Members Of Bcl-2 Protein Family Research Articles

Design, Synthesis, and Evaluation of BCL-2 Targeting PROTACs.

BCL-2, a member of the BCL-2 protein family, is an antiapoptotic factor that regulates the intrinsic pathway of apoptosis. Due to its aberrant activity, it is frequently implicated in haematopoietic cancers and represents an attractive target for the development of therapeutics that antagonize its activity. A selective BCL-2 inhibitor, venetoclax, was approved for treating chronic lymphocytic leukaemia, acute myeloid leukemia, and other haematologic malignancies, validating BCL-2 as an anticancer target. Since then, alternative therapeutic approaches to modulate the activity of BCL-2 have been explored, such as antibody-drug conjugates and proteolysis-targeting chimeras. Despite numerous research groups focusing on developing degraders of BCL-2 family member proteins, selective BCL-2 PROTACs remain elusive, as disclosed compounds only show dual BCL-xL/BCL-2 degradation. Herein, we report our efforts to develop BCL-2 degraders by incorporating two BCL-2 binding moieties into chimeric compounds that aim to hijack one of three E3 ligases: CRBN, VHL, and IAPs. Even though our project did not result in obtaining a potent and selective BCL-2 PROTAC, our research will aid in understanding the narrow chemical space of BCL-2 degraders.

Bcl-2 dependent modulation of Hippo pathway in cancer cells

IntroductionBcl-2 and Bcl-xL are the most studied anti-apoptotic members of Bcl-2 family proteins. We previously characterized both of them, not only for their role in regulating apoptosis and resistance to therapy in cancer cells, but also for their non-canonical functions, mainly including promotion of cancer progression, metastatization, angiogenesis, and involvement in the crosstalk among cancer cells and components of the tumor microenvironment. Our goal was to identify transcriptional signature and novel cellular pathways specifically modulated by Bcl-2.MethodsWe performed RNAseq analysis of siRNA-mediated transient knockdown of Bcl-2 or Bcl-xL in human melanoma cells and gene ontology analysis to identify a specific Bcl-2 transcriptional signature. Expression of genes modulated by Bcl-2 and associated to Hippo pathway were validated in human melanoma, breast adenocarcinoma and non-small cell lung cancer cell lines by qRT-PCR. Western blotting analysis were performed to analyse protein expression of upstream regulators of YAP and in relation to different level of Bcl-2 protein. The effects of YAP silencing in Bcl-2 overexpressing cancer cells were evaluated in migration and cell viability assays in relation to different stiffness conditions. In vitro wound healing assays and co-cultures were used to evaluate cancer-specific Bcl-2 ability to activate fibroblasts.ResultsWe demonstrated the Bcl-2-dependent modulation of Hippo Pathway in cancer cell lines from different tumor types by acting on upstream YAP regulators. YAP inhibition abolished the ability of Bcl-2 to increase tumor cell migration and proliferation on high stiffness condition of culture, to stimulate in vitro fibroblasts migration and to induce fibroblasts activation.ConclusionsWe discovered that Bcl-2 regulates the Hippo pathway in different tumor types, promoting cell migration, adaptation to higher stiffness culture condition and fibroblast activation. Our data indicate that Bcl-2 inhibitors should be further investigated to counteract cancer-promoting mechanisms.

Compatibility of Venetoclax and OPN51107 in Treatment of Mantle Cell Lymphoma

Introduction Mantle Cell Lymphoma (MCL) is the third most common form of B-Cell lymphoma and patients diagnosed tend to have poor long-term survival when compared to patients of other B-cell lymphoma subtypes. Deregulation of the apoptotic cellular machinery contributes to early acquirement of therapy resistance in MCL and contributes to the poor clinical outcomes observed in relapsed/refractory MCL patients. Developing therapeutic strategies using agents that favor a pro-apoptotic cellular environment are being investigated in pre-clinical and clinical studies. To this end, we evaluated the therapeutic effects of targeting the Bcl-2 related proteins using Venetoclax, a Bcl-2 inhibitor, and OPN51107, a BET inhibitor, in MCL pre-clinical models. OPN51107 targets BRD4 in B-cells, a protein involved in the preinitiation complex for transcription of miRNA inhibit the translation of BIM, a pro-apoptotic initiator in the Bcl-2 family. Methods Using a panel of MCL cell lines (HBL-2, Granta, Z-138, Mino, Jeko-1, and Rec-1) along with resistant cells of each cell line made to Cytarabine, we conducted pre-clinical targeting of Bcl-2 and BRD4 with Venetoclax and OPN51107 respectively. Single and double agent exposure experiments were performed at 72 hrs and coefficients of synergy were assessed using CalcuSyn. Synergy was determined from cellular viability using Cell Titer-Glo (Promega) and was read using a BioTek Synergy HTX multimode reader. During in vivo experiments, 6-week SCID mice were inoculated with 10 × 10 6 HBL-2 or Granta cells via tail vein injection. OPN51107 was administered at 5 and 20 mg/kg/dose PO Days (3-7, 10-14. And 17-21). Venetoclax was administered at 50 and 100 mg/kg/dose PO Days (3-7, 10-14, and 17-21). Bcl-2 family member protein expression was determined via western blot. Protein-protein interactions of members of the Bcl-2 family were also studied though Co-immunoprecipitation. Results In vitro exposure of MCL cells lines resulted in an up-regulation of BIM levels. Synergistic activity between Venetoclax and OPN51107 was observed in SCID mice bearing the Granta cells but not in animals inoculated with the HBL-2 cell line. The median survival for Granta bearing SCID mice treated with Venetoclax and OPN51107 was longer (26 days) when compared to animals treated with either Venetoclax (24 days) or OPN51107 (25 days) (P values: 0.000383 and 0.089 respectively). Ongoing studies are investigating protein to protein interactions between BIM and other Bcl-2 related proteins to further define the mechanisms responsible for the synergy observed between Venetoclax and OPN51107. Conclusion Previous in vitro activity presented a synergistic relationship on cell death from combination treatment of OPN51107 and Venetoclax. Our present research suggests that this relationship can also be replicated in vivo and further research has been done to help elucidate a possible mechanism of action responsible for the enhancement of activity seen. Continuing these directions to understand the synergy between OPN51107 and Venetoclax is necessary to advance therapies within MCL.

Molecular docking analysis of MCL-1 inhibitors for breast cancer management.

Myeloid leukemia 1 (MCL-1), a BCL-2 protein family member, acts as an anti-apoptotic protein by interacting with pro-apoptotic BCL-2 proteins. Its overexpression is frequently observed in numerous cancer types including breast cancer, and is closely linked to the initiation and progression of tumors as well as poor prognosis and resistance to therapeutic interventions. Here, a database of 3402 chemicals with established therapeutic activity against various diseases was chosen and systematically screened against the MCL-1 protein. Visual inspection and binding energy analysis revealed that the compounds OSU-03012, Raltitrexed, Ostarine (MK-2866), Dovitinib (TKI-258), and Varespladib (LY315920) had strong binding affinity for the MCL-1 protein. Notably, their binding affinity was higher than that of the control compounds. These compounds exhibited strong interactions with critical amino acid residues of the MCL-1 protein. Furthermore, these compounds shared several common amino acid residue interactions with the control compounds. These findings suggest that these compounds may be useful as MCL-1 inhibitors in the treatment of breast cancer. However, additional experimental validation is required to confirm these findings.

Polypeptide antibiotic actinomycin D induces Mcl-1 uncanonical downregulation in lung cancer cell apoptosis

AimsActinomycin (Act) D, a polypeptide antibiotic, is used clinically to inhibit the growth of malignant tumors. Act D binds to DNA at the transcription initiation complex to prevent the elongation of RNA. Act D causes DNA damage, growth inhibition, and cell death. Myeloid cell leukemia (Mcl-1) is an anti-apoptotic Bcl-2 family member protein, and the present study explored the effects and molecular mechanism of Act D-induced Mcl-1 downregulation. Main methodsHuman adenocarcinoma A549 cells were used to check the cytotoxic signaling pathways of Act D, particularly in apoptotic mechanism, in a cell-based study approach. Specific blockers targeting the apoptotic factors were examined for their possible roles. Key findingsWe found that Act D caused cell growth inhibition and apoptosis. Propidium iodide-based flow cytometric analysis and immunostaining confirmed cell apoptosis. Treatment with Act D caused DNA damage, followed by p53-independent cell death. Western blotting showed a significant decrease in Mcl-1 expression, mitochondrial transmembrane potential loss, and caspase-9/caspase-3 cascade activation. The proteasome inhibitor MG132 reversed Act D-induced Mcl-1 downregulation. However, pharmacological inhibition of glycogen synthase kinase-3, p53 expression, ER stress, autophagy, and vesicle acidification, which are Mcl-1-regulating signaling pathways, did not rescue these effects. Notably, Cullin-Ring E3 ligase partially mediated Mcl-1 downregulation. Administration of transforming growth factor-β induced mesenchymal cell differentiation, but Act D still decreased Mcl-1 and caused cell apoptosis. SignificanceAll of these data show a potential pro-apoptotic effect for Act D by facilitating Mcl-1 uncanonical downregulation.

PLX51107 Enhances the Anti-Tumor Activity of Targeted Therapy in Chronic Lymphocytic Leukemia When Combined with Venetoclax

Background: The development of drugs targeting BTK and BCL2 have dramatically improved the therapeutic landscape in chronic lymphocytic leukemia (CLL). However, resistance to these agents have been reported due to non-recurrent changes in oncogenic pathways and gene expression signatures. The bromodomain and extra terminal (BET) family proteins (BRD2, BRD3, BRD4 and BRDT) are epigenetic reader proteins that recognize acetylated lysine residues in histones. They play a critical role in mediating gene transcription and have been considered as highly promising targets in several diseases including cancer. Of these, the BRD4 protein is highly enriched in super enhancers and regulate gene transcription of relevant oncogenes such as MYC, CDK genes, cyclin-D1, BCL2 and MCL-1. BRD4 inhibitors (BRD4i) block the transcription of these key oncogenes through the displacement of BRDs and other epigenetic modifiers from chromatin. In this study, we investigated the preclinical activity of BRD4i PLX51107, and their cooperative role with BCL2 inhibitor Venetoclax in CLL. Methods: 56 primary CLL cells were isolated via histopaque-1077 (Sigma-Aldrich, St Louis MO) from peripheral blood apheresis samples from consenting adults using Roswell Park Comprehensive Cancer Center protocol CIC-01-16. Peripheral blood mononuclear cells (PBMC) were seeded at 3 x106/mL in a 384 or 6 well tissue culture plates and exposed to different doses of PLX51107 (1-20uM) as a single agent or in combination with Venetoclax (1-100nM) for 48 and 72hrs (Selleck Chemicals, Houston TX). Cell viability was assessed using Cell Titer-glo (Promega, Madison WI) with all data being normalized to untreated controls. Synergy was calculated using Calcusyn (Cambridge, UK) software to determine Coefficient of Synergy (CI) values. Anti-tumor effect was also analyzed by induction of apoptosis using Annexin-V/PI staining by flow cytometry at 72hrs. Cells pellets were saved for western blotting to determine expression of BRD4 members and different members of the B-cell receptor, NF-kB pathway as also the BCL-2 family of proteins. Bcl-2 family profiling was performed via Milliplex Bcl-2 family protein panels 1 and 2 to look at overall changes in Bcl-2 family expression. Results: Single agent exposure to PLX51107 displayed time and dose dependent induction of apoptosis of CLL cells (Figure 1a). Next, we examined the interaction between the PLX51107 and Venetoclax. In 72hr exposure the combination of PLX51107 with Venetoclax with an ATM deletion was synergistic in 4 of 8 samples and 4 of 7 samples with p53 deletion. Annexin/PI staining yielded apoptotic cell death in the single agent and combination exposure with Venetoclax. Western blotting showed a modulation of both BRD4 proteins as well as Bcl-2 family member proteins (Figure 1b). Bcl-2 family profiling via Milliplex panels demonstrated the ratio of Bcl-2/Bcl x/L was much higher in samples with a high Venetoclax IC50 (136/1) compared to Venetoclax low IC50 (54/1). (Figure 1) -A) Samples with p53 deletion and corresponding synergy. (B) BCL2 family protein expression of CLL sample 214c. Conclusions: Our data indicates that a) PLX51107 has an antitumor effect in CLL cells; b) the combinational treatment of Venetoclax with PLX51107 highly improved the apoptotic effect of single agent treatments. A better understanding of the key signaling pathways for CLL cells' proliferation and survival impacted by BRD4i in combination with Venetoclax would provide a proof-of-concept rationale for studies validating BRD4i as epigenetic approach to target BCR signaling in CLL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Amyloid aggregates exert cell toxicity causing irreversible damages in the endoplasmic reticulum

Amyloid oligomers and fibrils are protein aggregates that cause an onset and progression of many neurodegenerative diseases, diabetes type 2 and systemic amyloidosis. Although a growing body of evidence shows that oligomers and fibrils trigger mitochondrial dysfunction simultaneously enhancing production of reactive oxygen species, exact mechanisms by which these protein aggregates exert their toxicities remain unclear. In this study, we used advanced microscopic and spectroscopic methods to examine topography and structure of insulin aggregates grown in the lipid-free environment, as well as in the presence of major classes of phospho- and sphingolipids. We also employed a set of molecular markers to determine the extent to which insulin aggregates induce a damage of cell endoplasmic reticulum (ER), an important cell organelle used for calcium storage, protein synthesis and folding. Our results show that insulin aggregates activate the expression of Activating Transcription Factor 6 (ATF6), a transmembrane protein that is involved in unfolded protein response (UPR) of the stressed ER. At the same time, two other ER transmembrane proteins, Inositol Requiring 1 (IRE1α) and eLF2a, the product of PKR-like ER kinase (PERK), exhibited very low expression levels. Furthermore, amyloid aggregates trigger an expression of the 78-kDa glucose-regulated protein GRP78, which is also involved in the UPR. We also observed UPR-induced expression of a proapoptotic transcription factor CHOP, which, in turn, regulates expression of caspase 3 kinase and BCL2 protein family members, including the ER localized Bax. These findings show that insulin oligomers and fibrils induce UPR-associated ER stress and ultimately fatal changes in cell homeostasis.

Virus-Mediated Inhibition of Apoptosis in the Context of EBV-Associated Diseases: Molecular Mechanisms and Therapeutic Perspectives.

Epstein-Barr virus (EBV), the representative of the Herpesviridae family, is a pathogen extensively distributed in the human population. One of its most characteristic features is the capability to establish latent infection in the host. The infected cells serve as a sanctuary for the dormant virus, and therefore their desensitization to apoptotic stimuli is part of the viral strategy for long-term survival. For this reason, EBV encodes a set of anti-apoptotic products. They may increase the viability of infected cells and enhance their resistance to chemotherapy, thereby contributing to the development of EBV-associated diseases, including Burkitt’s lymphoma (BL), Hodgkin’s lymphoma (HL), gastric cancer (GC), nasopharyngeal carcinoma (NPC) and several other malignancies. In this paper, we have described the molecular mechanism of anti-apoptotic actions of a set of EBV proteins. Moreover, we have reviewed the pro-survival role of non-coding viral transcripts: EBV-encoded small RNAs (EBERs) and microRNAs (miRNAs), in EBV-carrying malignant cells. The influence of EBV on the expression, activity and/or intracellular distribution of B-cell lymphoma 2 (Bcl-2) protein family members, has been presented. Finally, we have also discussed therapeutic perspectives of targeting viral anti-apoptotic products or their molecular partners.

Abstract 6257: Mutations in the ASXL1 and ZRSR2 genes are associated with the response to the combination of alvocidib and 5-azacytidine in higher-risk myelodysplastic syndromes

Abstract The hypomethylating agent 5-azacytidine (5-Aza) is a standard-of-care for patients with higher-risk myelodysplastic syndromes (MDS). Although an initial response is induced in approximately 50% of 5-Aza treated patients, subsequent relapse is almost certain. Recently, inhibitors of anti-apoptotic BCL-2 protein family members have shown therapeutic potential in acute myeloid leukemia (AML) and higher-risk MDS. Alvocidib (Alv), a CDK9 inhibitor and indirect transcriptional repressor of the anti-apoptotic factor MCL-1, has shown anti-leukemic effects in a phase 1 study of patients with AML (Lee DJ et al, Expert Opin Investig Drugs 2019; Zeidner JF et al, Leuk Res 2015). A phase 1b/2 study with Alv and 5-Aza or decitabine in higher-risk patients with MDS was recently completed (NCT03593915); however, biomarkers for response to the Alv and 5-Aza combination are not well characterized. To identify potential biomarkers of response, we performed a comprehensive in vitro assessment of Alv and 5-Aza combination using a clinically well-characterized cohort of n=45 higher-risk patients with MDS and n=11 healthy controls (HC). CD34+ cells were purified from bone marrow (BM) aspirates using positive selection with MACS beads. CD34+ cells of HC were obtained from femur head replacement surgery bone specimens. After 4 days of expansion in SFEM II medium containing StemSpan Myeloid Expansion Supplement, cells were treated with 5-Aza for 48h, Alv for 24h or their combination (5-Aza for 48h followed by Alv for 24h). Cell viability was determined using CellTiter-Glo (CTG) and Annexin-V apoptosis assays. MCL-1 dependency of MDS samples was assessed using MS1 peptide-based assay. Recurrent myeloid neoplasia mutations in 67 genes were assessed in BM mononuclear cells using NGS panel deep sequencing. The combination of 5-Aza+Alv had an additive cytotoxic effect on CD34+ MDS cells in CTG assay (median cell viability = 74%, 73.8% and 55% for 5-Aza, Alv and combination respectively, p<0.0001). In Annexin-V apoptosis assay, MDS samples were more sensitive to the combination treatment compared to HC (median % of apoptotic and dead cells = 36.6% for MDS vs 25.6% for HC, p=0.0288). MCL-1 dependency inversely and not significantly correlated with CD34+ cell viability in CTG assays (Spearman r=-0.37, p=0.1119). In contrast, we found significant associations between ASXL1 and ZRSR2 mutations and higher sensitivity of MDS samples to 5-Aza+Alv combination (p=0.008 and p=0.0005 in univariable analysis respectively). ZRSR2 mutations also retained an independent impact on cell viability in multivariable analysis (p=0.035). Overall, we provide pre-clinical support for the use of 5-Aza+Alv combination for higher-risk MDS and identified ASXL1 and ZRSR2 mutations as potential genetic biomarkers of augmented response. Citation Format: Vladimir Ryabov, Nanni Schmitt, Qingyu Xu, Alexander Streuer, Johann-Christoph Jann, Alina Wein, Eva Altrock, Verena Nowak, Nadine Weimer, Julia Obländer, Iris Palme, Ahmed Jawhar, Ali Darwich, Patrick Wuchter, Christel Weiss, Georgia Metzgeroth, Jason M. Foulks, Laurenz Steiner, Mohamad Jawhar, Wolf-Karsten Hofmann, Daniel Nowak. Mutations in the ASXL1 and ZRSR2 genes are associated with the response to the combination of alvocidib and 5-azacytidine in higher-risk myelodysplastic syndromes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6257.

Soluble egg antigen of Schistosoma japonicum induces macrophage apoptosis in mice

To investigate the dynamic changes of macrophage numbers and apoptosis during Schistosoma japonicum infection, and to investigate the possible mechanisms of macrophage apoptosis induced by S. japonicum soluble egg antigen (SEA). C57BL/6 mice at ages of 6~8 weeks were randomly divided into 4 groups, including three experimental groups and a normal control group. Each mouse in the experimental groups was infected with (12 ± 1) cercariae of S. japonicum via the abdominal skin, and all mice in an experimental group were sacrificed 3, 5, 8 weeks post-infection, respectively, while mice in the control group were not infected with S. japonicum cercariae and sacrificed on the day of S. japonicum infection in the experimental group. Mouse liver specimens and peritoneal exudation cells were sampled in each group, and the dynamic changes of macrophage numbers and apoptosis were detected. Mouse peritoneal macrophages were isolated, purified and treated with S. japonicum SEA, PBS and ovalbumin (OVA) in vitro, and the macrophage apoptosis was detected using flow cytometry. The mRNA and protein expression of BCL-2 protein family members were determined in macrophages using real-time quantitative PCR (qP-CR) and Western blotting assays, and the activation of caspase 3 was determined using flow cytometry and Western blotting. In addition, macrophages were in vitro treated with S. japonicum SEA in presence of a caspase inhibitor, H2O2 or N-acetyl-L-cysteine, and the apoptosis of macrophages was detected using flow cytometry. The total macrophage numbers continued to increase in mouse liver [(0.873 ± 0.106) × 106, (2.737 ± 0.460) × 106 and (3.107 ± 0.367) × 106 cells, respectively; F = 81.900, P < 0.01] and peritoneal specimens [(5.282 ± 1.136) × 105, (7.500 ± 1.200) × 105 and (12.800 ± 0.800) × 105 cells, respectively; F = 55.720, P < 0.01] 3, 5 and 8 weeks post-infection with S. japonicum, and the numbers of apoptotic macrophages also continued to increase in mouse liver [(0.092 ± 0.018) × 106, (0.186 ± 0.025) × 106 and (0.173 ± 0.0270) × 106 cells; F = 57.780, P < 0.01] and peritoneal specimens [(0.335 ± 0.022) × 105, (0.771 ± 0.099) × 105 and (1.094 ± 0.051) × 105 cells; F = 49.460, P < 0.01] 3, 5 and 8 weeks post-infection with S. japonicum. The apoptotic rate of SEA-treated macrophages [(24.330 ± 0.784)%] was significantly higher than that of PBS-[(18.500 ± 1.077)%] and OVA-treated macrophages [(18.900 ± 1.350)%] (both P values < 0.01). There were no significant differences in the mRNA or protein expression of Bcl-2 [Bcl - 2 mRNA expression: (1.662 ± 0.943) vs. (1.000 ± 0.000), t = 1.215, P > 0.05; BCL protein expression: (0.068 ± 0.004) vs. (0.070 ± 0.005), t = 0.699, P > 0.05], Bax [Bax mRNA expression: (0.711 ± 0.200) vs. (1.000 ± 0.000), t = 2.507, P > 0.05; BAX protein expression: (0.089 ± 0.005) vs. (0.097 ± 0.003), t = 2.232, P > 0.05] and Bak [Bak mRNA expression: (1.255 ± 0.049) vs. (1.00 ± 0.00), t = 0.897, P > 0.05; BAK protein expression: (0.439 ± 0.048) vs. (0.571 ± 0.091), t = 2.231, P > 0.05] between in SEA- and PBS-treated macrophages. S. japonicum SEA induced macrophage apoptosis in the presence of a caspase inhibitor (F = 0.411, P > 0.05); however, SEA failed to induce macrophage apoptosis in the presence of H2O2 or NAC (F = 11.880 and 9.897, both P values < 0.05). S. japonicum SEA may induce macrophage apoptosis through promoting reactive oxygen species expression during S. japonicum infections in mice.

Design, synthesis and biological evaluation of hydantoin derivatives as Mcl-1 selective inhibitors

As a member of Bcl-2 protein family, myeloid cell leukemia-1 (Mcl-1) plays a critical role in cell apoptosis and has become a promising anti-cancer drug target. Herein, we designed and synthesized a series of hydantoin derivatives as novel Mcl-1 inhibitors based on our previously developed lead compound. Among them, compound M23 and M24 exhibited good binding affinities against Mcl-1 with Ki values of 0.49 μM and 0.33 μM respectively. Especially, compound M23 exhibited good selectivity over Bcl-xL, whereas compound M24 possessed good selectivity over both Bcl-2 and Bcl-xL. Furthermore, we also investigated the effects of these new Mcl-1 inhibitors on cell proliferation, apoptosis and mitochondrial membrane potential, as well as the stability in plasma.

The deubiquitinating enzyme USP20 regulates the stability of the MCL1 protein

Chemoresistance is a major obstacle faced by oesophageal cancer patients and is synonymous with a poor prognosis. MCL1 is a pivotal member of the anti-apoptotic Bcl-2 protein family, which has been found to play an important role in cell survival, proliferation, differentiation and chemoresistance. Thus, it might be an ideal target for treating oesophageal cancer patients. Although it is known that MCL1 is degraded via the ubiquitin-proteasome system, the deubiquitylating enzyme (DUB) responsible for stabilizing MCL1 remains elusive to date. Herein, we demonstrate that Ubiquitin-Specific Protease 20 (USP20) is a novel regulator of the apoptotic signaling pathway. Moreover, USP20 could regulate the deubiquitination of MCL1 to, in turn, regulate its stability. Increased expression of USP20 was correlated with increased levels of MCL1 protein in human patient samples. In addition, depletion of USP20 could increase the polyubiquitination of MCL1, thereby increasing the conversion rate of MCL1 and the sensitivity of cells to chemotherapy. Overall, our findings indicate that the USP20-MCL1 axis might play a key role in the apoptotic signaling pathway.

Bcl-2-like protein-10 increases aggressive features of melanoma cells

Aim:B-cell lymphoma-2 (Bcl-2)-like protein-10 (Bcl2L10) is the less studied member of Bcl-2 family proteins, with the controversial role in different cancer histotypes. Very recently, Bcl2L10 expression in melanoma tumor specimens and its role in melanoma response to therapy have been demonstrated. Here, the involvement of Bcl2L10 on the in vitro and in vivo properties associated with melanoma aggressive features has been investigated.Methods:Endogenous Bcl2L10 protein expression was detected by western blotting analysis in a panel of patient-derived and commercially available human melanoma cells. In vitro assays to evaluate clonogenicity, cell proliferation, cell migration, cell invasion, and in vitro capillary-like structure formation [vasculogenic mimicry (VM)] have been performed by using human melanoma cells stably overexpressing Bcl2L10 or transiently transfected for loss/gain function of Bcl2L10, grown under two- or three-dimensional (3D) conditions Xenograft melanoma model was employed to evaluate in vivo tumor growth and angiogenesis.Results:Results demonstrated that Bcl2L10 acts as an inducer of in vitro cell migration, invasion, and VM, while in vitro cell proliferation, in vivo tumor growth, as well as colony formation properties were not affected. Dissecting different signaling pathways, it was found that Bcl2L10 positively affects the phosphorylation of extracellular-signal-regulated kinase (ERK) and the expression of markers of cell invasion, such as urokinase plasminogen activator receptor (uPAR) and matrix metalloproteinases (MMPs). Of note, Bcl2L10-dependent in vitro migration, invasion, and VM are linked to uPAR. Bcl2L10 also negatively regulates the intracellular calcium level. Finally, reduced invasion capability in 3D spheroid invasion assay of melanoma cells transiently overexpressing Bcl2L10 was observed after treatment with inhibitors of MMPs and uPAR.Conclusions:Overall, data reported in this paper provide evidence supporting a positive role of Bcl2L10 in melanoma aggressive features.

Targeting the anti-apoptotic Bcl-2 family proteins: machine learning virtual screening and biological evaluation of new small molecules.

Bcl-2 family anti-apoptotic proteins are overexpressed in several hematological and solid tumors, and contribute to tumor formation, progression, and resistance to therapy. They represent a promising therapeutic avenue to explore for cancer treatment. Venetoclax, a Bcl-2 inhibitor is currently used for hematological malignancies or is undergoing clinical trials for either hematological or solid tumors. Despite these progresses, ongoing efforts are focusing on the identification and development of new molecules targeting Bcl-2 protein and/or other family members.Methods: Machine learning guided virtual screening followed by surface plasmon resonance, molecular docking and pharmacokinetic analyses were performed to identify new inhibitors of anti-apoptotic members of Bcl-2 family and their pharmacokinetic profile. The sensitivity of cancer cells from different origin to the identified compounds was evaluated both in in vitro (cell survival, apoptosis, autophagy) and in vivo (tumor growth in nude mice) preclinical models.Results: IS20 and IS21 were identified as potential new lead compounds able to bind Bcl-2, Bcl-xL and Mcl-1 recombinant proteins. Molecular docking investigation indicated IS20 and IS21 could bind into the Beclin-1 BH3 binding site of wild type Bcl-2, Bcl-xL and Mcl-1 proteins. In particular, although the IS21 docked conformation did not show a unique binding mode, it clearly showed its ability in flexibly adapting to either BH3 binding sites. Moreover, both IS20 and IS21 reduced cell viability, clonogenic ability and tumor sphere formation, and induced apoptosis in leukemic, melanoma and lung cancer cells. Autophagosome formation and maturation assays demonstrated induction of autophagic flux after treatment with IS20 or IS21. Experiments with z-VAD-fmk, a pan-caspase inhibitor, and chloroquine, a late-stage autophagy inhibitor, demonstrated the ability of the two compounds to promote apoptosis by autophagy. IS21 also reduced in vivo tumor growth of both human leukemia and melanoma models.Conclusion: Virtual screening coupled with in vitro and in vivo experimental data led to the identification of two new promising inhibitors of anti-apoptotic proteins with good efficacy in the binding to recombinant Bcl-2, Bcl-xL and Mcl-1 proteins, and against different tumor histotypes.

Preclinical Assessment of Alvocidib in Combination with 5-Azacytidine in High-Risk Myelodysplastic Syndromes

Hypomethylating therapy with 5-azacytidine (5-Aza) is a standard-of-care for patients with higher-risk myelodysplastic syndromes (MDS). Response is induced in approximately 50% of 5-Aza treated patients. However, despite robust efficacy in responders, relapse is almost certain. Recently, inhibitors of anti-apoptotic BCL-2 protein family members have shown potent activity against AML and higher-risk MDS in combination with 5-Aza. Alvocidib (Alv), a cyclin-dependent kinase 9 inhibitor and indirect transcriptional repressor of the anti-apoptotic BCL-2 family member MCL-1, has shown anti-leukemic effects in combination with 5-Aza in a phase 1 study of AML (Lee DJ et al, Expert Opin Investig Drugs 2019; Zeidner JF et al, Leuk Res 2015). Additionally, Alv has entered a phase 1b/2 study in patients with higher-risk MDS (NCT03593915). In order to possibly identify biomarkers of response, we performed a comprehensive pre-clinical in vitro assessment of Alv combined with 5-Aza using a clinically well-characterized cohort of n=40 MDS (high risk) patients and n=11 healthy controls.CD34+ HSCs were purified from bone marrow (BM) aspirates using positive selection with MACS microbeads. CD34+ cells of healthy controls were obtained from femur head replacement surgery bone specimens. Hematopoietic stem cells (HSCs) were expanded for four days in StemSpan SFEM II medium containing StemSpan Myeloid Expansion Supplement (Stem Cell Technologies) and treated with 5-Aza for 48h, Alv for 24h or their sequential combination (5-Aza for 48h followed by Alv for 24h). Cell viability was determined using CellTiter-Glo and Annexin-V apoptosis assays. MDS recurrent mutations in BM mononuclear cells were assessed using myeloid NGS panel deep sequencing containing 67 genes.The combination of 5-Aza+Alv showed an additive cytotoxic effect on CD34+ MDS cells in CellTiter- Glo cell viability assays (median cell viability = 74%, 73.8% and 55% for 5-Aza, Alv and combination respectively, p<0.0001). In annexin-V apoptotic assay, MDS cells were more sensitive to the cytotoxic effect of the combination treatment compared to healthy CD34+ cells (median % of apoptotic and dead cells = 36.6% for MDS vs 25.6% for healthy group, p=0.0288). Of note, the presence of ASXL1 and ZRSR2 mutations was associated with higher cytotoxic activity of 5-Aza+Alv combination. In particular, ZRSR2 mutations had an independent impact on the cell viability in a multivariable analysis (p=0.035). Overall, we provided pre-clinical support for the use of 5-Aza+Alv combination for higher risks MDS and identified ASXL1 and ZRSR2 mutations as potential genetic biomarkers of response. DisclosuresSchmitt: Affimed GmbH: Research Funding. Jawhar: Celgene: Other: Travel support; Takeda: Honoraria, Other: Travel support; Stemline: Consultancy, Honoraria; Blueprint Medicines: Honoraria; Novartis: Consultancy, Honoraria, Other: Travel support, Speakers Bureau. Foulks: Sumitomo Dainippon Pharma Oncology: Patents & Royalties: WO2021102343A1; Sumitomo Dainippon Pharma Oncology: Patents & Royalties: CA3103995A1; Sumitomo Dainippon Pharma Oncology: Patents & Royalties: US11040038B2. Hofmann: Amgen: Honoraria; BMS: Honoraria; Novartis: Honoraria. Nowak: Celgene: Honoraria; Takeda: Honoraria; Affimed: Research Funding; Pharmaxis: Current holder of individual stocks in a privately-held company, Research Funding; AbbVie: Other: Investigator on funded clinical trial; Tolero Pharma, Pharmaxis, Apogenix: Research Funding.

The manipulation of apoptosis for cancer therapy using BH3-mimetic drugs.

Apoptosis is a form of programmed cell death that is regulated by the balance between prosurvival and proapoptotic BCL-2 protein family members. Evasion of apoptosis is a hallmark of cancer that arises when this balance is tipped in favour of survival. One form of anticancer therapeutic, termed 'BH3-mimetic drugs', has been developed to directly activate the apoptosis machinery in malignant cells. These drugs bind to and inhibit specific prosurvival BCL-2 family proteins, thereby mimicking their interaction with the BH3 domains of proapoptotic BCL-2 family proteins. The BCL-2-specific inhibitor venetoclax is approved by the US Food and Drug Administration and many regulatory authorities worldwide for the treatment of chronic lymphocytic leukaemia and acute myeloid leukaemia. BH3-mimetic drugs targeting other BCL-2 prosurvival proteins have been tested in preclinical models of cancer, and drugs targeting MCL-1 or BCL-XL have advanced into phase I clinical trials for certain cancers. As with all therapeutics, efficacy and tolerability need to be carefully balanced to achieve a therapeutic window whereby there is significant anticancer activity with an acceptable safety profile. In this Review, we outline the current state of BH3-mimetic drugs targeting various prosurvival BCL-2 family proteins and discuss emerging data regarding primary and acquired resistance to these agents and approaches that may overcome this. We highlight issues that need to be addressed to further advance the clinical application of BH3-mimetic drugs, both alone and in combination with additional anticancer agents (for example, standard chemotherapeutic drugs or inhibitors of oncogenic kinases), for improved responses in patients with cancer.

THE MCL-1 INHIBITOR AZD5991 DISPLAYS A PROMISING TARGETED THERAPY FOR ACUTE MYELOID LEUKEMIA

MCL-1 protein is an anti-apoptotic member of BCL-2 protein family. In hematological neoplasms, increased MCL-1 gene expression or protein activity is related to disease relapse, poor prognosis and resistance to chemotherapy, especially to Venetoclax, an BCL-2 inhibitor. The present study aimed at evaluating the MCL-1 gene expression in AML patients as well as investigating the effects of MCL-1 protein inhibition in myeloid cells. Analysis of MCL-1 gene expression was performed in a cohort of 286 AML patients (OHSU/Nature 2018 study at cbioportal.org). Leukemia myeloid cell lines (U937, KG1a, OCI-AML3) were treated with increasing concentrations of the MCL-1 inhibitor compound AZD5991 (AstraZeneca) for 48, 72 and 96h for determination of the 50% inhibiting concentration (IC 50 ). Apoptosis (annexin-V stain), reactive oxygen species (ROS) production (DFCHA dye) and autophagy (acridine orange dye) were assessed by flow cytometry, after 48h of treatment. Coculture assays were performed by adding leukemia cells onto a monolayer of HS5 mesenchymal stromal cells, followed by treatment with AZD5991 for 48h. Statistical analyzes were performed using ANOVA, Mann-Whitney or Kruskal Wallis tests. p-value < .05 was considered significant. Increased MCL-1 gene expression in AML patients with adverse cytogenetic risk was observed when compared to patients with favorable or intermediate cytogenetic risk (p < .001). All leukemia cell lines showed dose and time-dependent sensitivity with AZD5991. IC 50 values of 4.3, 3.4 and 9.2μM in U937; 20.0, 15.2 and 14.9μM in KG1a; and 550, 380 and 420nM in OCI-AML3, for 48, 72 and 96h, respectively. Treatment with AZD5991 (0.5-20μM) in OCI-AML3, U937 and KG1a cells significantly reduced cell viability (69.2%, 50.3%, 40.6%) and increased apoptosis (68.2%, 90.1%, 92.2%) rate in a time and dose-dependent manner, with greater intensity in OCI-AML3 cells. AZD5991 was able to reduce ROS production at 15.1% and 17.2% in U937, 10.3% and 5.6% in KG1a, 59.4% and 45.8% in OCI-AML3, in monoculture and coculture, respectively (p < .05). Similarly, there was a reduction in the formation of acidic vesicles (autophagic process) at 43.0% and 19.0% in U937, 40.5% and 19.0% in KG1a, 62.6% and 18.8% in OCI-AML3, in monoculture and coculture, respectively (p < .05). Increased cell percentage in G0/G1 phases of cell cycle was also observed. Western blot analyses showed decreased MCL-1, Bax and Bak and increased Bim. MCL-1 overexpression is related with AML progression, and associated with poor prognosis (Li et al. Onco Targets Ther. 2019;12:3295-3304). Our analysis from OHSU data bank showed that patients with adverse cytogenetic risk presented higher MCL-1 gene expression that could induce resistance to pharmacological therapies. Our in vitro studies demonstrated that the MCL1 inhibitor AZD5991 induced apoptosis and reduced viability of leukemia cells, possibly by MCL-1-Bak complex disruption and subsequent Bak-dependent mitochondrial apoptotic pathway activation. The diminished MCL-1 availability in mitochondria, probably caused lower ROS production and impaired autophagy (Mukhopadhyay et al. Apoptosis. 2014;19:555-66). Therefore, these findings provide important tools for further investigations and highlight that MCL-1 inhibitor could be a promising compound for AML treatment due to its potent antitumor activity and may represent a potential therapeutic strategy. FAPESP e CNPq.

Immunomolecular evaluation of dihydroartemisinin effects on apoptosis in chronic lymphocytic leukemia cell lines

IntroductionDihydroartemisinin (DHA), a semi-synthetic derivative of artemisinin, has recently shown to induce apoptosis in many types of cancer cells. In this study, we aimed to determine the effects of DHA on apoptosis in human chronic lymphocytic leukemia (CLL) cell lines. MethodsThe cells were treated separately and combined by DHA and Fludurabine (FLU) during 24, 48 and 72 hours. The cell viabilities determined by XTT method. Following separate and combined treatment of IC50 concentrations of DHA and FLU to the cells during 24 hours, the cells were analyzed by flow cytometry to determine the effects on apopotis staining with AnnexinV FITC and PI. mRNA and protein expression levels of TCTP, Mcl-1, Bcl-2, Bax and Caspase-3 were analyzed to find out the molecular mechanisms of apoptosis by using quantitative real-time PCR and flow cytometric methods. ResultsTreatment with DHA alone or in combination with FLU induced apoptosis in a dose dependent manner in CLL cells. DHA alone was more effective than FLU alone or combined treatment with DHA and FLU. Our results suggest that Bcl-2 protein family member Bax was active in the apoptotic response of CLL cells after DHA treatment. Moreover, the apoptotic response induced by DHA was independent from the p53 mutation status of the CLL cells. ConclusionDHA might be a potential anti-cancer therapeutic for CLL.

Kinome-wide RNAi screening for mediators of ABT-199 resistance in breast cancer cells identifies Wee1 as a novel therapeutic target

Antiapoptotic and proapoptotic BCL-2 protein family members regulate mitochondrial apoptotic pathway. Small molecule inhibitors of antiapoptotic BCL-2 proteins including BCL-2-specific inhibitor ABT-199 (Venetoclax) are in clinical development. However, the efficiency of ABT-199 as a single agent in solid tumors is limited. We performed a high-throughput RNAi kinome screen targeting 691 kinases to identify potentially targetable kinases to enhance ABT-199 response in breast cancer cells. Our studies identified Wee1 as the primary target kinase to overcome resistance to ABT-199. Depletion of Wee1 by siRNA-mediated knockdown or inhibition of Wee1 by the small molecule Wee1 inhibitor AZD1775 sensitized SKBR3, MDA-MB-468, T47D and CAMA-1 breast cancer cells to ABT-199 along with decreased MCL1. BH3-only proteins PUMA and BIM functionally contribute to apoptosis signaling following co-targeting BCL-2 and Wee1. Suppression of Wee1 function increased mitochondrial cell death priming. Furthermore, we found that Wee1 inhibition altered MCL1 phosphorylation and protein stability, which led to HUWE1-mediated MCL1 degradation. Our findings suggest that Wee1 inhibition can overcome resistance to ABT-199 and provide a rationale for further translational investigation of BCL-2 inhibitor/Wee1 inhibitor combination in breast cancer.

Redox Imbalance and Mitochondrial Release of Apoptogenic Factors at the Forefront of the Antitumor Action of Mango Peel Extract.

Today, an improved understanding of cancer cell response to cellular stress has become more necessary. Indeed, targeting the intracellular pro-oxidant/antioxidant balance triggering the tumor commitment to cell demise could represent an advantageous strategy to develop cancer-tailored therapies. In this scenario, the present study shows how the peel extract of mango—a tropical fruit rich in phytochemicals with nutraceutical properties—can affect the cell viability of three colon cancer cell lines (HT29, Caco-2 and HCT116), inducing an imbalance of cellular redox responses. By using hydro-alcoholic mango peel extract (MPE), we observed a consistent decline in thiol group content, which was accompanied by upregulation of MnSOD—a mitochondrial scavenger enzyme that modulates the cellular response against oxidative damage. Such an effect was the consequence of an early production of mitochondrial superoxide anions that appeared after just 30 min of exposure of colon cancer cells to MPE. The effect was accompanied by mitochondrial injury, consisting of the dissipation of mitochondrial membrane potential and a decrease in the level of proteins localized in the mitochondrial membrane—such as voltage-dependent anion-selective channel (VDAC1), mitofilin, and some members of Bcl-2 family proteins (Mcl-1, Bcl-2 and Bcl-XL)—with the mitochondrial release of apoptogenic factors (cytochrome C and AIF). The analysis of the cytotoxic effects exerted by the different constituents of MPE (gallic acid, mangiferin, citric acid, quinic acid, pentagalloyl glucose, and methyl gallate) allowed us to identify those phytochemicals responsible for the observed anticancer effects, sustaining their future employment as chemopreventive or therapeutic agents.