Anti-apoptotic Bcl-2 Family Proteins Research Articles

Resistance of osteosarcoma cells to the proapoptotic effects of carfilzomib involves activation of mitogen activated protein kinase pathways.

What is the central question of this study? Carfilzomib, a second-generation proteasome inhibitor approved for the treatment of multiple myeloma, shows efficacy against osteosarcoma. However, drug resistance remains a major challenge. What is the role of carfilzomib-induced changes in mitogen-activated protein kinase (MAPK) pathways in the sensitivity of osteosarcoma cells to the proapoptotic effects of the drug? What is the main finding and its importance? The dose-dependent antiapoptotic effects in osteosarcoma are associated with activation of MAPK signalling. Combinational targeting of MAPK signalling pathways can synergistically enhance carfilzomib-induced cell apoptosis, suggesting that MAPK inhibitors in combination with proteasome inhibitors can serve as a novel therapeutic tool for osteosarcoma. Osteosarcoma is the most common primary bone malignancy. Despite efforts to improve outcomes, the overall survival rates for osteosarcoma have remained unchanged over the past three decades. In this study, we assessed the proapoptotic effects of the second-generation proteasome inhibitor carfilzomib on osteosarcoma and investigated the potential mechanisms underlying the synergistic proapoptotic action when combined with mitogen-activated protein kinase (MAPK) inhibitors. We found that carfilzomib alone significantly inhibited cell proliferation and induced apoptosis in a dose-dependent manner, characterized by the induction of cleaved caspase 3 and poly (ADP-ribose) polymerase. More importantly, focusing on the changes of antiapoptotic B-cell lymphoma 2 (Bcl-2) family members and signalling pathways, we found a striking induction of myeloid cell leukaemia 1 (Mcl-1) and the activation of MAPK pathways. Furthermore, we observed that combinational targeting of the MAPK pathways using the specific inhibitors U0126, SP600125 or SB203580 synergistically enhanced carfilzomib-induced cell apoptosis. Notably, we found that the combinational inhibition of extracellular signal-regulated kinase or c-Jun N-terminal kinase MAPK pathways significantly decreased the expression of the three antiapoptotic Bcl-2 family proteins, and in particular this reversed induction of Mcl-1 by carfilzomib. Collectively, our findings show that activation of the MAPK pathways contributes to the mechanisms of drug resistance to carfilzomib. In addition, the synergistic proapoptotic action of MAPK and proteasome inhibitors in osteosarcoma cells suggests that combinational therapy with both drug types may serve as a novel strategy for the clinical management of osteosarcoma.

A Cerium Vanadate Nanozyme with Specific Superoxide Dismutase Activity Regulates Mitochondrial Function and ATP Synthesis in Neuronal Cells.

Nanoparticles that functionally mimic the activity of metal-containing enzymes (metallo-nanozymes) are of therapeutic importance for treating various diseases. However, it is still not clear whether such nanozymes can completely substitute the function of natural enzymes in living cells. In this work, we show for the first time that a cerium vanadate (CeVO4 ) nanozyme can substitute the function of superoxide dismutase 1 and 2 (SOD1 and SOD2) in the neuronal cells even when the natural enzyme is down-regulated by specific gene silencing. The nanozyme prevents the mitochondrial damage in SOD1- and SOD2-depleted cells by regulating the superoxide levels and restores the physiological levels of the anti-apoptotic Bcl-2 family proteins. Furthermore, the nanozyme effectively prevents the mitochondrial depolarization, leading to a significant improvement in the cellular levels of ATP under oxidative stress.

Antiapoptotic Bcl-2 family proteins BCL-xL and MCL-1 integrate neural progenitor survival and proliferation during postnatal cerebellar neurogenesis

The tendency of brain cells to undergo apoptosis in response to exogenous events varies across neural development, with apoptotic threshold dependent on proliferation state. Proliferative neural progenitors show a low threshold for apoptosis, while terminally differentiated neurons are relatively refractory. To define the mechanisms linking proliferation and apoptotic threshold, we examined the effect of conditionally deleting Bcl2l1, the gene that codes the antiapoptotic protein BCL-xL, in cerebellar granule neuron progenitors (CGNPs), and of co-deleting Bcl2l1 homologs, antiapoptotic Mcl-1, or pro-apoptotic Bax. We found that cerebella in conditional Bcl2l1-deleted (Bcl-xLcKO) mice were severely hypoplastic due to the increased apoptosis of CGNPs and their differentiated progeny, the cerebellar granule neurons (CGNs). Apoptosis was highest as Bcl-xLcKO CGNPs exited the cell cycle to initiate differentiation, with proliferating Bcl-xLcKO CGNPs relatively less affected. Despite the overall reduction in cerebellar growth, SHH-dependent proliferation was prolonged in Bcl-xLcKO mice, as more CGNPs remained proliferative in the second postnatal week. Co-deletion of Bax rescued the Bcl-xLcKO phenotype, while co-deletion of Mcl-1 enhanced the phenotype. These findings show that CGNPs require BCL-xL to regulate BAX-dependent apoptosis, and that this role can be partially compensated by MCL-1. Our data further show that BCL-xL expression regulates MCL-1 abundance in CGNPs, and suggest that excessive MCL-1 in Bcl-xLcKO mice prolongs CGNP proliferation by binding SUFU, resulting in increased SHH pathway activation. Accordingly, we propose that BCL-xL and MCL-1 interact with each other and with developmental mechanisms that regulate proliferation, to adjust the apoptotic threshold as CGNPs progress through postnatal neurogenesis to CGNs.

A decision process for drug discovery in retinoblastoma.

Intraocular retinoblastoma treatment has changed radically over the last decade, leading to a notable improvement in ocular survival. However, eyes that relapse remain difficult to treat, as few alternative active drugs are available. More challenging is the scenario of central nervous system (CNS) metastasis, in which almost no advancements have been made. Both clinical scenarios represent an urgent need for new drugs. Using an integrated multidisciplinary approach, we developed a decision process for prioritizing drug selection for local (intravitreal [IVi], intrathecal/intraventricular [IT/IVt]), systemic, or intra-arterial chemotherapy (IAC) treatment by means of high-throughput pharmacological screening of primary cells from two patients with intraocular tumor and CNS metastasis and a thorough database search to identify clinical and biopharmaceutical data. This process identified 169 compounds to be cytotoxic; only 8 are FDA-approved, lack serious toxicities and available for IVi administration. Four of these agents could also be delivered by IT/IVt. Twelve FDA-approved drugs were identified for systemic delivery as they are able to cross the blood-brain barrier and lack serious adverse events; four drugs are of oral usage and six compounds that lack vesicant or neurotoxicity could be delivered by IAC. We also identified promising compounds in preliminary phases of drug development including inhibitors of survivin, antiapoptotic Bcl-2 family proteins, methyltransferase, and kinesin proteins. This systematic approach may be applied more broadly to prioritize drugs to be repurposed or to identify novel hits for use in retinoblastoma treatment.

BCL2A1: A Novel Target in Refractory Acute Myeloid Leukemia with FLT3-ITD/D835 Dual Mutations

Internal tandem duplications in the juxtamembrane domain of FMS-like tyrosine kinase 3 gene (FLT3-ITD) and missense mutations in the gene's tyrosine-kinase domain (FLT3-TKD) play critical roles in the pathophysiology of acute myeloid leukemia (AML). Recent study revealed that AML cells resistant to quizartinib, a type II TKI, consist of heterogeneous clonal populations harboring wild-type FLT3 as well as FLT3-ITD, FLT3-TKD and FLT3-ITD/TKD mutations, and on- and off-target mechanisms may contribute to the resistance to TKIs. To overcome the heterogeneous resistance mechanisms of FLT3-ITD and TKD mutations, various combinatorial therapies have been investigated. For example, BCL-2 inhibition in the presence of TKIs increased survival a murine FLT3-ITD AML model, and a phase Ib/II clinical trial of a combination of quizartinib with venetoclax, a BCL2 inhibitor is ongoing in relapsed/refractory FLT3-mutant AML patients (NCT03735875). To dissect underlying mechanisms of drug-resistance and exploring new targets in refractory AML with FLT3-ITD and TKD mutations, we investigated alterations of transcriptome signatures by analyzing AML samples with FLT3-ITD/D835 dual mutations. Previously, we reported BCL2A1 transcriptomes were upregulated in primary AML cells with FLT3-ITD/D835 dual mutations compared to cells with FLT3-ITD mutations only. This was recapitulated in the MV4-11 cells harboring FLT3-ITD/D835 dual mutations after 6 month-exposure to quizartinib. The MV4-11 cells with the FLT3-ITD/D835 dual mutations became resistant to quizartinib, and the cells also became resistant to venetoclax, a BCL2 inhibitor (Yamatani et al. ASH 2019). In this study, we further investigated BCL2A1 as new target in refractory AML with FLT3-ITD/D835 dual mutations. First, we examined whether overexpression of BCL2A1 induces drug-resistance in MV4-11 and Molm13 cell lines with FLT3-ITD. While parental MV4-11 and Molm13 cells are sensitive to venetoclax and quizartinib, MV4-11 and Molm13 cells transfected with lentivirus carrying BCL2A1 became resistant to venetoclax (IC50: MV4-11 with BCL2A1 over-expression >1000 nM vs. mock vector 0.71 nM; Molm13 with over-expression >1000 nM vs. mock vector 0.38 nM, 72h). In contrast, the sensitivity to quizartinib was retained in the BCL2A1 overexpressing MV4-11 and Molm13 cells. These findings indicate that the overexpression of BCL2A1 could play a role in the acquired resistance to venetoclax, but not to quizartinib. Bromodomain-containing protein 4 (BRD4), a family member of bromodomain and extra-terminal motif (BET) is known to transcriptionally modulate BCL2A1 gene expression. Next, we examined effects of CPI-0610, a BET inhibitor, on MV4-11 cells with FLT3-ITD or the FLT3-ITD/D835 dual mutation. CPI-0610 inhibited cell growth of MV4-11 cells by inducing apoptosis irrespective of co-existing FLT3 mutations (IC50: FLT3-ITD/D835, 255 nM vs. FLT3-ITD, 191 nM, 72h). Immunoblotting analyses confirmed that BET inhibition by CPT-0610 decreased the expression of BCL2A1 in MV4-11 cells FLT3-ITD/D835. WIn conclusion, transcriptome analysis and molecular pharmacological approaches identified alterations in the anti-apoptotic BCL2 family proteins in double-mutant FLT3 leukemias. BCL2A1 upregulation might be involved in drug resistance of FLT3-ITD/D835 dual mutant AML cells, and could be a promising new target in refractory AML with FLT3-ITD/D835 dual mutations. Disclosures Konopleva: Cellectis: Research Funding; Eli Lilly: Research Funding; Calithera: Research Funding; Sanofi: Research Funding; Ascentage: Research Funding; AstraZeneca: Research Funding; Agios: Research Funding; Stemline Therapeutics: Consultancy, Research Funding; Kisoji: Consultancy; Genentech: Consultancy, Research Funding; F. Hoffmann La-Roche: Consultancy, Research Funding; Forty-Seven: Consultancy, Research Funding; AbbVie: Consultancy, Research Funding; Reata Pharmaceutical Inc.;: Patents & Royalties: patents and royalties with patent US 7,795,305 B2 on CDDO-compounds and combination therapies, licensed to Reata Pharmaceutical; Rafael Pharmaceutical: Research Funding; Ablynx: Research Funding; Amgen: Consultancy. Carter:Syndax: Research Funding; AstraZeneca: Research Funding; Amgen: Research Funding; Ascentage: Research Funding. Andreeff:Daiichi-Sankyo; Breast Cancer Research Foundation; CPRIT; NIH/NCI; Amgen; AstraZeneca: Research Funding; Centre for Drug Research & Development; Cancer UK; NCI-CTEP; German Research Council; Leukemia Lymphoma Foundation (LLS); NCI-RDCRN (Rare Disease Clin Network); CLL Founcdation; BioLineRx; SentiBio; Aptose Biosciences, Inc: Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding; Daiichi-Sankyo; Jazz Pharmaceuticals; Celgene; Amgen; AstraZeneca; 6 Dimensions Capital: Consultancy.

Preclinical Efficacy and Biological Effects of Venetoclax and Ixazomib in Combination in Lymphoma Cells

Introduction. Bcl-2 family proteins comprise anti-apoptotic and pro-apoptotic proteins. Interaction between these proteins, as well as severe regulation of their expression, mediates cell survival and can quickly induce cell death. Venetoclax is Bcl-2-targeting that has shown preclinical and clinical activity in hematologic malignancies. Due to the development of resistance and the loss of dependence on the target protein, the monotherapy may be insufficient for maximal effectiveness. To circumvent the resistance mechanisms, many preclinical studies have shown that combination of venetoclax with other agents may represent a more effective therapeutic strategy. Ubiquitin-proteasome signaling pathway is a potential target that plays an important role in the proteolysis of key regulatory proteins. Proteasome inhibitors include ixazomib that inhibits cell growth and induces apoptosis in hematological malignancies cells resistant to conventional therapies and bortezomib. Objective: To analyze the preclinical efficacy and associated biological effects of venetoclax combined with ixazomib in a panel of lymphoma cell lines with diverse expression levels of Bcl-2 and other Bcl-2 family proteins. Methods: 12 lymphoma cell lines including FL (RL, WSU-NHL, Karpas422), MCL (Jeko1, Granta519), DLBCL (OCI-LY3, OCI-LY18), CTCL (Hut-78), ALCL (Karpas299), HL (L1236, L540), CLL (Mec1) and two MCL primary patient samples were exposed to venetoclax (0.01 - 8 µM) and ixazomib (10 - 2000 nM) alone for 24 - 72 hours to calculate IC50. Subsequently, lymphoma cells were exposed to venetoclax (0.015 - 25 nM) in combination with ixazomib (0.015 - 0.5 nM) for 24 hours. Cell viability was determined by MTT. Coefficient of synergy (combination index - CI) was calculated using CalcuSyn. Cell cycle and induction of apoptosis were evaluated by flow cytometry and changes in Bcl-2 family members, caspase activation and AKT phosphorylation were determined by western blotting. Results. In vitro, venetoclax and ixazomib alone induced cell death in a dose- and time-dependent manner against lymphoma cell lines. The IC50 is between 0.5 and 8 µM for venetoclax and between 12 and 1250 nM for ixazomib. The combination of venetoclax (0.03, 0.06, 12.5, 25 nM) with ixazomib (0.03, 0.06, 0.25, 0.5 nM) produced a synergistic effect (CI < 1) after 24 h of treatment in the most lymphoma cells lines leading to inhibition of cell growth and induction of apoptosis between 26 % and 59 % accompanied by increased with cleavage of caspases-3, -9 and PARP. We observed an additive effect (CI = 1) in Jeko1 (MCL) and MEC1 cells (CLL) and antagonist effect (CI > 1) Hut-78 cells (CTCL). Synergistic effect has been seen in two MCL primary patient samples (CI = 0.5 - 0.7). In sensitive lymphoma cells, the combination abrogated colony formation in the methylcellulose medium. When lymphoma cell lines were co-cultured with mesenchymal stromal cells with both drugs we observed a decrease of cell viability and a fraction of apoptotic cells indicating that drug combination may overcome the tumor promoting effects of stromal cells. The apoptosis induced in FL and Granta519 cells (MCL) by drug combination was accompanied by partial downregulation of Bcl-2 and strong upregulation of Bax, Bad, Bim and Noxa proteins. Jeko-1 cells were less sensitive to venetoclax-ixazomib combination-induced apoptosis. Western blot analysis showed a differential expression of Bcl-2, Mcl-1 and Bcl-XL proteins in FL, MCL and HL cell lines. Jeko-1 cells showed a normal expression of Bcl-2 and Mcl-1 proteins and high Bcl-xL protein level. Co-expression of related anti-apoptotic Bcl-2 family proteins could limit activity of treatment. Combined treatment induced G0/G1 cell cycle arrest and increased the sub-G1 population that was linked by the upregulation of p27 and p21. In addition, in RL, WSU-NHL and Granta519, enhanced cell death is associated with AKT inactivation and with a reduction of p-4EBP1, leading to decreased levels of c-MYC. Conclusion. Venetoclax exhibits strong synergistic activity with ixazomib in lymphoma cells. Studies are still ongoing and signaling pathways that promote the combination of venetoclax with ixazomib are to be analyzed. These data offer a rationale to continue exploring venetoclax-ixazomib combination and suggest that suppression of Bcl-2 family protein driven survival signaling may be one important mechanism for combination synergy. Disclosures No relevant conflicts of interest to declare.

Ex Vivo Drug Sensitivity and Functional Genomics Platform Identifies Novel Combinations Targeting Intrinsic and Extrinsic Apoptotic Signaling Pathways in Multiple Myeloma

Introduction: Despite some long-term remissions, eventual drug resistance in most patients remains a critical obstacle in the treatment of multiple myeloma (MM). The development of new drugs/drug combinations with novel mechanisms of action are needed for continued improvement in patient outcomes. Initiation of tumor cell death via activation of the intrinsic (mitochondrial) and/or extrinsic (death receptor) apoptotic signaling pathways has been shown to be an effective therapeutic strategy in MM. Venetoclax (Ven) is a selective, small-molecule inhibitor of BCL-2 that exhibits clinical activity in MM cells, particularly in patients harboring the t(11;14) translocation. Navitoclax (Nav) is a small-molecule that targets multiple antiapoptotic BCL-2 family proteins, including BCL-XL, BCL-2, and BCL-W to initiate the intrinsic apoptotic pathway. Eftozanermin alfa (Eftoza) is a novel, second generation TRAIL receptor agonist that induces cell death via death receptor pathways and is under investigation in multiple solid and heme malignancies. In addition, the pan-BET inhibitor mivebresib (Miv) and the BDII selective BET inhibitor ABBV-744 have shown synergistic activity with Ven in cell line models of multiple heme malignancies. Results reported here describe ex vivo drug sensitivities and functional genomic analyses of Ven, Nav, Eftoza, Miv, and ABBV-744 alone or in combination with standard-of-care agents, including bortezomib, carfilzomib, panobinostat, daratumumab, or pomalidomide. Methods: A high-throughput ex vivo drug screening assay using a coculture system of bone marrow (BM)-derived MM and stromal cells was used to assess the sensitivity of MM patient tumor cells (Figure 1A). Paired whole exome sequencing (WES) and RNA sequencing (RNA-seq) analyses were performed. Results: Primary MM patient specimens (n=52) were evaluated in the ex vivo platform, including treatment-naïve, early relapse (1-3 prior lines), and late relapse (4-8 prior lines) patients treated with proteasome inhibitors, immunomodulatory drugs, and monoclonal antibodies. As expected, t(11;14)-positive MM patient specimens were more sensitive than wildtype to Ven ex vivo (D AUC, -18.6, P=0.002), however MM cells harboring amp(1q) were more resistant than wildtype (D AUC, +5.07, P=0.032), suggesting MCL1 (1q21 gene locus) is a key resistance factor to Ven single-agent activity in MM. Gene set enrichment analysis identified B-cell receptor signaling (normalized enrichment score (NES), 1.96, adjusted P=0.010) and MYC pathway (NES, 1.95, adjusted P=0.010) overexpression as predictors of increased sensitivity to Ven ex vivo. A t(11;14) gene expression signature was also generated using a penalized regression model approach in an additional MMWG/ORIEN MM patient cohort (n=155). The t(11;14) predictive gene expression signature was confirmed by correlation with Ven AUC in the ex vivo model. Additional pathway analyses were performed to identify potential predictive markers of sensitivity/resistance for each single agent and drug combination. Although ex vivo activity of Nav was higher in t(11;14) specimens compared to non-t(11;14) (D AUC, -17.8, P=0.011), ex vivo activity in non-t(11;14) specimens was also observed, indicating additional anti-MM activity by cotargeting of BCL-XL and BCL-2. Both Miv and ABBV-744 showed single-agent activity ex vivo, however Miv demonstrated higher activity (median LD50=88.4nM), suggesting that pan-BET inhibition is more effective than BDII-specific BET inhibition in MM. Finally, a novel drug-combination effect analysis was used that identified novel synergistic ex vivo combinations including Ven and panobinostat (P=0.0013) and Eftoza with bortezomib (P=1.8E-7) or carfilzomib (P=7E-4). Additionally, single-agent induction of macrophage-mediated phagocytosis was observed in both Ven and daratumumab, which was synergistic when the 2 drugs were combined (Figure 1B). Conclusion: An ex vivo functional genomic screen of MM patient specimens demonstrated the usefulness of this approach to identify candidate drugs and potential predictive biomarkers for continued evaluation in clinical trials. This approach confirmed known mechanisms of drug sensitivity and identified new ones, including a novel characterized immune-mediated synergy between Ven and daratumumab, and potential combination strategy for Eftoza and proteasome inhibitors. Figure 1 Disclosures Siqueira Silva: Karyopharm: Research Funding; NIH/NCI: Research Funding; AbbVie: Research Funding. Kulkarni:M2GEN: Current Employment. Mitchell:AbbVie: Other: payment for bioinformatics analysis, Research Funding; M2GEN: Current Employment, Research Funding. Dai:Cygnal Therapeutics: Current Employment; M2GEN: Ended employment in the past 24 months. Hampton:M2GEN: Current Employment. Lu:AbbVie: Current Employment, Current equity holder in publicly-traded company. Modi:AbbVie: Current Employment, Other: may own stock or stock options. Motwani:AbbVie: Current Employment, Current equity holder in publicly-traded company. Harb:AbbVie: Current Employment, Other: may hold stock or stock options. Ross:AbbVie: Current Employment, Current equity holder in publicly-traded company. Shain:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; GlaxoSmithKline: Speakers Bureau; Sanofi/Genzyme: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Karyopharm: Research Funding, Speakers Bureau; AbbVie: Research Funding; Takeda: Honoraria, Speakers Bureau; Janssen: Honoraria, Speakers Bureau; Amgen: Speakers Bureau; Adaptive: Consultancy, Honoraria; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. OffLabel Disclosure: While this is a preclinical study, venetoclax for treatment of multiple myeloma is not an approved indication

Lycorine hydrochloride inhibits cell proliferation and induces apoptosis through promoting FBXW7-MCL1 axis in gastric cancer

BackgroundLycorine hydrochloride (LH), an alkaloid extracted from the bulb of the Lycoris radiata, is considered to have anti-viral, anti-malarial, and anti-tumorous effects. At present, the underlying mechanisms of LH in gastric cancer remain unclear. MCL1, an anti-apoptotic protein of BCL2 family, is closely related to drug resistance of tumor. Therefore, MCL1 is considered as a potential target for cancer treatment.MethodsThe effect of LH on gastric cancer was assessed in vitro (by MTT, BrdU, western blotting…) and in vivo (by immunohistochemistry).ResultsIn this study, we showed that LH has an anti-tumorous effect by down-regulating MCL1 in gastric cancer. Besides, we unveiled that LH reduced the protein stability of MCL1 by up-regulating ubiquitin E3 ligase FBXW7, arrested cell cycle at S phase and triggered apoptosis of gastric cancer cells. Meanwhile, we also demonstrated that LH could induce apoptosis of the BCL2-drug-resistant-cell-lines. Moreover, PDX (Patient-Derived tumor xenograft) model experiment proved that LH combined with HA14–1 (inhibitor of BCL2), had a more significant therapeutic effect on gastric cancer.ConclusionsThe efficacy showed in our data suggests that lycorine hydrochloride is a promising anti-tumor compound for gastric cancer.

A "Three-in-One" Multifunctional Probe for Bcl-2/Mcl-1 Profiling and Visualizing in Situ.

Bcl-2 and Mcl-1, the two arms of the anti-apoptotic Bcl-2 family proteins, have been identified as key regulators of apoptosis and effective therapeutic targets of cancer. However, no small-molecular probe is capable of profiling and visualizing both Bcl-2 and Mcl-1 simultaneously in situ. Herein, we report a multifunctional molecular probe (BnN3 -OPD-Alk) by a "three-in-one" molecular designing strategy, which integrated the Bcl-2/Mcl-1 binding ligand, fluorescent reporter group and photoreactive group azido into the same scaffold. BnN3 -OPD-Alk exhibited sub-micromolar affinities to Bcl-2/Mcl-1 and bright green self-fluorescence. It was then successfully applied for Bcl-2/Mcl-1 labeling, capturing, enriching, and bioimaging both in vitro and in cells. This strategy could facilitate the precise early diagnosis and effective therapy of dual Bcl-2/Mcl-1-related diseases.

Isolinderalactone Induces Cell Death via Mitochondrial Superoxide- and STAT3-Mediated Pathways in Human Ovarian Cancer Cells.

The mortality rate of ovarian cancer (OC) worldwide increases with age. OC is an often fatal cancer with a curative rate of only 20–30%, as symptoms often appear after disease progression. Studies have reported that isolinderalactone (ILL), a furanosesquiterpene derivative extracted from the dried root of Lindera aggregata, can inhibit several cancer cell lines’ growth. However, the molecular mechanisms underlying ILL activities in human OC cells remain unexplored. This study investigated the antitumor activities of ILL in human OC cells by inducing mitochondrial superoxide (mtSO) and JAK-signal transducer and activator of transcription 3 (STAT3)-dependent cell death. ILL caused cell death in SKOV-3 and OVCAR-3 cells and increased the cell proportion in the subG1 phase. Additionally, ILL significantly induced mtSO production and reduced ROS production. Moreover, ILL downregulated mitochondrial membrane potential and the expression levels of anti-apoptotic Bcl-2 family proteins and superoxide dismutase (SOD)2. Results showed that ILL decreased phosphorylation of serine 727 and tyrosine 705 of STAT3 and expression of survivin, a STAT3-regulated gene. Furthermore, ILL-induced cell death was reversed by pretreatment of Mito-TEMPO, a mitochondria-specific antioxidant. These results suggest that ILL induces cell death by upregulation of mtSO, downregulation of mitochondrial SOD2, and inactivation of the STAT3-mediated pathway.

Targeting Bcl-2 Family Proteins: What, Where, When?

Proteins of the Bcl-2 family are known as regulators of apoptosis, one of the most studied forms of programmed cell death. The Bcl-2 protein family is represented by both pro- and antiapoptotic members. Antiapoptotic proteins are often exploited by tumor cells to avoid their death, thus playing an important role in carcinogenesis and in acquisition of resistance to various therapeutic agents. Therefore, antiapoptotic proteins represent attractive targets for cancer therapy. A detailed investigation of interactions between Bcl-2 family proteins resulted in the development of highly selective inhibitors of individual antiapoptotic members. These agents are currently being actively studied at the preclinical and clinical stages and represent a promising therapeutic strategy, which is highlighted by approval of venetoclax, a selective inhibitor of Bcl-2, for medical use. Meanwhile, inhibition of antiapoptotic Bcl-2 family proteins has significant therapeutic potential that is yet to be revealed. In the coming era of precision medicine, a detailed study of the mechanisms responsible for the sensitivity or resistance of tumor cells to various therapeutic agents, as well as the search for the most effective combinations, is of great importance. Here, we discuss mechanisms of how the Bcl-2 family proteins function, principles of their inhibition by small molecules, success of this approach in cancer therapy, and, eventually, biochemical features that can be exploited to improve the use of Bcl-2 family inhibitors as anticancer drugs.

IL-17B Regulates Endothelial Cell Apoptosis in Venous Thrombosis

Venous thromboembolism (VTE) is the comorbidity of deep vein thrombosis (DVT) and pulmonary embolism (PE); it is an urgent public health problem. The primary cause of venous thrombosis is endothelial dysfunction caused by vascular injury and inflammation or overexpressed procoagulant factors. Previous studies have shown that vascular endothelial cell apoptosis is involved in venous thrombosis, causing vascular wall damage. The pro-inflammatory cytokine interleukin-17 (Il-17A) induces endothelial cell apoptosis and promotes thrombosis. However, it remains unclear whether other IL-17 family cytokines are involved in thrombus formation. Among the IL-17 family, IL-17B is less well-characterized. Several studies have reported that IL-17B could stimulate TNF-α, IL-1, and IL-6 expression in macrophages. Furthermore, IL-17B induced activation of the ERK1/2 pathway, upregulating Bcl-2 family anti-apoptotic proteins in breast tumors. However, it is unclear whether IL-17B is involved in thrombus formation by regulating endothelial apoptosis. Therefore, this study aimed to examine whether IL-17B could affect endothelial apoptosis by promoting thrombus formation.

A redox switch regulates the structure and function of anti-apoptotic BFL-1.

Apoptosis is regulated by BCL-2 family proteins. Anti-apoptotic members suppress cell death by deploying a surface groove to capture the critical BH3 α-helix of pro-apoptotic members. Cancer cells hijack this mechanism by overexpressing anti-apoptotic BCL-2 family proteins to enforce cellular immortality. We previously identified and harnessed a unique cysteine (C55) in the groove of anti-apoptotic BFL-1 to selectively neutralize its oncogenic activity using a covalent stapled-peptide inhibitor. Here, we find that disulfide-bonding between a native cysteine pair at the groove (C55) and C-terminal α9 helix (C175) of BFL-1 operates as a redox switch to control the accessibility of the anti-apoptotic pocket. Reducing the C55-C175 disulfide triggers α9 release, which promotes mitochondrial translocation, groove exposure for BH3 interaction, and inhibition of mitochondrial permeabilization by pro-apoptotic BAX. C55-C175 disulfide-formation in an oxidative cellular environment abrogates the ability of BFL-1 to bind BH3 domains. Thus, we identify a mechanism of conformational control of BFL-1 by an intramolecular redox switch.

Abstract A73: BCL-XL dependency in a subset of ovarian clear-cell carcinoma cell lines

Abstract Clear-cell ovarian carcinoma is a rare histologic subtype of epithelial ovarian cancers. Clear-cell ovarian cancers tend to be less responsive to platinum-based chemotherapy compared to high-grade serous ovarian cancers. In a dataset of large-scale CRISPR knockout and RNAi screens of cancer cell lines (depmap.org), a subset of ovarian clear-cell carcinoma cell lines were sensitive to knockout or knockdown of BCL2L1, encoding antiapoptotic protein BCL-XL. Most clear-cell ovarian cancer cell lines also express BCL2L1 mRNA and protein. We therefore tested a panel of ovarian cancer cell lines for response to BH3 mimetic drugs, which inhibit different members of the BCL-2 family of antiapoptotic proteins. Initial data showed that a subset of ovarian clear-cell carcinoma cell lines was sensitive to single-agent BCL-XL inhibition with A1331852 and dual BCL-2/BCL-XL inhibition with ABT-263. In contrast, these cell lines were relatively insensitive to single-agent BCL-2 inhibition or MCL1 inhibition. We are currently applying BH3 profiling, a functional assay for apoptotic priming, to test the response of ovarian clear-cell carcinoma cell lines to BCL-XL inhibition using a BCL-XL specific peptide, HRK. Collectively, these studies suggest that ovarian clear-cell carcinomas may be sensitive to BCL-XL inhibition, either alone or in combination with chemotherapy. Citation Format: Elizabeth H. Stover, Maya K. Gelbard, Ursula A. Matulonis, Matthew Meyerson. BCL-XL dependency in a subset of ovarian clear-cell carcinoma cell lines [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A73.

Combining triptolide with ABT-199 is effective against acute myeloid leukemia through reciprocal regulation of Bcl-2 family proteins and activation of the intrinsic apoptotic pathway

Bcl-2 inhibitors display an effective activity in acute myeloid leukemia (AML), but its clinical efficacy as a monotherapy was limited in part owing to failure to target other antiapoptotic Bcl-2 family proteins, such as Mcl-1. In this context, the combination strategy may be a promising approach to overcome this barrier. Here, we report the preclinical efficacy of a novel strategy combining ABT-199 with triptolide (TPL), a natural product extracted from a traditional Chinese medicine, in AML. Combination treatment exhibited markedly increased cytotoxicity in leukemic cells irrespective of p53 status while largely sparing normal cells of the hematopoietic lineage. Moreover, co-administration of ABT-199 with TPL dramatically suppressed leukemia progression as well as prolonged animal survival in a xenograft AML model. The potentiated effect of ABT-199 and TPL against AML was associated with activation of the mitochondrum-related intrinsic apoptotic pathway through a mechanism reciprocally modulating Bcl-2 family proteins. In this case, TPL not only downregulated Mcl-1 but also upregulated proapoptotic BH3-only proteins, thereby overcoming the resistance toward ABT-199. Conversely, ABT-199 abrogated Bcl-2-mediated cytoprotection against TPL. Together, these findings suggest that the regimen combining TPL and ABT-199 might be active against AML by inducing robust apoptosis through reciprocal regulation of anti- and proapoptotic Bcl-2 family proteins, therefore providing a strong rationale for the clinical investigation of this combination regimen for the treatment of AML.

The (+)-Brevipolide H Displays Anticancer Activity against Human Castration-Resistant Prostate Cancer: The Role of Oxidative Stress and Akt/mTOR/p70S6K-Dependent Pathways in G1 Checkpoint Arrest and Apoptosis.

Because conventional chemotherapy is not sufficiently effective against prostate cancer, various examinations have been performed to identify anticancer activity of naturally occurring components and their mechanisms of action. The (+)-brevipolide H, an α-pyrone-based natural compound, induced potent and long-term anticancer effects in human castration-resistant prostate cancer (CRPC) PC-3 cells. Flow cytofluorometric analysis with propidium iodide staining showed (+)-brevipolide H-induced G1 arrest of cell cycle and subsequent apoptosis through induction of caspase cascades. Since Akt/mTOR pathway has been well substantiated in participating in cell cycle progression in G1 phase, its signaling and downstream regulators were examined. Consequently, (+)-brevipolide H inhibited the signaling pathway of Akt/mTOR/p70S6K. The c-Myc inhibition and downregulation of G1 phase cyclins were also attributed to (+)-brevipolide H action. Overexpression of myristoylated Akt significantly rescued mTOR/p70S6K and downstream signaling under (+)-brevipolide H treatment. ROS and Ca2+, two key mediators in regulating intracellular signaling, were determined, showing that (+)-brevipolide H interactively induced ROS production and an increase of intracellular Ca2+ levels. The (+)-Brevipolide H also induced the downregulation of anti-apoptotic Bcl-2 family proteins (Bcl-2 and Bcl-xL) and loss of mitochondrial membrane potential, indicating the contribution of mitochondrial dysfunction to apoptosis. In conclusion, the data suggest that (+)-brevipolide H displays anticancer activity through crosstalk between ROS production and intracellular Ca2+ mobilization. In addition, suppression of Akt/mTOR/p70S6K pathway associated with downregulation of G1 phase cyclins contributes to (+)-brevipolide H-mediated anticancer activity, which ultimately causes mitochondrial dysfunction and cell apoptosis. The data also support the biological significance and, possibly, clinically important development of natural product-based anticancer approaches.

Measurements of Mcl-1 protein dependencies using dimerization-specific antibodies as predictive biomarkers for BH-3 mimetic class therapies in AML.

e19505 Background: The anti-apoptotic Bcl-2 family proteins facilitate pro-survival and resistance to anti-cancer therapies. Measuring the function of these proteins has shown utility in predicting response to treatment. A biomarker platform that assesses the functionality of these proteins by measuring BH3 mediated signaling potential in individual patients’ cancers, could provide a potential guiding platform for treating AML. In AML venetoclax is becoming widely prescribed and highly effective in combination with other drugs. Recent data indicate that Mcl-1 dependence is a resistance factor to venetoclax and that methods for identifying this could provide guidance for combination treatments. We are developing a technology to directly measure the occurrence of heterodimers of Mcl-1, or Bcl-xL, bound to pro-apoptotic BH3-only protein Bim. These measurements are combined in algorithms developed to indicate the cancer cell apoptotic priming state and offer great potential for identifying best AML treatment options. Methods: Monoclonal antibodies against conformation-specific epitopes induced in Mcl-1/Bim and Bcl-xL/Bim protein complexes were made. Selective bonding of Heterodimer Specific Mcl-1 Bim (HSMCB) and Heterodimer Specific Bcl-xL Bim (HSBXB) mabs were confirmed using ELISA, fluorescence polarization, immunofluorescence microscopy and flow, and by immunohistochemistry in genetically defined cell lines and in AML patient biopsied samples. Results: We show that HSMCB signal depends on both Mcl-1 and Bim protein levels while HSBXB requires Bc-xL and Bim levels. The relative signals of HSMCB to unbound Mcl-1 signal ([HsMcB]/[Mcl-1]) provide a biomarker for Mcl-1 dependence. We carefully established binding metrics that correlate to drug response in cell lines. The pharmacological disruption of these complexes is monitored by the ratiometric readouts that serve as predictive biomarkers for BH-3 mimetic drugs in AML cells. Conclusions: Mcl-1 dependence is a predictive biomarker for venetoclax resistance and for response to Mcl-1 targeted therapies. Flow cytometric and IHC based measurements of a heterodimer complex offer a direct and simpler approach that harbors potential for use in clinical settings. Additional antibodies targeting Mcl-1/Bak and Bcl-2/Bim complexes are being tested.

MCL-1Matrix maintains neuronal survival by enhancing mitochondrial integrity and bioenergetic capacity under stress conditions

Mitochondria play a crucial role in neuronal survival through efficient energy metabolism. In pathological conditions, mitochondrial stress leads to neuronal death, which is regulated by the anti-apoptotic BCL-2 family of proteins. MCL-1 is an anti-apoptotic BCL-2 protein localized to mitochondria either in the outer membrane (OM) or inner membrane (Matrix), which have distinct roles in inhibiting apoptosis and promoting bioenergetics, respectively. While the anti-apoptotic role for Mcl1 is well characterized, the protective function of MCL-1 Matrix remains poorly understood. Here, we show MCL-1OM and MCL-1Matrix prevent neuronal death through distinct mechanisms. We report that MCL-1Matrix functions to preserve mitochondrial energy transduction and improves respiratory chain capacity by modulating mitochondrial oxygen consumption in response to mitochondrial stress. We show that MCL-1Matrix protects neurons from stress by enhancing respiratory function, and by inhibiting mitochondrial permeability transition pore opening. Taken together, our results provide novel insight into how MCL-1Matrix may confer neuroprotection under stress conditions involving loss of mitochondrial function.

Inhibition of BCL2 Family Members Increases the Efficacy of Copper Chelation in BRAFV600E-Driven Melanoma.

The principal unmet need in BRAFV600E-positive melanoma is lack of an adequate therapeutic strategy capable of overcoming resistance to clinically approved targeted therapies against oncogenic BRAF and/or the downstream MEK1/2 kinases. We previously discovered that copper (Cu) is required for MEK1 and MEK2 activity through a direct Cu-MEK1/2 interaction. Repurposing the clinical Cu chelator tetrathiomolybdate (TTM) is supported by efficacy in BRAFV600E-driven melanoma models, due in part to inhibition of MEK1/2 kinase activity. However, the antineoplastic activity of Cu chelators is cytostatic. Here, we performed high-throughput small-molecule screens to identify bioactive compounds that synergize with TTM in BRAFV600E-driven melanoma cells. Genetic perturbation or pharmacologic inhibition of specific members of the BCL2 family of antiapoptotic proteins (BCL-W, BCL-XL, and MCL1) selectively reduced cell viability when combined with a Cu chelator and induced CASPASE-dependent cell death. Further, in BRAFV600E-positive melanoma cells evolved to be resistant to BRAF and/or MEK1/2 inhibitors, combined treatment with TTM and the clinically evaluated BCL2 inhibitor, ABT-263, restored tumor growth suppression and induced apoptosis. These findings further support Cu chelation as a therapeutic strategy to target oncogene-dependent tumor cell growth and survival by enhancing Cu chelator efficacy with chemical inducers of apoptosis, especially in the context of refractory or relapsed BRAFV600E-driven melanoma. SIGNIFICANCE: This study unveils a novel collateral drug sensitivity elicited by combining copper chelators and BH3 mimetics for treatment of BRAFV600E mutation-positive melanoma.

Mapping mitochondrial respiratory chain deficiencies by respirometry: Beyond the Mito Stress Test

Cell-based respirometers, such as the Seahorse Extracellular Flux Analyzer, are valuable tools to assess the functionality of mitochondria within adherent neurons, as well as other cell types. The Mito Stress Test is the most frequently employed protocol of drug additions to evaluate mitochondrial bioenergetic function. Sequential exposure of cells to an ATP synthase inhibitor such as oligomycin and an uncoupler such as FCCP cause changes in oxygen consumption rate that allow estimation of the cellular efficiency and capacity for mitochondrial ATP synthesis. While a useful first step in assessing whether an experimental treatment or genetic manipulation affects mitochondrial energetics, the Mito Stress Test does not identify specific sites of altered respiratory chain function. This article discusses limitations of the Mito Stress Test, proposes a refined protocol for comparing cell populations that requires independent drug titrations at multiple cell densities, and describes a stepwise series of respirometry-based assays that “map” locations of electron transport deficiency. These include strategies to test for cytochrome c release, to probe the functionality of specific electron transport chain complexes within intact or permeabilized cells, and to measure NADH oxidation by the linked activity of Complexes I, III, and IV. To illustrate utility, we show that although UK5099 and ABT-737 each decrease the spare respiratory capacity of cortical neurons, the stepwise assays reveal different underlying mechanisms consistent with their established drug targets: deficient Complex I substrate supply induced by the mitochondrial pyruvate carrier inhibitor UK5099 and cytochrome c release induced by the anti-apoptotic BCL-2 family protein inhibitor ABT-737.