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Although clinical outcomes in chronic lymphocytic leukemia (CLL) have greatly improved with several approved small molecular inhibitors, acquired resistance does occur, leading to disease progression and eventual death. Thus, the effort to explore novel inhibitors and combination therapeutic regimens is needed. The inhibition of MDM2-p53 interaction to restore p53 function has been regarded as a potential strategy for treating different cancers. We investigated the effects of novel MDM2 inhibitor APG-115 in CLL. We found that APG-115 treatment upregulated the expression of p53, MDM2, and p21 at the mRNA and protein level. APG-115 inhibited cell proliferation, induced apoptosis, and arrested the cell cycle at G0/G1 stage. Moreover, APG-115 inhibited the expression of BCL-2, BCLxL, and MCL-1, and suppressed the activation of AKT and ERK signaling pathways. APG-115 combined with the BCL2 inhibitor, ABT-199 (venetoclax), led to further inhibition of the expression of BCL-2 family anti-apoptotic proteins and consequently enhanced cell death. Collectively, this study demonstrates that APG-115 activates p53 and thus inhibits multiple pro-survival mechanisms, which provides a rational explanation for APG-115 efficiency in inducing cell apoptosis in CLL. The synergistic effect of APG-115 with ABT-199 suggested a potential combination application in CLL therapy. [ABSTRACT FROM AUTHOR]

The present study compared vacuum drum drying (VDD) and conventional spray drying (SD) for solidifying crystalline ABT-199 nanosuspensions into redispersible oral drug products. The aim was to optimize formulation compositions and process conditions to maintain nanoparticle size after tablet redispersion. The impact of drug load (22%, 33%, 44%) and type of drying protectant (mannitol, mannitol/trehalose mix (1:1), trehalose) on redispersibility and material powder properties were investigated. Moreover, compression analysis was performed assessing the influence of compaction pressure on primary nanocrystal redispersibility and tablet disintegration. Higher drug loads and lower drying protectant levels resulted in particle growth, confirming a drug load dependence on redispersibility behavior. Notably, all drying protectants showed similar protection properties at properly chosen drying process parameters (Tg-dependent), except when VDD was used for mannitol formulations. Differences between the applied drying processes were observed in terms of downstream processing and tabletability: mannitol-containing formulations solidified via VDD showed an improved processability compared to formulations with trehalose. In conclusion, VDD is a promising drying technique that offers advantageous downstream processability compared to SD and represents an attractive novel processing technology for the pharmaceutical industry. As demonstrated in the present study, VDD combines higher yields with a leaner manufacturing process flow. The improved bulk properties provide enhanced tabletability and enable direct compression. [ABSTRACT FROM AUTHOR]

Transformed follicular lymphoma (t-FL) is an aggressive malignancy that is refractory and rapidly progressing with poor prognosis. There is currently no effective treatment. High-throughput screening (HTS) platforms are used to profile the sensitivity or toxicity of hundreds of drug molecules, and this approach is applied to identify potential effective treatments for t-FL. We randomly selected a compound panel from the School of Pharmaceutical Sciences Xiamen University, tested the effects of the panel on the activity of t-FL cell lines using HTS and the CCK-8 assay, and identified compounds showing synergistic anti-proliferative activity with the Bcl-2 inhibitor venetoclax (ABT-199). Bioinformatics tools were used to analyze the potential synergistic mechanisms. The single-concentration compound library demonstrated varying degrees of activity across the t-FL cell lines evaluated, of which the Karpas422 cells were the most sensitive, but it was the cell line with the least synergy with ABT-199. We computationally identified 30 drugs with synergistic effects in all cell lines. Molecularly, we found that the targets of these 30 drugs didn't directly regulate Bcl-2 and identified 13 medications with high evidence value above.9 of coordination with ABT-199, further confirming TP53 may play the largest role in the synergistic effect. Collectively, these findings identified the combined regimens of ABT-199 and further suggested that the mechanism is far from directly targeting Bcl-2, but rather through the regulation and synergistic action of p53 and Bcl-2. This study intended to reveal the best synergistic scheme of ABT-199 through HTS to more quickly inform the treatment of t-FL. [ABSTRACT FROM AUTHOR]

Acyl-CoA synthetase long chain family member 5 (ACSL5), is a member of the acyl-CoA synthetases (ACSs) family that activates long chain fatty acids by catalyzing the synthesis of fatty acyl-CoAs. The dysregulation of ACSL5 has been reported in some cancers, such as glioma and colon cancers. However, little is known about the role of ACSL5 in acute myeloid leukemia (AML). We found that the expression of ACSL5 was higher in bone marrow cells from AML patients compared with that from healthy donors. ACSL5 level could serve as an independent prognostic predictor of the overall survival of AML patients. In AML cells, the ACSL5 knockdown inhibited cell growth both in vitro and in vivo. Mechanistically, the knockdown of ACSL5 suppressed the activation of the Wnt/β-catenin pathway by suppressing the palmitoylation modification of Wnt3a. Additionally, triacsin c, a pan-ACS family inhibitor, inhibited cell growth and robustly induced cell apoptosis when combined with ABT-199, the FDA approved BCL-2 inhibitor for AML therapy. Our results indicate that ACSL5 is a potential prognosis marker for AML and a promising pharmacological target for the treatment of molecularly stratified AML. [ABSTRACT FROM AUTHOR]

To investigate the effect of Apatinib (an inhibitor targeting VEGFR‐2) enhances chemosensitivity of ABT‐199 on diffuse large B‐cell lymphoma (DLBCL). Viability assay and flow cytometric assay for determining apoptosis, cell cycle, mitochondrial membrane potential, reactive oxygen species and immunoblotting were used to explore the combination effect in DLBCL cell lines, DLBCL patient samples, and DLBCL mouse models. RNA sequencing assay helped identify mechanisms of ABT‐199 plus Apatinib. The results show that ABT‐199 combined with Apatinib inhibited cell proliferation, reduced colony‐forming capacity, and induced apoptosis and cell cycle arrest in DLBCL cells. Mechanistically, the combination therapy inhibited tumour cell growth and promoted tumour cell death by regulating EDN1 and MAPK‐related pathways and activating the intrinsic apoptotic pathway. The effect of the combination therapy was also validated in primary DLBCL blasts and xenograft mouse models. Our findings indicate that Apatinib enhances the chemosensitivity of ABT‐199 and might serve as a promising therapeutic strategy for DLBCL.

Venetoclax, the best established BH3-mimetic, is a practice-changing proapoptotic drug in blood cancers in adults. In paediatrics the data are fewer but exciting results were recently presented in relapsed or refractory leukaemias demonstrating significant clinical activity. Importantly, the in-terventions could be potentially molecularly guided as vulnerabilities to BH3-mimetics were re-ported. Currently venetoclax is not incorporated into paediatric treatment schedules in Poland but it has been already used in patients that failed conventional therapy in Polish paediatric haemato-oncology departments. The aim of the study was to gather clinical data and correlates of all paediatric patients treated so far with venetoclax in Poland. We set out to gather this experience to help choose the right clinical context for the drug and stimulate further research. The questionnaire regarding the use of venetoclax was sent to all 18 Polish paediatric haemato-oncology centres. The data as available in November 2022 were gathered and analysed for the diagnoses, triggers for the intervention, treatment schedules, outcomes and molecular associations. We received response from 11 centres, 5 of which administered venetoclax to their patients. Clinical benefit, in most cases consistent with hematologic complete remission (CR), was reported in 5 patients out of ten, whereas 5 patient did not show clinical benefit from the intervention. Importantly, patients with CR included subtypes expected to show venetoclax vulnerability, such as poor-prognosis ALL with TCF::HLF fusion. We believe BH3-mimetics have clinical activity in children and should be available to pae-diatric haemato-oncology practitioners in well-selected applications. [ABSTRACT FROM AUTHOR]

Objective: Chronic lymphoid leukemia (CLL) is the most common type of leukemia among adults. Increased levels of Mcl-1 and Bcl-xL is linked to resistance to Bcl-2 inhibitors including ABT-199. In this study, we investigated the effect of miRNA-16-1 on apoptosis and sensitivity of the CLL cells to ABT-199.Materials and Methods: In this experimental study, the Mcl-1 and Bcl-2 expression were measured using qualitative reverse transcription-polymerase chain reaction (qRT-PCR) and western blotting. The effect of treatments on cell survival and growth were explored with MTT assay and Trypan blue assay, respectively. The drug interaction was evaluated using combination index analysis. Apoptosis was assessed by ELISA cell death and caspase-3 activity assays.Results: MiRNA-16-1 markedly inhibited the expression of Mcl-1 and Bcl-2 in a time dependent manner (P

Although clinical outcomes of CLL have improved with the use of BCL-2 inhibitor, ABT-199, acquired resistance eventually occurs in many cases, which leads to CLL disease progression. Thus, understanding the mechanisms that mediate this relapse is important to design improved therapies. Herein, we report that cytokine IFN-γ, secreted by dysfunctional T cells, enhanced CLL cells resistance to ABT-199. IFN-γ stimulation significantly increased the expression of BCL-2, MCL-1 and BCL-xL. Blocking JAK1/2-STAT3 signaling pathway impaired the expression of these anti-apoptotic proteins after IFN-γ stimulation. The combination of ABT-199 with JAK1/2 inhibitor Ruxolitinib or STAT3 inhibitors Stattic and C188-9 increased malignant B cell death. In summary, we show that IFN-γ enhanced CLL cells resistance to ABT-199 at least in part by up-regulating BCL-2, MCL-1 and BCL-xL expression via JAK1/2-STAT3 pathway, and thus blocking this pathway with inhibitors increased ABT-199 efficiency to induce CLL cell apoptosis, suggesting a potential therapeutically relevant combination to overcome ABT-199 resistance. [ABSTRACT FROM AUTHOR]

ABT-199 (venetoclax) is the first-in-class selective B-cell lymphoma 2 (BCL2) inhibitor, which is known to be ineffective towards liver cancer cells. Here, we investigated the efficacy and the underlying molecular processes of the sensitization effect of kaempferol isolated from persimmon leaves (KPL) on the ABT-199-resistant HepG2 cells. The effects of various doses of KPL coupled with ABT-199 on the proliferation of HepG2 cells and on the H22 liver tumorbearing mouse model were examined, as well as the underlying mechanisms. Our findings showed that ABT-199 alone, in contrast to KPL, had no significant impact on hepatoma cell growth, both in vitro and in vivo. Interestingly, the combination therapy showed significantly higher anti-hepatoma efficacy. Mechanistic studies revealed that combining KPL and ABT-199 may promote both early and late apoptosis, as well as decrease the mitochondrial membrane potential in HepG2 cells. Western blot analysis showed that combination of KPL and ABT-199 significantly reduced the expression of the anti-apoptotic proteins Bcl-2, Bcl-xL, and Mcl-1, raised the expression of Bax and cleaved caspase 3, and enhanced cytochrome C release and Bax translocation. Therefore, KPL combined with ABT-199 has a potential application prospect in the treatment of hepatocellular carcinoma. [ABSTRACT FROM AUTHOR]

Introduction: ABT-199 (venetoclax) is a BCL-2 suppressor with pronounced effects on acute myeloid leukemia (AML). However, its usefulness as a monotherapy or in combination with hypomethylating medicines like azacitidine is debatable due to acquired resistance. Usnic acid, a dibenzofuran extracted from lichen Usnea diffracta Vain, exhibits anticancer properties and may counteract multidrug resistance in leukemia cells., Objective: This study investigated whether usnic acid at low-cytotoxicity level could enhance sensitivity of AML cells with acquired resistance to ABT-199 by targeting the integrated stress response pathways., Methods: To investigate the combined effects on AML cells, we used a cell viability test, flow cytometry to quantify apoptosis, cell cycle analysis, and mitochondrial membrane potential measurement. RNA-seq and immunoblot were used to determine the potential mechanisms of ABT-199 + usnic acid combination., Results: Usnic acid, at a low cytotoxicity level, successfully restored ABT-199 sensitivity in AML cell lines that had developed ABT-199 resistance and increased ABT-199's antileukemic activity in a xenograft model. Mechanistically, the combination of usnic acid and ABT-199 cooperated to boost the expression of the integrated stress response (ISR)-associated genes ATF4, CHOP, and NOXA through the heme-regulated inhibitor kinase (HRI), while also promoting the degradation of the anti-apoptotic protein MCL-1. ISRIB, a compound that blocks the ISR, was able to reverse the growth suppression and cell death, the increase in expression of genes related with the ISR, and the inhibition of MCL-1 protein caused by combination therapy. Additionally, the downregulation of MCL-1 was linked to an increase in MCL-1 phosphorylation at serine 159 and subsequent destruction by the proteasome., Conclusion: In summary, usnic acid improves chemosensitivity to ABT-199 by triggering the integrated stress response, leading to decreased levels of MCL-1 protein, suggesting a potential treatment for AML cases resistant to Bcl-2 inhibitors., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Production and hosting by Elsevier B.V.)

Objective: Chronic lymphoid leukemia (CLL) is the most common type of leukemia among adults. Increased levels of Mcl-1 and Bcl-xL is linked to resistance to Bcl-2 inhibitors including ABT-199. In this study, we investigated the effect of miRNA-16-1 on apoptosis and sensitivity of the CLL cells to ABT-199. Materials and Methods: In this experimental study, the Mcl-1 and Bcl-2 expression were measured using qualitative reverse transcription-polymerase chain reaction (qRT-PCR) and western blotting. The effect of treatments on cell survival and growth were explored with MTT assay and Trypan blue assay, respectively. The drug interaction was evaluated using combination index analysis. Apoptosis was assessed by ELISA cell death and caspase-3 activity assays. Results: MiRNA-16-1 markedly inhibited the expression of Mcl-1 and Bcl-2 in a time dependent manner (P<0.05, relative to blank control). Pretreatment with miRNA-16-1 synergistically suppressed the cell growth and survival and reduced the half-maximal inhibitory concentration (IC50) value of ABT-199. Moreover, miRNA-16-1 markedly augmented the apoptotic effect of ABT-199 in CLL cells (P<0.05). Conclusion: Our findings propose that miRNA-16-1 act in concert with ABT-199 to exert synergistic anticancer efficacy against CLL, which is attributed to the inhibition of Bcl-2 and Mcl-1. This may propose a promising strategy for CLL resistant patients. [ABSTRACT FROM AUTHOR]

Selective BCL2 inhibitor ABT-199 has been approved to treat hematological malignancies including acute myeloid leukemia (AML). However, acquired drug resistance and severe side effects occur after extended treatment limiting the clinical usage of ABT-199. Here, we successfully encapsulated pure ABT-199 in amphiphilic mPEG- b -PTMC 169 block copolymer, forming mPEG-b-PTMC 169 @ABT-199 nanoparticles (abbreviated as PEG-ABT-199), which presented better aqueous dispersion and higher efficiency of loading and encapsulation than pure ABT-199. We then compared the anti-leukemic ability of pure ABT-199 and PEG-ABT-199 in vitro and in vivo. PEG-ABT-199 had a lower IC50 value compared with pure ABT-199 in MV4-11 and MOLM-13 cell lines. In addition, PEG-ABT-199 significantly induced apoptosis and decreased colony number than pure ABT-199. Most importantly, PEG-ABT-199 markedly reduced leukemic burden, inhibited the infiltration of leukemic blasts in the spleen, and extended the overall survival (OS) in MLL-AF9-transduced murine AML compared with free ABT-199. Meanwhile, the blank PEG169 NP was non-toxic to normal hematopoiesis in vitro and in vivo , suggesting that PEG169 NP is a safe carrier. Mechanistically, PEG-ABT-199 enhanced mitochondria-targeted delivery of ABT-199 to trigger the collapse of mitochondrial membrane potential (MMP), the release of cytochrome c (cyt-c), and mitochondria-based apoptosis. In conclusion, our results suggest that PEG-ABT-199 has more vital anti-leukemic ability than pure ABT-199. PEG-ABT-199 has potential application in clinical trials to alleviate side effects and improve anti-leukemia ability. ATB-199, an orally selective inhibitor for BCL2 protein, presents marked activity in relapsed or refractory AML, T-ALL, and CLL patients. However, ABT-199 resistance severely limits the further clinical usage because of off-target effects, non-specific toxicities, and low delivery of drugs. To reduce the side-effects and improve the solubility and bioavailability, ABT-199 was encapsulated into the amphiphilic mPEG- b -PTMC block copolymer by co-assembly method to obtain mPEG- b -PTMC@ABT-199 nanoparticles (PEG-ABT-199). PEG-ABT-199 has several advantages compared with pure ABT-199. 1.PEG-ABT-199 presents better aqueous dispersion and higher efficiencies of loading and encapsulation than pure ABT-199. 2. PEG-ABT-199 substantially enhances the anti-leukemic ability in vitro and in vivo compared with pure ABT-199. 3. PEG-ABT-199 has little effects on normal cells. 4. PEG-ABT-199 can reduce treatment cost. [Display omitted] [ABSTRACT FROM AUTHOR]

Venetoclax, the best established BH3-mimetic, is a practice-changing proapoptotic drug in blood cancers in adults. In paediatrics the data are fewer but exciting results were recently presented in relapsed or refractory leukaemias demonstrating significant clinical activity. Importantly, the in-terventions could be potentially molecularly guided as vulnerabilities to BH3-mimetics were re-ported. Currently venetoclax is not incorporated into paediatric treatment schedules in Poland but it has been already used in patients that failed conventional therapy in Polish paediatric haemato-oncology departments. The aim of the study was to gather clinical data and correlates of all paediatric patients treated so far with venetoclax in Poland. We set out to gather this experience to help choose the right clinical context for the drug and stimulate further research. The questionnaire regarding the use of venetoclax was sent to all 18 Polish paediatric haemato-oncology centres. The data as available in November 2022 were gathered and analysed for the diagnoses, triggers for the intervention, treatment schedules, outcomes and molecular associations. We received response from 11 centres, 5 of which administered venetoclax to their patients. Clinical benefit, in most cases consistent with hematologic complete remission (CR), was reported in 5 patients out of ten, whereas 5 patient did not show clinical benefit from the intervention. Importantly, patients with CR included subtypes expected to show venetoclax vulnerability, such as poor-prognosis ALL with TCF::HLF fusion. We believe BH3-mimetics have clinical activity in children and should be available to pae-diatric haemato-oncology practitioners in well-selected applications.

Simple Summary: Multiple myeloma is a non-curable disease and new therapeutic approaches are needed. PAPTP and PCARBTP, two novel mitochondria-specific inhibitors of the Kv1.3 ion channel, are effective in killing cultured myeloma cell lines and myeloma cells isolated from patient punctates, while healthy bone marrow cells are not affected. Cell death occurs through the classical mitochondrial apoptotic pathway, and further treatment with venetoclax, a BCL-2 inhibitor, has a clear synergistic effect. We identify Kv1.3 channels as a new therapeutic target for the treatment of multiple myeloma. Despite several new developments in the treatment of multiple myeloma, all available therapies are only palliative without curative potential and all patients ultimately relapse. Thus, novel therapeutic options are urgently required to prolong survival of or to even cure myeloma. Here, we show that multiple myeloma cells express the potassium channel Kv1.3 in their mitochondria. The mitochondrial Kv1.3 inhibitors PAPTP and PCARBTP are efficient against two tested human multiple myeloma cell lines (L-363 and RPMI-8226) and against ex vivo cultured, patient-derived myeloma cells, while healthy bone marrow cells are spared from toxicity. Cell death after treatment with PAPTP and PCARBTP occurs via the mitochondrial apoptotic pathway. In addition, we identify up-regulation of the multidrug resistance pump MDR-1 as the main potential resistance mechanism. Combination with ABT-199 (venetoclax), an inhibitor of Bcl2, has a synergistic effect, suggesting that mitochondrial Kv1.3 inhibitors could potentially be used as combination partner to venetoclax, even in the treatment of t(11;14) negative multiple myeloma, which represent the major part of cases and are rather resistant to venetoclax alone. We thus identify mitochondrial Kv1.3 channels as druggable targets against multiple myeloma. [ABSTRACT FROM AUTHOR]

Although clinical outcomes in chronic lymphocytic leukemia (CLL) have greatly improved with several approved small molecular inhibitors, acquired resistance does occur, leading to disease progression and eventual death. Thus, the effort to explore novel inhibitors and combination therapeutic regimens is needed. The inhibition of MDM2-p53 interaction to restore p53 function has been regarded as a potential strategy for treating different cancers. We investigated the effects of novel MDM2 inhibitor APG-115 in CLL. We found that APG-115 treatment upregulated the expression of p53, MDM2, and p21 at the mRNA and protein level. APG-115 inhibited cell proliferation, induced apoptosis, and arrested the cell cycle at G0/G1 stage. Moreover, APG-115 inhibited the expression of BCL-2, BCL-xL, and MCL-1, and suppressed the activation of AKT and ERK signaling pathways. APG-115 combined with the BCL2 inhibitor, ABT-199 (venetoclax), led to further inhibition of the expression of BCL-2 family anti-apoptotic proteins and consequently enhanced cell death. Collectively, this study demonstrates that APG-115 activates p53 and thus inhibits multiple pro-survival mechanisms, which provides a rational explanation for APG-115 efficiency in inducing cell apoptosis in CLL. The synergistic effect of APG-115 with ABT-199 suggested a potential combination application in CLL therapy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer HW declared a shared affiliation with the author YL to the handling editor at the time of review., (Copyright © 2024 Cui, Shao, Yang, Xin, Liu, Zhang, Sun, Chen, Shen, Meng and Chen.)

Idiopathic pulmonary fibrosis (IPF) is a distressing lung disorder with poor prognosis and high mortality rates. Limited therapeutic options for IPF is a major clinical challenge. Well-known for its anti-apoptotic properties, B-cell lymphoma 2 (Bcl-2) plays a critical role in the pathology of malignancies and inflammatory diseases, including IPF. In this study, we aimed to investigate the therapeutic effect of a Bcl-2 homology domain 3 mimetic inhibitor, ABT-199, on bleomycin (BLM)-induced pulmonary fibrosis in mice, and explore possible underlying mechanism. The lung inflammation and fibrosis model was established by intratracheal instillation of a single dose of BLM. We observed elevated Bcl-2 in the alveolar macrophages and fibroblasts derived from BLM-instilled mice from day 7. Further, we obtained in vivo evidence that early therapeutic treatment with Bcl-2 inhibitor ABT-199 from day 3, and late treatment from day 10, both alleviated airway inflammation and lung fibrosis induced by BLM. Our data suggest that ABT-199 might be an effective antifibrotic agent that interferes with profibrogenic cells, which may be a promising therapy in the treatment of clinical IPF patients.

BackgroundExisting research shows that ABT-199, as a first-line drug, have been widely used in hematological malignancies, especially in leukemia, but the clinical efficacy of single drug therapy was limited part of the reason was that BCL-2 inhibitors failure to target other anti-apoptotic BCL-2 family proteins, such as MCL-1. In this case, combination therapy may be a promising way to overcome this obstacle. Here, we investigate the preclinical efficacy of a new strategy combining ABT-199 with homoharringtonine (HHT), a selective inhibitor of MCL-1 may be a promising approach for AML treatment as these two molecules are important in apoptosis.MethodsA Cell Counting Kit-8 (CCK8) assay and flow cytometry were used to determine the half-maximal inhibitory concentration (IC50) value and cell apoptosis rate, respectively. The flow cytometry results showed that combined treatment with HHT and ABT-199 caused apoptosis in AML patient samples (n=5) but had no effect on normal healthy donor samples (n=11). Furthermore, we used a Western blot assay to explore the mechanism underlying the efficacy of HHT combined with ABT-199. Finally, antileukemic activity was further evaluated in vivo xenograft model.ResultsOur results indicated that ABT-199 combined with HHT significantly inhibited cell growth and promoted apoptosis in both AML cell lines and primary AML tumors in a dose- and time-dependent manner. Moreover, HHT combined with ABT-199 suppressed AML cell growth and progression in vivo xenograft model.ConclusionsOur research found that HHT combined with ABT-199 exerted its anti-leukemia effect by inducing apoptosis through the treatment of AML in vitro and in vivo.

Current standard treatment of patients with hairy cell leukemia (HCL), a chronic B-cell neoplasia of low incidence that affects the elderly, is based on the administration of purine analogs such as cladribine. This chemotherapy approach shows satisfactory responses, but the disease relapses, often repeatedly. Venetoclax (ABT-199) is a Bcl-2 inhibitor currently approved for the treatment of chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML) in adult patients ineligible for intensive chemotherapy. Given that HCL cells express Bcl-2, our aim was to evaluate venetoclax as a potential therapy for HCL. We found that clinically relevant concentrations of venetoclax (0.1 and 1 µM) induced primary HCL cell apoptosis in vitro as measured by flow cytometry using Annexin V staining. As microenvironment induces resistance to venetoclax in CLL, we also evaluated its effect in HCL by testing the following stimuli: activated T lymphocytes, stromal cells, TLR-9 agonist CpG, and TLR-2 agonist PAM3. We found decreased levels of venetoclax-induced cytotoxicity in HCL cells exposed for 48 h to any of these stimuli, suggesting that leukemic B cells from HCL patients are sensitive to venetoclax, but this sensitivity can be overcome by signals from the microenvironment. We propose that the combination of venetoclax with drugs that target the microenvironment might improve its efficacy in HCL.

Current standard treatment of patients with hairy cell leukemia (HCL), a chronic B-cell neoplasia of low incidence that affects the elderly, is based on the administration of purine analogs such as cladribine. This chemotherapy approach shows satisfactory responses, but the disease relapses, often repeatedly. Venetoclax (ABT-199) is a Bcl-2 inhibitor currently approved for the treatment of chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML) in adult patients ineligible for intensive chemotherapy. Given that HCL cells express Bcl-2, our aim was to evaluate venetoclax as a potential therapy for HCL. We found that clinically relevant concentrations of venetoclax (0.1 and 1 µM) induced primary HCL cell apoptosis in vitro as measured by flow cytometry using Annexin V staining. As microenvironment induces resistance to venetoclax in CLL, we also evaluated its effect in HCL by testing the following stimuli: activated T lymphocytes, stromal cells, TLR-9 agonist CpG, and TLR-2 agonist PAM3. We found decreased levels of venetoclax-induced cytotoxicity in HCL cells exposed for 48 h to any of these stimuli, suggesting that leukemic B cells from HCL patients are sensitive to venetoclax, but this sensitivity can be overcome by signals from the microenvironment. We propose that the combination of venetoclax with drugs that target the microenvironment might improve its efficacy in HCL. [ABSTRACT FROM AUTHOR]

Background: Existing research shows that ABT-199, as a first-line drug, have been widely used in hematological malignancies, especially in leukemia, but the clinical efficacy of single drug therapy was limited part of the reason was that BCL-2 inhibitors failure to target other anti-apoptotic BCL-2 family proteins, such as MCL-1. In this case, combination therapy may be a promising way to overcome this obstacle. Here, we investigate the preclinical efficacy of a new strategy combining ABT-199 with homoharringtonine (HHT), a selective inhibitor of MCL-1 may be a promising approach for AML treatment as these two molecules are important in apoptosis. Methods: A Cell Counting Kit-8 (CCK8) assay and flow cytometry were used to determine the half-maximal inhibitory concentration (IC50) value and cell apoptosis rate, respectively. The flow cytometry results showed that combined treatment with HHT and ABT-199 caused apoptosis in AML patient samples (n=5) but had no effect on normal healthy donor samples (n=11). Furthermore, we used a Western blot assay to explore the mechanism underlying the efficacy of HHT combined with ABT-199. Finally, antileukemic activity was further evaluated in vivo xenograft model. Results: Our results indicated that ABT-199 combined with HHT significantly inhibited cell growth and promoted apoptosis in both AML cell lines and primary AML tumors in a dose- and time-dependent manner. Moreover, HHT combined with ABT-199 suppressed AML cell growth and progression in vivo xenograft model. Conclusions: Our research found that HHT combined with ABT-199 exerted its anti-leukemia effect by inducing apoptosis through the treatment of AML in vitro and in vivo. [ABSTRACT FROM AUTHOR]

Purpose: The clinical outcome of head and neck squamous cell carcinoma (HNSCC) remains poor, partly due to the presence of resistant cancer stem cells (CSCs) which are responsible of recurrences. CSCs have low EGFR expression and, conversely, overexpress the anti-apoptotic Bcl-2 protein, which is involved in resistance to apoptosis and the invasion/migration capacities of tumour cells. Methods: The combination therapy of ABT-199, a Bcl-2 inhibitor, cetuximab an EGFR inhibitor, and radiation using an HNSCC model (SQ20B cell line) and its corresponding CSC subpopulation were evaluated in vitro (2D/3D cell proliferation; invasion/migration and apoptosis using videomicroscopy) and in vivo. Results: Cetuximab strongly inhibited 2D and 3D cell proliferation, as well as invasion/migration, only in non-CSC-SQ20B cells, whereas ABT-199 selectively inhibited these mechanisms in SQ20B/CSCs. The combination of irradiation + cetuximab + ABT-199 increased the inhibition of the 2D and 3D cell proliferation, invasion/migration, and resistance to apoptosis in both cell sub-populations. In addition, in a nude mouse model with heterotopic tumour xenograft, a treatment combining cetuximab + ABT-199 with fractional irradiation strongly delayed the tumour growth and increased in vivo lifespan without side effects. Conclusion: Based on the present results, this triple combination therapy may represent a new opportunity for testing in clinical trials, particularly in locally advanced HNSCC. [ABSTRACT FROM AUTHOR]

Tristan Knight,1,2 Holly Edwards,3,4 Jeffrey W Taub,1–2,4 Yubin Ge2–41Division of Pediatric Hematology and Oncology, Department of Pediatrics, Children’s Hospital of Michigan, Detroit, MI, USA; 2Department of Pediatrics, Wayne State University School of Medicine, Detroit, MI, USA; 3Department of Oncology, Wayne State University School of Medicine, Detroit, MI, USA; 4Molecular Therapeutics Program, Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, MI, USAAbstract: Venetoclax (ABT-199), a BH3-mimetic and selective BCL-2 inhibitor, was recently approved by the US Food and Drug Administration (FDA) for the treatment of acute myeloid leukemia (AML) in adult patients aged 75 years or older, or otherwise unable to tolerate intensive induction chemotherapy, in combination with either hypomethylating agents or low-dose cytarabine. In this review article, we discuss venetoclax’s mechanism of action, in relation to both the BCL-2 protein family in general and BH3-mimetic activity in particular. We then outline the pharmacological advances that preceded and facilitated its development, as well as providing an overview of key preclinical and clinical studies which lead to its use first in chronic lymphoid leukemia (CLL), then in small lymphocytic leukemia (SLL), and subsequently in AML. Finally, we seek to offer an overview of the challenges and opportunities encountered as venetoclax moves into more widespread use, including its use and activity against leukemia initiating cells and oxidative phosphorylation.Keywords: ABT-199, acute myeloid leukemia, apoptosis, BCL-2 inhibitor, BH3-mimetic, venetoclax

Ibrutinib is a small molecule drug that targets Bruton's tyrosine kinase in B‐cell malignancies and is highly efficient at killing mantle cell lymphoma and chronic lymphocytic leukemia. However, the anti‐cancer activity of ibrutinib against solid tumors, such as non‐small cell lung cancer (NSCLC), remains low. To improve the cytotoxicity of ibrutinib towards lung cancer, we synthesized a series of ibrutinib derivatives, of which Ibr‐7 exhibited superior anti‐cancer activity to ibrutinib, especially against epithelial growth factor receptor (EGFR) wild‐type NSCLC cell lines. Ibr‐7 was observed to dramatically suppress the mammalian target of Rapamycin complex 1 (mTORC1)/S6 signaling pathway, which is only slightly affected by ibrutinib, thus accounting for the superior anti‐cancer activity of Ibr‐7 towards NSCLC. Ibr‐7 was shown to overcome the elevation of Mcl‐1 caused by ABT‐199 mono‐treatment, and thus exhibited a significant synergistic effect when combined with ABT‐199. In conclusion, we used a molecular substitution method to generate a novel ibrutinib derivative, termed Ibr‐7, which exhibits enhanced anti‐cancer activity against NSCLC cells as compared with the parental compound.

Colorectal cancer (CRC) is one of the most common malignancies worldwide and is associated with poor prognosis and high mortality. Despite advances in treatment with chemotherapy, CRC remains a major cause of drug resistance-related cancer deaths. One of the main reasons for such resistance is dysregulation of Mcl-1 expression. In this study, we identified LZT-106 as a novel kinase inhibitor that was able to bind to CDK9 with potent inhibitory ability, and indirectly regulate the expression of Mcl-1. However, different regulatory profiles were observed between LZT-106 and the well-studied CDK9 inhibitor flavopiridol with regards to Mcl-1 inhibition. Via Western blotting, real-time PCR and immunoprecipitation, we confirmed that LZT-106 was also able to target GSK-3β signaling and facilitate the degradation of Mcl-1. And LZT-106 was shown to synergize with ABT-199 to induce apoptosis even in the RKO cell line that overexpressed Mcl-1. Finally, LZT-106 significantly inhibited tumor growth in a xenograft mouse model with minimal toxicity. Overall, our findings suggest that LZT-106 is a promising candidate drug for the treatment of patients with CRC. [ABSTRACT FROM AUTHOR]

Development of resistance to chemo- and radiotherapy in patients suffering from advanced cervical cancer narrows the therapeutic window for conventional therapies. Previously we reported that a combination of the selective BCL-2 family inhibitors ABT-263 and A-1210477 decreased cell proliferation in C33A, SiHa and CaSki human cervical cancer cell lines. As ABT-263 binds to both BCL-2 and BCL-XL with high affinity, it was unclear whether the synergism of the drug combination was driven either by singly inhibiting BCL-2 or BCL-XL, or inhibition of both. In this present study, we used the BCL-2 selective inhibitor ABT-199 and the BCL-XL selective inhibitor A1331852 to resolve the individual antitumor activities of ABT-263 into BCL-2 and BCL-XL dependent mechanisms. A-1210477 was substituted for the orally bioavailable S63845. Four cervical cancer cell lines were treated with the selective BCL-2 family inhibitors ABT-199, A1331852 and S63845 alone and in combination using 2-dimensional (2D) and 3-dimensional (3D) cell culture models. The SiHa, C33A and CaSki cell lines were resistant to single agent treatment of all three drugs, suggesting that none of the BCL-2 family of proteins mediate survival of the cells in isolation. HeLa cells were resistant to single agent treatment of ABT-199 and A1331852 but were sensitive to S63845 indicating that they depend on MCL-1 for survival. Co-inhibition of BCL-2 and MCL-1 with ABT-199 and S63845, inhibited cell proliferation of all cancer cell lines, except SiHa. However, the effect of the combination was not as pronounced as combination of A1331852 and S63845. Co-inhibition of BCL-XL and MCL-1 with A1331852 and S63845 significantly inhibited cell proliferation of all four cell lines. Similar data were obtained with 3-dimensional spheroid cell culture models generated from two cervical cancer cell lines in vitro. Treatment with a combination of A1331852 and S63845 resulted in inhibition of growth and invasion of the 3D spheroids. Collectively, our data demonstrate that the combination of MCL-1-selective inhibitors with either selective inhibitors of either BCL-XL or BCL-2 may be potentially useful as treatment strategies for the management of cervical cancer.

As a proapoptotic death effect domain (DED)-containing protein, DED-containing DNA-binding protein (DEDD) has been demonstrated to inhibit tumor growth, invasion and metastasis in our previous studies. Here, we demonstrated that knockdown of DEDD in MCF-7 cells resulted in characteristic drug resistance to doxorubicin and paclitaxel, and overexpression of DEDD in MDA-MB-231 cells increased their sensitivity to doxorubicin and paclitaxel. The expression levels of DEDD were positively correlated with Bcl-2 in breast cancer cell lines as well as in human breast cancer tissue. Knockdown of DEDD downregulated the transcriptional activity of the bcl-2 gene and shortened the time for Bcl-2 degradation. DEDD interacts with and stabilizes Bcl-2, and breast cancer cells with low DEDD expression were more sensitive to treatment with a BH3 mimetic, ABT-199, than were those with high DEDD expression. In total, our findings highlight a new strategy for treating breast cancer with no/low DEDD expression by targeting Bcl-2 with the BH3 mimetic ABT-199. • Low DEDD expression in breast cancer cells resulted in drug resistance to doxorubicin and paclitaxel. • DEDD is positively correlated with Bcl-2 in human breast cancer tissues. • DEDD interacts with and stabilizes Bcl-2. • Breast cancer cells with lower DEDD expression were more sensitive to ABT-199. [ABSTRACT FROM AUTHOR]

Abstract Background MicroRNAs (miRs) are involved in tumor progression by regulating tumor cells and tumor microenvironment. MiR21 is overexpressed in diffuse large B-cell lymphoma (DLBCL) and its biological impact on tumor microenvironment remains unclear. Methods MiR21 was assessed by quantitative RT-PCR in patients with newly diagnosed DLBCL. The mechanism of action of miR21 on lymphoma progression and tumor angiogenesis was examined in vitro in B-lymphoma cell lines and in vivo in a murine xenograft model. Results Serum miR21 was significantly elevated in patients and associated with advanced disease stage, International Prognostic Index indicating intermediate-high and high-risk, and increased tumor angiogenesis. When co-cultured with immune cells and endothelial cells, miR21-overexpressing B-lymphoma cells were resistant to chemotherapeutic agents, but sensitive to Bcl-2 inhibitor ABT-199, irrespective of Bcl-2 expression on lymphoma cells. In both co-culture systems of Bcl-2positive and Bcl-2negative B-lymphoma cells, miR21 induced inducible co-stimulator (ICOS) expression on regulatory T (Treg) cells. Through crosstalking with Treg cells by ICOS ligand (ICOSL), endothelial cells were activated, resulting in stimulation of Bcl-2 expression and vessel formation. ABT-199 directly targeted Bcl-2 on endothelial cells, induced endothelial cell apoptosis and inhibited tumor angiogenesis. In a murine xenograft model established with subcutaneous injection of B-lymphoma cells, ABT-199 particularly retarded the growth of miR21-overexpressing tumors, consistent with the induction of endothelial cell apoptosis and inhibition of tumor angiogenesis. Conclusions As a serum oncogenic biomarker of B-cell lymphoma, miR21 indicated B-lymphoma cell sensitivity to ABT-199 via ICOS/ICOSL-mediated interaction of Treg cells with endothelial cells.

Introduction: Change in the balance of Bcl-2 family proteins is one of the main reasons for resistance of tumor cells to ABT-199. In this study, the effect of dihydroartemisinin on cell growth, apoptosis and sensitivity of the AML cells to ABT-199 was investigated., Methods: Cell proliferation and survival were assessed by trypan blue staining and MTT assay, respectively. Cell apoptosis was measured by Hoechst 33342 staining and caspase-3 activity assay. The expression levels of Bcl-2, Mcl-1 and Bax mRNA were tested by qRT-PCR., Results: Our data showed that combination therapy significantly reduced the IC50 value and synergistically decreased the AML cell survival and growth compared with dihydroartemisinin or ABT-199 alone. Treatment with each of ABT-199 or dihydroartemisinin alone clearly enhanced the Bax mRNA expression and inhibited the expression of Mcl-1 and Bcl-2 mRNA. Inhibition of Mcl-1 mRNA by dihydroartemisinin was associated with enhancement of apoptosis induced by ABT-199 in AML cells., Conclusion: In conclusion, dihydroartemisinin not only triggers the intrinsic pathway of apoptosis, but also can increase the sensitivity of the AML cells to ABT-199 via suppression of Mcl-1 expression.

Graphical abstract Abstract ABT-737, ABT-263 (Navitoclax) and ABT-199 (Venetoclax) are under intensive preclinical and clinical investigation as treatments for hematologic and other malignancies. These small molecules mimic pro-death B-cell lymphoma-2 (Bcl-2) Homology 3 (BH3) domain-only proteins. They also bear a structural resemblance to certain sigma (σ) receptor ligands. Moreover, the Bcl-2 and σ receptor protein families are both located primarily at the endoplasmic reticulum, mediate cell death and survival through protein–protein interactions, and physically associate. Accordingly, we examined the ability of the ABT series of BH3 mimetics to interact with σ receptors using radioligand-binding techniques. Negative allosteric modulation of [3H](+)-pentazocine, an agonist, binding to σ 1 receptors in guinea pig brain membranes was observed for ABT-737, ABT-263 and ABT-199. Findings included reduction of specific binding to distinct plateaus in concentration-dependent fashion, significant slowing of radioligand dissociation kinetics, and decreases in radioligand affinity with no or modest changes in maximal receptor densities. Using a ternary complex model, dissociation constants (K X) for modulator binding to the σ 1 receptor ranged from 1 to 2.5 μM, while negative cooperativity factors (α), representing the changes in affinity of ligand and modulator when bound as a ternary complex with the receptor, ranged from 0.15 to 0.42. These observations were extended and reinforced by studies using intact small cell (NCI-H69) and non-small cell (NCI-H23) lung cancer cells, and by using an antagonist σ 1 receptor radioligand, E - N -1-(3′-[125I]iodoallyl)- N ′-4-(3″,4″-dimethoxyphenethyl)piperazine, in mouse brain membranes. By contrast, exploratory studies indicate marked enhancement of the σ 2 receptor binding of [3H]1,3-di-(o -tolyl)guanidine/(+)-pentazocine in NCI-H23 cells and guinea pig brain membranes. These findings raise intriguing questions regarding mechanism and potential functional outcomes. [ABSTRACT FROM AUTHOR]

Ibrutinib is a small molecule drug that targets Bruton's tyrosine kinase in B‐cell malignancies and is highly efficient at killing mantle cell lymphoma and chronic lymphocytic leukemia. However, the anti‐cancer activity of ibrutinib against solid tumors, such as non‐small cell lung cancer (NSCLC), remains low. To improve the cytotoxicity of ibrutinib towards lung cancer, we synthesized a series of ibrutinib derivatives, of which Ibr‐7 exhibited superior anti‐cancer activity to ibrutinib, especially against epithelial growth factor receptor (EGFR) wild‐type NSCLC cell lines. Ibr‐7 was observed to dramatically suppress the mammalian target of Rapamycin complex 1 (mTORC1)/S6 signaling pathway, which is only slightly affected by ibrutinib, thus accounting for the superior anti‐cancer activity of Ibr‐7 towards NSCLC. Ibr‐7 was shown to overcome the elevation of Mcl‐1 caused by ABT‐199 mono‐treatment, and thus exhibited a significant synergistic effect when combined with ABT‐199. In conclusion, we used a molecular substitution method to generate a novel ibrutinib derivative, termed Ibr‐7, which exhibits enhanced anti‐cancer activity against NSCLC cells as compared with the parental compound. Ibr‐7 exhibits antitumor activity against EGFR wild‐type and mutant NSCLC cells via inhibition of the mTOR‐S6 pathway; the parent compound of Ibr‐7, ibrutinib, does not affect this pathway. The anti‐proliferation effect of Ibr‐7 against freshly cultured primary lung cancer cells was greater than that of ibrutinib and AZD‐9291, irrespective of pathological patterns or EGFR mutation status. [ABSTRACT FROM AUTHOR]

Abstract Elimination of airway inflammatory cells is essential for asthma control. As Bcl-2 protein is highly expressed on the mitochondrial outer membrane in inflammatory cells, we chose a Bcl-2 inhibitor, ABT-199, which can inhibit airway inflammation and airway hyperresponsiveness by inducing inflammatory cell apoptosis. Herein, we synthesized a pH-sensitive nanoformulated Bcl-2 inhibitor (Nf-ABT-199) that could specifically deliver ABT-199 to the mitochondria of bronchial inflammatory cells. The proof-of-concept study of an inflammatory cell mitochondria-targeted therapy using Nf-ABT-199 was validated in a mouse model of allergic asthma. Nf-ABT-199 was proven to significantly alleviate airway inflammation by effectively inducing eosinophil apoptosis and inhibiting both inflammatory cell infiltration and mucus hypersecretion. In addition, the nanocarrier or Nf-ABT-199 showed no obvious influence on cell viability, airway epithelial barrier and liver function, implying excellent biocompatibility and with non-toxic effect. The nanoformulated Bcl-2 inhibitor Nf-ABT-199 accumulates in the mitochondria of inflammatory cells and efficiently alleviates allergic asthma. [ABSTRACT FROM AUTHOR]

Background and Aims: Pancreatic cancer is an aggressive malignancy with poor prognosis. Gemcitabine is the standard chemotherapeutic drug used to treat the disease; however, it has a low response rate. Therefore, there is an urgent need to develop new and safe therapies to enhance sensitivity to gemcitabine in treating pancreatic cancer.Methods: The synergistic effect of gemcitabine combined with specific B cell CLL/lymphoma 2 (Bcl-2) inhibitor ABT-199 against pancreatic cancer was tested using cell viability, cell cycle, and apoptosis assays in vitro and in an MIA Paca-2 xenograft model in vivo. Its underlying mechanism was explored using western blotting analysis of Bcl-2 family proteins.Results: ABT-199 not only enhanced the effect of gemcitabine on cell growth inhibition but also promoted gemcitabine-induced apoptosis in pancreatic cancer cell lines. Gemcitabine decreased the expression of anti-apoptotic protein Mcl-1 but increased the expression of anti-apoptotic protein Bcl-2. ABT-199 downregulated the gemcitabine-induced production of Bcl-2 and increased the expression of pro-apoptotic protein Bcl-2 interacting protein (BIM). Mouse xenograft experiments also confirmed the synergistic effect of gemcitabine and ABT-199 on tumor growth inhibition and the induction of tumor cell apoptosis.Conclusion: Our results indicated that ABT-199 improved the anti-tumor effect of gemcitabine on pancreatic cancer by downregulating gemcitabine-induced overexpression of Bcl-2. ABT-199 has already been investigated in phase 3 clinical trials for chronic lymphocytic leukemia; therefore, it may serve as a potential drug to improve the sensitivity of pancreatic cancer to gemcitabine. [ABSTRACT FROM AUTHOR]

The antiapoptotic Bcl‐2 family proteins play critical roles in resistance to chemotherapy in acute myeloid leukaemia (AML). The Bcl‐2‐selective inhibitor ABT‐199 (Venetoclax) shows promising antileukaemic activity against AML, though Mcl‐1 limits its antileukaemic activity. XPO1 is a nuclear exporter overexpressed in AML cells and its inhibition decreases Mcl‐1 levels in cancer cells. Thus, we hypothesized that the XPO1‐selective inhibitor KPT‐330 (Selinexor) can synergize with ABT‐199 to induce apoptosis in AML cells through down‐regulation of Mcl‐1. The combination of KPT‐330 and ABT‐199 was found to synergistically induce apoptosis in AML cell lines and primary patient samples and cooperatively inhibit colony formation capacity of primary AML cells. KPT‐330 treatment decreased Mcl‐1 protein after apoptosis initiation. However, binding of Bim to Mcl‐1 induced by ABT‐199 was abrogated by KPT‐330 at the same time as apoptosis initiation. KPT‐330 treatment increased binding of Bcl‐2 to Bim but was overcome by ABT‐199 treatment, demonstrating that KPT‐330 and ABT‐199 reciprocally overcome apoptosis resistance. Mcl‐1 knockdown and overexpression confirmed its critical role in the antileukaemic activity of the combination. In summary, KPT‐330 treatment, alone and in combination with ABT‐199, modulates Mcl‐1, which plays an important role in the antileukaemic activity of the combination. [ABSTRACT FROM AUTHOR]

• Flavokawain B (FKB) induced proliferation inhibition and apoptosis in multiple B-cell lymphoma cell lines in vitro through the PI3K/Akt axis. • FKB synergistically potentiated the effect of the BCL-2 inhibitor ABT-199. • FKB suppressed the growth of different types of B-cell lymphoma in vivo. B-cell lymphoma, which originates from B cells at diverse differentiation stages, is the most common non-Hodgkin lymphoma with tremendous treatment challenges and unsatisfactory clinical outcomes. Flavokawain B (FKB), a naturally occurring chalcone extracted from kava, possesses promising anticancer properties. However, evidence on the effects of FKB on hematological malignancies, particularly lymphomas, remains scarce. This study aimed to investigate the antilymphoma effect of FKB and its underlying mechanisms. Proliferation assays, flow cytometry, and western blotting were employed to determine whether and how FKB affected B-cell lymphoma cell lines in vitro. Xenograft mouse models were established to evaluate the antilymphoma efficacy of FKB in vivo. FKB reduced the viability of a panel of B-cell lymphoma cell lines in a dose- and time-dependent manner. Mitochondrial apoptosis was markedly induced by FKB, as evidenced by an increased percentage of annexin V-positive cells, a loss of mitochondrial membrane potential, and cleavage of caspase-3 and PARP. Moreover, FKB inhibited BCL-XL expression and synergized with the BCL-2 inhibitor ABT-199. Mechanistically, FKB treatment decreased the phosphorylation of Akt, mammalian target of rapamycin (mTOR), glycogen synthase kinase-3β (GSK3β), and ribosomal protein S6 (RPS6). Pharmacological blockage of phosphoinositide 3-kinase (PI3K), Akt, or GSK3β potentiated the activity of FKB, indicating the involvement of the PI3K/Akt cascade in FKB-mediated inhibitory effects. In mouse xenograft models, the intraperitoneal administration of FKB significantly decreased lymphoma growth, accompanied by diminished mitosis and Ki-67 staining of tumor tissues. Our data demonstrate the robust therapeutic potential of FKB in the treatment of B-cell lymphoma. [Display omitted] [ABSTRACT FROM AUTHOR]

Anti-apoptotic Bcl-2-family members are frequently dysregulated in both blood and solid cancers, contributing to their survival despite ongoing oncogenic stress. Yet, such cancer cells often are highly dependent on Bcl-2 for their survival, a feature that is exploited by so-called BH3-mimetic drugs. Venetoclax (ABT-199) is a selective BH3-mimetic Bcl-2 antagonist that is currently used in the clinic for treatment of chronic lymphocytic leukemia patients. Unfortunately, venetoclax resistance has already emerged in patients, limiting the therapeutic success. Here, we examined strategies to overcome venetoclax resistance. Therefore, we used two diffuse large B-cell lymphoma (DLBCL) cell lines, Riva WT and venetoclax-resistant Riva (VR). The latter was obtained by prolonged culturing in the presence of venetoclax. We report that Riva VR cells did not become more sensitive to BIRD-2, a peptide targeting the Bcl-2 BH4 domain, and established cross-resistance towards BDA-366, a putative BH4-domain antagonist of Bcl-2. However, we found that Bcl-XL, another Bcl-2-family protein, is upregulated in Riva VR, while Mcl-1 expression levels are not different in comparison with Riva WT, hinting towards an increased dependence of Riva VR cells to Bcl-XL. Indeed, Riva VR cells could be resensitized to venetoclax by A-1155463, a selective BH3 mimetic Bcl-XL inhibitor. This is underpinned by siRNA experiments, demonstrating that lowering Bcl-XL-expression levels also augmented the sensitivity of Riva VR cells to venetoclax. Overall, this work demonstrates that Bcl-XL upregulation contributes to acquired resistance of DLBCL cancer cells towards venetoclax and that antagonizing Bcl-XL can resensitize such cells towards venetoclax.

Cells from patients with acute myeloid leukemia (AML) that remain dormant and protected by stromal cells may escape effects of chemotherapy. We modeled dormancy in vitro and investigated the ability of Bcl-2 inhibitors ABT-199 and ABT-737 to overcome chemoprotection of dormant cells. CD34-enriched primary AML cells with aberrant leukemia-associated phenotypes (LAPs) were cultured on stromal cells. The chemosensitivity of dormant (PKH26high), CD34+, LAP+ cells was ascertained by 5-colour flow cytometric counting after 12 d. The PKH26high, CD34+, LAP + subset retained clonogenic capacity. The dormant fraction was completely resistant to Ara-C (p = .007). However, ABT-199 and ABT-737 were able to reduce the dormant fraction by 84% and 80%, respectively, of their effects on proliferating counterparts. In conclusion, we have elaborated a system for quantifying chemosensitivity in LAP+ dormant leukemia cells, thought to contribute to disease relapse, and shown sensitivity of dormant LAP+ cells to ABT-199 and ABT-737 in this system. [ABSTRACT FROM AUTHOR]

Background Aberrant overexpression of Bcl-2 protein has been detected in 80% of nasopharyngeal carcinoma (NPC), and Bcl-2 family proteins are implicated in both NPC oncogenesis and chemotherapy resistance. Previous studies have shown that while treatment of NPC cells with Bcl-2 family inhibitors alone is rarely effective, concomitant treatment with a cytotoxic reagent such as cisplatin can increase efficacy through a synergistic effect. The aim of the current work was to determine how we might increase the efficacy of Bcl-2 family inhibitors in the absence of cytotoxic reagents, which are associated with negative side effect profiles. Methods We assessed cell proliferation in Bcl-2 high-expressing NPC cells by CCK-8 assay after treatment with the Bcl-2 inhibitor ABT-199 and/or the Mcl-1 inhibitor S63845. Apoptotic induction by ABT-199 was evaluated by Annexin V-FITC and PI double staining. We also evaluated Bcl-2 family protein expression (Bim, Mcl-1, Bcl-xL, Noxa) after treatment with ABT-199 by western blotting. Finally, xenografted Balb/c nude mice were used to test ABT-199 efficacy in vivo , H&E and immunohistochemistry assay were used to analyze tumor samples. Results ABT-199 effectively induced NPC cell apoptosis in vitro and in the xenograft model. Following ABT-199 treatment in NPC cells, upregulation of Mcl-1 and Bcl-xL can lead to drug resistance, while concomitant Noxa overexpression partially neutralized the Mcl-1-caused resistance. Given that ABT-199 induces apoptosis in NPC cells through the Bcl-2/Noxa/Mcl-1 axis, treatment avenues further targeting this pathway should be promising. Indeed, the newly developed Mcl-1 inhibitor S63845 in combination with ABT-199 had a synergistic effect on NPC cell apoptosis. Conclusion Bcl-2 inhibition in NPC cells with ABT-199 triggers apoptosis through the Bcl-2/Noxa/Mcl-1 axis, and dual inhibition of the anti-apoptotic Bcl-2 family proteins Bcl-2 and Mcl-1 provided a strong synergistic effect without the need for adjunctive cytotoxic agent treatment with cisplatin. [ABSTRACT FROM AUTHOR]

Graphical abstract Abstract Targeting Bcl-2 with ABT-199 (Venetoclax) shows limited single-agent activity against many cancers in both preclinical and clinical investigations. Combination therapies have attracted great attention. The principal purpose of this study was to investigate the mechanism of synergism between ABT-199 and paclitaxel. Moreover, we analyzed the biomarker to identify tumors which are most likely to respond to this combination. We evaluated the effect of this combination in a panel of nine cancer cell lines including cervical cancer, lung cancer, ovarian cancer, lymphoma, leukemia and breast cancer. Combination index (CI) assay showed that four of nine call lines exhibited synergistic respond to ABT-199/paclitaxel combination due to enhanced intrinsic apoptosis. However, paclitaxel-induced Bcl-2 phosphorylation impaired the synergistic effect by impeding the freeing of Bax and Bim by ABT-199 because ABT-199 cannot hit phosphorylated Bcl-2 (pBcl-2). By means of a correlation analysis of JNK level with CI value in combination with overexpressing or silencing JNK protein in cancer cells, we identified basal JNK1 level as a potential biomarker for predicting the level of pBcl-2 upon paclitaxel treatment, and thus for predicting a synergistic response. A cut-off value of 0.37 for relative JNK1 expression level was determined using receiver operating characteristic (ROC) analysis to distinguish between synergistic and non-synergistic response cancers. A more accurate and valid cut-off value for JNK1 will be gained based on a large-scale clinical samples analysis. [ABSTRACT FROM AUTHOR]

Abstract: Venetoclax has been approved in the United States, Europe, Canada, and Australia for appropriate patients with difficult‐to‐treat chronic lymphocytic leukemia (CLL). The objective of this phase 1 study was to evaluate the pharmacokinetics of venetoclax in Chinese subjects to inform the dose selection of venetoclax in a phase 2 study of patients with relapsed/refractory (R/R) CLL in China. Twelve healthy first‐generation Han Chinese subjects received a single 100‐mg dose of venetoclax following a low‐fat breakfast. Pharmacokinetic parameters were estimated using noncompartmental methods. After a single dose of venetoclax in healthy Chinese subjects, the median time to peak concentration was 6 hours (range, 4 to 6 hours), and the mean ± SD Cmax, AUCinf, and terminal half‐life were 1.0 ± 0.32 μg/mL, 12.6 ± 5.4 μg·h/mL, and 18.4 ± 2.97 hours, respectively. On average, venetoclax Cmax and AUCinf values were 94% and 66% higher, respectively, in Chinese subjects compared with those observed historically for non‐Asian subjects receiving the same dose. Based on these pharmacokinetic results and the established exposure–response relationship of venetoclax in non‐Asian CLL subjects, a 400‐mg once‐daily dosage regimen was selected for evaluating the venetoclax pharmacokinetics, efficacy, and safety in the venetoclax phase 2 open‐label study in Chinese subjects with R/R CLL. [ABSTRACT FROM AUTHOR]

Treatment of acute myeloid leukemia (AML) patients is still hindered by resistance and relapse, resulting in an overall poor survival rate. Recently, combining specific B-cell lymphoma (Bcl)-2 inhibitors with compounds downregulating myeloid cell leukemia (Mcl)-1 has been proposed as a new effective strategy to eradicate resistant AML cells. We show here that 1(R), 6(S), 1’(R), 6’(S), 11(R), 17(S)-fistularin-3, a bromotyrosine compound of the fistularin family, isolated from the marine sponge Suberea clavata, synergizes with Bcl-2 inhibitor ABT-199 to efficiently kill Mcl-1/Bcl-2-positive AML cell lines, associated with Mcl-1 downregulation and endoplasmic reticulum stress induction. The absolute configuration of carbons 11 and 17 of the fistularin-3 stereoisomer was fully resolved in this study for the first time, showing that the fistularin we isolated from the marine sponge Subarea clavata is in fact the (+)-11(R), 17(S)-fistularin-3 stereoisomer keeping the known configuration 1(R), 6(S), 1’(R), and 6’(S) for the verongidoic acid part. Docking studies and in vitro assays confirm the potential of this family of molecules to inhibit DNA methyltransferase 1 activity.

The artimisinins are a class of antimalarial compounds whose antiparasitic activity is mediated by induction of reactive oxygen species (ROS). Herein, we report that among the artimisinins, artesunate (ARTS), an orally bioavailable compound has the most potent antileukemic activity in AML models and primary patients’ blasts. ARTS was most cytotoxic to the FLT3-ITD+ AML MV4-11 and MOLM-13 cells (IC 50 values of 1.1 and 0.82 μM respectively), inhibited colony formation in primary AML and MDS cells and augmented cytotoxicity of chemotherapeutics. ARTS lowered cellular BCL-2 level via ROS induction and increased the cytotoxicity of the BCL-2 inhibitor venetoclax (ABT-199). ARTS treatment led to cellular and mitochondrial ROS accumulation, double stranded DNA damage, loss of mitochondrial membrane potential and induction of the intrinsic mitochondrial apoptotic cascade in AML cell lines. The antileukemic activity of ARTS was further confirmed in MV4-11 and FLT3-ITD+ primary AML cell xenografts as well as MLL-AF9 syngeneic murine AML model where ARTS treatment resulted in significant survival prolongation of treated mice compared to control. Our results demonstrate the potent preclinical antileukemic activity of ARTS as well as its potential for a rapid transition to a clinical trial either alone or in combination with conventional chemotherapy or BCL-2 inhibitor, for treatment of AML. [ABSTRACT FROM AUTHOR]

Anti-apoptotic B cell-lymphoma-2 (Bcl-2) proteins are emerging as therapeutic targets in a variety of cancers for precision medicines, like the BH3-mimetic drug venetoclax (ABT-199), which antagonizes the hydrophobic cleft of Bcl-2. However, the impact of venetoclax on intracellular Ca 2+ homeostasis and dynamics in cell systems has not been characterized in detail. Here, we show that venetoclax did not affect Ca 2+ -transport systems from the endoplasmic reticulum (ER) in permeabilized cell systems. Venetoclax (1 μM) did neither trigger Ca 2+ release by itself nor affect agonist-induced Ca 2+ release in a variety of intact cell models. Among the different cell types, we also studied two Bcl-2-dependent cancer cell models with a varying sensitivity towards venetoclax, namely SU-DHL-4 and OCI-LY-1, both diffuse large B-cell lymphoma cell lines. Acute application of venetoclax did also not dysregulate Ca 2+ signaling in these Bcl-2-dependent cancer cells. Moreover, venetoclax-induced cell death was independent of intracellular Ca 2+ overload, since Ca 2+ buffering using BAPTA-AM did not suppress venetoclax-induced cell death. This study therefore shows that venetoclax does not dysregulate the intracellular Ca 2+ homeostasis in a variety of cell types, which may underlie its limited toxicity in human patients. Furthermore, venetoclax-induced cell death in Bcl-2-dependent cancer cells is not mediated by intracellular Ca 2+ overload. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech. [ABSTRACT FROM AUTHOR]

In 1988, the BCL-2 protein was found to promote cancer by limiting cell death rather than enhancing proliferation. This discovery set the wheels in motion for an almost 30 year journey involving many international research teams that has recently culminated in the approval for a drug, ABT-199/venetoclax/Venclexta that targets this protein in the treatment of cancer. This review will describe the long and winding path from the discovery of this protein and understanding the fundamental process of apoptosis that BCL-2 and its numerous homologues control, through to its exploitation as a drug target that is set to have significant benefit for cancer patients. [ABSTRACT FROM AUTHOR]

The first-in-class selective B-cell lymphoma 2 (Bcl-2) inhibitor, ABT-199 (venetoclax), is well-known for having no effect on liver cancer cells. Here, we looked into the effectiveness and underlying molecular mechanisms of kaempferol derived from persimmon leaves' (KPL) sensitization effect on ABT-199-resistant HepG2 cells. The proliferation of HepG2 cells and the H22 liver tumour bearing animal model were both studied, along with the underlying mechanisms, in relation to different dosages of KPL combined with ABT-199. According to our research, ABT-199 alone, as opposed to KPL, had no discernible effect on the development of hepatoma cells either in vitro or in vivo. It's interesting that the combined therapy shown noticeably greater anti-hepatoma effectiveness. In HepG2 cells, combining KPL and ABT-199 may increase both early and late apoptosis and lower the mitochondrial membrane potential, according to mechanistic investigations. ABT-199 and KPL dramatically decreased the expression of the antiapoptotic proteins Bcl-2, Bcl-xL, and Mcl-1, increased the expression of Bax and cleaved caspase 3, and improved cytochrome C release and Bax translocation, according to a Western blot study. As a result, the combination of KPL and ABT-199 may one day be used to treat hepatocellular cancer. [ABSTRACT FROM AUTHOR]

Members of the Bcl-2 family are major regulators of apoptosis in mammalian cells and hence infection-induced perturbations in their expression could result into elimination of the parasites or creation of a niche favoring survival. In this investigation, we uncover a novel role of host Bcl-2 in sustaining Leishmania donovani infection. A rapid two-fold increase in Bcl-2 expression occurred in response to parasite challenge. Downregulation of post infection Bcl-2 increase using siRNA or functional inhibition using Bcl-2 small molecule inhibitors interfered with intracellular parasite survival confirming the necessity of elevated Bcl-2 during infection. An increased nitric oxide (NO) response and reduced parasitic burden was observed upon Bcl-2 inhibition, where restitution of the NO response accounted for parasite mortality. Mechanistic insights revealed a major role of elevated Th2 cytokine IL-13 in parasite induced Bcl-2 expression via the transcription factor STAT-3, where blocking at the level of IL-13 receptor or downstream kinase JAK-2 dampened Bcl-2 induction. Increase in Bcl-2 was orchestrated through TLR-2-MEK-ERK signaling and changes in TLR-2 levels affected parasite uptake. In a mouse model of visceral leishmaniasis (VL), Bcl-2 inhibitors partially restored the anti-microbial NO response by at least two-fold increase that resulted in significantly reduced parasite burden. Interestingly, monocytes derived from the peripheral blood of six out of nine human VL subjects demonstrated Bcl-2 expression at significantly higher levels and sera from these patients showed only marginally quantifiable nitrites. Collectively, our study for the first time reveals a pro-parasitic role of host Bcl-2 and the capacity of host-derived IL-13 to modulate NO levels during infection via Bcl-2. Here, we propose Bcl-2 inhibition as a possible therapeutic intervention for VL.