Apoptosis-based treatment of glioblastomas with ABT-737, a novel small molecule inhibitor of Bcl-2 family proteins

Defects in the apoptotic signaling cascades contribute to the poor therapeutic response of malignant gliomas. As glioblastomas are characterized by high expression levels of anti-apoptotic Bcl-2 family proteins, we studied the effects of the novel Bcl-2 inhibitor, ABT-737, on malignant glioma cells. ABT-737 treatment released the pro-apoptotic Bax protein from its binding partner Bcl-2 and potently induced apoptotic cell death in glioblastoma cells in vitro and in vivo. The local administration of ABT-737 prolonged the survival in an intracranial glioma xenograft model. Downregulation of Mcl-1 and overexpression of Bcl-2 sensitized the cells to ABT-737-mediated apoptosis. Moreover, ABT-737 potentiated the cytotoxicity of the chemotherapeutic drugs vincristine and etoposide, and of the death ligand TRAIL. As glioma stem cells may play a crucial role for the tumor progression and the resistance to treatment in glioblastomas, we investigated the effects of ABT-737 on the subpopulation of glioma cells exhibiting stem cell characteristics. Inhibition of proliferation and induction of apoptosis by ABT-737 were less efficient in glioma stem cells than in non-stem cell-like glioma cells. As the resistance of glioma stem cells was associated with high Mcl-1 expression levels, ABT-737 treatment combined with downregulation of Mcl-1 could represent a promising novel approach in glioblastoma treatment.     

Influence of Bcl-2 family members on the cellular response of small-cell lung cancer cell lines to ABT-737

ABT-737 is a novel and potent Bcl-2 antagonist with single-agent activity against small-cell lung cancer (SCLC) cell lines. Here, we evaluated the contribution of Bcl-2 family members to the in vitro cellular response of several SCLC cell lines to ABT-737. Relatively higher levels of Bcl-2, Bcl-X(L), Bim and Noxa, and lower levels of Mcl-1 characterized na?ve SCLC cell lines that were sensitive to ABT-737. Conversely, a progressive decrease in the relative levels of Bcl-2 and Noxa and a progressive increase in Mcl-1 levels characterized the increased resistance of H146 cells following chronic exposure to ABT-737. Knockdown of Mcl-1 with small interfering RNA sensitized two resistant SCLC cell lines H196 and DMS114 to ABT-737 by enhancing the induction of apoptosis. Likewise, up-regulation of Noxa sensitized H196 cells to ABT-737. Combination treatment with DNA-damaging agents was extremely synergistic with ABT-737 and was associated with the down-regulation of Mcl-1 and the up-regulation of Noxa, Puma, and Bim in H196 cells. Thus, SCLC cells sensitive to ABT-737 expressed the target proteins Bcl-2 and Bcl-X(L), whereas Mcl-1 and factors regulating Mcl-1 function seem to contribute to the overall resistance of SCLC cells to ABT-737. Overall, these observations provide further insight as to the mechanistic bases for ABT-737 efficacy in SCLC and will be helpful for profiling patients and aiding in the rational design of combination therapies.     

The IGF-1 receptor inhibitor picropodophyllin potentiates the anti-myeloma activity of a BH3-mimetic

The ABT-analogous 737, 263 and 199 are BH3 mimetics showing potent anti-myeloma (MM) activity, but only on defined molecular subgroups of MM patients presenting a Bcl-2^high/Mcl-1^low profile. IGF-1 is a major survival factor in MM regulating the expression of Bcl-2 proteins and might therefore be a resistance factor to these ABT-analogous. We first show that IGF-1 protected human MM cell lines (HMCLs) against ABT-737. Concurrently, the IGF-1 receptor inhibitor picropodophyllin (PPP) synergistically sensitized HMCL, primary human MM and murine 5T33MM cells to ABT-737 and ABT-199 by further decreasing cell viability and enhancing apoptosis. Knockdown of Bcl-2 by shRNA protected MM cells to ABT-737, while Mcl-1 shRNA sensitized the cells. PPP overcame the Bcl-2 dependency of ABT-737, but failed to completely overcome the protective effect of Mcl-1. In vivo, co-treatment of 5T33MM bearing mice significantly decreased tumor burden and prolonged overall survival both in a prophylactic and therapeutic setting. Interestingly, proteasome inhibitor resistant CD138− 5T33MM cells were more sensitive to ABT-737, whereas PPP alone targeted the CD138+ cells more effectively. After co-treatment, both subpopulations were targeted equally. Together, the combination of an IGF-1R inhibitor and an ABT-analogue displays synergistic anti-myeloma activity providing the rational for further (pre)clinical testing.     

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.     

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.     

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.     

Predominant requirement of Bax for apoptosis in HCT116 cells is determined by Mcl-1's inhibitory effect on Bak

The intrinsic mitochondrial apoptotic pathway acts through two core pro-apoptotic proteins Bax (Bcl2-associated X protein) and Bak (Bcl2-antagonist/killer 1). Although Bax and Bak seem to have redundant roles in apoptosis, accumulating evidence also suggests that they might not be interchangeable under certain conditions, at least in some human cell lines. Here we report the generation of Bak knockout as well as BaxBak double knockout HCT116 human colon carcinoma cells. We show that Bak is dispensable for apoptosis induced by a variety of stimuli including ABT-737 but not for fluorouracil-induced apoptosis. In addition, Bax deficiency only provides partial protection against camptothecin and cisplatin-induced apoptosis and no protection against killing by Puma or ABT-737 plus Noxa overexpression. Moreover, Bak is activated normally in response to many chemotherapeutic drugs in the presence of Bax, but remains kept in check by Mcl-1 in the absence of Bax. Our data suggest that Bax and Bak are functionally redundant, but they are counteracted by distinct anti-apoptotic Bcl-2 family proteins in different species.     

Mcl-1 is an important therapeutic target for oral squamous cell carcinomas

Oral and oropharyngeal cancers are the sixth most common cancers worldwide. Despite intensive investigation, oral squamous cell carcinomas (OSCC) represent a clinical challenge resulting in significant morbidity and mortality. Resistance to cell death is common in OSCC and is often mediated by the Bcl-2 family proteins. Among all anti-apoptotic Bcl-2 family members, Mcl-1 functions as a major survival factor, particularly in solid cancers. Despite the confirmed importance of Mcl-1 in several neoplasms, the role of Mcl-1 in OSCC survival has yet to be explored. In this study, we found that knocking down Mcl-1 sensitized OSCC cells to ABT-737, which binds to Bcl-2/Bcl-x_L but not Mcl-1. We report for the first time that a BH3 mimetic, Sabutoclax, which functions as an inhibitor of all anti-apoptotic Bcl-2 proteins, induced cancer-specific cell death in an Mcl-1-dependent manner through both apoptosis and toxic mitophagy. In vivo studies demonstrated that Sabutoclax alone decreased tumor growth in a carcinogen-induced tongue OSCC mouse model. In a combination regimen, Sabutoclax and COX-2 inhibitor, Celecoxib, synergistically inhibited the growth of OSCC in vitro and also significantly reduced OSCC tumor growth in vivo. Overall, these results identify Mcl-1 as a therapeutic prospective target in OSCC.     

GDC-0941 sensitizes breast cancer to ABT-737 in vitro and in vivo through promoting the degradation of Mcl-1

The present study showed that GDC-0941 potently sensitized breast cancer to ABT-737 in vitro and in vivo. ABT-737 exhibited limited lethality in breast cancer cells; however, when combined with GDC-0941, it displayed strong synergistic cytotoxicity and enhanced caspase-mediated apoptosis. GDC-0941 promoted proteasomal degradation of Mcl-1, of which the overexpression has been validated to confer ABT-737 resistance, thereby enhanced the anticancer efficacy of ABT-737. Furthermore, the combination of GDC-0941 and ABT-737 exerted increased anti-tumor efficacy on MDA-MB-231 xenograft models. Overall, our data described unprecedentedly the promising therapeutic potential and underlying mechanisms of combining GDC-0941 with ABT-737 in treating breast cancer.     

Mcl-1 down-regulation potentiates ABT-737 lethality by cooperatively inducing Bak activation and Bax translocation

The Bcl-2 antagonist ABT-737 targets Bcl-2/Bcl-xL but not Mcl-1, which may confer resistance to this novel agent. Here, we show that Mcl-1 down-regulation by the cyclin-dependent kinase (CDK) inhibitor roscovitine or Mcl-1-shRNA dramatically increases ABT-737 lethality in human leukemia cells. ABT-737 induces Bax conformational change but fails to activate Bak or trigger Bax translocation. Coadministration of roscovitine and ABT-737 untethers Bak from Mcl-1 and Bcl-xL, respectively, triggering Bak activation and Bax translocation. Studies employing Bax and/or Bak knockout mouse embryonic fibroblasts (MEFs) confirm that Bax is required for ABT-737+/-roscovitine lethality, whereas Bak is primarily involved in potentiation of ABT-737-induced apoptosis by Mcl-1 down-regulation. Ectopic Mcl-1 expression attenuates Bak activation and apoptosis by ABT-737+roscovitine, whereas cells overexpressing Bcl-2 or Bcl-xL remain fully sensitive. Finally, Mcl-1 knockout MEFs are extremely sensitive to Bak conformational change and apoptosis induced by ABT-737, effects that are not potentiated by roscovitine. Collectively, these findings suggest down-regulation of Mcl-1 by either CDK inhibitors or genetic approaches dramatically potentiate ABT-737 lethality through cooperative interactions at two distinct levels: unleashing of Bak from both Bcl-xL and Mcl-1 and simultaneous induction of Bak activation and Bax translocation. These findings provide a mechanistic basis for simultaneously targeting Mcl-1 and Bcl-2/Bcl-xL in leukemia.     

'Seed' analysis of off-target siRNAs reveals an essential role of Mcl-1 in resistance to the small-molecule Bcl-2/Bcl-XL inhibitor ABT-737

ABT-737 is a subnanomolar inhibitor of the antiapoptotic proteins Bcl-2, Bcl-X(L) and Bcl-w. Although ABT-737 triggers extensive cell death in many small-cell lung carcinoma (SCLC) cell lines, some of the SCLC cell lines and the majority of the cancer cell lines derived from other solid tumors were found to be resistant to ABT-737. To better understand the mechanism of resistance to ABT-737, we screened a short interfering RNA library consisting of short interfering RNA against 4000 'druggable' targets in an SCLC-derived cell line, NCI-H196. By comparing the knockdowns with phenotypes, all of the three top 'hits' from the screen were found to result from off-target gene silencing. Interestingly, the three off-target siRNAs were found to knock down an antiapoptotic Bcl-2 family protein Mcl-1 owing to the complementation between their seed regions with the 3' untranslated region (3' UTR) of Mcl-1. Furthermore, reducing the level of Mcl-1 using siRNAs or the small-molecule compounds Bay43-9006 and Seliciclib was sufficient to overcome the resistance to ABT-737 in the resistant SCLC cell line and cancer cell lines derived from other solid tumors. These results provide further evidence that Mcl-1 is the major factor that causes resistance to ABT-737 in cancer cells derived from diverse solid tumors, and the combination of Mcl-1 downregulating agents with ABT-737 could be potent therapeutic regimens for patient with ABT-737-resistant SCLC and many other types of solid tumors.     

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.     

Targeting multiple arms of the apoptotic regulatory machinery

ABT-737 targets Bcl-2/Bcl-xL but not Mcl-1, which confers resistance to this novel agent. Here, we summarize recent findings indicating that Mcl-1 represents a critical determinant of ABT-737 sensitivity and resistance, and that Mcl-1 down-regulation by various pharmacologic agents or genetic approaches dramatically increases ABT-737 lethality in diverse malignant cell types. These findings also show that the multidomain proapoptotic proteins Bax and Bak play important functional roles in ABT-737-mediated apoptosis, and that Bak activation is essential in potentiation of ABT-737 lethality by agents that down-regulate Mcl-1. Collectively, these findings suggest a novel therapeutic strategy targeting multiple arms of the apoptotic machinery.     

Deciphering the molecular events necessary for synergistic tumor cell apoptosis mediated by the histone deacetylase inhibitor vorinostat and the BH3 mimetic ABT-737

The concept of personalized anticancer therapy is based on the use of targeted therapeutics through in-depth knowledge of the molecular mechanisms of action of these agents when used alone and in combination. We have identified the apoptotic proteins and pathways necessary for synergistic tumor cell apoptosis and in vivo antitumor responses seen when the HDAC inhibitor vorinostat is combined with the BH3-mimetic ABT-737 in lymphomas overexpressing Bcl-2. Vorinostat "primes" tumors overexpressing Bcl-2 for rapid ABT-737-mediated apoptosis by inducing expression of the BH3-only gene bmf. Moreover, these synergistic effects of vorinostat/ABT-737 were blunted in cells with an inactive p53 pathway or in cells lacking expression of the p53 target gene, noxa. These studies show the important and complex functional interaction between specific proapoptotic BH3-only proteins and the BH3-mimetic compound ABT-737 and provide the most comprehensive functional link between tumor genotype and the apoptotic and therapeutic effects of HDACi combined with ABT-737.     

Activation of apoptosis signaling eliminates CD34+ progenitor cells in blast crisis CML independent of response to tyrosine kinase inhibitors

Despite being highly effective for newly diagnosed chronic myeloid leukemia (CML), imatinib not only is inactive against quiescent CML stem cells, but also has limited activity against blast crisis (BC) CML. The relative activity of Bcr-Abl and the expression levels of antiapoptotic proteins in proliferating and quiescent CD34(+) BC CML progenitor cells and the effects of targeting antiapoptotic proteins in these cells are unknown. Here we report higher levels of p-CrkL in quiescent than in proliferating CD34(+) progenitor cells and comparable expression levels of Bcl-2, Bcl-xL, Mcl-1 and XIAP in the two populations in BC CML. Inhibition of Bcl-2/Bcl-xL by ABT-737 in cells from patients with tyrosine kinase inhibitor (TKI)-resistant BC CML promoted apoptosis in quiescent CD34(+) progenitor cells with an efficacy similar to that in proliferating cells. Combination of ABT-737 with imatinib (which decreases Mcl-1 levels) or triptolide (which decreases Mcl-1 and XIAP) synergistically induced death of both proliferating and quiescent CD34(+) progenitor cells obtained from TKI-resistant BC CML patients. These results suggest that antiapoptotic proteins are critical targets in BC CML and that activation of apoptosis signaling can eliminate both proliferating and quiescent CD34(+) progenitor cells in BC CML, independent of response to TKIs.     

Bak and Mcl-1 are essential for Temozolomide induced cell death in human glioma

Temozolomide (TMZ) is an alkylating agent used for the treatment of glioblastoma multiforme (GBM), the main form of human brain tumours in adults. It has been reported that TMZ induced DNA lesions that subsequently trigger cell death but the actual mechanisms involved in the process are still unclear. We investigated the implication of major proteins of the Bcl-2 family in TMZ-induced cell death in GBM cell lines at concentrations closed to that reached in the brain during the treatments. We did not observe modulation of autophagy at these concentrations but we found an induction of apoptosis. Using RNA interference, we showed that TMZ induced apoptosis is dependent on the pro-apoptotic protein Bak but independent of the pro-apoptotic protein Bax. Apoptosis was not enhanced by ABT-737, an inhibitor of Bcl-2/Bcl-Xl/Bcl-W but not Mcl-1. The knock-down of Mcl-1 expression increased TMZ induced apoptosis. Our results identify a Mcl-1/Bak axis for TMZ induced apoptosis in GBM and thus unravel a target to overcome therapeutic resistance toward TMZ.     

Gene Therapy with MiRNA-Mediated Targeting of Mcl-1 Promotes the Sensitivity of Non-Small Cell Lung Cancer Cells to Treatment with ABT-737

Background: Despite the dramatic efficacy of ABT-737, a large percentage of cancer cells ultimately become resistance to this drug. Evidences show that over-expression of Mcl-1 is linked to ABT-737 resistance in NSCLC cells. The aim of this study was to investigate the effect of miRNA-101 on Mcl-1 expression and sensitivity of the A549 NSCLC cells to ABT-737. Methods: After miRNA-101 transfection, the Mcl-1 mRNA expression levels were quantified by RT-qPCR. Trypan blue staining was used to explore the effect of miRNA-101 on cell growth. The cytotoxic effects of miRNA-101 and ABT-737, alone and in combination, were measured using MTT assay. The effect of drugs combination was determined using the method of Chou-Talalay. Cell death was assessed using cell death detection ELISA assay kit. Results: Results showed that miRNA-101 markedly suppressed the expression of Mcl-1 mRNA in a time dependent manner, which led to A549 cell proliferation inhibition and enhancement of apoptosis (p < 0.05, relative to blank control). Pretreatment with miRNA-101 synergistically decreased the cell survival rate and lowered the IC50 value of ABT-737. Furthermore, miRNA-101 dramatically enhanced the apoptotic effect of ABT-737. Negative control miRNA had no remarkable effect on cellular parameters. Conclusions: Our findings propose that suppression of Mcl-1 by miRNA-101 can effectively inhibit the cell growth and sensitize A549 cells to ABT-737. Therefore, miRNA-101 can be considered as a potential therapeutic target in patients with non-small cell lung cancer.