B-cell Lymphoma-2 Family Research Articles

Targeting multifunctional magnetic nanowires for drug delivery in cancer cell death: an emerging paradigm.

Apoptosis, often known as programmed cell death is a mechanism used by numerous species to maintain tissue homeostasis. The process leading to cell death is complicated because it requires the stimulation of caspases. According to several studies, nanowires have important medical benefits, can kill cells by adhering to cancer cells, destroying them, and killing the entire cell using a triple attack that integrates vibration, heat, and drug delivery to trigger apoptosis. The sewage effluents and industrial, fertilizer and organic wastes decomposition can produce elevated levels of chemicals in the environment which may interrupt the cell cycle and activate apoptosis. The purpose of this review is to give a thorough summary of the evidence that is currently available on apoptosis. Current review discussed topics like the morphological and biochemical alterations that occur during apoptosis, as well as the various mechanisms that cause cell death, including the intrinsic (or mitochondrial), extrinsic (or death receptor), and intrinsic endoplasmic reticulum pathway. The apoptosis reduction in cancer development is mediated by (i) an imbalance between pro- and anti-apoptotic proteins, such as members of the B-cell lymphoma-2 (BCL2) family of proteins, tumour protein 53 and inhibitor of apoptosis proteins, (ii) a reduction in caspase activity, and (iii) impaired death receptor signalling. This review does an excellent task of outlining the function of nanowires in both apoptosis induction and targeted drug delivery for cancer cells. A comprehensive summary of the relevance of nanowires synthesised for the purpose of inducing apoptosis in cancer cells has been compiled collectively.

Inhibition of antiapoptotic BCL-2 proteins with ABT-263 induces fibroblast apoptosis, reversing persistent pulmonary fibrosis.

Patients with progressive fibrosing interstitial lung diseases (PF-ILDs) carry a poor prognosis and have limited therapeutic options. A hallmark feature is fibroblast resistance to apoptosis, leading to their persistence, accumulation, and excessive deposition of extracellular matrix. A complex balance of the B cell lymphoma 2 (BCL-2) protein family controlling the intrinsic pathway of apoptosis and fibroblast reliance on antiapoptotic proteins has been hypothesized to contribute to this resistant phenotype. Examination of lung tissue from patients with PF-ILD (idiopathic pulmonary fibrosis and silicosis) and mice with PF-ILD (repetitive bleomycin and silicosis) showed increased expression of antiapoptotic BCL-2 family members in α-smooth muscle actin-positive fibroblasts, suggesting that fibroblasts from fibrotic lungs may exhibit increased susceptibility to inhibition of antiapoptotic BCL-2 family members BCL-2, BCL-XL, and BCL-W with the BH3 mimetic ABT-263. We used 2 murine models of PF-ILD to test the efficacy of ABT-263 in reversing established persistent pulmonary fibrosis. Treatment with ABT-263 induced fibroblast apoptosis, decreased fibroblast numbers, and reduced lung collagen levels, radiographic disease, and histologically evident fibrosis. Our studies provide insight into how fibroblasts gain resistance to apoptosis and become sensitive to the therapeutic inhibition of antiapoptotic proteins. By targeting profibrotic fibroblasts, ABT-263 offers a promising therapeutic option for PF-ILDs.

Iridium(III) complexes inhibit the proliferation and migration of BEL-7402 cells through the PI3K/AKT/mTOR signaling pathway

Iridium(III) complexes are largely studied as anti-cancer complexes due to their excellent anti-cancer activity. In this article, two new iridium(III) complexes [Ir(piq)2(THPIP)]PF6 (THPIP = 2,4-di-tert-butyl-6-(1H-imidazo[4,5-f][1,10]phenanthrolin-2-yl)phenol, piq = deprotonated 1-phenylisoquinoline) (Ir1) and [Ir(bzq)2(THPIP)]PF6 (bzq = deprotonated benzo[h]quinolone) (Ir2) were synthesized. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays showed that complex Ir1 exhibits moderate activity (IC50 = 29.9 ± 4.6 μM) and Ir2 shows high cytotoxicity (IC50 = 9.8 ± 1.8 μM) against BEL-7402 cells. Further studies on the mechanism showed that Ir1 and Ir2 induced apoptosis by changing the mitochondrial membrane potential, Ca2+ release, ROS accumulation, and cell cycle arrest at the S phase. The complexes can effectively inhibit cell colony formation and migration. The expression of B-cell lymphoma-2 (Bcl-2) family proteins, PI3K (phosphatidylinositol 3-kinase), AKT (protein kinase B), mTOR (mammalian target of rapamycin), and p-mTOR was studied by immunoblotting. Complexes Ir1 and Ir2 downregulated the expression of anti-apoptotic protein Bcl-2 and increased the expression of autophagy-related proteins of Beclin-1 and LC3-II. Further experiments showed that the complexes inhibited the production of glutathione (GSH) and increased the amounts of malondialdehyde (MDA). Fluorescence of HMGB1 was significantly increased. We also investigated the effect of the complexes on the expression of genes using RNA-sequence analysis, we further calculated the lowest binding energies between the complexes and proteins using molecular docking. Taken together, the above results indicated that complexes Ir1 and Ir2 induce apoptosis in BEL-7402 cells through a ROS-mediated mitochondrial dysfunction and inhibition of the PI3K/AKT/mTOR signaling pathway.

The endoplasmic reticulum stress and B cell lymphoma-2 related ovarian killer participate in docosahexaenoic acid-induced adipocyte apoptosis in grass carp (Ctenopharyngodon idellus).

Docosahexaenoic acid (DHA) lessens adipose tissue lipid deposition partly by inducing adipocyte apoptosis in grass carp, but the underlying mechanism remains unclear. Endoplasmic reticulum (ER) stress and unfolded protein response (UPR) is the novel pathway for inducing apoptosis. This study aimed to explore the potential role of ER stress in DHA-induced apoptosis in grass carp (Ctenopharyngodon idellus) adipocytes. DHA induced apoptosis by deforming the nuclear envelope, condensing the chromatin, and increasing the expression of apoptosis-related proteins and genes in vivo and in vitro (P < 0.05). However, the ER stress inhibitor, 4-phenylbutyric acid (4-PBA), effectively suppressed DHA-induced apoptosis (P < 0.05), indicating that ER stress mediates DHA-induced adipocyte apoptosis. Furthermore, we observed that 200 μM DHA significantly up-regulates the transcripts of B cell lymphoma-2 (BCL-2) related ovarian killer (BOK) in vitro (P < 0.05). BOK is a pro-apoptotic protein in the BCL-2 family, which governs the mitochondria apoptosis pathway. Hence, we hypothesized that BOK might be an important linker between ER stress and apoptosis. We cloned and identified two grass carp BOK genes, BOKa and BOKb, which encode peptides of 213 and 216 amino acids, respectively. BOKa primarily localizes in ER and mitochondria in the cytoplasm, while BOKb localizes in the nucleus and cytoplasm of grass carp adipocytes. Moreover, 200 μM DHA treatment up-regulated the mRNA expression of BOKa and BOKb, whereas 4-PBA suppressed the DHA-induced expressions. These results raised the possibility that BOK participates in DHA-induced adipocyte apoptosis through ER stress signaling, in line with its localization in ER and mitochondria. Two UPR branches, the inositol-requiring enzyme 1 (IRE1α) and activating transcription factor 6 (ATF6) signaling pathways, are possibly important in DHA-induced adipocyte apoptosis, unlike protein kinase RNA-activated-like ER kinase. The study also emphasized the roles of BOKa and BOKb in IRE1α- and ATF6-mediated apoptosis. This work is the first to elucidate the importance of the ER stress-BOK pathway during adipocyte apoptosis in teleost.

Low Bcl-2 is a robust biomarker of sensitivity to nab-paclitaxel in Ewing sarcoma

Nanoparticle albumin-bound paclitaxel (nab-paclitaxel) shows potent preclinical anticancer activity in pediatric solid tumors such as Ewing sarcoma, rhabdomyosarcoma and neuroblastoma, but responses in clinical trials have been modest. In this work, we aimed to discover a rational biomarker-based approach to select the right candidate patients for this treatment. We assessed the efficacy of nab-paclitaxel in 27 patient-derived xenografts (PDX), including 14 Ewing sarcomas, five rhabdomyosarcomas and several other pediatric solid tumors. Response rate (partial or complete response) was remarkable in rhabdomyosarcomas (four of five) and Ewing sarcomas (four of 14). We addressed several predictive factors of response to nab-paclitaxel such as the expression of the secreted protein acidic and rich in cysteine (SPARC), chromosomal stability of cancer cells and expression of antiapoptotic members of the B-cell lymphoma-2 (Bcl-2) family of proteins such as Bcl-2, Bcl-xL, Bcl-W and Mcl-1. Protein (immunoblotting) and gene expression of SPARC correlated positively, while immunoblotting and immunohistochemistry expression of Bcl-2 correlated negatively with the efficacy of nab-paclitaxel in Ewing sarcoma PDX. The negative correlation of Bcl-2 immunoblotting signal and activity was especially robust (r = 0.8352; P = 0.0007; Pearson correlation). Consequently, we evaluated pharmacological strategies to inhibit Bcl-2 during nab-paclitaxel treatment. We observed that the Bcl-2 inhibitor venetoclax improved the activity of nab-paclitaxel in highly resistant Bcl-2-expressing Ewing sarcoma PDX. Overall, our results suggest that low Bcl-2 expression could be used to select patients with Ewing sarcoma sensitive to nab-paclitaxel, and Bcl-2 inhibitors could improve the activity of this drug in Bcl-2-expressing Ewing sarcoma.

Neuroprotective effects of chlorogenic acid by controlling the Bcl-2 family protein in a stroke animal model.

Ischemic stroke causes severe brain damage and high mortality. Chlorogenic acid is a phenolic compound that has neuroprotective properties. B-cell lymphoma-2 (Bcl-2) family proteins are important for apoptosis regulation. Bcl-2 and Bcl-xL are proteins that inhibit apoptosis, and Bax and Bad induce apoptosis. In this study, we investigated whether chlorogenic acid exerts a neuroprotective effect against ischemic stroke damage by regulating Bcl-2 family proteins. We performed middle cerebral artery occlusion (MCAO) to induce ischemic stroke in adult male rats. The animals were intraperitoneally injected with normal saline as a vehicle or chlorogenic acid (30 mg/kg) 2 hr after MCAO. Cerebral cortex tissue was collected 24 hr after MCAO damage. MCAO damage caused histopathological changes and increased the number of terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick end labeling-positive cells, while chlorogenic acid attenuated these changes. RT-qPCR and Western blot results showed decreases in Bcl-2 and Bcl-xL expression and an increase in Bax and Bad expression in MCAO animals. However, chlorogenic acid treatment attenuated these changes due to MCAO damage. The interaction of Bax with Bcl-2 or Bcl-xL decreased in MCAO animals, and the binding of Bad with Bcl-2 or Bcl-xL increased. However, chlorogenic acid treatment reduced these changes. Chlorogenic acid also prevented MCAO-induced increases in caspase-3 and caspase-9 expression. This study provides evidence that chlorogenic acid has neuroprotective effects against MCAO damage by modulating Bcl-2 family proteins including Bcl-2, Bcl-xL, Bax, and Bad. Furthermore, chlorogenic acid regulates the interaction between Bcl-2 family proteins. In conclusion, chlorogenic acid contributes to neuroprotection against ischemic stroke damage by controlling Bcl-2 family proteins.

Cancer-cell-membrane-camouflaged supramolecular self-assembly of antisense oligonucleotide and chemodrug for targeted combination therapy.

The anti-apoptotic B-cell lymphoma-2 (Bcl-2) family of proteins are critical regulators of cell death that are overexpressed in many cancer cells, especially in multi-drug resistant cancer cells. Combinatorial gene- and chemotherapies using antisense oligonucleotides (ASOs) to suppress the expression of Bcl-2-family mRNA and restore the sensitivity of the cell to chemodrugs provide a promising pathway for anticancer treatment. However, intrinsic differences between macromolecular ASOs and small molecular chemodrugs make their co-delivery challenging. Moreover, extraneous carriers may induce immunogenicity and inflammation problems. Herein, we develop a targeted nanodrug delivery system using the cationic amphiphilic chemodrug mitoxantrone (Mito), which interacts with Bcl-2 ASO through electrostatic interaction and self-assembles into nanoparticles (NP[Bcl-2/Mito]), whose size can be controlled by regulating the ratio of ASO and Mito. NP[Bcl-2/Mito] can protect the ASO from degradation during delivery and combine gene- and chemotherapies to improve the anticancer effect. Furthermore, cancer cell membranes (CCMs) derived from homologous tumors were used to camouflage NP[Bcl-2/Mito] (NP[Bcl-2/Mito]@CCM) to achieve immune escape and tumor targeting. Both in vitro and in vivo assessments demonstrate the excellent performance of NP[Bcl-2/Mito]@CCM for drug-resistant breast tumor therapy. This CCM-camouflaged ASO/chemodrug nanoplatform provides a promising pathway for the targeted delivery of ASOs and chemodrugs for tumor combination therapy.

B-cell lymphoma-2 family proteins-activated proteases as potential therapeutic targets for influenza A virus and severe acute respiratory syndrome coronavirus-2: Killing two birds with one stone?

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has led to a global health emergency. There are many similarities between SARS-CoV-2 and influenza A virus (IAV); both are single-stranded RNA viruses infecting airway epithelial cells and have similar modes of replication and transmission. Like IAVs, SARS-CoV-2 infections poses serious challenges due to the lack of effective therapeutic interventions, frequent appearances of new strains of the virus, and development of drug resistance. New approaches to control these infectious agents may stem from cellular factors or pathways that directly or indirectly interact with viral proteins to enhance or inhibit virus replication. One of the emerging concepts is that host cellular factors and pathways are required for maintaining viral genome integrity, which is essential for viral replication. Although IAVs have been studied for several years and many cellular proteins involved in their replication and pathogenesis have been identified, very little is known about how SARS-CoV-2 hijacks host cellular proteins to promote their replication. IAV induces apoptotic cell death, mediated by the B-cell lymphoma-2 (Bcl-2) family proteins in infected epithelia, and the pro-apoptotic members of this family promotes viral replication by activating host cell proteases. This review compares the life cycle and mode of replication of IAV and SARS-CoV-2 and examines the potential roles of host cellular proteins, belonging to the Bcl-2 family, in SARS-CoV-2 replication to provide future research directions.

Promotion of diet-induced obesity and metabolic syndromes by BID is associated with gut microbiota.

A growing body of evidence has indicated an expanding functional network of B-cell lymphoma 2 (BCL-2) family proteins beyond regulation of cell death and survival. Here, we examined the role and mechanisms of BH3 interacting-domain death agonist (BID), a pro-death BCL-2 family member, in the development of diet-induced metabolic dysfunction. Mice deficient in bid (bid-/- ) were resistant to high-fat diet (HFD)-induced obesity, hepatic steatosis, and dyslipidemia with an increased insulin sensitivity. Indirect calorimetry analysis indicated that bid deficiency increased metabolic rate and decreased respiratory exchange ratio, suggesting a larger contribution of lipids to overall energy expenditure. While expression of several genes related to lipid accumulation was only increased in wild-type livers, metabolomics analysis revealed a consistent reduction in fatty acids but an increase in certain sugars and Krebs cycle intermediates in bid-/- livers. Gut microbiota (GM) analysis indicated that HFD induced gut dysbiosis with differential patterns in wild-type and in bid-/- mice. Notably, abrogation of GM by antibiotics during HFD feeding eliminated the beneficial effects against obesity and hepatic steatosis conferred by the bid deficiency. Conclusion: These results indicate that the protective role of bid-deficiency against diet-induced metabolic dysfunction interacts with the function of GM.

SOHO State of the Art Updates and Next Questions | Mechanisms of Resistance to BCL2 Inhibitor Therapy in Chronic Lymphocytic Leukemia and Potential Future Therapeutic Directions.

Chronic lymphocytic leukaemia (CLL) constitutively overexpresses B-cell lymphoma 2 (BCL2) with consequent dysregulation of intrinsic apoptosis leading to abnormal cellular survival. Therapeutic use of BCL2 inhibitors (BCL2i, eg, venetoclax) in CLL, as both continuous monotherapy or in fixed duration combination, has translated scientific rationale into clinical benefit with significant rates of complete responses, including those without detectable minimal residual disease. Unlike with chemotherapy, response rates to venetoclax do not appear to be influenced by pre-existing chromosomal abnormalities or somatic mutations present, although the duration of response observed remains shorter for those with traditional higher risk genetic aberrations. This review seeks to describe both the disease factors that influence primary venetoclax sensitivity/resistance and those resistance mechanisms that may be acquired secondary to BCL2i therapy in CLL. Baseline venetoclax-sensitivity or -resistance is influenced by the expression of BCL2 relative to other BCL2 family member proteins, microenvironmental factors including nodal T-cell stimulation, and tumoral heterogeneity. With selection pressure applied by continuous venetoclax exposure, secondary resistance mechanisms develop in oligoclonal fashion. Those mechanisms described include acquisition of BCL2 variants, dynamic aberrations of alternative BCL2 family proteins, and mutations affecting both BAX and other BH3 proteins. In view of the resistance described, this review also proposes future applications of BCL2i therapy in CLL and potential means by which BCL2i-resistance may be abrogated.

The role of BCL-2 family proteins in regulating apoptosis and cancer therapy.

Apoptosis, as a very important biological process, is a response to developmental cues or cellular stress. Impaired apoptosis plays a central role in the development of cancer and also reduces the efficacy of traditional cytotoxic therapies. Members of the B-cell lymphoma 2 (BCL-2) protein family have pro- or anti-apoptotic activities and have been studied intensively over the past decade for their importance in regulating apoptosis, tumorigenesis, and cellular responses to anticancer therapy. Since the inflammatory response induced by apoptosis-induced cell death is very small, at present, the development of anticancer drugs targeting apoptosis has attracted more and more attention. Consequently, the focus of this review is to summarize the current research on the role of BCL-2 family proteins in regulating apoptosis and the development of drugs targeting BCL-2 anti-apoptotic proteins. Additionally, the mechanism of BCL-2 family proteins in regulating apoptosis was also explored. All the findings indicate the potential of BCL-2 family proteins in the therapy of cancer.

Transcriptional signatures of the BCL2 family for individualized acute myeloid leukaemia treatment

BackgroundAlthough anti-apoptotic proteins of the B-cell lymphoma-2 (BCL2) family have been utilized as therapeutic targets in acute myeloid leukaemia (AML), their complicated regulatory networks make individualized therapy difficult. This study aimed to discover the transcriptional signatures of BCL2 family genes that reflect regulatory dynamics, which can guide individualized therapeutic strategies.MethodsFrom three AML RNA-seq cohorts (BeatAML, LeuceGene, and TCGA; n = 451, 437, and 179, respectively), we constructed the BCL2 family signatures (BFSigs) by applying an innovative gene-set selection method reflecting biological knowledge followed by non-negative matrix factorization (NMF). To demonstrate the significance of the BFSigs, we conducted modelling to predict response to BCL2 family inhibitors, clustering, and functional enrichment analysis. Cross-platform validity of BFSigs was also confirmed using NanoString technology in a separate cohort of 47 patients.ResultsWe established BFSigs labeled as the BCL2, MCL1/BCL2, and BFL1/MCL1 signatures that identify key anti-apoptotic proteins. Unsupervised clustering based on BFSig information consistently classified AML patients into three robust subtypes across different AML cohorts, implying the existence of biological entities revealed by the BFSig approach. Interestingly, each subtype has distinct enrichment patterns of major cancer pathways, including MAPK and mTORC1, which propose subtype-specific combination treatment with apoptosis modulating drugs. The BFSig-based classifier also predicted response to venetoclax with remarkable performance (area under the ROC curve, AUROC = 0.874), which was well-validated in an independent cohort (AUROC = 0.950). Lastly, we successfully confirmed the validity of BFSigs using NanoString technology.ConclusionsThis study proposes BFSigs as a biomarker for the effective selection of apoptosis targeting treatments and cancer pathways to co-target in AML.

Natural Products as Mcl-1 Inhibitors: A Comparative Study of Experimental and Computational Modelling Data

The human myeloid leukemia cell differentiation protein (hMcl-1) is an anti-apoptotic multi-partner protein, belonging to the B-cell lymphoma-2 (Bcl-2) family of proteins. Studies have linked hMcl-1 alleviated expression with resistance to hemopoietic chemotherapeutics, which makes it a key drug target in blood cancers. However, most of the developed small- to medium-sized hMcl-1 inhibitors have typical off-target activity towards other members of the Bcl-2 family. To improve the hMcl-1 inhibitor design, especially exploring a suitable scaffold with pharmacophoric features, we focused on natural hMcl-1 inhibitors. To date, seven classes of natural compounds have been isolated, which display a low micromolar affinity for hMcl-1 and have limited biophysical studies. We screened hMcl-1 co-crystal structures, and identified nine co-crystal structures of hMcl-1 protein, which were later evaluated by multiple receptor conformations (which indicates that the differences between hMcl-1 in crystal structures are low (RMSD values between 0.52 and 1.13 Å, average RMSD of 0.638–0.888 Å, with a standard deviation of 0.102–0.185Å)), and multiple ligand conformations (which led to the selection of the PDB structure, 3WIX (RMSD value = 0.879 Å, standard deviation 0.116 Å), to accommodate various Mcl-1 ligands from a range of co-crystal PDB files) methods. Later, the three adopted docking methods were assessed for their ability to reproduce the conformation bound to the crystal as well as predict trends in Ki values based on calculated RMSD and docking energies. Iterative docking and clustering of the docked pose within ≤1.0 Å was used to evaluate the reproducibility of the adopted docking methods and compared with their experimentally determined hMcl-1 affinity data.

Lisaftoclax (APG-2575) Is a Novel BCL-2 Inhibitor with Robust Antitumor Activity in Preclinical Models of Hematologic Malignancy.

Development of B-cell lymphoma 2 (BCL-2)-specific inhibitors poses unique challenges in drug design because of BCL-2 homology domain 3 (BH3) shared homology between BCL-2 family members and the shallow surface of their protein-protein interactions. We report herein discovery and extensive preclinical investigation of lisaftoclax (APG-2575). Computational modeling was used to design "lead" compounds. Biochemical binding, mitochondrial BH3 profiling, and cell-based viability or apoptosis assays were used to determine the selectivity and potency of BCL-2 inhibitor lisaftoclax. The antitumor effects of lisaftoclax were also evaluated in several xenograft models. Lisaftoclax selectively binds BCL-2 (Ki < 0.1 nmol/L), disrupts BCL-2:BIM complexes, and compromises mitochondrial outer membrane potential, culminating in BAX/BAK-dependent, caspase-mediated apoptosis. Lisaftoclax exerted strong antitumor activity in hematologic cancer cell lines and tumor cells from patients with chronic lymphocytic leukemia, multiple myeloma, or Waldenström macroglobulinemia. After lisaftoclax treatment, prodeath proteins BCL-2‒like protein 11 (BIM) and Noxa increased, and BIM translocated from cytosol to mitochondria. Consistent with these apoptotic activities, lisaftoclax entered malignant cells rapidly, reached plateau in 2 hours, and significantly downregulated mitochondrial respiratory function and ATP production. Furthermore, lisaftoclax inhibited tumor growth in xenograft models, correlating with caspase activation, poly (ADP-ribose) polymerase 1 cleavage, and pharmacokinetics of the compound. Lisaftoclax combined with rituximab or bendamustine/rituximab enhanced antitumor activity in vivo. These findings demonstrate that lisaftoclax is a novel, orally bioavailable BH3 mimetic BCL-2-selective inhibitor with considerable potential for the treatment of certain hematologic malignancies.

Natural Dibenzo[b,f]oxepines, Pacharin and Bauhiniastatin-1, Isolated from Bauhinia acuruana Induce Apoptosis on Breast Cancer Cells via MCL-1 Protein Reduction.

Herein, we describe the antiproliferative effects of two natural dibenzo [b,f]oxepines, pacharin and bauhiniastatin-1, isolated from Bauhinia acuruana on a breast cancer cell line and the mode of action underlying the cytotoxicity. Both compounds were cytotoxic in a panel of six tumor lines analyzed by the MTT assay, and IC50 values ranged from 7.8 to 45.1 µM, including human breast adenocarcinoma (MCF-7) cells. In contrast, none of the compounds were cytotoxic on normal human peripheral blood mononuclear cells (IC50 > 100 µM). Human breast adenocarcinoma (MCF-7) cells treated with pacharin or bauhiniastatin-1 20 µM for 24 h presented a reduction in cell volume and intensification of chromatin condensation, DNA fragmentation, and apoptotic cells. These findings became more evident after 48 h of exposure. Antiapoptotic B-cell lymphoma-2 family members, such as myeloid cell leukemia-1 and B-cell lymphoma-extra large, are important targets in cancer cells since their overexpression confers resistance to cancer treatments. A significant reduction of the myeloid cell leukemia-1 protein levels in human breast adenocarcinoma (MCF-7) cells after 24 h of treatment with pacharin or bauhiniastatin-1 at 20 µM was observed, while the B-cell lymphoma-extra large protein content was reduced in bauhiniastatin-1-treated cells at 40 µM only. The cytotoxic effects of pacharin and bauhiniastatin-1 are likely linked to myeloid cell leukemia-1 inhibition, which leads to the apoptosis of breast adenocarcinoma cells.

Selective inhibition of MCL1 overcomes venetoclax resistance in a murine model of myelodysplastic syndromes.

Treatment for myelodysplastic syndromes (MDS) remains insufficient due to clonal heterogeneity and lack of effective clinical therapies. Dysregulation of apoptosis is observed across MDS subtypes regardless of mutations and represents an attractive therapeutic opportunity. Venetoclax (VEN), a selective inhibitor of anti-apoptotic protein B-cell lymphoma- 2 (BCL2), has yielded impressive responses in older patients with acute myeloid leukemia (AML) and high risk MDS. BCL2 family anti-apoptotic proteins BCL-XL and induced myeloid cell leukemia 1 (MCL1) are implicated in leukemia survival, and upregulation of MCL1 is seen in VEN-resistant AML and MDS. We determined in vitro sensitivity of MDS patient samples to selective inhibitors of BCL2, BCL-XL and MCL1. While VEN response positively correlated with MDS with excess blasts, all MDS subtypes responded to MCL1 inhibition. Treatment with combined VEN + MCL1 inhibtion was synergistic in all MDS subtypes without significant injury to normal hematopoiesis and reduced MDS engraftment in MISTRG6 mice, supporting the pursuit of clinical trials with combined BCL2 + MCL1 inhibition in MDS.

Synthesis and characterization of polypyridine ruthenium(II) complexes and anticancer efficacy studies in vivo and in vitro

In this article, ligand IPP (IPP = 4-(1H-imidazo[4,5-f][1,10]phenanthrolin-2-yl)-N,N-diphenylaniline) and its three Ru(II) complexes: [Ru(bpy)2(IPP)](ClO4)2 (1) (bpy = 2,2′-bipyridine), [Ru(dmbpy)2(IPP)](ClO4)2 (2) (dmbpy = 4,4′-dimethyl-2,2′-bipyridine), and [Ru(phen)2(IPP)](ClO4)2 (3) (phen = 1,10-phenanthroline) were synthesized and characterized. The anticancer activity in vitro of the complexes was investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. The scratching and colony-forming experiments confirmed the complexes 1, 2, 3 interfered with the proliferation and migration ability of cells. The accumulation of the complexes in cells was researched and we found that these complexes directly accumulated in mitochondria, then the complexes cause a decline of the mitochondrial membrane potential and induce an increase of intracellular reactive oxygen species (ROS) levels. The growth of B16 cells were inhibited by 1, 2 and 3 at G0/G1 phase. Apoptosis was induced through mitochondrial pathway and the expression of apoptosis-related factors was regulated. In addition, the complexes promoted the transition of poly(ADP-ribose)polymerase (PARP) into the cleaved form (Cleaved PARP), downregulated the anti-apoptotic proteins, and upregulated the pro-apoptotic proteins. Consequently, complexes 1, 2 and 3 exerted their anticancer activity by regulating B-cell lymphoma-2 (Bcl-2) family proteins. Complex 2 showed excellent antitumor effects with a high inhibitory rate of 65.95% in vivo. Taken together, the complexes cause apoptosis in B16 cells through a ROS-mediated mitochondrial dysfunction pathway.

In silico mutational analysis to identify the role and pathogenicity of BCL-w missense variants

BackgroundIntrinsic pathway of apoptosis is generally mediated by BCL-2 (B cell lymphoma 2) family of proteins; they either induce or inhibit the apoptosis. Overexpression of BCL-2 in cancer cell may lead to delay in apoptosis. BCL-w is the pro-survival member of the BCL-2 family. BCL2L2 gene is present on chromosome number 14 in humans, and it encodes BCL-w protein; BCL-w protein is 193 amino acids residues in length. Interactions among the BCL-2 proteins are very specific. The fate of cell is determined by the ratio of pro-apoptotic proteins to pro-survival proteins. BCL-w promotes cell survival. Studies suggested that overexpression of BCL-w protein is associated with many cancers including DLBCL, BL, colorectal cancers, gastric cancers, and many more. The cause of overexpression is translocations or gene amplification which will subsequently result in cancerous activity. ProcessFor in-silico analysis, BCL2L2 gene was retrieved from UniProt (UniProt ID: Q92843). 54 missense variants have been collected in BCL-w proteins from COSMIC database. Different tools were used to detect the deleteriousness of the variants. ResultIn silico mutational study reveals how the non-synonymous mutations directly affect the protein’s native structure and its function. Variant mutational analysis with PolyPhen-2 revealed that out of 55 variants, 28 of the missense mutations was probably damaging with a score ranging from 0.9 to 1, while 24 variants were benign with a score ranging from 0 to 0.4. ConclusionsThis in silico work aims to determine how missense mutations in BCL-w protein affect the activity of the protein, the stability of the protein, and to determine the pathogenicity of the variants. Prediction of pathogenicity of variants will reveal if the missense mutation has a damaging effect on the native structure of protein or not. Prediction of protein stability will reveal whether the mutation has a stabilizing or destabilizing effect on the protein.

Abstract 5377: Decitabine sensitizes diffuse large B cell lymphoma cells to venetoclax

Abstract Introduction: Despite recent advances in the therapy of diffuse large B cell lymphoma (DLBCL), many patients are still not cured, and therefore new therapeutic combinations are needed. The anti-apoptotic B cell lymphoma 2 (BCL-2) gene is commonly dysregulated in DLBCL due to various mechanisms such as chromosomal translocation t(14;18)(q32.3;q21.3), copy number alterations and gene amplifications; however, targeting BCL-2 with a selective inhibitor, venetoclax, led to response in only a minority of patients. Thus, we sought to identify a novel combination partner for venetoclax to improve response to therapy. Previously with dynamic BH3 profiling, we found that DNA hypomethylating agents (HMA) could increase BCL2 dependence in DLBCL cells. Here, we investigate the activity of the HMA decitabine with venetoclax in DLBCL. Methods: We utilized BH3 profiling, a functional assay to assess the mitochondrial priming of cells for apoptosis and the dependence of cells on various anti-apoptotic BCL-2 family proteins. Other standard techniques included Western blot, cell cycle (BrdU/7-AAD), CellTiter-Glo, and Seahorse XF cell mito stress test. The DLBCL cell lines (TMD8, HBL1, OCI-Ly3, OCI-Ly1, Karpas 422, SUDHL4, Toledo, OCI-Ly7) and double hit lymphoma (DHL) patient-derived xenograft cell lines were used to investigate the in vitro anti-cancer activity of decitabine and venetoclax. Results: Through BH3 profiling, we found heterogenous dependence of DLBCL, consistent with the variable expression of multiple anti-apoptotic proteins in DLBCL assessed by Western blot. DLBCL cells were less sensitive to single BH3 mimetic treatment (venetoclax, S63 (MCL-1i) and A133 (BCL-xLi)). Using dynamic BH3 profiling (which assesses the change in priming induced by ex vivo drug treatment), we found that decitabine primes DLBCL cells for apoptosis and increases BCL-2 dependence. Consequently, decitabine increases the sensitivity of DLBCL cells to venetoclax, as the combination of decitabine and venetoclax synergistically induces apoptosis and suppresses cell proliferation. Decitabine has previously been shown to increase TGF-β signaling in DLBCL by restoring the expression of a downstream target, SMAD1. Interestingly, we found that inhibition of TGF-β signaling only partially antagonizes the anti-cancer activity of decitabine, implicating other anti-cancer mechanisms of decitabine. Indeed, we found that decitabine also induces DNA damage and leads to cell cycle arrest in DLBCL cells. Furthermore, decitabine promotes activation of the key apoptotic effector proteins, BAK and BAX. Additionally, we found that this combination also suppresses oxidative phosphorylation, thus restricting the energy supply for DLBCL cells. Conclusions: The HMA decitabine sensitizes DLBCL cells to venetoclax in vitro through increasing apoptotic priming and BCL-2 dependence. This combination is worthy of further study in in vivo model systems. Citation Format: Matthew S. Davids, Fen Zhu, Stephen J. Chong, Jennifer Crombie, Liam Hackett, May C. Collins. Decitabine sensitizes diffuse large B cell lymphoma cells to venetoclax [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 5377.

Enhanced in vitro cytotoxicity and antitumor activity in vivo of iridium(III) complexes liposomes targeting endoplasmic reticulum and mitochondria

In this paper, two new iridium(III) complexes [Ir(ppy)2(CBIP)](PF6) (ppy = 2-phenylpyridine, CBIP = 2-(4′-chloro-(1,1′-biphenyl))-1H-imidazo[4,5-f][1,10]phenanthroline) (Ir1) and [Ir(piq)2(CBIP)](PF6) (piq = 1-phenylisoquinoline) (Ir2) were synthesized and characterized. The anticancer activity of the complexes against cancer A549, HepG2, SGC-7901, BEL-7402, HeLa and LO2 cells was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. Unexpectedly, the complexes exhibit no or low cytotoxic activity toward the selected cancer cells. To increase the anticancer activity, complexes Ir1 and Ir2 were encapsulated into the liposome to form Ir1lipo and Ir2lipo, while Ir1lipo and Ir2lipo show high cytotoxic efficacy against BEL-7402, SGC-7901 and HeLa cells and Ir2lipo displays moderate cytotoxic activity against A549 and HepG2. The anticancer mechanism was explored through wound healing, cell cycle arrest, apoptosis, the change of mitochondrial membrane potential and antitumor activity in vivo. The antitumor in vivo showed that Ir1Lipo (3.9 mg/kg) exhibited significant antitumor activity with an inhibitory rate of 62.16%. Additionally, the expression of B-cell lymphoma-2 family proteins was studies by western blotting analysis. The results demonstrate that Ir1lipo and Ir2lipo induce apoptosis in BEL-7402 cells via endoplasmic reticulum stress-mitochondrial pathway.