Bcl-2 Homology Domain Research Articles

Pro-death activity of a BH3 domain in an aquaporin from the protozoan parasite Leishmania.

The decision of a given cell to undergo cell death is determined, in most cases, by the interplay among pro- and anti-apoptotic members of the Bcl-2 family of proteins. Anti-apoptotic proteins such as Bcl-2, Bcl-XL, and MCL-1 maintain cell survival by binding and sequestering their pro-apoptotic counterparts, which can be classified as Bcl-2 homology 3 (BH3)-only or (BH1–BH3) multi-domain proteins.

Orphan Nuclear Receptor NR4A1 Binds a Novel Protein Interaction Site on Anti-apoptotic B Cell Lymphoma Gene 2 Family Proteins

B cell lymphoma gene 2 (Bcl-2) family proteins are key regulators of programmed cell death and important targets for drug discovery. Pro-apoptotic and anti-apoptotic Bcl-2 family proteins reciprocally modulate their activities in large part through protein interactions involving a motif known as BH3 (Bcl-2 homology 3). Nur77 is an orphan member of the nuclear receptor family that lacks a BH3 domain but nevertheless binds certain anti-apoptotic Bcl-2 family proteins (Bcl-2, Bfl-1, and Bcl-B), modulating their effects on apoptosis and autophagy. We used a combination of NMR spectroscopy-based methods, mutagenesis, and functional studies to define the interaction site of a Nur77 peptide on anti-apoptotic Bcl-2 family proteins and reveal a novel interaction surface. Nur77 binds adjacent to the BH3 peptide-binding crevice, suggesting the possibility of cross-talk between these discrete binding sites. Mutagenesis of residues lining the identified interaction site on Bcl-B negated the interaction with Nur77 protein in cells and prevented Nur77-mediated modulation of apoptosis and autophagy. The findings establish a new protein interaction site with the potential to modulate the apoptosis and autophagy mechanisms governed by Bcl-2 family proteins.

Relation of the BIM deletion polymorphism to intrinsic EGFR-TKI resistance of Chinese patients with EGFR mutant advanced non-small cell lung cancer.

e14095Background: In order to identify the influence of BCL2-like 11 (BIM) gene deletion polymorphism, which results in expression of BIM isoforms lacking the proapoptotic BCL2-homology domain 3 (B...

Gamma-tocotrienol acts as a BH3 mimetic to induce apoptosis in neuroblastoma SH-SY5Y cells

Bcl-2 family proteins are crucial regulators of apoptosis. Both pro- and antiapoptotic members exist, and overexpression of the latter facilitates evasion of apoptosis in many cancer types. Bcl-2 homology domain 3 (BH3) mimetics are small molecule inhibitors of antiapoptotic Bcl-2 family members, and these inhibitors are promising anticancer agents. In this study, we report that gamma-tocotrienol (γT3), an isomer of vitamin E, can inhibit Bcl-2 to induce apoptosis. We demonstrate that γT3 induces cell death in human neuroblastoma SH-SY5Y cells by depolarising the mitochondrial membrane potential, enabling release of cytochrome c to the cytosol and increasing the activities of caspases-9 and -3. Treatment of cells with inhibitors of Bax or caspase-9 attenuated the cell death induced by γT3. Simulated docking analysis suggested that γT3 binds at the hydrophobic groove of Bcl-2, while a binding assay showed that γT3 competed with a fluorescent probe to bind at the hydrophobic groove. Our data suggest that γT3 mimics the action of BH3-only protein by binding to the hydrophobic groove of Bcl-2 and inducing apoptosis via the intrinsic pathway in a Bax- and caspase-9-dependent manner.

Endoplasmic reticulum stress-mediated upregulation of miR-29a enhances sensitivity to neuronal apoptosis.

Disturbance of homeostasis within the endoplasmic reticulum (ER) lumen leads to the accumulation of unfolded and misfolded proteins. This results in the activation of an evolutionary conserved stress response termed ER stress that, if unresolved, induces apoptosis. Previously the Bcl-2 homology domain 3-Only Protein Puma was identified as a mediator of ER stress-induced apoptosis in neurons. In the search of alternative contributors to ER stress-induced apoptosis, a downregulation of the anti-apoptotic Bcl-2 family protein Mcl-1 was noted during ER stress in both mouse cortical neurons and human SH-SY5Y neuroblastoma cells. Downregulation of Mcl-1 was associated with an upregulation of microRNA-29a (miR-29a) expression, and subsequent experiments showed that miR-29a targeted the 3'-untranslated region of the anti-apoptotic Bcl-2 family protein, Mcl-1. Inhibition of miR-29a expression using sequence-specific antagomirs or the overexpression of Mcl-1 decreased cell death following tunicamycin treatment, while gene silencing of Mcl-1 increased cell death. miR-29a did not alter the signalling branches of the ER stress response, rather its expression was controlled by the ER stress-induced transcription factor activating-transcription-factor-4 (ATF4). The current data demonstrate that the ATF4-mediated upregulation of miR-29a enhances the sensitivity of neurons to ER stress-induced apoptosis.

Affinity purification-mass spectrometry analysis of bcl-2 interactome identified SLIRP as a novel interacting protein.

Members of the bcl-2 protein family share regions of sequence similarity, the bcl-2 homology (BH) domains. Bcl-2, the most studied member of this family, has four BH domains, BH1–4, and has a critical role in resistance to antineoplastic drugs by regulating the mitochondrial apoptotic pathway. Moreover, it is also involved in other relevant cellular processes such as tumor progression, angiogenesis and autophagy. Deciphering the network of bcl-2-interacting factors should provide a critical advance in understanding the different functions of bcl-2. Here, we characterized bcl-2 interactome by mass spectrometry in human lung adenocarcinoma cells. In silico functional analysis associated most part of the identified proteins to mitochondrial functions. Among them we identified SRA stem–loop interacting RNA-binding protein, SLIRP, a mitochondrial protein with a relevant role in regulating mitochondrial messenger RNA (mRNA) homeostasis. We validated bcl-2/SLIRP interaction by immunoprecipitation and immunofluorescence experiments in cancer cell lines from different histotypes. We showed that, although SLIRP is not involved in mediating bcl-2 ability to protect from apoptosis and oxidative damage, bcl-2 binds and stabilizes SLIRP protein and regulates mitochondrial mRNA levels. Moreover, we demonstrated that the BH4 domain of bcl-2 has a role in maintaining this binding.

Abstract IA19: Targeting the BCL-2 family in breast cancer

Abstract The steps taken to evade cell death are a recognized hallmark of cancer. Intense efforts over the last three decades have provided important insights into the molecular mechanisms that govern programmed cell death and how tumors recalibrate key survival pathways to sustain their growth. The pro-survival protein BCL-2 is overexpressed in approximately 75% of breast cancer, where it is has emerged as an important prognostic marker. Indeed, BCL-2 expression is a key component of the Oncotype DX assay. BCL-2 overexpression is even more prominent in luminal tumors (~85%), reflecting its estrogen-responsiveness. Other key pro-survival family members, MCL-1 and BCL-XL, are also commonly expressed across breast cancer subtypes. This group of pro-survival guardian proteins play a critical role in keeping pro-apoptotic effector proteins (such as BAX and BAK) in check. The effector proteins are essential for commitment to apoptosis, which occurs following mitochondrial cytochrome c release and caspase activation, resulting in cell death. BCL-2 guardian proteins also neutralize a group of sensor proteins (such as BIM). The sensor proteins are triggered by distinct cytotoxic stimuli (such as chemotherapy) to activate the effectors BAX and BAK. The sensor proteins are termed BH3 only proteins, as they lack the BCL-2 Homology (BH) domains BH1, BH2 and BH4 found in the two other subgroups. While BCL-2 is an important prognostic marker in breast cancer, its precise role as a predictive marker of tumor response has yet to be properly clarified. Nevertheless, there is increasing evidence that augmented levels of pro-survival BCL-2 proteins can prime tumors for death induced by conventional chemotherapy or endocrine therapy. This is due to the high occupation of pro-survival BCL-2 proteins by pro-apoptotic BH3 proteins, which can be activated by therapy, thereby committing tumor cells to apoptosis. Small molecule inhibitors termed BH3 mimetics that bind and neutralize BCL-2 pro-survival proteins have recently been described. These are showing considerable promise in early phase studies of lymphoid malignancies. We recently explored the feasibility of targeting luminal B tumors in combination therapy comprising endocrine therapy (tamoxifen) and a BH3 mimetic (ABT-737 or ABT-199) using patient derived xenograft (PDX) models of primary breast cancer. Tumor response and overall survival were significantly improved by combination therapy, when compared to tamoxifen alone. Moreover, synergy between BH3 mimetics and PI3K/mTOR inhibitors could be exploited by concomitant targeting of both survival pathways, a strategy that appeared both safe and effective. Since the potent and selective BCL-2 inhibitor ABT-199 was effective in these models (despite abundant BCL-XL expression), BCL-2 may be a crucial target for some luminal tumors, where we speculate it could provide a suitable companion biomarker for patient selection. This work has formed the basis for a Phase 1b study of BCL-2 inhibition with ABT-199 (venetoclax) in combination with tamoxifen in metastatic ER-positive breast cancer (ISRCTN98335443). The prospects for the development of BCL-XL and MCL-1 inhibitors will also be discussed. Citation Format: Geoffrey J. Lindeman, Delphine Merino, François Vaillant, James R. Whittle, Sheau Wen Lok, Kylie Shackleton, Jane E. Visvader. Targeting the BCL-2 family in breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr IA19.

NGF sensitizes TrkA SH-SY5Y neuroblastoma cells to TRAIL-induced apoptosis.

We report a novel pro-apoptotic function for nerve growth factor (NGF) and its tropomyosin-related kinase A (TrkA) receptor in sensitizing TRAIL (TNF-related apoptotis-inducing ligand)-resistant SH-SY5Y neuroblastoma (NB) cells to TRAIL-induced apoptosis, resulting in the abrogation of anchorage-independent tumourigenic growth in vitro. We show that the TRAIL-resistant SH-SY5Y phenotype is cFLIP (cellular FLICE-like inhibitory protein) dependent and not due to low-level functional TRAIL receptor or caspase expression or an inhibitory equilibrium between functional and decoy TRAIL receptors or B-cell lymphoma 2 (Bcl-2) and BH3-only (Bcl-2 homology domain 3-only) family proteins. NGF sensitization of SH-SY5Y cells to TRAIL-induced apoptosis was dependent upon TrkA expression, activation and subsequent sequestration of cFLIP. This reduces cFLIP recruitment to TRAIL-activated death receptors and increases the recruitment of caspase-8, leading to TRAIL-induced, caspase-dependent, type II apoptosis via the intrinsic mitochondrial pathway. This effect was temporary, inhibited within 6 h by nuclear factor-κ binding (NF-κB)-mediated increase in myeloid cell leukaemia-1 (Mcl-1) expression, abrogated by transient cFLIP or B-cell lymphoma-extra large (Bcl-xL) overexpression and optimized by NF-κB and Mcl-1 inhibitors. This novel mechanism adds an important pro-apoptotic immunological dimension to NGF/TrkA interaction that may not only help to explain the association between TrkA expression, better prognosis and spontaneous remission in NB, but also provides a novel potential pro-apoptotic therapeutic use for NGF, TRAIL and inhibitors of NF-κB and/or Mcl-1 in favourable and unfavourable NBs that express TrkA and exhibit cFLIP-mediated TRAIL resistance.

Small Molecule Bda-366 As a Bcl2-BH4 Antagonist for Multiple Myeloma Therapy

Multiple myeloma (MM) is the most common hematological malignancy in USA, characterized with excessive abnormal clonal plasma cells in the bone marrow. Despite the great improvement in MM treatment by targeting the normal plasma cell biology of the cells during the last decade, the disease still remains incurable. B-cell lymphoma-2 (Bcl2) protein has four domains: BH1 (Bcl2 homology domain 1), BH2, BH3 and BH4, and plays critical roles in promoting the survival and drug-resistant of MM cells. Unlike the BH1, BH2, and BH3 domains, the BH4 region is responsible for the anti-apoptotic function of Bcl2 protein. Current BH3-mimetic and other Bcl2 inhibitors have been trailed in clinic but showed limited therapeutic efficacy. We recently identified a small molecule BDA-366 as a Bcl2-BH4 domain antagonist with potent anti-MM effect. BDA-366 bound to the BH4 resulted in the decrease of Bcl2 phosphorylation and the exposure of the BH3 domain as detected by anti-Bcl2-BH3 antibodies. Consequently, BDA-366 treatment induced robust apoptotic death on both human MM cell lines (RPMI8226 and U266) and primary MM cells from patients. In NOD-scid/IL2Rγnull (NSG) murine xenograft model, administration of BDA-366 (20mg/kg/day, 5 doses) markedly suppressed the growth of human MM tumor cells in vivo. We propose that BDA-366 as a novel BH4-based anti-MM agent could provide a more efficient pharmacological approach to treat MM. DisclosuresNo relevant conflicts of interest to declare.

SLAMF1 regulation of chemotaxis and autophagy determines CLL patient response

Chronic lymphocytic leukemia (CLL) is a variable disease; therefore, markers to identify aggressive forms are essential for patient management. Here, we have shown that expression of the costimulatory molecule and microbial sensor SLAMF1 (also known as CD150) is lost in a subset of patients with an aggressive CLL that associates with a shorter time to first treatment and reduced overall survival. SLAMF1 silencing in CLL-like Mec-1 cells, which constitutively express SLAMF1, modulated pathways related to cell migration, cytoskeletal organization, and intracellular vesicle formation and recirculation. SLAMF1 deficiency associated with increased expression of CXCR4, CD38, and CD44, thereby positively affecting chemotactic responses to CXCL12. SLAMF1 ligation with an agonistic monoclonal antibody increased ROS accumulation and induced phosphorylation of p38, JNK1/2, and BCL2, thereby promoting the autophagic flux. Beclin1 dissociated from BCL2 in response to SLAMF1 ligation, resulting in formation of the autophagy macrocomplex, which contains SLAMF1, beclin1, and the enzyme VPS34. Accordingly, SLAMF1-silenced cells or SLAMF1(lo) primary CLL cells were resistant to autophagy-activating therapeutic agents, such as fludarabine and the BCL2 homology domain 3 mimetic ABT-737. Together, these results indicate that loss of SLAMF1 expression in CLL modulates genetic pathways that regulate chemotaxis and autophagy and that potentially affect drug responses, and suggest that these effects underlie unfavorable clinical outcome experienced by SLAMF1(lo) patients.

Myeloid leukemia factor 1 interfered with Bcl-XL to promote apoptosis and its function was regulated by 14-3-3.

Myeloid leukemia factor 1 (MLF1) was involved in t(3;5) chromosomal rearrangement and aberrantly expressed in myelodysplastic syndromes/acute myeloid leukemia patients. Ex vivo experiments showed that the lymphocytes from the Mlf1-deficient mice were more resistant to apoptotic stimulations than the wild-type cells. Furthermore, the ectopically expressed MLF1 induced apoptosis in the cell models. These findings revealed that MLF1 was required for the cells to respond to the apoptotic stimulations. Ex vivo experiments also demonstrated that cytokine withdrawal significantly up-regulated Mlf1's expression and promoted its association with B cell lymphoma-extra large (Bcl-XL) in the lymphocytes, at the same time reduced the association of Bax with Bcl-XL The same effects were also observed in the cells that over-expressed MLF1. However, these effects were observed in Mlf1 null lymphocytes as well as the cells over-expressing Bcl-XL. In addition, MLF1's proapoptosis could be completely prevented by co-expression of Bcl-XL and significantly attenuated in Bax/Bak double null cells. These data, taken together, strongly suggested that in response to the stresses, up-regulated Mlf1 promoted its association with Bcl-XL and reduced the available Bcl-XL for associating with Bax, which resulted in releasing Bax from the Bcl-XL and apoptosis in turn. Lastly, we showed that MLF1 was negatively regulated by 14-3-3 and revealed that 14-3-3 bound to MLF1 and physically blocked MLF1's Bcl-2 homology domain 3 (BH3) as well as Bcl-XL from associating with MLF1. Our findings suggested that ectopically expressed MLF1 could be responsible for the pathological apoptosis in early myelodysplastic syndrome (MDS) patients.

Targeting Bcl2 in cancer.

The decision phase of apoptosis is mainly regulated by the Bcl-2 family, which renders these proteins potential targets for cancer therapy through apoptotic mechanisms. Bcl2 family members have homology clustered within four conserved Bcl2 homology (BH) domains (BH1, BH2, BH3 and BH4). Only the antiapoptotic proteins, such as Bcl2, Bcl-XL, Bcl-w and A1, bear the NH2-terminal BH4 domain [1]. Mcl-1 does not have a typical BH4 domain but has a helical BH4-like domain which is located between the PEST region and the BH3 domain. The BH1, BH2 and BH3 domains form the surface binding pocket of Bcl2 which mediates protein-protein interactions involving Bcl2 family members. Mutations in this hydrophobic surface binding pocket abolish the antiapoptotic function of Bcl2, demonstrating its requirement in the biological function of Bcl2. To interfere with Bcl2 function, it is necessary to impede binding to the hydrophobic cleft. Synthetic peptides that bind this surface pocket of Bcl-XL and Bcl2 have been shown to induce apoptosis in vitro. The hydrocarbon-stapled BH3 peptide not only induces apoptosis but also possesses potent antitumor activity in vivo. Recently, an approach called “structure-activity relationship by nuclear magnetic resonance (SAR by NMR)” has been used for the discovery of novel Bcl2/Bcl-XL inhibitors [2]. In this approach, the relatively large site to be targeted is divided into two smaller half-sites that are individually targeted by small molecules. The two lead molecules are then chemically linked to improve the binding affinity. Subsequent iterative chemical manipulations to improve affinity and decrease binding to human serum albumin, guided by NMR and three-dimensional structures of antiapoptotic proteins, yielded the small molecules ABT-737 and ABT-263, which bind to the hydrophobic pocket of Bcl2 or Bcl-XL with high-affinity and subsequently disrupt the antiapoptotic function of Bcl2 and Bcl-XL with potent anti-tumor effect [2]. Thus, such small molecules that directly target Bcl2 and/or the Bcl2-like proteins by mimicking the BH3 domain should be highly effective anticancer drugs. However, ABT-737 and ABT-263 can induce thrombocytopenia due to their inhibitory effects on both Bcl2 and Bcl-XL [3]. To generate a more selective Bcl2 inhibitor, a tethered indole was incorporated into ABT-263 to fill the P4 hot spot, which specifically interacts with aspartic acid (Asp 103) of Bcl2 but not Glu96 of Bcl-XL, leading to the generation of the Bcl2-selective inhibitor ABT-199 [4]. Since ABT-199 did not cause thrombocytopenia in vivo [4], this suggests that selective inhibition of Bcl2 may benefit the development of improved Bcl2 antagonists. In addition to the hydrophobic surface binding pocket, the NH2-terminal BH4 domain (aa-6-31) of Bcl2 is also required for its antiapoptotic function [5]. The BH4 domain of Bcl2 can interact with multiple molecules, including Bax, CED-4, Ras, PP2A, PP2B, IP3 receptor (IP3R), and others. Since only the prosurvival Bcl2 family members possess a conserved N-terminal region denoted BH4, this suggests a critical role of this amphipathic helix for their survival activity. Intriguingly, either caspase-mediated cleavage or mutagenic removal of the BH4 domain not only completely abolishes the antiapoptotic activity of Bcl2 but also results in a conversion of Bcl-2 to a Bax-like death effector [6]. The BH4 domain peptide has been reported to exert antiapoptotic activity in vivo, which provides direct evidence that the BH4 domain contributes to the survival function of the prosurvival Bcl2 family members. Since the BH4 domain is critical for the antiapoptotic function of Bcl2, this amphipathic helix should also be an ideal structural target for the screening of small molecules that may bind to this domain and interfere with Bcl2's survival activity. Solution structure of the BH4 domain exhibits multiple potential binding pockets for small molecule docking [7]. Recently, we chose the BH4 domain of Bcl2 as the docking site for screening of small molecules and identified BDA-366 as a Bcl2 BH4 antagonist that is distinct from previous BH3 mimetics. BDA-366 selectively targets the BH4 domain of Bcl2 and converts Bcl2 from a survival molecule to a cell death inducer through a conformational change (BH3 exposure) (Figure ​(Figure1).1). BDA-366 not only induces apoptosis but also autophagic cell death of cancer cells by disruption of Bcl2 activity. BDA-366 demonstrates potent antitumor activity in lung cancer xenografts derived from either a lung cancer cell line or a patient-derived small cell lung cancer tumor [8]. Figure 1 Proposed model of Bcl2 BH4 antagonist BDA-366 induction of apoptosis in cancer cells In summary, BH3 mimetics (ABT-263 and ABT-199) and the BH4 antagonist (BDA-366) are two different classes of Bcl2 inhibitors that target Bcl2 at the hydrophobic binding pocket or BH4 domain, respectively. Disruption of Bcl2's antiapoptotic function via BH3 mimetics or the BH4 antagonist may represent attractive strategies for cancer treatment.

Bax/Bak-dependent, Drp1-independent Targeting of X-linked Inhibitor of Apoptosis Protein (XIAP) into Inner Mitochondrial Compartments Counteracts Smac/DIABLO-dependent Effector Caspase Activation

Efficient apoptosis requires Bax/Bak-mediated mitochondrial outer membrane permeabilization (MOMP), which releases death-promoting proteins cytochrome c and Smac to the cytosol, which activate apoptosis and inhibit X-linked inhibitor of apoptosis protein (XIAP) suppression of executioner caspases, respectively. We recently identified that in response to Bcl-2 homology domain 3 (BH3)-only proteins and mitochondrial depolarization, XIAP can permeabilize and enter mitochondria. Consequently, XIAP E3 ligase activity recruits endolysosomes into mitochondria, resulting in Smac degradation. Here, we explored mitochondrial XIAP action within the intrinsic apoptosis signaling pathway. Mechanistically, we demonstrate that mitochondrial XIAP entry requires Bax or Bak and is antagonized by pro-survival Bcl-2 proteins. Moreover, intramitochondrial Smac degradation by XIAP occurs independently of Drp1-regulated cytochrome c release. Importantly, mitochondrial XIAP actions are activated cell-intrinsically by typical apoptosis inducers TNF and staurosporine, and XIAP overexpression reduces the lag time between the administration of an apoptotic stimuli and the onset of mitochondrial permeabilization. To elucidate the role of mitochondrial XIAP action during apoptosis, we integrated our findings within a mathematical model of intrinsic apoptosis signaling. Simulations suggest that moderate increases of XIAP, combined with mitochondrial XIAP preconditioning, would reduce MOMP signaling. To test this scenario, we pre-activated XIAP at mitochondria via mitochondrial depolarization or by artificially targeting XIAP to the intermembrane space. Both approaches resulted in suppression of TNF-mediated caspase activation. Taken together, we propose that XIAP enters mitochondria through a novel mode of mitochondrial permeabilization and through Smac degradation can compete with canonical MOMP to act as an anti-apoptotic tuning mechanism, reducing the mitochondrial contribution to the cellular apoptosis capacity.

Structure-Function Analysis of the Mcl-1 Protein Identifies a Novel Senescence-regulating Domain

Unlike other antiapoptotic Bcl-2 family members, Mcl-1 also mediates resistance to cancer therapy by uniquely inhibiting chemotherapy-induced senescence (CIS). In general, Bcl-2 family members regulate apoptosis at the level of the mitochondria through a common prosurvival binding groove. Through mutagenesis, we determined that Mcl-1 can inhibit CIS even in the absence of its apoptotically important mitochondrion-localizing domains. This finding prompted us to generate a series of Mcl-1 deletion mutants from both the N and C termini of the protein, including one that contained a deletion of all of the Bcl-2 homology domains, none of which impacted anti-CIS capabilities. Through subsequent structure-function analyses of Mcl-1, we identified a previously uncharacterized loop domain responsible for the anti-CIS activity of Mcl-1. The importance of the loop domain was confirmed in multiple tumor types, two in vivo models of senescence, and by demonstrating that a peptide mimetic of the loop domain can effectively inhibit the anti-CIS function of Mcl-1. The results from our studies appear to be highly translatable because we discerned an inverse relationship between the expression of Mcl-1 and of various senescence markers in cancerous human tissues. In summary, our findings regarding the unique structural properties of Mcl-1 provide new approaches for targeted cancer therapy.

HTLV-1-associated adult T cell leukemia is highly susceptible to Navitoclax due to enhanced Bax expression.

Over-expression of Bcl-2, Bcl-xL and Bcl-w is frequently associated with cancer resistance to chemotherapy. Navitoclax (ABT-263), an orally bio-available small-molecule mimetic of the Bcl-2 homology domain 3, specifically inhibits Bcl-2, Bcl-xL and Bcl-w. Despite promising results obtained from the clinical trials, the use of Navitoclax in patients is dose-limited due to induction of death of platelets via inhibition of Bcl-xL and subsequent thrombocytopenia. This side effect limits the use of Navitoclax in low doses and to very sensitive tumors. In this study, we show that HTLV-1-associated adult T-cell leukemia/lymphoma (ATL) cells, which over-express Bcl-2, Bcl-xL and Bcl-w, show a 10- to 20-fold higher sensitivity (EC50 = ∼ 25-50 nM) to Navitoclax compared to non-HTLV-1-associated leukemic cells (EC50 = ∼ 1 μM). Investigation of the molecular mechanisms revealed that the HTLV-1 oncogenic protein Tax up-regulates expression of the pro-apoptotic protein Bax which enhances the therapeutic efficacy of Navitoclax. In addition, we show that agents that inhibit the transcription elongation or translation initiation such as Wogonin and Roc-A can further decrease the effective dose of Navitoclax. Our study suggests that HTLV-1 ATL may be a good candidate disease for low dose Navitoclax therapy and probably with less risk of thrombocytopenia.

Abstract 489: Loss of NOXA expression by INI1/SNF5 loss impaired sensitivity to chemotherapeutic agents in malignant rhabdoid tumor in vitro and iv vivo

Abstract Background: We previously showed that INI1/SNF5 reexpression increased transcriptional activity at the NOXA locus in malignant rhabdoid tumor (MRT) cell lines resulting in NOXA protein production. In other cancer cells, the binding between NOXA and MCL-1 induces apoptosis after exposure to chemotherapeutic agents. Therefore, we hypothesized that loss of NOXA alters the apoptotic pathway in MRTs and contributes to their resistance to chemotherapeutic agents. Methods: The TTC549 and KP-MRT-RY MRT cell lines were transfected with the pcDNA 3.1(+)-wild type hNOXA plasmid, a kind gift from Dr. Kelekar, University of Minnesota, using Lipofectamine reagent and then selected with G418. To generate MCL-1 knockdown MRT cell lines, we transfected a small interfering (si) RNA targeting MCL-1 using Lipofectamine reagent. Cells were plated in growth medium for 24 hrs and then treated with serial dilutions of doxorubicin (DOX) and/or TW-37. To generate mice xenografts for both TTC549 MCL-1 stable knockdown (KD) and control cell lines, we established two independently derived MCL-1 stable knockdown TTC549 cell lines using lentiviral vectors encoding a shRNA targeting MCL-1 and a negative control cell line (pLKO.1). We injected either 5 × 106 TTC549 MCL-1 KD or control cell lines s.c. into athymic mice. One week after injection, mice were treated once daily with DOX (15mg/kg) or vehicle alone for five days. Results: In both NOXA expressing and MCL-1 KD MRT cells, we observed a dramatic reduction of the IC50 values of DOX treatment. Moreover, apoptosis was also observed at 48 hrs after DOX treatment at concentrations less than the IC50 values for control cells. Immunoprecipitation of MCL-1 showed that expressed NOXA associated with MCL-1 in MRT cells. Furthermore, we observed that mean tumor volume in the DOX-treated MCL-1 KD group was significantly smaller than that in the vehicle-treated MCL-1 knockdown group. Next, we assessed the effects of TW-37, an inhibitor that binds to the Bcl-2 homology domain 3 (BH3) of proteins including MCL-1, in combination with DOX. Using the combination index, we found that DOX and TW-37 displayed modest synergy in both MRT cell lines (CI<1) in vitro and in vivo. Discussion: Ectopic expression of NOXA or knockdown of MCL-1 increased DOX sensitivity in MRT cell lines. These results indicate that loss of INI1/SNF5-mediated regulation of NOXA expression impairs an apoptotic pathway leading to repression of MCL-1 followed by the resistance to chemotherapeutic agents. We also found that TW-37, a BH3 only protein mimetic, has a synergistic effect with DOX on MRT cell lines in vitro and in vivo, providing a potential foundation for molecular targeting therapy for MRT. Citation Format: Kazutaka Ouchi, Yasumichi Kuwahara, Tomoko Iehara, Eiichi Konishi, Hajime Hosoi. Loss of NOXA expression by INI1/SNF5 loss impaired sensitivity to chemotherapeutic agents in malignant rhabdoid tumor in vitro and iv vivo. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 489. doi:10.1158/1538-7445.AM2015-489

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.

Inhibition of MCL-1 by obatoclax sensitizes Sp2/0-Ag14 hybridoma cells to glutamine deprivation-induced apoptosis.

For several cancer cell types, the lack of an adequate supply of the amino acidl-glutamine (Gln) triggers apoptosis, a phenomenon termed Gln addiction. In this report, we examined the role of the anti-apoptotic proteins of the B-cell lymphoma 2 (BCL-2) protein family in the survival of Sp2/0-Ag14 (Sp2/0) mouse hybridoma cells, a cell line that undergoes apoptosis within minutes of Gln deprivation. Western blot analysis revealed that myeloid cell leukaemia 1 (MCL-1) was expressed at much higher levels than BCL-2, B-cell lymphoma extra-large and BCL-2-like protein 2 making it the prominent pro-survival BCL-2 family member in this hybridoma. Gln deprivation triggered a progressive decrease in MCL-1 protein levels, which coincided with the decrease in Sp2/0 cell survival. Moreover, Sp2/0 cells were much more sensitive to the broad Bcl-2 homology domain-3 (BH3) mimetic obatoclax (which targets MCL-1) than to the more selective drug ABT-737 (which does not target MCL-1). Finally, we show that obatoclax sensitizes Sp2/0 cells to apoptosis following Gln starvation. All together, the data presented here reveal that modulation of the pro-survival protein MCL-1 is an important step in the sequence of events leading to the initiation of apoptosis in Gln-starved Sp2/0 cells. Cancer cells require an adequate supply ofl-glutamine for their survival. Using a mouse hybridoma cell line that is exquisitely sensitive to glutamine starvation, we show that the levels of the pro-survival BCL-2 family protein MCL-1 decrease upon glutamine starvation in a manner that correlates with the loss of cell viability. Moreover, inhibiting MCL-1 with the drug obatoclax sensitizes hybridoma cells to glutamine starvation. Thus, in some cancer cells, glutamine starvation triggers the inactivation of pro-survival proteins. Our data suggest that the combined inhibition of glutamine biosynthesis pathways and BCL-2 proteins may prove effective against some cancers.

Identification, characterization and functional analysis of anti-apoptotic protein BCL-2-like gene from pufferfish, Takifugu obscurus, responding to bacterial challenge

Apoptosis plays a crucial role in many biological processes, including development, cellular homeostasis and immune responses. The BCL-2 family is a key regulator of the mitochondrial response to apoptotic signals in the intrinsic pathway. In this study, we identified and characterized the cDNA and expression pattern of pufferfish BCL-2 (PfBCL-2). The full-length cDNA of PfBCL-2 was 1412 bp with an open reading frame of 657 bp encoding a putative protein of 219 amino acids (Accession no: KP898414). The calculated molecular mass of the PfBCL-2 was 24.2 kDa with a predicted isoelectric point of 5.27. The deduced PfBCL-2 protein exhibited four highly conserved BCL-2 homology domains, suggesting that PfBCL-2 may play a similar role in the apoptotic-signaling pathway as in other species. Real-time PCR results showed that PfBCL-2 transcript was expressed in a wide range of tissues but exhibited the greatest level of expression in blood. Transcriptional responses of PfBCL-2 exhibited different spatial and temporal expression profiles in liver and blood after bacterial infection. PfBcl-2 transcript was significantly up-regulated in liver at 6, 12, 24 and 48 h (with maximum induction at 48 h) and was up-regulated in blood at 3, 6, 12 and 24 h (with maximum induction at 12 h). Meanwhile, recombinant PfBCL-2 fused with His6 tag was efficiently expressed in Escherichia coli BL21 (DE3) and purified using Ni-nitrilotriacetic acid resin. Western blot analysis indicated that its protein level appeared to be elevated during the initial bacterial infection. These results suggest that PfBCL-2 plays important roles in immune responses against bacteria challenge.

AT101 Down‐regulates CXLC12 mRNA and Secreted Protein in Malignant Peripheral Nerve Sheath Tumor cells

Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive, Schwann cell‐derived neoplasms of the peripheral nervous system. MPNSTs have recently been shown to possess an autocrine CXCL12/CXCR4 signaling loop that promotes tumor cell proliferation and survival. Importantly, the CXCL12/CXCR4 axis is driven by availability of the CXCL12 ligand rather than CXCR4 receptor levels. Accordingly, pharmacological reduction of CXCL12 expression and/or secretion could be a potential chemotherapeutic option for patients with MPNSTs or other malignancies wherein the CXCL12/CXCR4 axis is relevant. AT101 is a polyphenolic cottonseed derivative with well‐established Bcl‐2 homology domain 3 (BH3)‐ mimetic properties. We recently reported that AT101 can also chelate intracellular iron and therefore act as a hypoxia mimetic. Importantly, we have observed that AT101 causes a marked reduction in CXCL12 mRNA and secreted protein levels in cultured MPNST cells. This property of AT101 was recapitulated by other BH3‐mimetics (ABT‐737, obatoclax and subatoclax) but not by desferrioxamine (DFO), an iron chelator and known hypoxia‐mimetic. These data indicate that CXCL12 reduction is likely a function of AT101′s BH3‐mimetic property rather than its iron chelation ability. Ongoing studies are aimed at addressing the mechanism by which AT101 and other BH3‐mimetics reduce CXCL12 mRNA and protein in MPNST cells.