrin receptor. SCLC lines are variably sensitive to the BH-3 only peptide mimetic, ABT-263. To determine their sensitivity to toxin-based reagents, we incubated four representative SCLC lines with a model Pseudomonas exotoxin-based immunotoxin directed to the transferrin receptor. Remarkably in 4-of-4 lines, there was little evidence of immunotoxin-mediated cytotoxicity despite near complete inhibition of protein synthesis. However, when combinations of ABT-263 and immunotoxin were added to the ABT-263-resistant cell lines (H196 and H69AR), there was synergistic killing as evidenced by increased activation of caspase 3/7, annexin V staining, and loss of cell integrity. Synergistic killing was evident at 6 hr and correlated with loss of Mcl-1. This synergy was also noted when the closely related compound ABT-737 was combined with the same immunotoxin. To establish that the synergy seen in tissue culture could be achieved in vivo, H69AR cells were grown as tumors in nude mice and shown to be susceptible to the killing action of an immunotoxin-ABT-737 combination but not to either agent alone. When immunotoxin-ABT combinations were added to ABT-263-sensitive lines (H146 and H1417), killing was additive. Our data support combination approaches for treating ABT-263-resistant SCLC with ABT-263 and a second agent that provides synergistic killing action.

Abstract P042: Loss of Mcl-1 in the Heart Leads to Rapid Mitochondrial Dysfunction and Development of Heart Failure

Mcl-1 is an anti-apoptotic Bcl-2 family protein that is expressed at high levels in the heart. However, the functional importance of Mcl-1 in the heart has not been investigated. To study the function of Mcl-1 in cardiac myocytes, we generated inducible, myocyte-specific Mcl-1 knock-out mice using a tamoxifen-driven loxP/α-MHC-Cre-recombinase system. We found that deletion of Mcl-1 by tamoxifen injections resulted in rapid development of cardiac hypertrophy, pulmonary edema and cardiac dysfunction in adult mice. Although Mcl-1 is known to inhibit apoptosis in cells, Mcl-1 deficient hearts were TUNEL negative and showed no activation of caspase-3. The anti-apoptotic proteins Bcl-2 and Bcl-X L have also been reported to inhibit autophagy, but we found no increase in autophagy upon loss of Mcl-1. Western blot analysis of other Bcl-2 family members revealed that Bcl-2 was significantly increased in Mcl-1 deficient hearts, suggesting a compensatory mechanism. Interestingly, ultrastructural analysis revealed the presence of swollen mitochondria, ruptured cells, and intracellular contents in the extracellular space. These signs are characteristics of necrotic cell death. Mcl-1 deficient hearts also exhibited reduced mitochondrial respiration and significantly lower cellular ATP levels compared to wild type, suggesting that the loss of Mcl-1 impairs mitochondrial function. Moreover, mitochondria isolated from Mcl-1-/- hearts showed pronounced swelling at baseline that could be alleviated by polyethylene glycol-induced shrinkage. Calcium release experiments also showed higher baseline Ca 2+ levels in mitochondria from Mcl-1-/- hearts compared to wild type, suggesting that Mcl-1 deletion increases permeability to Ca 2+ . Our data suggest that loss of Mcl-1 in cardiac myocytes leads to rapid mitochondrial impairment and necrotic cell death. Thus, in addition to its anti-apoptotic role, Mcl-1 has an essential but unidentified role in maintaining mitochondrial function in cardiac myocytes.

2-Deoxyglucose Induces Noxa-Dependent Apoptosis in Alveolar Rhabdomyosarcoma

Alveolar and embryonal rhabdomyosarcomas are childhood tumors that do not respond well to current chemotherapies. Here, we report that the glycolytic inhibitor 2-deoxyglucose (2-DG) can efficiently promote cell death in alveolar, but not embryonal, rhabdomyosarcoma cell lines. Notably, 2-DG also induced cell differentiation accompanied by downregulation of PAX3/FOXO1a, the chromosome translocation-encoded fusion protein that is a central oncogenic driver in this disease. Cell death triggered by 2-DG was associated with its ability to activate Bax and Bak. Overexpression of the antiapoptotic Bcl-2 homologues Bcl-x(L) and Mcl-1 prevented apoptosis, indicating that cell death proceeds through the mitochondrial pathway. Mechanistic investigations indicated that Mcl-1 downregulation and Noxa upregulation were critical for 2-DG-induced apoptosis. In addition, 2-DG promoted eIF2α phosphorylation and inactivation of the mTOR pathway. Mcl-1 loss and cell death were prevented by downregulation of the endoplasmic reticulum (ER) stress-induced protein ATF4 and by incubating cells in the presence of mannose, which reverted 2-DG-induced ER stress but not ATP depletion. Thus, energetic stresses created by 2-DG were not the primary cause of cell death. Together, our findings suggest that glycolysis inhibitors such as 2-DG may be highly effective in treating alveolar rhabdomyosarcoma and that Noxa could offer a prognostic marker to monitor the efficacy of such agents.

Characterization of Cholix Toxin-induced Apoptosis in HeLa Cells

Cholix toxin (Cholix) is a novel ADP-ribosylating cytotoxin produced by Vibrio cholerae, which utilizes eukaryotic elongation factor 2 as a substrate and acts by a mechanism similar to that of diphtheria toxin and Pseudomonas exotoxin A. First it was found that Cholix-treated HeLa cells exhibited caspase-dependent apoptosis, whereas intestinal cells such as Caco-2, HCT116, and RKO did not. Here we investigated Cholix-induced cell death signaling pathways in HeLa cells. Cholix-induced cytochrome c release into cytosol was initiated by specific conformational changes of pro-apoptotic Bak associated with Bax. Silencing of bak/bax genes or bak gene alone using siRNA significantly suppressed cytochrome c release and caspase-7 activation, but not activation of caspases-3 and -9. Although pretreatment with a caspase-8 inhibitor (Z-IETD-FMK) reduced Cholix-induced cytochrome c release and activation of caspases-3, -7, and -9, cytotoxicity was not decreased. Pretreatment with Z-YVAD-FMK, which inhibits caspase-1, -4, and -5, suppressed not only cytochrome c release, activation of caspase-3, -7, -8, or -9, and PARP cleavage, but also cytotoxicity, indicating that caspase-1, -4, and -5 activation is initiated at an early stage of Cholix-induced apoptosis and promotes caspase-8 activation. These results show that the inflammatory caspases (caspase-1, -4, and -5) and caspase-8 are responsible for both mitochondrial signals and other caspase activation. In conclusion, we showed that Cholix-induced caspase activation plays an essential role in generation of apoptotic signals, which are mediated by both mitochondria-dependent and -independent pathways.

Interleukin-7 Inhibits Tumor-Induced CD27−CD28− Suppressor T Cells: Implications for Cancer Immunotherapy

We have previously reported that many types of tumors can induce changes in human T cells that lead to the acquisition of suppressive function and phenotypic alterations resembling those found in senescent T cells. In the present study, we find a role for interleukin 7 (IL-7) in protecting T cells from these changes and further define involved signaling pathways. We evaluated the ability of IL-7 treatment to prevent the gain of suppressive function and phenotypic alterations in human T cells after a short coculture with tumor cells in vitro. We then used inhibitors of components of the phosphoinositide 3-kinase (PI3K)/AKT pathway and short interfering RNA knockdown of Mcl-1 and Bim to evaluate the role of these signaling pathways in IL-7 protection. We found that IL-7 inhibits CD27/CD28 loss and maintains proliferative capacity, IL-2 production, and reduced suppressive function. The protective ability of IL-7 depended on activation of the PI3K/AKT pathway, which inhibited activation of glycogen synthase kinase 3β, which, in turn, prevented the phosphorylation and loss of Mcl-1. We further showed a key role for Mcl-1 in that its knockdown or inhibition abrogated the effects of IL-7. In addition, knockdown of the Mcl-1 binding partner and proapoptotic protein Bim protected T cells from these dysfunctional alterations. These observations confirm the role for Bcl-2 family members in cytokine signaling and suggest that IL-7 treatment in combination with other immunotherapies could lead to new clinical strategies to maintain normal T-cell function and reduce tumor-induced generation of dysfunctional and suppressor T cells.

Navitoclax (ABT-263) Accelerates Apoptosis during Drug-Induced Mitotic Arrest by Antagonizing Bcl-xL

Combining microtubule-targeting antimitotic drugs with targeted apoptosis potentiators is a promising new chemotherapeutic strategy to treat cancer. In this study, we investigate the cellular mechanism by which navitoclax (previously called ABT-263), a Bcl-2 family inhibitor, potentiates apoptosis triggered by paclitaxel and an inhibitor of kinesin-5 (K5I, also called a KSP inhibitor), across a panel of epithelial cancer lines. By using time-lapse microscopy, we showed that navitoclax has little effect on cell death during interphase, but strongly accelerates apoptosis during mitotic arrest, and greatly increases the fraction of apoptosis-resistant cells that die. By systematically knocking down individual Bcl-2 proteins, we determined that Mcl-1 and Bcl-xL are the primary negative regulators of apoptosis during prolonged mitotic arrest. Mcl-1 levels decrease during mitotic arrest because of an imbalance between synthesis and turnover, and turnover depends in part on the MULE/HUWE1 E3 ligase. The combination of Mcl-1 loss with inhibition of Bcl-xL by navitoclax causes rapid apoptosis in all lines tested. Variation in expression levels of Mcl-1 and Bcl-xL largely determines variation in response to antimitotics alone, and antimitotics combined with navitoclax, across our panel. We concluded that Bcl-xL is a critical target of Bcl-2 family inhibitors for enhancing the lethality of antimitotic drugs in epithelial cancers, and combination treatment with navitoclax and a spindle specific antimitotic, such as a K5I, might be more effective than paclitaxel alone.

Molecular analysis of neutrophil spontaneous apoptosis reveals a strong role for the pro-apoptotic BH3-only protein Noxa

Neutrophils enter the peripheral blood from the bone marrow and die after a short time. Molecular analysis of spontaneous neutrophil apoptosis is difficult as these cells die rapidly and cannot be easily manipulated. We use conditional Hoxb8 expression to generate mouse neutrophils and test the regulation of apoptosis by extensive manipulation of B-cell lymphoma protein 2 (Bcl-2)-family proteins. Spontaneous apoptosis was preceded by downregulation of anti-apoptotic Bcl-2 proteins. Loss of the pro-apoptotic Bcl-2 homology domain (BH3)-only protein Bcl-2-interacting mediator of cell death (Bim) gave some protection, but only neutrophils deficient in both BH3-only proteins, Bim and Noxa, were strongly protected against apoptosis. Function of Noxa was at least in part neutralization of induced myeloid leukemia cell differentiation protein (Mcl-1) in neutrophils and progenitors. Loss of Bim and Noxa preserved neutrophil function in culture, and apoptosis-resistant cells remained in circulation in mice. Apoptosis regulated by Bim- and Noxa-driven loss of Mcl-1 is thus the final step in neutrophil differentiation, required for the termination of neutrophil function and neutrophil-dependent inflammation.

HBx Sensitizes Cells to Oxidative Stress-induced Apoptosis by Accelerating the Loss of Mcl-1 Protein via Caspase-3 Cascade

BackgroundOxidative stress has been implicated in the pathogenesis of a wide spectrum of human diseases, including Hepatitis B virus (HBV)-related liver disease. Hepatitis B virus X protein (HBx) is a key regulator of HBV that exerts pleiotropic activity on cellular functions. Recent studies showed that HBx alters mitochondrial membrane potential, thereby sensitizing cells to pro-apoptotic signals. However, it remains largely unknown whether susceptibility of hepatocytes could be disturbed by HBx under oxidative stress conditions. The purpose of this study is to determine the apoptotic susceptibility of HBx-expressing hepatocytes upon exposure to pro-oxidant stimuli in vitro and in vivo and explore its underlying mechanism.ResultsAlthough expression of HBx itself did not activate apoptotic signaling, it significantly enhanced oxidative stress-induced cell death both in vitro and in vivo. Interestingly, this phenomenon was associated with a pronounced reduction of protein levels of Mcl-1, but not other anti-apoptotic Bcl-2 members. Importantly, enforced expression of Mcl-1 prevented HBx-triggered cell apoptosis; conversely, specific knockdown of Mcl-1 exacerbated HBx-induced apoptosis upon exposure to oxidative stress. Furthermore, inhibition of caspase-3 not only abrogated HBx-triggered apoptotic killing but also blocked HBx-induced Mcl-1 loss. Additionally, expression of HBx and Mcl-1 was found to be inversely correlated in HBV-related hepatocellular carcinogenesis (HCC) tissues.ConclusionsOur findings indicate that HBx exerts pro-apoptotic effect upon exposure to oxidative stress probably through accelerating the loss of Mcl-1 protein via caspase-3 cascade, which may shed a new light on the molecular mechanism of HBV-related hepatocarcinogenesis.

Abstract LB-277: Dynamic functional analysis of the response of cancer cell lines to the drug UNBS1450

Abstract The analysis of the dynamics of cellular responses to drugs is currently bounded by inherent limitations of the methods most often used to assess levels of promoter activity and protein abundance or activation. The sensitivity of these assays is limited by the averaging of analyte over many cells, which can produce an average that is not accurately descriptive of the wide range of activities of members of the population examined. The ability of these assays to indicate the trajectory of the various responses is limited by a very infrequent sampling rate, frequently 2–4 time points spread over many hours. These limits are further exacerbated by populations of treated cells' inhomogeneity in the timing of their own response to the drugs. To improve the determination of the functional dynamics of cells' responses to drugs, PBS-Bio has developed technology that allows gathering data from living cells using fluorescent reporter technology. This technology allows measurements to be made serially on single populations of cells at the individual cell level, providing both high sensitivity in terms of when even a small fraction of the cells exhibit significant shifts of a particular activity, the rate of shift of cells in the population to this different activity and the ability to temporally order the timing of events relative to each other. Experimentation on two cell lines, A549 and PC3, using a variety of drugs that affect different cellular processes and a set of fluorescent reporters that provide information on a variety of cellular processes it was possible to show that the modified cardenolide drug, UNBS1450 (Unibioscreen), provoked a very early reduction in the expression of the MCL1 and MYC genes, followed by induction of a strong apoptotic response in both cell lines. As loss of MCL1 provoked by other means has been shown to be sufficient to induce apoptosis in both these lines, a very strong case can be made for UNBS1450's ability to provoke Mcl1 protein reduction as the drug's mode of apoptotic induction. This finding would also implicate Mcl1 as a useful biomarker of the possible efficacy of UNBS1450. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-277. doi:10.1158/1538-7445.AM2011-LB-277

Abstract 2138: Vinblastine acutely sensitizes hematopoietic cancers to Bcl-2 targeting drugs

Abstract Chronic lymphocytic leukemia (CLL) characteristically maintains high levels of the anti-apoptotic protein Bcl-2, thus protecting them from apoptotic death and leading to an accumulation of mature resting B cells. Many other hematopoietic malignancies including acute promyelocytic leukemia (APL) also depend upon Bcl-2 family proteins for survival, suggesting that Bcl-2 proteins are excellent targets for anti-cancer therapies. We have previously shown that some leukemia and lymphoma cell lines undergo rapid apoptosis (≤6 h) when incubated with the microtubule disrupting agent vinblastine. Several other cell lines undergo this rapid apoptosis when vinblastine is combined with various approaches that target the anti-apoptotic proteins Bcl-2 or Mcl-1. These approaches include incubation with ABT-737 that inhibits Bcl-2 and Bcl-X, and SCH727965 (dinaciclib) that inhibits CDK7/9, thereby inhibiting translation which results in rapid loss of Mcl-1. In this study, we have assessed the ability of these agents alone or in combination to induce apoptosis in freshly isolated CLL and APL cells. Incubation with vinblastine alone induced variable apoptosis within 6 h (5-60%). ABT-737 induced rapid and almost complete apoptosis in CLL and in 50% of APL cells ex vivo. However, when combined with vinblastine, a 10-fold lower concentration of ABT-737 was required to induce complete apoptosis. Dinaciclib alone also induced variable levels of apoptosis (5-90%), but again the combination with vinblastine induced extensive apoptosis at lower concentrations of both drugs. Normal peripheral blood mononuclear cells were resistant to single agent or combination treatments. Sensitivity to ABT-737, dinaciclib or vinblastine was independent of CD38 and ZAP70 status and previous clinical treatment (untreated or relapsed patients). These results suggest that combinations of vinblastine and ABT-737 or dinaciclib are more effective than single agent therapy. Furthermore, these combinations may overcome resistance to individual drug therapies in patients with CLL or APL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2138. doi:10.1158/1538-7445.AM2011-2138

Abstract 183: Loss of Bad phosphorylation and Mcl-1 expression is necessary for rapid apoptosisinduced by combination of TGFα-PE and ZSTK474 in prostate cancer cells

Abstract Advanced androgen-independent prostate cancer is notoriously resistant to conventional systemic therapies, and at the moment there is no effective protocol for hormone-refractory advanced prostate cancer affected patients. One of possible mechanisms of such resistance is activation of the anti-apoptotic signaling PI3K/Akt pathway that is constitutively active in the 60% of advanced prostate cancers. However, multiple inhibitors of PI3K are just well studied and some have just gone on to clinical trials, but unfortunately they showed limited efficacy against prostate cancer alone. Our recent experiments has shown remarkable synergy in inducing rapid and massive apoptosis when PI3K inhibitor ZSTK474 is used in combination with Pseudomonas aeruginosa exotoxin A fragment fused with Transforming Growth Factor Alpha (TGFα-PE38). Time lapse video microscopy of prostate cancer C4-2 cell line has shown the substantial cell death within 4-6 hours with combined administration, compared to limited cell death in single agents-treated cells. Analysis of PARP and cleaved-caspase 3 fragment by Western blotting confirmed that cell death occurs via apoptosis. Quantitation of caspase 3 and 7 activity with luminescent AFC-DEVD substrate confirmed synergy in apoptosis activation by combination of TGFα-PE38 and ZSTK474, while single agents even in higher concentration did not induce substantial apoptosis within 6 hours time period. Analysis of the mechanisms underlying this synergy has shown that PI3K inhibitor ZSTK474 triggers Bad dephosphorylation, while TGFα-PE38 reduces expression levels of Mcl-1. A slight increase of Bim protein was detected and related with ZSTK474 administration. No major variations were detected in the expression levels of pro- and anti-apoptotic proteins Bax, Bcl-2 and Bcl-XL. To address the role of Bad phosphorylation and Mcl-1 expression in apoptosis induction by PI3K inhibitor ZSTK474 and TGFα-PE38, we examined apoptosis in C4-2 cells that express phosphorylation-deficient mutant Bad (BAD2SA). Results indicated that the expression of BAD2SA sensitized C4-2 cells to apoptosis induced by TGFα-PE38. Experiments that address the role of Mcl-1 loss in apoptosis induced by TGFα-PE38 are ongoing. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 183. doi:10.1158/1538-7445.AM2011-183

Hepatitis B Virus X Protein Enhances Cisplatin-Induced Hepatotoxicity via a Mechanism Involving Degradation of Mcl-1

Hepatitis B virus X protein (HBx) is implicated in the pathogenesis of hepatitis B virus (HBV)-associated liver diseases. However, whether HBx has the ability to disturb the susceptibility of hepatocytes to common chemotherapeutic agents remains incompletely understood. Here we demonstrate that HBx enhances cisplatin-induced hepatotoxicity by a mechanism involving degradation of Mcl-1, an antiapoptotic member of the Bcl-2 family. Ectopic expression of HBx sensitized hepatocytes to cisplatin-induced apoptosis, which was accompanied by a marked downregulation of Mcl-1 but not of Bcl-2 or Bcl-xL. Overexpression of Mcl-1 prevented HBx-induced proapoptotic and proinflammatory effects during cisplatin treatment both in vitro and in vivo. HBx-induced dysregulation of Mcl-1 resulted mainly from posttranslational degradation rather than transcription repression. Moreover, a caspase-3 inhibitor effectively abrogated HBx-enhanced Mcl-1 degradation and cell death. Importantly, antioxidants blocked activation of caspase-3 and acceleration of Mcl-1 loss, as well as cell death, in HBx-expressing hepatocytes upon cisplatin exposure in vitro and in vivo. Collectively, these data implicate oxidative stress-dependent caspase-3-mediated degradation of Mcl-1 as a mechanism contributing to HBx-mediated sensitization of cisplatin-induced hepatotoxicity. A combination of cisplatin and antioxidants might provide more advantage than cisplatin alone in the treatment of cancer patients with chronic HBV infection.

NO signaling in exercise training-induced anti-apoptotic effects in human neutrophils

Short-lived neutrophils play a predominant role in innate immunity, the effects of exercise training on neutrophil survival is unclear. In this study, we investigated the underlying mechanisms of training effects on human neutrophil apoptosis. Healthy male subjects were trained on a cycling ergometer for 8 weeks and followed by 4 weeks of detraining. Blood neutrophils were collected before exercise, after training, and after detraining. Comparing with pre-exercise specimens, neutrophils collected after training showed reduced apoptosis rate, which partially returned after detraining. Various intracellular proteins, including iNOS, Mcl-1, A1, Grp78, and IL-8, were upregulated by training, and they remained high after detraining. Upregulated iNOS was closely correlated with these anti-apoptotic molecules in neutrophils. Furthermore, the possible mechanism by which iNOS suppressed apoptosis was explored. Neutrophil apoptosis was accelerated by blocking and retarded by stimulating the endogenous iNOS activity. As an anti-apoptosis mediator of NO signaling, the Mcl-1 level dropped by depletion of the major NO downstream molecule cGMP and such loss of Mcl-1 was avoidable when supplying exogenous NO. Upon activation of NO-cGMP signaling, neutrophils held increased Mcl-1 expression and delayed apoptosis. Collectively, our results suggested that exercise training may retard neutrophil apoptosis by upregulating the iNOS-NO-cGMP-Mcl-1 pathway.

Enhancing mda-7/IL-24 therapy in renal carcinoma cells by inhibiting multiple protective signaling pathways using sorafenib and by Ad.5/3 gene delivery

We have determined whether an adenovirus that comprises the tail and shaft domains of a serotype 5 virus and the knob domain of a serotype 3 virus expressing MDA-7/IL-24, Ad.5/3-mda-7, more effectively infects and kills renal carcinoma cells (RCCs) compared to a serotype 5 virus, Ad.5-mda-7. RCCs are a tumor cell type that generally does not express the receptor for the type 5 adenovirus; the coxsakie and adenovirus receptor (CAR). Ad.5/3-mda-7 infected RCCs to a much greater degree than Ad.5-mda-7. MDA-7/IL-24 protein secreted from Ad.5/3-mda-7-infected RCCs induced MDA-7/IL-24 expression and promoted apoptosis in uninfected "bystander" RCCs. MDA-7/IL-24 killed both infected and bystander RCCs via CD95 activation. Knockdown of intracellular MDA-7/IL-24 in uninfected RCCs blocked the lethal effects of conditioned media. Infection of RCC tumors in one flank, with Ad.5/3-mda-7, suppressed growth of infected tumors and reduced the growth rate of uninfected tumors implanted on the opposite flank. The toxicity of the serotype 5/3 recombinant adenovirus to express MDA-7/IL-24 was enhanced by combined molecular or small molecule inhibition of MEK1/2 and PI3K; inhibition of mTOR, PI3K and MEK1/2; or use of the multi-kinase inhibitor sorafenib. In RCCs, combined inhibition of cytoprotective cell signaling pathways enhanced the MDA-7/IL-24-induction of CD95 activation, with greater mitochondrial dysfunction due to loss of MCL-1 and BCL-XL expression, and tumor cell death. Treatment of RCC tumors in vivo with sorafenib also enhanced Ad.5/3-mda-7 toxicity and prolonged animal survival. Future combinations of these approaches hold promise for developing a more effective therapy for kidney cancer.

MCL-1 is a stress sensor that regulates autophagy in a developmentally regulated manner

Apoptosis has an important role during development to regulate cell number. In differentiated cells, however, activation of autophagy has a critical role by enabling cells to remain functional following stress. In this study, we show that the antiapoptotic BCL-2 homologue MCL-1 has a key role in controlling both processes in a developmentally regulated manner. Specifically, MCL-1 degradation is an early event not only following induction of apoptosis, but also under nutrient deprivation conditions where MCL-1 levels regulate activation of autophagy. Furthermore, deletion of MCL-1 in cortical neurons of transgenic mice activates a robust autophagic response. This autophagic response can, however, be converted to apoptosis by either reducing the levels of the autophagy regulator Beclin-1, or by a concomitant activation of BAX. Our results define a pathway whereby MCL-1 has a key role in determining cell fate, by coordinately regulating apoptosis and autophagy.

Bcl-XL Is the primary mediator of p21 protection against hyperoxia-induced cell death

ABSTRACTA tight balance between anti- and proapoptotic members of the Bcl-2 family controls cell survival and death. Exposure to hyperoxia shifts this balance towards a prodeath state that ultimately activates Bak- and Bax-dependent cell death. Mechanisms underlying this shift are undefined; however, the cell cycle inhibitor p21 delays the loss of antiapoptotic Mcl-1 and Bcl-XL, and protects against hyperoxia. Here, H1299 human lung adenocarcinoma cells are used to investigate how these and other members of the Bcl-2 family cooperate with p21 to protect against hyperoxia. Expression of antiapoptotic Mcl-1 and Bcl-XL, but not Bcl-2 or A1, declined during hyperoxia, whereas proapoptotic Bak, but not Bax, increased. Conditional overexpression of p21 selectively delayed the loss of Mcl-1 and Bcl-XL, without affecting expression of the other members. siRNA knockdown of Mcl-1 and Bcl-XL sensitized cells to hyperoxia, but only the loss of Bcl-XL ablated the protective effects of p21. Conversely, overexpression of Mcl-1 and Bcl-XL protected against hyperoxia, but only Bcl-XL bound Bak and Bax. Altogether, these data suggest that Bcl-XL is the primary mediator by which p21 protects against hyperoxia-induced Bak/Bax-dependent cell death.