Pro-apoptotic BH3-only Protein Research Articles

Arsenic trioxide induces Noxa-dependent apoptosis in rhabdomyosarcoma cells and synergizes with antimicrotubule drugs

The prognosis of metastatic or relapsed rhabdomyosarcoma (RMS) is poor, highlighting the need of new treatment options. In the present study, we evaluated the in vitro efficacy of arsenic trioxide (ATO) in RMS, a FDA-approved drug used in pediatric leukemia. Here, we report that ATO exerts antitumor activity against RMS cells both as single agent and in combination with microtubule-targeting drugs. Monotherapy with ATO reduces cell viability, triggers apoptosis and suppresses clonogenic survival of RMS cells, at least in part, by transcriptional induction of the proapoptotic BH3-only protein Noxa. siRNA-mediated knockdown of Noxa significantly rescues ATO-mediated cell death, demonstrating that Noxa is required for cell death. Also, ATO suppresses endogenous Hedgehog (Hh) signaling, as it significantly reduces Gli1 transcriptional activity and expression levels of several Hh target genes. Furthermore, we identify synergistic induction of apoptosis by ATO together with several antimicrotubule agents including vincristine (VCR), vinblastine and eribulin. The addition of the broad-range caspase inhibitor zVAD.fmk or overexpression of the antiapoptotic protein Bcl-2 significantly reduce ATO/VCR-induced cell death, indicating that the ATO/VCR combination triggers caspase-dependent apoptosis via the mitochondrial pathway. In summary, ATO exerts antitumor activity against RMS, especially in combination with antimicrotubule drugs. These findings have important implications for the development of novel therapeutic strategies for RMS.

Abstract 2981: DNA damaging agent-induced apoptosis is controled by MCL-1 phosphorylation and degradation mediated by the Noxa/MCL-1/CDK2 complex

Abstract DNA damaging agents, such as cisplatin and etoposide, are employed for the treatment of a wide array of solid tumors, but the prolonged use of chemotherapeutic drugs is limited by their toxicity and by the development of resistance. To overcome these major roadblocks to improved prognosis requires mechanism-based therapeutic strategies that maximize the antitumor effect of drugs while limiting their toxicities. These agents exert anticancer effects via multiple mechanisms, yet their most prominent mode of action involves the generation of DNA lesions followed by activation of the DNA damage response and induction of the BCL-2 family-dependent mitochondrial apoptosis. However, the signaling pathways that connect to BCL-2-family-regulated cell death are not entirely clear. Noxa, a BH3-only pro-apoptotic BCL-2 family protein, causes apoptosis by specifically interacting with the anti-apoptotic BCL-2 family protein MCL-1 to induce its proteasome-mediated degradation. We show here that the DNA damaging agents cisplatin and etoposide upregulate Noxa expression, which is required for the phosphorylation of MCL-1 at Ser64/Thr70 sites, proteasome-dependent degradation, and as a consequence, apoptosis. Noxa-induced MCL-1 phosphorylation at these sites occurs at the mitochondria and is primarily regulated by CDK2. MCL-1 and CDK2 form a stable complex and Noxa binds to this complex to facilitate the phosphorylation of MCL-1. When Ser64 and Thr70 of MCL-1 are substituted with alanine, the mutated MCL-1 is neither phosphorylated nor ubiquitinated, and becomes more stable than the wild-type protein. As a consequence, this mutant can inhibit apoptosis induced by Noxa overexpression or cisplatin treatment. These results indicate that Noxa-mediated MCL-1 phosphorylation followed by MCL-1 degradation is critical for apoptosis induced by DNA damaging agents through regulation of the Noxa/MCL-1/CDK2 complex. Of note, the identified phosphorylation sites are only observed in human MCL-1; thus, the regulatory mechanism shown here may be human-specific. It is of interest in the future to analyze whether CDK2 expression/activity correlates to cisplatin-resistance in patient samples. Citation Format: Wataru Nakajima, June Y. Lee, Nicolas Maxim, Kanika Sharma, Mark A. Hicks, Thien-Trang Vu, Angela Luu, William Andrew Yeudall, Nobuyuki Tanaka, Hisashi Harada. DNA damaging agent-induced apoptosis is controled by MCL-1 phosphorylation and degradation mediated by the Noxa/MCL-1/CDK2 complex. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2981.

BH3-only protein Bim predicts advanced stage of cutaneous melanoma.

Bim having strong pro-apoptotic effects belongs to the BH3-only proteins of the Bcl-2 protein family and contributes to survival pathways in cancer cells. We aimed to investigate Bim protein expression in cutaneous melanoma (CM). Bim protein expression was assessed by immunohistochemistry in primary and metastatic melanomas and correlated with clinical and histopathological features. The Bim immunoreactivity score of the primary melanomas investigated (4.6 ± 1.5) was significantly (P < 0.0001) higher than that observed in metastases (2.8 ± 1.1). Low Bim expression was significantly associated with primary nodular melanoma type (P = 0.005). Moreover, Bim expression was significantly inversely correlated with tumour thickness (r = -0.36; P = 0.0035), advanced stage of disease (stage III and IV; r = -0.60; P < 0.0001), disease relapse (r = -0.18; P = 0.034) and disease-related death (r = -0.19; P = 0.026). Advanced stage of disease was independently predicted by low Bim expression (P = 0.0010, odds ratio: 0.22, 95% CI: 0.10-0.56) on multivariate analysis; however, Bim was not shown to be an independent predictor for disease relapse (P = 0.40) and disease-related death (P = 0.77). Our data demonstrate that Bim protein expression is significantly inversely correlated with melanoma features that are associated with worse prognosis. We have shown that Bim protein expression in CM is an independent predictor for advanced disease confirming that this pro-apoptotic BH3-only protein might be a potent biomarker and promising therapeutic target.

Induction of Apoptosis by the Nonstructural Protein 4 and 10 of Porcine Reproductive and Respiratory Syndrome Virus.

Infection by most viruses triggers apoptosis in host cells, and viruses manipulate this cell response to promote viral replication, virus spread, and cell killing. Porcine reproductive and respiratory syndrome virus (PRRSV) has been shown to induce apoptosis both in vitro and in vivo, while the regulatory roles of PRRSV-encoded products in apoptosis are not fully understood. In the present study, we first showed a biphasic apoptosis regulation by a highly pathogenic PRRSV strain JXwn06. It was indicated that PRRSV infection delays apoptosis at early infection but activates apoptosis at late infection in MARC-145 cells. In PRRSV-infected MARC-145 cells, procaspase-8, -9 and -12 were activated at late infection, demonstrating the involvements of death receptor pathway, mitochondrial pathway and endoplasmic reticulum (ER) stress pathway in inducing apoptosis. PRRSV was also shown to induce a similar apoptosis process in pulmonary alveolar macrophages (PAMs) with an early initiation. Next, the PRRSV-encoded apoptosis inducers were screened, indicating that the nonstructural protein (Nsp) 4 and Nsp10 of PRRSV are pro-apoptotic. In the presence of Nsp4, it was confirmed that procaspase-8, -9 and -12 were cleaved, and Nsp4 facilitates the cleavage of procaspase-9 by activating B-cell lymphoma 2 interacting mediator of cell death (Bim), a pro-apoptotic protein. In addition, Nsp4 was shown to induce the degradation of an anti-apoptotic protein, B-cell lymphoma-extra large (Bcl-xL). Nsp10 was shown to activate procaspase-8 and -9 but procaspase-12 and to upregulate the expression of BH3-only pro-apoptotic protein BH3 interacting-domain death agonist (Bid) and its active form, truncated Bid (tBid). Clearly, the participation of both activated caspase-8 and Bid is required for Nsp10-induced apoptosis, indicating a crosstalk between extrinsic- and mitochondria-dependent pathways. Together, our findings suggest that PRRSV infection regulates apoptosis in a two-phase manner and activates all three apoptotic pathways; the Nsp4 and Nsp10 of PRRSV function as apoptosis inducers with different molecular basis.

Bax/Bak activation in the absence of Bid, Bim, Puma, and p53.

How BH3-only proteins activate Bax/Bak, the two gateway proteins of the mitochondria-dependent apoptotic pathway, remains incompletely understood. Although all pro-apoptotic BH3-only proteins are known to bind/neutralize the anti-apoptotic Bcl-2 proteins, the three most potent ones, Bid (tBid), Bim, and Puma, possess an additional activity of directly activating Bax/Bak in vitro. This latter activity has been proposed to be responsible for triggering Bax/Bak activation following apoptotic stimulation. To test this hypothesis, we generated Bid−/−Bim−/−Puma−/− (TKO), TKO/Bax−/−/Bak−/− (PentaKO), and PentaKO/Mcl-1−/− (HexaKO) HCT116 cells through gene editing. Surprisingly, although the TKO cells were resistant to several apoptotic stimuli, robust apoptosis was induced upon the simultaneous inactivation of Bcl-xL and Mcl-1, two anti-apoptotic Bcl-2 proteins known to suppress Bax/Bak activation and activity. Importantly, such apoptotic activity was completely abolished in the PentaKO cells. In addition, ABT-737, a BH3 mimetic that inhibits Bcl-xL/Bcl-w/Bcl-2, induced Bax activation in HexaKO cells reconstituted with endogenous level of GFP-Bax. Further, by generating TKO/p53−/− (QKO) cells, we demonstrated that p53, a tumor suppressor postulated to directly activate Bax, is not required for Bid/Bim/Puma-independent Bax/Bak activation. Together, these results strongly suggest that the direct activation activities of Bid (tBid), Bim, Puma, and p53 are not essential for activating Bax/Bak once the anti-apoptotic Bcl-2 proteins are neutralized.

Loss of PUMA (BBC3) does not prevent thrombocytopenia caused by the loss of BCL-XL (BCL2L1).

Apoptosis is required to maintain tissue homeostasis in multicellular organisms. Platelets, the anucleate cells that are essential for blood clotting, are a prime example. Their brief life span in the circulation is regulated by the intrinsic apoptosis pathway. Pro-survival BCL-XL (also termed BCL2L1) is essential for platelet viability. It functions to restrain the pro-apoptotic BCL-2 family members BAK (also termed BAK1) and BAX, the essential mediators of intrinsic apoptosis. Genetic deletion or pharmacological inhibition of BCL-XL results in thrombocytopenia. Conversely, deletion of BAK in platelets doubles their circulating life span. However, what triggers platelet apoptosis invivo remains unclear. The pro-apoptotic BH3-only proteins are essential for initiating apoptosis in nucleated cells, and there is some evidence to suggest they also play a role in platelet biology. We investigated whether PUMA (also termed BBC3), a potent BH3-only protein that can inhibit all pro-survival BCL-2 family members as well as directly activate BAX, regulates the death of platelets. Surprisingly, loss of PUMA had no impact on the loss of platelets caused by loss of BCL-XL. It therefore remains to be established whether other BH3-only proteins play a critical role in induction of apoptosis in platelets or whether their death is controlled solely by the interactions between BCL-XL with BAK and BAX.

DNA damaging agent-induced apoptosis is regulated by MCL-1 phosphorylation and degradation mediated by the Noxa/MCL-1/CDK2 complex.

Noxa, a BH3-only pro-apoptotic BCL-2 family protein, causes apoptosis by specifically interacting with the anti-apoptotic protein MCL-1 to induce its proteasome-mediated degradation. We show here that the DNA damaging agents cisplatin and etoposide upregulate Noxa expression, which is required for the phosphorylation of MCL-1 at Ser64/Thr70 sites, proteasome-dependent degradation, and apoptosis. Noxa-induced MCL-1 phosphorylation at these sites occurs at the mitochondria and is primarily regulated by CDK2. MCL-1 and CDK2 form a stable complex and Noxa binds to this complex to facilitate the phosphorylation of MCL-1. When Ser64 and Thr70 of MCL-1 are substituted with alanine, the mutated MCL-1 is neither phosphorylated nor ubiquitinated, and becomes more stable than the wild-type protein. As a consequence, this mutant can inhibit apoptosis induced by Noxa overexpression or cisplatin treatment. These results indicate that Noxa-mediated MCL-1 phosphorylation followed by MCL-1 degradation is critical for apoptosis induced by DNA damaging agents through regulation of the Noxa/MCL-1/CDK2 complex.

Inactivation of prosurvival Bcl-2 proteins activates Bax/Bak through the outer mitochondrial membrane.

The mechanism of Bax/Bak activation remains a central question in mitochondria-dependent apoptotic signaling. While it is established that all proapoptotic Bcl-2 homology 3 (BH3)-only proteins bind and neutralize the anti-apoptotic Bcl-2 family proteins, how this neutralization leads to Bax/Bak activation has been actively debated. Here, genome editing was used to generate cells deficient for all eight proapoptotic BH3-only proteins (OctaKO) and those that lack the entire Bcl-2 family (Bcl-2 allKO). Although the OctaKO cells were resistant to most apoptotic stimuli tested, they underwent Bax/Bak-dependent and p53/Rb-independent apoptosis efficiently when both Bcl-xL and Mcl-1, two anti-apoptotic Bcl-2 proteins, were inactivated or eliminated. Strikingly, when expressed in the Bcl-2 allKO cells, both Bax and Bak spontaneously associated with the outer mitochondrial membrane (OMM) through their respective helix 9, and this association triggered their homo-oligomerization/activation. Together, these results strongly suggest that the OMM, not BH3-only proteins or p53/Rb, is the long-sought-after direct activator of Bax/Bak following BH3-only-mediated neutralization of anti-apoptotic Bcl-2 proteins.

P53-upregulated-modulator-of-apoptosis (PUMA) deficiency affects food intake but does not impact on body weight or glucose homeostasis in diet-induced obesity.

BCL-2 proteins have been implicated in the control of glucose homeostasis and metabolism in different cell types. Thus, the aim of this study was to determine the role of the pro-apoptotic BH3-only protein, p53-upregulated-modulator-of-apoptosis (PUMA), in metabolic changes mediated by diet-induced obesity, using PUMA deficient mice. At 10 weeks of age, knockout and wild type mice either continued consuming a low fat chow diet (6% fat), or were fed with a high fat diet (23% fat) for 14–17 weeks. We measured body composition, glucose and insulin tolerance, insulin response in peripheral tissues, energy expenditure, oxygen consumption, and respiratory exchange ratio in vivo. All these parameters were indistinguishable between wild type and knockout mice on chow diet and were modified equally by diet-induced obesity. Interestingly, we observed decreased food intake and ambulatory capacity of PUMA knockout mice on high fat diet. This was associated with increased adipocyte size and fasted leptin concentration in the blood. Our findings suggest that although PUMA is dispensable for glucose homeostasis in lean and obese mice, it can affect leptin levels and food intake during obesity.

BCL2-modifying factor promotes germ cell loss during murine oogenesis.

Apoptosis plays a prominent role during ovarian development by eliminating large numbers of germ cells from the female germ line. However, the precise mechanisms and regulatory proteins involved in germ cell death are yet to be determined. In this study, we characterised the role of the pro-apoptotic BH3-only protein, BCL2-modifying factor (BMF), in germ cell apoptosis in embryonic and neonatal mouse ovaries. BMF protein was immunohistochemically localised to germ cells at embryonic days 15.5 (E15.5) and E17.5 and postnatal day 1 (PN1), coincident with entry into the meiotic prophase, but was undetectable at E13.5, and only present at low levels at PN3 and PN5. Consistent with this expression pattern, loss of BMF in female mice was associated with a decrease in apoptosis at E15.5 and E17.5. Furthermore, increased numbers of germ cells were found in ovaries from Bmf(-/-) mice compared with WT animals at E15.5 and PN1. However, germ cell numbers were comparable between Bmf(-/-) and WT ovaries at PN3, PN5 and PN10. Collectively, these data indicate that BMF mediates foetal oocyte loss and its action limits the maximal number of germ cells attained in the developing ovary, but does not influence the number of primordial follicles initially established in ovarian reserve.

Mechanisms of Acquired Resistance to Venetoclax in Preclinical AML Models

BH3-mimetic ABT-199 (venetoclax, VEN) is a selective small-molecule antagonist of the anti-apoptotic BCL-2 protein. It binds to BCL-2 specifically, causing the release of pro-apoptotic BAX and BH3-only proteins and induction of cell death. Our studies indicated that AML is a BCL-2 dependent disease that, in pre-clinical studies, responds robustly to VEN by induction of apoptotic cell death (Pan et al., Cancer Discovery 2014). As a single agent, VEN demonstrated clinical activity in relapsed/refractory AML, yet patients who initially responded ultimately developed resistance and progressed. In this study we investigated mechanisms of acquired resistance to VEN in preclinical AML models.First, we generated 5 VEN-resistant cell lines (OCI-AML2, Kasumi, KG-1, MV4;11 and Molm13; with VEN cell-killing IC50s of 0.021µM, 0.046µM, 0.073µM, 0.020µM and 0.050µM, respectively) by exposing the cells to gradually increasing VEN concentrations. The IC50s of resistant cells are 15.2µM, 5.7µM, 31.6µM, 11.4µM and 15.4µM (124-723-fold greater than their parental counterparts). Protein analysis of resistant cells using immunoblotting demonstrated increased expression of MCL-1, a known resistance factor to VEN, in 4 resistant cell lines (OCI-AML2, KG-1, Mv4;11 and Molm13); and BCL-XL increase in MV4;11 and Molm13 resistant cells. To characterize the functional role of MCL-1 and BCL-XL in resistance to VEN, we co-treated parental and resistant cells with novel MCL-1 and BCL-XL- selective inhibitors (A-1210477 and A-1155463). The combination of VEN with A-1210477 or A-1155463 showed synergistic growth inhibition in all 5 parental cell lines (combination indices (CI) for A-1210477 were 0.15-0.62; CI for A-1155463 were 0.33-0.51, except >3 for KG-1). Notably, 4 out of 5 resistant cell lines (OCI-AML2, Kasumi, MV4;11, Molm13) became more sensitive to MCL-1 selective inhibitor A-1155463 but not to BCL-XL inhibitor A-1210477. However, no further effects were seen in resistant cells when combined with VEN.We next compared sensitivity of three paired parental and resistant cell lines (OCI-AML2, MV4;11 and Molm13) to a library of 130 specific small-molecule inhibitors (Tyner, et.al.. Cancer Res. 2013). Cells were co-treated with VEN and each specific inhibitor, and drug target scores were calculated based on the IC50 of measured effectiveness of panel drugs against the cells. The screening revealed modulation of sensitivity to mTOR, MEK, and FLT3 pathways in resistant cells (Fig.1C). To confirm these findings, we next co-treated AML cells with VEN and specific inhibitors of the mTOR pathway (rapamycin and AZD2014) or MEK pathway (CI1040) in all 5 paired parental and resistant cell lines; or with FLT3 inhibitors (quizartinib and sorafenib) in parental and resistant MV4;11 and Molm13, which harbor FLT3-ITD. The combination of VEN and AZD2014 achieved synergistic effects in all 5 parental cell lines (CI AZD2014: 0.08-0.94), and VEN/rapamycin were synergistic in 3 parental cell lines (CI rapamycin: 0.00-0.55, except 1.76 for KG-1 and 1.59 for Molm13). Combination of VEN with CI1040 achieved synergy in OCI-AML2, Kasumi, MV4;11 and Molm13 parental cell lines (CI: 0.14-0.61). Finally, VEN/FLT3 inhibitors achieved synergistic effects in MV4;11 and Molm13 parental cell lines (CI quizartinib: 0.66-0.69; CI sorafenib: 0.64-0.71). The resistant cell lines exhibited sensitivity to these inhibitors as single agents, and no synergistic effects were seen when combined with VEN.We have further induced in vivo resistance in two primary AML xenografts by treating NSG mice engrafted with 2nd passage AML cells with 100 mg/kg Q.D. VEN for 4 weeks followed by harvest of leukemic cells that repopulated the mouse after treatment discontinuation. While the proteomics, gene expression (RNAseq) and drug screening assays are in progress, preliminary immunoblotting studies demonstrated decreased expression of BCL-XL and BCL-2 (Fig.1B).In summary, we identified multiple mechanisms of acquired resistance to VEN, which ultimately modulate the balance between pro- and anti-apoptotic BCL-2 family members. Our studies indicate that upfront combination of VEN with selective inhibitors of MCL-1, or with inhibitors of specific signaling pathways, can synergistically induce apoptosis in AML cells and conceivably prevent emergence of VEN resistance. [Display omitted] DisclosuresLeverson:AbbVie: Employment, Equity Ownership. Tyner:Aptose Biosciences: Research Funding; Constellation Pharmaceuticals: Research Funding; Janssen Pharmaceuticals: Research Funding; Array Biopharma: Research Funding; Incyte: Research Funding. Konopleva:Novartis: Research Funding; AbbVie: Research Funding; Stemline: Research Funding; Calithera: Research Funding; Threshold: Research Funding.

Dual Targeting of NOXA/MCL-1 Synergistically Induces Cell Death in Mantle Cell Lymphoma (MCL) Cells

Mantle cell lymphoma (MCL) cells are characterized by a discrepancy between high mRNA and low protein levels of the pro-apoptotic BH3-only protein NOXA. Modulation of NOXA protein expression has been described as a major mechanism of cell death induction in MCL cells. However, the efficiency of induction of apoptosis at lower concentrations is limited despite effective stabilization of NOXA.We therefore investigated whether the main binding partner of NOXA, the anti-apoptotic protein MCL-1 is co-regulated by these agents and whether dual targeting of NOXA and MCL-1 could be a promising strategy to enhance effectiveness of cell death induction in MCL cell lines.Screening a panel of compounds supposed to lead to NOXA stabilization in MCL, we identified the proteasome inhibitor Bortezomib, the fatty acid synthase inhibitor Orlistat and the ROS inducing agents Helenalin, a sesquesterpenone lactone from Arnica and the naphtoquinone derivative Menadione to kill MCL cells very effectively in a NOXA -dependent manner.Investigating the NOXA-/MCL-1-protein expression upon treatment with these agents, we could observe that at lower, sublethal concentrations of Bortezomib and Orlistat, NOXA protein was increased to a certain extent but also the anti-apoptotic MCL-1 was highly induced, counteracting induction of apoptosis.From this observation it has to be concluded that MCL-1 limits the apoptotic activity of NOXA stabilizing agents and dual targeting of MCL-1 and NOXA is required for optimal killing of MCL cells.In search for MCL-1 regulating agents we could identify the cdk-inhibitor Dinaciclib to be the most effective one. This compound rapidly downregulates Mcl-1 protein in a dose- and time-dependent manner presumably due to cdk 9-mediated inhibition of phosphorylation of the RNA-Polymerase II-subunit RPB1.To study the efficiency of dual targeting of NOXA/MCL-1 in killing of MCL cells we combined sublethal doses of Dinaciclib with NOXA stabilizing agents and observed a synergistic induction of apoptosis in MCL cells. Western Blot analysis showed that combination treatment decreased Mcl-1 and increased NOXA protein expression compared to single-agents. It could be shown that induction of cell death by treatment with the combined agents depends on NOXA as transfection of cells with NOXA-siRNA rescued cells from induction of apoptosis. Cell death upon treatment with Dinaciclib and ROS-inducing agents Helenalin and Menadione could furthermore be rescued by preincubation with the antioxidant GSH.Interestingly, combined treatment of cells with Orlistat and Dinaciclib killed most effectively when Dinaciclib was added for 8 hours after 16 hours of preincubation with the NOXA stabilizing agent. This observation contributes to the hypothesis that stabilization of NOXA protein leads to priming of MCL cells to induction of apoptosis by pharmacological downregulation of Mcl-1 with Dinaciclib.In summary, the NOXA-MCL-1 balance is critical for survival of MCL cells. Dual targeting of MCL-1 and NOXA efficiently kills MCL cells and appears to be of particular importance especially at lower concentrations of these compounds. These culture conditions most likely resemble the limited exposure after in vivo treatment. Therefore the combination of NOXA stabilizing agents with Dinaciclib appears to be a promising strategy to be tested in clinical trials. DisclosuresNo relevant conflicts of interest to declare.

Binding of Released Bim to Mcl-1 Is Responsible for Resistance to ABT-199 Which Can be Overcome By Combination with Daunorubicin or Cytarabine in Acute Myeloid Leukemia Cells

Acute myeloid leukemia (AML) is a malignant heterogeneous disease characterized by rapid clonal growth of myeloid lineage blood cells. This year there will be an estimated 20,830 new AML cases and an estimated 10,400 deaths from this deadly disease in the United States. Overall survival rates remain low despite advances in treatment with overall survival rates of 25% for adults and 65% for children. Resistance to frontline chemotherapy remains a major cause of treatment failure, highlighting the need for new therapies.Overexpression of the anti-apoptotic Bcl-2 family members is associated with chemoresistance in leukemic cell line models and with poor clinical outcome. Anti-apoptotic Bcl-2 family members, such as Bcl-2, Bcl-xL, and Mcl-1, sequester pro-apoptotic BH3-only proteins, such as Bim, which activate pro-apoptotic proteins Bax and Bak causing mitochondrial outer membrane permeabilization resulting in cytochrome c release and apoptosis. Thus, inhibition of anti-apoptotic Bcl-2 family members represents a promising approach for the treatment of AML. We previously demonstrated preclinical efficacy of a pan-Bcl-2 inhibitor, GX15-070, in combination with cytarabine in AML cell lines and primary patient samples. Another promising inhibitor, ABT-263, has shown preclinical efficacy, but has been associated with thrombocytopenia due to inhibition of Bcl-xL, thus much attention has been focused on inhibition of Bcl-2. ABT-199, a Bcl-2 selective inhibitor, has demonstrated encouraging results in AML, acute lymphoblastic leukemia, chronic lymphocytic leukemia, mantle cell lymphoma, multiple myeloma, and breast cancer. We previously demonstrated that ABT-199 has a wide range of activity in AML cells (Niu X, et al. Leukemia. 2014; 28: 1557-1560.) However, it has limited efficacy in Bcl-xL and Mcl-1 dependent malignancies. Thus, intrinsic drug resistance remains a concern. Understanding the molecular mechanisms of resistance to ABT-199 will allow for rationally designed combination regimens to increase its antileukemic efficacy.In this study, we investigated the molecular mechanism underlying intrinsic resistance to ABT-199 in AML cells. Immunoprecipitation of Bim from ABT-199 treated cells demonstrated decreased association with Bcl-2, but increased association with Mcl-1, without corresponding change in mitochondrial outer membrane potential. ABT-199 treatment resulted in increased levels of Mcl-1 protein and unchanged or decreased Mcl-1 transcript levels. shRNA knockdown of Bim almost completely abolished ABT-199 treatment-induced increase of Mcl-1 protein levels, suggesting that the association with Bim plays an important role in stabilizing Mcl-1 protein. AML cells treated with ABT-199 in the presence of the protein translation inhibitor cycloheximide resulted in significantly longer Mcl-1 half-life and treatment with the proteasome inhibitor MG-132 resulted in increased Mcl-1 protein level and no further enhancement was detected when treated with combined MG-132 and ABT-199, suggesting that ABT-199 affects Mcl-1 protein stability. Combining conventional chemotherapeutic agent cytarabine or daunorubicin with ABT-199 resulted in increased DNA damage, decreased Mcl-1 protein levels, decreased association of Mcl-1 with Bim, and synergistic induction of cell death compared to ABT-199 alone, in both AML cell lines and primary patient samples obtained from AML patients at diagnosis independent of their sensitivities to ABT-199, thus providing evidence that screening for ABT-199 resistance is not necessary.Our results demonstrate that sequestration of Bim by Mcl-1 is a mechanism of intrinsic ABT-199 resistance, and this mechanism of resistance can be overcome by combining ABT-199 with daunorubicin or cytarabine in AML cells. Our findings, though in a limited number of primary patient samples, provide new insights into the mechanism of ABT-199 resistance in AML cells and support the clinical development of the combination of daunorubicin or cytarabine and ABT-199 in the treatment of AML. DisclosuresNo relevant conflicts of interest to declare.

Combination with vorinostat overcomes ABT-263 (navitoclax) resistance of small cell lung cancer

ABSTRACTSmall cell lung cancer (SCLC) is an aggressive tumor type with high mortality. One promising approach for SCLC treatment would be to utilize agents targeting molecular abnormalities regulating resistance to apoptosis. BH3 mimetic antagonists, such as ABT-737 and its orally available derivative ABT-263 (navitoclax) have been developed to block the function of pro-survival BCL-2 family members. The sensitivity of SCLC to these drugs varies over a broad range in vitro and in clinical trials. We have previously shown that the expression of Noxa, a BH3-only pro-apoptotic BCL-2 family protein, is a critical determinant of sensitivity to ABT-737. Thus, pharmacological up-regulation of Noxa could enhance cell death induced by the BH3 mimetics. We find that the combination of ABT-263 and a HDAC inhibitor, vorinostat, efficiently induces apoptosis in a variety of SCLC cell lines, including ABT-263 resistant cell lines. Cell death induced by combined treatment is Noxa- and/or BIM-dependent in some cell lines but in others appears to be mediated by down-regulation of BCL-XL and release of BAK from BCL-XL and MCL-1. These results suggest that combination of HDAC inhibitors and BCL-2 inhibitors could be an alternative and effective regimen for SCLC treatment.

Poly (ADP-Ribose) Polymerase-1 Causes Mitochondrial Damage And Neuron Death Mediated By Bnip3: Journal of Neuroscience,, 2014;34(48):15975-15987

Hypoxia/ischemia is one of the major causes of mitochondrial dysfunction and neuronal cell death. So far, it has been reported that the DNA damage repair enzyme Poly (ADP-Ribose) Polymerase-1 (PARP1) gets activated during hypoxia/ischemia, leading to mitochondrial membrane permeability transition and caspase independent neuronal death mediated by nuclear translocation of the mitochondrial proapoptotic factor AIF. On the other hand, Bnip3, a proapoptotic BH3-only protein of the Bcl-2 family, is also found to be upregulated during hypoxia/ischemia, resulting in mitochondrial permeability transition and neuronal death in a manner similar to that of PARP1. This led the authors to hypothesize that the pathway of neuronal cell death induced by PARP1 and Bnip3 may be linked. Also, it is reported that PARP1 mediated NAD + depletion leads to inhibition of SIRT1, a NAD + dependent class III histone deacetylase enzyme which targets many transcription factors responsible for maintaining mitochondrial integrity including the FoxO (Fork-head box O) family of proteins. Thus, they also hypothesized that PARP1 mediated inhibition of SIRT1 may lead to derepression of FoxO3a resulting in upregulation of Bnip3 under hypoxic conditions.

CBIO-07COMBINED BH3 AND METABOLIC PROFILING AS A METHOD TO DEFINE THERAPEUTIC RESPONSE AND RESISTANCE IN GRADE IV ASTROCYTOMAS

BACKGROUND: Grade IV astrocytomas, formerly known as glioblastoma multiforme (GBM), are the most common primary brain tumors and have the highest mortality. The therapeutic standard for managing this malignancy remains a combination of surgery, chemotherapy, and radiotherapy; however, there is no cure, nor has there been any significant advancement in the clinical approach to GBM since this protocol was established in 2005. This study aims to better understand the molecular and metabolic characteristics of GBM to better define treatment groups and potentially identify new avenues for therapy. This study utilized continuous, commercially-available GBM cell lines U87, U118, A172, and H4, and examined the concentrations of Bcl-2 family proteins on mitochondria and metabolic activity for each of the cell lines. The measures were correlated to temozolamide (TMZ) sensitivity. RESULTS: Western blot analysis of pro-survival and pro-apoptotic Bcl-2 proteins revealed that U118 and U87 expressed high levels of Bcl-2, while A172 had increased Bcl-xL expression. Interestingly, Bcl-2 and Bcl-xL were not detected in H4 cells. Incidentally, pro-apoptotic BH3-only protein levels were increased in H4 and A172 cells. Metabolic analysis of the cell lines revealed that U118 cells were a glutaminolytic, while U87, A172, and H4 were classified as glycolytic. We found that U118 cells were the most resistant followed by U87, A172, and H4, respectively. CONCLUSIONS: We found that Bcl-2 protein profiling was a useful means to determine therapeutic response and resistance. This was enhanced by addingmetabolic stratification, wherein glycolytic cells were shown to be more sensitive TMZ than glutaminolytic cells. We will expand the number of cell lines in our study and increase the cellular profiling to include mitochondrial dynamics. Using this approach, we will produce a method to define GBM responses and outcomes based on the mitochondrial and metabolic context of individual tumors.

The role of BH3-only protein Bmf in the pathogenesis of dominant negative hepatocyte nuclear factor-1 –induced mature-onset diabetes of the young in transgenic mice

Maturity Onset Diabetes of the Young 3 (MODY3) is the most common monogenic form of diabetes, characterized by early age of onset (before the age of 25), autosomal dominant transmission and severe defect in insulin secretion [1,2]. MODY accounts for 2-5% of Non-Insulin Dependent Diabetes Mellitus (NIDDM) cases, with MODY3 identified as the most common and severe form, accounting for 65% of all MODY cases and results from loss of function mutations of the transcription factor Hepatocyte Nuclear Factor-1 α (HNF1α). As a result of this, pancreatic islets show reduction in glucose-stimulated insulin secretion response and in beta cell mass, hallmarks of MODY3. Previous work in this laboratory has shown that induction of dominant negative mutant-HNF1a expression results in bioenergetic stress, activation of AMPK and induction of pro-apoptotic BH3-only family protein, Bmf [3].

Effect of cAMP signaling on expression of glucocorticoid receptor, Bim and Bad in glucocorticoid-sensitive and resistant leukemic and multiple myeloma cells.

Stimulation of cAMP signaling induces apoptosis in glucocorticoid-sensitive and resistant CEM leukemic and MM.1 multiple myeloma cell lines, and this effect is enhanced by dexamethasone in both glucocorticoid-sensitive cell types and in glucocorticoid-resistant CEM cells. Expression of the mRNA for the glucocorticoid receptor alpha (GR) promoters 1A3, 1B and 1C, expression of mRNA and protein for GR, and the BH3-only proapoptotic proteins, Bim and Bad, and the phosphorylation state of Bad were examined following stimulation of the cAMP and glucocorticoid signaling pathways. Expression levels of GR promoters were increased by cAMP and glucocorticoid signaling, but GR protein expression was little changed in CEM and decreased in MM.1 cells. Stimulation of these two signaling pathways induced Bim in CEM cells, induced Bad in MM.1 cells, and activated Bad, as indicated by its dephosphorylation on ser112, in both cell types. This study shows that leukemic and multiple myeloma cells, including those resistant to glucocorticoids, can be induced to undergo apoptosis by stimulating the cAMP signaling pathway, with enhancement by glucocorticoids, and the mechanism by which this occurs may be related to changes in Bim and Bad expression, and in all cases, to activation of Bad.

Dynamics of the BH3-Only Protein Binding Interface of Bcl-xL

The balance and interplay between pro-death and pro-survival members of the B-cell lymphoma-2 (Bcl-2) family proteins play key roles in regulation of the mitochondrial pathway of programmed cell death. Recent NMR and biochemical studies have revealed that binding of the proapoptotic BH3-only protein PUMA induces significant unfolding of antiapoptotic Bcl-xL at the interface, which in turn disrupts the Bcl-xL/p53 interaction to activate apoptosis. However, the molecular mechanism of such regulated unfolding of Bcl-xL is not fully understood. Analysis of the existing Protein Data Bank structures of Bcl-xL in both bound and unbound states reveal substantial intrinsic heterogeneity at its BH3-only protein binding interface. Large-scale atomistic simulations were performed in explicit solvent for six representative structures to further investigate the intrinsic conformational dynamics of Bcl-xL. The results support that the BH3-only protein binding interface of Bcl-xL is much more dynamic compared to the rest of the protein, both unbound and when bound to various BH3-only proteins. Such intrinsic interfacial conformational dynamics likely provides a physical basis that allows Bcl-xL to respond sensitively to detailed biophysical properties of the ligand. The ability of Bcl-xL to retain or even enhance dynamics at the interface in bound states could further facilitate the regulation of its interactions with various BH3-only proteins such as through posttranslational modifications.

BCL-2 proteins: melanoma lives on the edge.

BCL-2 proteins: melanoma lives on the edge.