Conformational Change In Bcl-2 Research Articles

The Bcl-2 BH4 Antagonist Bda-366 Induces Apoptosis in CLL and DLBCL Cells through a Bcl-2 Independent Mechanism

Overexpression of the antiapoptotic protein Bcl-2 is considered primarily responsible for the increased apoptosis resistance and prolonged survival of the malignant B cells in chronic lymphocytic leukemia (CLL), follicular lymphoma and subsets of diffuse large B cell lymphoma (DLBCL). The importance of Bcl-2 overexpression is corroborated by the significant clinical activity of the recently approved Bcl-2 BH3 antagonist venetoclax. Venetoclax induces apoptosis by sequestering Bcl-2 from the pro-apoptotic Bcl-2 family members Bim, Bax and Bak, resulting in consequent Bax/Bak activation, mitochondrial outer membrane permeabilization and cytochrome c release. Although venetoclax is extremely toxic against CLL cells in vitro, resistance can arise in vivo because of overexpression of other anti-apoptotic Bcl-2 family members, such as Bcl-xL and Mcl-1.In the present study we investigated the activity of BDA-366, another small molecule Bcl-2 inhibitor that was recently reported to selectively induce apoptosis in Bcl-2-overexpressing lung cancer cells through a distinct mechanism (Han B et al, Cancer Cell. 2015;27:852-63). This drug was designed to selectively bind the BH4 domain of Bcl-2 and was reported to trigger apoptosis by inducing a conformational change in Bcl-2 that converts it into a pro-apoptotic protein. To investigate whether BDA-366 is also selectively toxic for CLL cells, we analyzed the viability of CLL (n=31) and normal peripheral blood mononuclear cells (PBMCs)(n=6) after 48 hours in culture with increasing concentrations of BDA-366. Annexin V/PI analysis showed that BDA-366 is significantly more toxic against CLL (LD50=1.11±0.46 mM) than normal PBMCs (LD50=2.03±0.31 mM, P<0.001). However, considerable variability was noted in the sensitivity of the different CLL samples, with approximatelly 50% of the cases demonstrating LD50 values in the nanomolar range and the remaining cases displaying sensitity similar to normal PBMC. To investigate the reasons for this variability, we correlated LD50 values with IGHV mutation status, ZAP-70 expression, cytogenetic abnormalities, and expression of various Bcl-2 family members, including Bcl-2, Bcl-xL, Mcl-1, BimEL, BimL, Bax, Bak and Hrk. The only positive finding from this analysis was the inverse correlation between LD50 and expression of BimL (n=23, r=-0.446, p=0.033), whereas no correlation was observed with Bcl-2 expression.Variable sensitivity to BDA-366 and absence of correlation with Bcl-2 expression was also observed in the analysis of a series of DLBCL and CLL cell lines. These cell lines showed LD50 values ranging from nanomolar (Pfeiffer, 0.19 µM; OCI-LY-18, 0.32 µM; OCI-LY-1, 0.33 µM; TOLEDO, 0.41 µM; OCI-Ly10, 0.50 µM; HT, 0.56 µM; Ri-1, 0.58 µM; and SU-DHL-6, 0.81 µM) to micromolar range (MEC1, 1.5 µM; HBL-1, 4.0 µM; Karpas, 4.34 µM; BJAB, 4.8 µM; and SU-DHL-4, 6.31 µM). Although most of the sensitive lines expressed Bcl-2, some had very low or even undetectable Bcl-2 expression (e.g., Pfeiffer and HT), suggesting that BDA-366 can induce apoptosis independently of Bcl-2. In support of this possibility, we observed that BDA-366 was equally effective against Bcl-2-negative HT cells and HT cells stably transfected with Bcl-2. In addition, transient overexpression of Bcl-2 in the DLBCL cell line BJAB and the CLL cell line MEC1 resulted in greater resistance to BDA-366, further suggesting that BDA-366 does not induce apoptosis by converting Bcl-2 into a proapoptotic protein.To determine whether mechanisms that induce resistance to venetoclax can also induce resistance to BDA-366, we investigated viability of BDA-366-treated CLL cells following stimulation with immobilized anti-IgM. Pre-treatment of CLL cells with immobilized anti-IgM resulted in increased resistance to both BDA-366 and venetoclax. Interestingly, the combination of BDA-366 and venetoclax was significantly more toxic than each agent alone (Figure 1), suggesting that combining these drugs could be a potential approach to overcome resistance induced by BCR signals.In conclusion, these data show that BDA-366 is selectively toxic at low nanomolar concentrations for a large proportion of primary CLL samples and DLBCL cell lines. The primary mechanism of action is still unclear, but the lack of a correlation with Bcl-2 expression suggests that it differs from the one initially proposed. [Display omitted] DisclosuresEfremov:Gilead: Honoraria.

Abstract 2922: Development of Bcl2 BH4 antagonist for cancer therapy

Abstract The BH4 domain of Bcl2 is required for its antiapoptotic function, thus constituting a promising anticancer target. We identified a novel small molecule Bcl2-BH4 domain-antagonist (BDA-366) that binds BH4 with high affinity and selectivity. BDA-366-Bcl2 binding induces conformational change in Bcl2 that abrogates its antiapoptotic function, converting it from a survival to a cell death inducer. BDA-366 induces regression of lung cancer xenografts derived from cell line and patient without significant normal tissue toxicity at effective doses. mTOR inhibition up-regulates Bcl2 in lung cancer cells and tumor tissues from clinical trial patients. Combined BDA-366 and RAD001 treatment exhibits strong synergy against lung cancer in vivo. Development of this Bcl2-BH4 antagonist may provide a novel strategy to improve lung cancer outcome. Citation Format: Bingshe Han, Dongkyoo Park, Rui Li, Maohua Xie, Taofeek Owonikoko, Gabriel Sica, Chunyong Ding, Jia Zhou, Andrew Magis, Suresh Ramalingam, Fadlo Khuri, Walter Curran, Xingming Deng. Development of Bcl2 BH4 antagonist for cancer therapy. [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 2922. doi:10.1158/1538-7445.AM2015-2922

Small-Molecule Bcl2 BH4 Antagonist for Lung Cancer Therapy

The BH4 domain of Bcl2 is required for its antiapoptotic function, thus constituting a promising anticancer target. We identified a small-molecule Bcl2-BH4 domain antagonist, BDA-366, that binds BH4 with high affinity and selectivity. BDA-366-Bcl2 binding induces conformational change in Bcl2 that abrogates its antiapoptotic function, converting it from a survival molecule to a cell death inducer. BDA-366 suppresses growth of lung cancer xenografts derived from cell lines and patient without significant normal tissue toxicity at effective doses. mTOR inhibition upregulates Bcl2 in lung cancer cells and tumor tissues from clinical trial patients. Combined BDA-366 and RAD001 treatment exhibits strong synergy against lung cancer in vivo. Development of this Bcl2-BH4 antagonist may provide a strategy to improve lung cancer outcome.

Nuclear receptor nur77 promotes cerebral cell apoptosis and induces early brain injury after experimental subarachnoid hemorrhage in rats.

Nur77 is a potent proapoptotic member of the nuclear receptor superfamily that is expressed predominantly in brain tissue. It has been demonstrated that Nur77 mediates apoptosis in multiple organs. Nur77-mediated early brain injury (EBI) involves a conformational change in BCL-2 and triggers cytochrome C (cytoC) release resulting in cellular apoptosis. This study investigates whether Nur77 can promote cerebral cell apoptosis after experimentally induced subarachnoid hemorrhage (SAH) in rats. Sprague Dawley rats were randomly assigned to three groups: 1) untreated group, 2) treatment control group, and 3) SAH group. The experimental SAH group was divided into four subgroups, corresponding to 12 hr, 24 hr, 48 hr, and 72 hr after experimentally induced SAH. It remains unclear whether Nur77 can play an important role during EBI after SAH as a proapoptotic protein in cerebral cells. Cytosporone B (Csn-B) was used to demonstrate that Nur77 could be enriched and used to aggravate EBI after SAH. Rats treated with Csn-B were given an intraperitoneal injection (13 mg/kg) 30 min after experimentally induced SAH. We found that Nur77 promotes cerebral cell apoptosis by mediating EBI and triggering a conformational change in BCL-2, resulting in cytoC release. Nur77 activity, along with cerebral cell apoptosis, peaked at 24 hr after SAH onset. After induction of SAH, an injection of Csn-B, an agonist for Nur77, enhanced the expression and function of Nur77. In summary, we have demonstrated the proapoptotic effect of Nur77 within cerebral cells, an effect that can be further exacerbated with Csn-B stimulation.

ALS-linked mutant SOD1 damages mitochondria by promoting conformational changes in Bcl-2

In mutant superoxide dismutase (SOD1)-linked amyotrophic lateral sclerosis (ALS), accumulation of misfolded mutant SOD1 in spinal cord mitochondria is thought to cause mitochondrial dysfunction. Whether mutant SOD1 is toxic per se or whether it damages the mitochondria through interactions with other mitochondrial proteins is not known. We previously identified Bcl-2 as an interacting partner of mutant SOD1 specifically in spinal cord, but not in liver, mitochondria of SOD1 mice and patients. We now show that mutant SOD1 toxicity relies on this interaction. Mutant SOD1 induces mitochondrial morphological changes and compromises mitochondrial membrane integrity leading to release of Cytochrome C only in the presence of Bcl-2. In cells, mouse and human spinal cord with SOD1 mutations, the binding to mutant SOD1 triggers a conformational change in Bcl-2 that results in the uncovering of its toxic BH3 domain and conversion of Bcl-2 into a toxic protein. Bcl-2 carrying a mutagenized, non-toxic BH3 domain fails to support mutant SOD1 mitochondrial toxicity. The identification of Bcl-2 as a specific target and active partner in mutant SOD1 mitochondrial toxicity suggests new therapeutic strategies to inhibit the formation of the toxic mutant SOD1/Bcl-2 complex and to prevent mitochondrial damage in ALS.

Protein phosphatase 2A inactivates Bcl2's antiapoptotic function by dephosphorylation and up-regulation of Bcl2-p53 binding

AbstractBcl2 is associated with chemoresistance and poor prognosis in patients with various hematologic malignancies. DNA damage-induced p53/Bcl2 interaction at the outer mitochondrial membrane results in a Bcl2 conformational change with loss of its antiapoptotic activity in interleukin-3–dependent myeloid H7 cells. Here we find that specific disruption of protein phosphatase 2A (PP2A) activity by either expression of small t antigen or depletion of PP2A/C by RNA interference enhances Bcl2 phosphorylation and suppresses cisplatin-stimulated p53/Bcl2 binding in association with prolonged cell survival. By contrast, treatment of cells with C2-ceramide (a potent PP2A activator) or expression of the PP2A catalytic subunit (PP2A/C) inhibits Bcl2 phosphorylation, leading to increased p53/Bcl2 binding and apoptotic cell death. Mechanistically, PP2A-mediated dephosphorylation of Bcl2 in vitro promotes its direct interaction with p53 as well as a conformational change in Bcl2. PP2A directly interacts with the BH4 domain of Bcl2 as a docking site to potentially “bridge” PP2A to Bcl2's flexible loop domain containing the target serine 70 phosphorylation site. Thus, PP2A may provide a dual inhibitory effect on Bcl2's survival function by both dephosphorylating Bcl2 and enhancing p53-Bcl2 binding. Activating PP2A to dephosphorylate Bcl2 and/or increase Bcl2/p53 binding may represent an efficient and novel approach for treatment of hematologic malignancies.

A Novel Peptide Containing a Nine Amino Acid Sequence from Nur77 Induces Bcl-2-Dependent Apoptosis in AML.

Nur77 (also known as TR3 and NGFI-B) is a nuclear orphan receptor that in response to various stimuli can translocate from the nucleus to the mitochondria, bind Bcl-2, and induce a conformational change in Bcl-2 that exposes its BH3 domain. This conformational change of Bcl-2 transforms this protein into a pro-apoptotic molecule that can induce cytochrome c release and apoptosis (Cell 116:527, 2004). Interestingly, in acute myeloid leukemia (AML) cell lines and blasts, Nur77 is absent. We have recently generated a nine amino acid peptide from the Nur77 protein (TR3) that is capable of inducing the same pro-apoptotic conformational change in Bcl-2 that Nur77 elicits (manuscript submitted). We tested the hypothesis that a cell-permeable version of this peptide can induce Bcl-2-dependent apoptosis in AML cells and AML stem cells. Primary AML samples (n=6) were incubated with 20 μM control or TR3 peptide for 24 hrs. Induction of apoptosis was measured as CD34+38−123+ PS/Annexin V+ by multiparametric flow cytometry. Our results demonstrate that the L-enantiomer form of TR3 indeed induces apoptosis in total AML cells (60%, p=0.001) and CD34+38−123+ stem cells (67%, p=0.001), as compared to control peptide. Bax expression did not affect the peptide's ability to induce apoptosis, as wild-type HCT116 cells and HCT116 cells deficient in Bax showed similar levels of apoptosis after treatment with the TR3 peptide. However, Bcl-2 expression was shown to be critical since lentiviral shRNA ablation of Bcl-2 in KG1 cells completely prevented induction of apoptosis by TR3 peptides. We conclude that the TR3 peptide is a potent inducer of apoptosis that mimics the action of Nur77 on Bcl-2 at the mitochondrial level. These results suggest that the Nur77 peptide or compounds that mimic this peptide may have utility as a novel therapeutic agent against Bcl-2 expressing cancers and leukemias.

Bcl2's flexible loop domain regulates p53 binding and survival.

p53 not only functions as a transcription factor but also has a direct, apoptogenic role at the mitochondria. We have discovered that DNA damage-induced p53-Bcl2 binding is associated with decreased Bcl2-Bax interaction and increased apoptotic cell death in a mechanism regulated by Bcl2's flexible loop regulatory domain (FLD), since purified p53 protein can disrupt the Bcl2/Bax complex by directly binding to a negative regulatory region of the FLD (amino acids [aa] 32 to 68). Deletion of the negative regulatory region (Delta32-68) abolishes Bcl2-p53 binding and enhances Bcl2's antiapoptotic function. Conversely, removal of a positive regulatory region (aa 69 to 87) of the FLD, which contains the Bcl2 phosphorylation site(s) T69, S70, and S87, enhances Bcl2-p53 binding and significantly abrogates Bcl2's survival activity. The phospho-mimetic T69E/S70E/S87E (EEE) but not the nonphosphorylatable T69A/S70A/S87A (AAA) Bcl2 mutant displays a reduced capacity to bind p53 and potently inhibits p53-induced cytochrome c release from isolated mitochondria. Furthermore, the FLD-only aa32-87 and aa32-68 peptides but not the aa69-87 peptide can directly bind p53 in vitro. p53-induced cytochrome c release occurs through a mechanism involving Bax's integral insertion into the outer mitochondrial membrane. Either DNA damage to cells or expression of p53 selectively targeted to the mitochondria results in Bcl2-p53 binding followed by exposure of Bcl2's BH3 domain in association with inactivation of Bcl2's antiapoptotic function, indicating a conformational change in Bcl2 can occur upon direct ligation of p53. Thus, Bcl2's FLD contains both positive and negative regulatory regions which functionally regulate Bcl2's antiapoptotic activity by affecting Bax or p53 binding.

ERK2 Prohibits Apoptosis-induced Subcellular Translocation of Orphan Nuclear Receptor NGFI-B/TR3

Transcription factor NGFI-B (neuronal growth factor-induced clone B), also called Nur77 or TR3, is an immediate early gene and an orphan member of the nuclear receptor family. The NGFI-B protein also has a function distinct from that of a transcription factor; it translocates to mitochondria to initiate apoptosis. Recently, it was demonstrated that NGFI-B interacts with Bcl-2 by inducing a conformational change in Bcl-2, converting it from protector to a killer. After exposing rat cerebellar granule neurons to glutamate (100 mum, 15 min), NGFI-B translocated to the mitochondria. Growth factors such as the epidermal growth factor activate the MAP kinase ERK, the activity of which may determine whether a cell survives or undergoes apoptosis. In the present study we found that the epidermal growth factor activated ERK2 in cerebellar granule neurons and that this activation prohibited glutamate-induced subcellular translocation of NGFI-B. Likewise, overexpressed active ERK2 resulted in a predominant nuclear localization of green fluorescent protein-tagged NGFI-B. Thus, activation of ERK2 may overcome apoptosis-induced subcellular translocation of NGFI-B. This finding represents a novel and rapid growth factor survival pathway that is independent of gene regulation.

A mechanism for the proapoptotic activity of ursodeoxycholic acid: effects on Bcl-2 conformation.

Ursodeoxycholic acid (UDCA), a relatively nontoxic bile acid, enhanced the apoptotic response of tumor cells to both photosensitizers that cause photodamage to Bcl-2 and to the nonpeptidic Bcl-2/Bcl-x(L) antagonist HA14-1. The latter agent binds to the surface pocket formed by the BH1, BH2 and BH3 domains of Bcl-2 and Bcl-x(L). Fluorescence polarization studies indicated that affinity of HA14-1 for Bcl-2 was enhanced in the presence of UDCA. Moreover, Bcl-2 photodamage was promoted by UDCA using a photosensitizing agent with affinity for the endoplasmic reticulum, a site of Bcl-2 localization. Fluorescence resonance energy transfer (FRET) studies revealed that the proximity of Bcl-2 to a hydrophobic photosensitizing agent embedded in liposomes was enhanced by UDCA. Since photodamage will occur only if a protein is in close contact with a photosensitizing agent, we propose that these findings support the hypothesis that UDCA causes a conformational change in Bcl-2, promoting HA14-1 binding and enhancing affinity for certain membrane-bound photosensitizers.

During Apoptosis Bcl-2 Changes Membrane Topology at Both the Endoplasmic Reticulum and Mitochondria

In healthy cells the antiapoptotic protein Bcl-2 adopts a topology typical of tail-anchored proteins with only the hydrophobic carboxyl terminus inserted into the membrane, as shown by labeling cell lysates with a membrane-impermeant sulfhydryl-specific reagent. Induction of apoptosis in cells triggered a change in the conformation of Bcl-2 such that cysteine 158 near the base of helix 5 inserted into the lipid bilayer of both endoplasmic reticulum and mitochondria where it was protected from labeling. Addition of a peptide corresponding to the BH3 domain of the proapoptotic protein Bim to cell lysates triggered a similar conformational change in Bcl-2, demonstrating that preexisting, membrane-bound Bcl-2 proteins change topology.