Human Melanoma Cells under Endoplasmic Reticulum Stress Are More Susceptible to Apoptosis Induced by the BH3 Mimetic Obatoclax

Past studies have shown that melanoma cells have largely adapted to endoplasmic reticulum (ER) stress, and this is associated with up-regulation of the antiapoptotic proteins Bcl-2 and Mcl-1. In this report, we show that the BH3 mimetic obatoclax potently overcomes resistance of melanoma cells to apoptosis induced by ER stress. Obatoclax, as a single agent at nanomolar concentrations, was relatively ineffective in the induction of apoptosis in melanoma cells, but treatment with obatoclax at these concentrations in combination with the ER stress inducer tunicamycin (TM) or thapsigargin markedly enhanced apoptotic cell death. This was primarily because of the inhibition of Mcl-1 by obatoclax, in that cotreatment with TM and another BH3 mimetic ABT737, which does not antagonize Mcl-1, caused only minimal increases in apoptosis. Moreover, overexpression of Mcl-1 inhibited apoptosis to greater degrees than overexpression of Bcl-2. In addition to direct inhibition of Mcl-1 by obatoclax, the combination of obatoclax and TM caused strong up-regulation of the BH3-only protein Noxa. Small RNA interference knockdown of Noxa partially inhibited apoptosis induced by cotreatment with obatoclax and TM. Similarly, knockdown of Bak also blocked induction of apoptosis by the compounds. The Mcl-1/Bak interaction seemed to be disrupted more efficiently in melanoma cells cotreated with obatoclax and TM. Taken together, these results identify obatoclax as a potent agent that overcomes resistance of melanoma cells to ER stress-induced apoptosis and seem to have important implications in the use of BH3 mimetics in the treatment of melanoma.     

Mcl-1 Phosphorylation defines ABT-737 resistance that can be overcome by increased NOXA expression in leukemic B cells

ABT-737 is a small molecule Bcl-2 homology (BH)-3 domain mimetic that binds to the Bcl-2 family proteins Bcl-2 and Bcl-xL and is currently under investigation in the clinic. In this study, we investigated potential mechanisms of resistance to ABT-737 in leukemia cell lines. Compared with parental cells, cells that have developed acquired resistance to ABT-737 showed increased expression of Mcl-1 in addition to posttranslational modifications that facilitated both Mcl-1 stabilization and its interaction with the BH3-only protein Bim. To sensitize resistant cells, Mcl-1 was targeted by two pan-Bcl-2 family inhibitors, obatoclax and gossypol. Although gossypol was effective only in resistant cells, obatoclax induced cell death in both parental and ABT-737-resistant cells. NOXA levels were increased substantially by treatment with gossypol and its expression was critical for the gossypol response. Mechanistically, the newly generated NOXA interacted with Mcl-1 and displaced Bim from the Mcl-1/Bim complex, freeing Bim to trigger the mitochondrial apoptotic pathway. Together, our findings indicate that NOXA and Mcl-1 are critical determinants for gossypol-mediated cell death in ABT-737-resistant cells. These data therefore reveal novel insight into mechanisms of acquired resistance to ABT-737.     

BH3 Mimetics Reactivate Autophagic Cell Death in Anoxia-Resistant Malignant Glioma Cells

Here, we investigated the specific roles of Bcl-2 family members in anoxia tolerance of malignant glioma. Flow cytometry analysis of cell death in 17 glioma cell lines revealed drastic differences in their sensitivity to oxygen withdrawal (<0.1% O₂). Cell death correlated with mitochondrial depolarization, cytochrome C release, and translocation of green fluorescent protein (GFP)-tagged light chain 3 to autophagosomes but occurred in the absence of caspase activation or phosphatidylserine exposure. In both sensitive and tolerant glioma cell lines, anoxia caused a significant up-regulation of BH3-only genes previously implicated in mediating anoxic cell death in other cell types (BNIP3, NIX, PUMA, and Noxa). In contrast, we detected a strong correlation between anoxia resistance and high expression levels of antiapoptotic Bcl-2 family proteins Bcl-xL, Bcl-2, and Mcl-1 that function to neutralize the proapoptotic activity of BH3-only proteins. Importantly, inhibition of both Bcl-2 and Bcl-xL with the small-molecule BH3 mimetics HA14-1 and BH3I-2′ and by RNA interference reactivated anoxia-induced autophagic cell death in previously resistant glioma cells. Our data suggest that endogenous BH3-only protein induction may not be able to compensate for the high expression of antiapoptotic Bcl-2 family proteins in anoxia-resistant astrocytomas. They also support the conjecture that BH3 mimetics may represent an exciting new approach for the treatment of malignant glioma.     

Noxa up-regulation and Mcl-1 cleavage are associated to apoptosis induction by bortezomib in multiple myeloma

Targeting the ubiquitin-proteasome pathway has emerged as a potent anticancer strategy. Bortezomib, a specific proteasome inhibitor, has been approved for the treatment of relapsed or refractory multiple myeloma. Multiple myeloma cell survival is highly dependent on Mcl-1 antiapoptotic molecules. In a recent study, proteasome inhibitors induced Mcl-1 accumulation that slowed down their proapoptotic effects. Consequently, we investigated the role of Bcl-2 family members in bortezomib-induced apoptosis. We found that bortezomib induced apoptosis in five of seven human myeloma cell lines (HMCL). Bortezomib-induced apoptosis was associated with Mcl-1 cleavage regardless of Mcl-1L accumulation. Furthermore, RNA interference mediated Mcl-1 decrease and sensitized RPMI-8226 HMCL to bortezomib, highlighting the contribution of Mcl-1 in bortezomib-induced apoptosis. Interestingly, an important induction of Noxa was found in all sensitive HMCL both at protein and mRNA level. Concomitant to Mcl-1 cleavage and Noxa induction, we also found caspase-3, caspase-8, and caspase-9 activation. Under bortezomib treatment, Mcl-1L/Noxa complexes were highly increased, Mcl-1/Bak complexes were disrupted, and there was an accumulation of free Noxa. Finally, we observed a dissociation of Mcl-1/Bim complexes that may be due to a displacement of Bim induced by Noxa. Thus, in myeloma cells, the mechanistic basis for bortezomib sensitivity can be explained mainly by the model in which the sensitizer Noxa can displace Bim, a BH3-only activator, from Mcl-1, thus leading to Bax/Bak activation.     

Hyperforin induces apoptosis of chronic lymphocytic leukemia cells through upregulation of the BH3-only protein Noxa

We previously reported that hyperforin, a phloroglucinol purified from Hypericum perforatum, induces the mitochondrial pathway of caspase-dependent apoptosis in chronic lymphocytic leukemia (CLL) cells ex vivo, and that this effect is associated with upregulation of Noxa, a BH3-only protein of the Bcl-2 family. Here, we investigated the role of this upregulation in the pro-apoptotic activity of hyperforin in the cells of CLL patients and MEC-1 cell line. We found that the increase in Noxa expression is a time- and concentration-dependent effect of hyperforin occurring without change in Noxa mRNA levels. A post-translational regulation is suggested by the capacity of hyperforin to inhibit proteasome activity in CLL cells. Noxa silencing by siRNA reduces partially hyperforin-elicited apoptosis. Furthermore, treatment with hyperforin, which has no effect on the expression of the prosurvival protein Mcl-1, induces the interaction of Noxa with Mcl-1 and the dissociation of Mcl-1/Bak complex, revealing that upregulated Noxa displaces the proapoptotic protein Bak from Mcl-1. This effect is accompanied with Bak activation, known to allow the release of apoptogenic factors from mitochondria. Our data indicate that Noxa upregulation is one of the mechanisms by which hyperforin triggers CLL cell apoptosis. They also favor that new agents capable of mimicking specifically the BH3-only protein Noxa should be developed for apoptosis-based therapeutic strategy in CLL.     

Mechanisms of antileukemic activity of the novel Bcl-2 homology domain-3 mimetic GX15-070 (obatoclax)

In this study, we investigated the mechanism of apoptosis induction of obatoclax (GX15-070), a novel Bcl-2 homology domain-3 (BH3) mimetic, in acute myeloid leukemia (AML) cell lines and primary AML samples. Obatoclax inhibited cell growth of HL-60, U937, OCI-AML3, and KG-1 cell lines. Apoptosis induction contributed to the observed antiproliferative effects at concentrations of this agent that mirror its affinity for antiapoptotic Bcl-2 proteins. We show that obatoclax can promote the release of cytochrome c from isolated leukemia cell mitochondria and that apoptosis induced by this agent is preceded by the release of Bak from Mcl-1, liberation of Bim from both Bcl-2 and Mcl-1, and the formation of an active Bak/Bax complex. Notably, apoptosis was diminished, but not fully prevented, in the absence of Bak/Bax or Bim, suggesting that obatoclax has additional targets that contribute to its cytotoxicity. At growth inhibitory doses that did not induce apoptosis or decrease viability, obatoclax induced an S-G(2) cell-cycle block. Obatoclax induced apoptosis in AML CD34+ progenitor cells with an average IC(50) of 3.59 +/- 1.23 micromol/L although clonogenicity was inhibited at concentrations of 75 to 100 nmol/L. Obatoclax synergized with the novel BH3 mimetic ABT-737 to induce apoptosis in OCI-AML3 cells and synergistically induced apoptosis in combination with AraC in leukemic cell lines and in primary AML samples. In conclusion, we show that obatoclax potently induces apoptosis and decreases leukemia cell proliferation and may be used in a novel therapeutic strategy for AML alone and in combination with other targeted agents and chemotherapeutics.     

The BH3-only protein Noxa is stimulated during apoptosis of chronic lymphocytic leukemia cells triggered by M2YN, a new plant-derived extract

Deficiency of apoptosis is a hallmark of chronic lymphocytic leukemia (CLL) cells. M2Yn is a natural extract from plants of central Asia, identified for its antiangiogenic properties and its ability to block the migration of malignant cells. Here, we report that in vitro treatment of cells derived from CLL patients with M2Yn results in internucleosomal DNA fragmentation, phosphatidylserine externalization, mitochondrial membrane depolarization, caspase-3 activation and cleavage of the caspase substrate PARP-1. The extents of these effects depend on the patients and are mostly comparable to those of flavopiridol or hyperforin, two known plant-derived apoptosis inducers of CLL cells. M2Yn does not modulate Mcl-1 expression, while downregulation of this antiapoptotic protein is involved in the action of flavopiridol. By contrast, M2Yn, like hyperforin, upregulates the Noxa protein, possibly by inhibiting proteasomal activity. This BH3-only protein is known to trigger the activation of the pro-apoptotic protein Bak through displacement of the Mcl-1/Bak complex at the mitochondrial membrane, as actually observed here in M2Yn-treated cells. Our data, therefore, show that M2Yn can induce the caspase-dependent mitochondrial pathway of apoptosis in CLL cells via a mechanism resembling that of hyperforin. Our data also confirm that the BH3-only protein Noxa is a relevant target for CLL therapy.     

Cyclin E/Cdk2-dependent phosphorylation of Mcl-1 determines its stability and cellular sensitivity to BH3 mimetics

Cyclin E/Cdk2 kinase activity is frequently deregulated in human cancers, resulting in impaired apoptosis. Here, we show that cyclin E/Cdk2 phosphorylates and stabilizes the pro-survival Bcl-2 family protein Mcl-1, a key cell death resistance determinant to the small molecule Bcl-2 family inhibitors ABT-199 and ABT-737, mimetics of the Bcl-2 homology domain 3 (BH3). Cyclin E levels were elevated and there was increased association of cyclin E/Cdk2 with Mcl-1 in ABT-737-resistant compared to parental cells. Cyclin E depletion in various human tumor cell-lines and cyclin E^−/− mouse embryo fibroblasts showed decreased levels of Mcl-1 protein, with no change in Mcl-1 mRNA levels. In the absence of cyclin E, Mcl-1 ubiquitination was enhanced, leading to decreased protein stability. Studies with Mcl-1 phosphorylation mutants show that cyclin E/Cdk2-dependent phosphorylation of Mcl-1 residues on its PEST domain resulted in increased Mcl-1 stability (Thr92, and Thr163) and Bim binding (Ser64). Cyclin E knock-down restored ABT-737 sensitivity to acquired and inherently resistant Mcl-1-dependent tumor cells. CDK inhibition by dinaciclib resulted in Bim release from Mcl-1 in ABT-737-resistant cells. Dinaciclib in combination with ABT-737 and ABT-199 resulted in robust synergistic cell death in leukemic cells and primary chronic lymphocytic leukemia patient samples. Collectively, our findings identify a novel mechanism of cyclin E-mediated Mcl-1 regulation that provides a rationale for clinical use of Bcl-2 family and Cdk inhibitors for Mcl-1-dependent tumors.     

Deubiquitinase USP9x Confers Radioresistance through Stabilization of Mcl-1

Myeloid cell leukemia sequence 1 (Mcl-1), an antiapoptotic member of the Bcl-2 family, is often overexpressed in tumor cells limiting the therapeutic success. Mcl-1 differs from other Bcl-2 members by its high turnover rate. Its expression level is tightly regulated by ubiquitylating and deubiquitylating enzymes. Interaction of Mcl-1 with certain Bcl-2 homology domain 3 (BH3)-only members of the Bcl-2 family can limit the access to Mcl-1 ubiquitin ligase E3 and stabilizes the antiapoptotic protein. In addition, the overexpression of the deubiquitinase ubiquitin-specific protease 9x (USP9x) can result in the accumulation of Mcl-1 by removing poly-ubiquitin chains from Mcl-1 preventing its proteasomal degradation. Analyzing radiation-induced apoptosis in Jurkat cells, we found that Mcl-1 was downregulated more efficiently in sensitive parental cells than in a resistant subclone. The decline of Mcl-1 correlated with cell death induction and clonogenic survival. Knockdown of BH3-only proteins Bim, Puma, and Noxa did not affect Mcl-1 level or radiation-induced apoptosis. However, ionizing radiation resulted in activation of USP9x and enhanced deubiquitination of Mcl-1 in the radioresistant cells preventing fast Mcl-1 degradation. USP9x knockdown enhanced radiation-induced decrease of Mcl-1 and sensitized the radioresistant cells to apoptosis induction, whereas USP9x knockdown alone did not change Mcl-1 level in unirradiated cells. Together, our results indicate that radiation-induced activation of USP9x inhibits Mcl-1 degradation and apoptosis resulting in increased radioresistance.     

BH3 mimetic ABT-737 potentiates TRAIL-mediated apoptotic signaling by unsequestering Bim and Bak in human pancreatic cancer cells

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been shown to induce mitochondrial apoptotic signaling that can be negatively regulated by prosurvival Bcl-2 proteins. ABT-737 is a small-molecule BH3 mimetic that binds to and antagonizes Bcl-2/Bcl-x(L) but not Mcl-1. We show that ABT-737 can synergistically enhance TRAIL-mediated cytotoxicity in human pancreatic cancer cell lines. ABT-737 was shown to enhance TRAIL-induced apoptosis as shown by DNA fragmentation, activation of caspase-8 and Bid, and cleavage of caspase-3 and poly(ADP-ribose) polymerase. A Bax conformational change induced by TRAIL was enhanced by ABT-737. ABT-737 disrupted the interaction of Bak with Bcl-x(L) in both cell lines. Furthermore, ABT-737 untethered the proapoptotic BH3-only protein Bim from its sequestration by Bcl-x(L) or Bcl-2. Bim small hairpin RNA (shRNA) was shown to attenuate caspase-3 cleavage and to reduce the cytotoxic effects of TRAIL plus ABT-737 compared with shRNA control cells. Finally, Mcl-1 shRNA potentiated caspase-3 cleavage by ABT-737 and enhanced its cytotoxic effects. Taken together, ABT-737 augments TRAIL-induced cell killing by unsequestering Bim and Bak and enhancing a Bax conformational change induced by TRAIL. These findings suggest a novel strategy to enhance cross-talk between the extrinsic and intrinsic apoptotic pathways to improve therapeutic efficacy against pancreatic cancer.     

TGFbeta-mediated apoptosis of Burkitt's lymphoma BL41 cells is associated with the relocation of mitochondrial BimEL

In this study, we showed that the transforming growth factor beta (TGFbeta)-mediated apoptosis of Burkitt's lymphoma BL41 cells is dependent on the BH3-only protein Bim. In contrast to what has been observed with other cell types, TGFbeta activation did not promote Bim upregulation in BL41 cells, but instead resulted in Bim release from the mitochondria. Indeed, Bim levels were high in healthy BL41 cells, in which they dimerized with the Bcl-2-like protein Mcl-1 at the mitochondrial surface. In healthy and TGFbeta-activated BL41 cells, unlike in epithelial cells or hepatocytes, Bim did not associate with Bcl-2 or Bcl-xL. TGFbeta activation of BL41 cells triggered the p38-dependent activation of caspase-8, causing the cleavage of Mcl-1 and the transfer of Bim from the mitochondria to the cytoskeleton. In addition to mitochondrial activation, this relocation of Bim may facilitate the complete demise of a cell death that is beyond the commitment point to apoptosis and may represent a hallmark of the TGFbeta-mediated apoptosis of human lymphoma B cells.     

Apoptosis induced by histone deacetylase inhibitors in leukemic cells is mediated by Bim and Noxa.

Several histone deacetylase inhibitors (HDACi), which have recently entered early clinical trials, exert their anticancer activity in part through the induction of apoptosis although the precise mechanism of this induction is not known. Induction of apoptosis by structurally diverse HDACi in primary cells from patients with chronic lymphocytic leukemia (CLL) and different leukemic cell lines was mediated by the Bcl-2 regulated intrinsic pathway and demonstrated a requirement for de novo protein synthesis. A marked time-dependent induction of the pro-apoptotic BH3-only proteins, Bim, Noxa and Bmf was observed, which preceded the induction of apoptosis. A key role for both Bim and Noxa was proposed in HDACi-mediated apoptosis based on our findings that siRNA for Bim and Noxa but not Bmf largely prevented the HDACi-induced loss in mitochondrial membrane potential, caspase processing and phosphatidylserine externalization. Noxa, induced by HDACi, in CLL cells and tumor cell lines, bound extensively to Mcl-1, a major anti-apoptotic Bcl-2 family member present in CLL cells. Our data strongly suggests that HDACi induce apoptosis primarily through inactivation of anti-apoptotic Bcl-2 family members by increases in Bim and Noxa and highlights these increases as a potential clinical target for CLL/lymphoma therapy.     

The BH3 mimetic ABT-737 targets selective Bcl-2 proteins and efficiently induces apoptosis via Bak/Bax if Mcl-1 is neutralized

Since apoptosis is impaired in malignant cells overexpressing prosurvival Bcl-2 proteins, drugs mimicking their natural antagonists, BH3-only proteins, might overcome chemoresistance. Of seven putative BH3 mimetics tested, only ABT-737 triggered Bax/Bak-mediated apoptosis. Despite its high affinity for Bcl-2, Bcl-x(L), and Bcl-w, many cell types proved refractory to ABT-737. We show that this resistance reflects ABT-737's inability to target another prosurvival relative, Mcl-1. Downregulation of Mcl-1 by several strategies conferred sensitivity to ABT-737. Furthermore, enforced Mcl-1 expression in a mouse lymphoma model conferred resistance. In contrast, cells overexpressing Bcl-2 remained highly sensitive to ABT-737. Hence, ABT-737 should prove efficacious in tumors with low Mcl-1 levels, or when combined with agents that inactivate Mcl-1, even to treat those tumors that overexpress Bcl-2.     

The Bcl-2 apoptotic switch in cancer development and therapy

Impaired apoptosis is both critical in cancer development and a major barrier to effective treatment. In response to diverse intracellular damage signals, including those evoked by cancer therapy, the cell's decision to undergo apoptosis is determined by interactions between three factions of the Bcl-2 protein family. The damage signals are transduced by the diverse 'BH3-only' proteins, distinguished by the BH3 domain used to engage their pro-survival relatives: Bcl-2, Bcl-x(L), Bcl-w, Mcl-1 and A1. This interaction ablates pro-survival function and allows activation of Bax and Bak, which commit the cell to apoptosis by permeabilizing the outer membrane of the mitochondrion. Certain BH3-only proteins (e.g. Bim, Puma) can engage all the pro-survival proteins, but others (e.g. Bad, Noxa) engage only subsets. Activation of Bax and Bak appears to require that the BH3-only proteins engage the multiple pro-survival proteins guarding Bax and Bak, rather than binding to the latter. The balance between the pro-survival proteins and their BH3 ligands regulates tissue homeostasis, and either overexpression of a pro-survival family member or loss of a proapoptotic relative can be oncogenic. Better understanding of the Bcl-2 family is clarifying its role in cancer development, revealing how conventional therapy works and stimulating the search for "BH3 mimetics" as a novel class of anticancer drugs.     

Akt-dependent glucose metabolism promotes Mcl-1 synthesis to maintain cell survival and resistance to Bcl-2 inhibition

Most cancer cells utilize aerobic glycolysis, and activation of the phosphoinositide 3-kinase/Akt/mTOR pathway can promote this metabolic program to render cells glucose dependent. Although manipulation of glucose metabolism may provide a means to specifically eliminate cancer cells, mechanistic links between cell metabolism and apoptosis remain poorly understood. Here, we examined the role and metabolic regulation of the antiapoptotic Bcl-2 family protein Mcl-1 in cell death upon inhibition of Akt-induced aerobic glycolysis. In the presence of adequate glucose, activated Akt prevented the loss of Mcl-1 expression and protected cells from growth factor deprivation-induced apoptosis. Mcl-1 associated with and inhibited the proapoptotic Bcl-2 family protein Bim, contributing to cell survival. However, suppression of glucose metabolism led to induction of Bim, decreased expression of Mcl-1, and apoptosis. The proapoptotic Bcl-2/Bcl-xL/Bcl-w inhibitor, ABT-737, shows clinical promise, but Mcl-1 upregulation can promote resistance. Importantly, inhibition of glucose metabolism or mTORC1 overcame Mcl-1-mediated resistance in diffuse large B cell leukemic cells. Together these data show that Mcl-1 protein synthesis is tightly controlled by metabolism and that manipulation of glucose metabolism may provide a mechanism to suppress Mcl-1 expression and sensitize cancer cells to apoptosis.