B-cell Lymphoma-2 Family Proteins Research Articles

Abstract 5852: Tumor gene expression patterns affecting response to BCl-2 inhibitor venetoclax in acute myeloid leukemia

Abstract Background: Venetoclax (VEN), a B-cell lymphoma-2 (BCL-2) inhibitor, is an important drug in acute myeloid leukemia (AML) treatment regimens. However, resistance to VEN-based therapy is a common challenge in AML management. Some factors contributing to resistance include dysregulation of BCL-2 family proteins, mutations in the TP53 gene, activating kinase mutations, and oxidative phosphorylation activity. Despite these insights, other mechanisms of resistance remain elusive. We aim to analyze tumor gene expression patterns associated with treatment response in patients who received VEN-based therapy. Methods: Molecular and clinical data of the patients were obtained as part of the Oncology Research Information Exchange Network (ORIEN) Avatar dataset. RNA-Seq data from diagnostic Bone Marrow were processed to remove batch effects, and differential expression analysis was performed using DESeq2, while correcting for age at sample collection and sex of patients. Results: We included 16 individuals treated with VEN-based therapy. Median age at the time of specimen collection was 65 years (interquartile range, 58-69), 9 (56%) male, 11 (69%) had active disease despite treatment with VEN. VEN-based therapy was used as 1st line, 2nd line, 3rd line, 4th line or more in 3, 4, 4, 2, and 3 patients, respectively. We found a total of 332 genes differentially expressed (fdr<0.05 and abs(log2FoldChange)>2.0) among patients having no evidence of active disease and those with no active disease at the time of last contact. Among those genes, we found HOXA7 and PRAME of particular interest. HOXA7 has been previously implicated in Bromodomain and Extraterminal Domain Inhibitors (BETi) inhibitor resistance in AML. This makes HOXA7 a potentially universal mechanism of chemotherapy resistance in AML. PRAME has been associated with leukemia cell survival, and its knockdown has been shown to induce apoptosis in K562 cells, which is only expressed in AML cells but not in normal hematopoietic cells. Pathway analysis found that the top two enriched pathways for the differentially expressed genes were MET activates PTK2 signaling (R-HSA-8874081) and Metabolism of Angiotensinogen to Angiotensins(R-HSA-2022377). Conclusions: This study highlights some molecular mechanisms that give rise to AML resistance to VEN. Our results indicate that HOXA7 may potentially be a universal mechanism of chemotherapy resistance not limited to VEN-based therapy. Similarly, pathways enriched in resistant cells could be targeted to prevent or treat resistance to VEN-based therapy. These and other findings may help us identify resistance mechanisms in all AML cases vs those specific to VEN-based therapy, allowing the development of treatment strategies based on treatment mechanism. Also, analysis of these mechanisms can aid therapeutic decision-making and the development of effective treatment strategies to improve outcomes in AML. Citation Format: Juan Antonio Raygoza Garay, Seongseok Yun, Afaf Osman, Reshma Ramlal, David Feith, Michelle Churchman, Prajwal Dhakal. Tumor gene expression patterns affecting response to BCl-2 inhibitor venetoclax in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5852.

B-cell lymphoma-2 family proteins in the crosshairs: Small molecule inhibitors and activators for cancer therapy.

The B-cell lymphoma-2 (BCL-2) family of proteins plays a crucial role in the regulation of apoptosis, offering a dual mechanism for its control. Numerous studies have established a strong association between gene disorders of these proteins and the proliferation of diverse cancer cell types. Consequently, the identification and development of drugs targeting BCL-2 family proteins have emerged as a prominent area in antitumor therapy. Over the last two decades, several small-molecules have been designed to modulate the protein-protein interactions between anti- and proapoptotic BCL-2 proteins, effectively suppressing tumor growth and metastasis in vivo. The primary focus of research has been on developing BCL-2 homology 3 (BH3) mimetics to target antiapoptotic BCL-2 proteins, thereby competitively releasing proapoptotic BCL-2 proteins and restoring the blocked intrinsic apoptotic program. Additionally, for proapoptotic BCL-2 proteins, exogenous small molecules have been explored to activate cell apoptosis by directly interacting with executioner proteins such as BCL-2-associated X protein (BAX) or BCL-2 homologous antagonist/killer protein (BAK). In this comprehensive review, we summarize the inhibitors and activators (sensitizers) of BCL-2 family proteins developed over the past decades, highlighting their discovery, optimization, preclinical and clinical status, and providing an overall landscape of drug development targeting these proteins for therapeutic purposes.

Hesperidin ameliorates impairment in hippocampal neural stem cells related to apoptosis induced by methotrexate in adult rats

Neurogenesis is a process of generating neural stem cells (NSCs) as functional neurons can be decreased after chemotherapy treatments. Methotrexate (MTX) is a folate antagonist that is used for cancer treatment but has negative effects, including oxidative stress, neuronal apoptosis and cognitive impairments. Hesperidin (Hsd), a flavonoid found in citrus fruits, has antioxidant and neuroprotection properties. This study investigated whether Hsd could attenuate impairments of hippocampal neural stem cells related to apoptosis induced by MTX. Spraque-Dawley rats (n = 24) were divided into 4 groups: (1) Vehicle group received propylene glycol (21 days) and 0.9% normal saline (day 8 and 15), (2) Hsd group received 100 mg/kg (21 days), (3) MTX group received 75 mg/kg (days 8 and 15) and (4) MTX+Hsd group received MTX, 75 mg/kg (day 8 and 15) and Hsd 100 mg/kg (21 days). Our results showed that MTX decreased hippocampal neural stem cells including SRY (sex determining region Y)-box 2 (SOX2) and nestin. MTX diminished vascular related (VR) Ki-67 positive cells in the hippocampus but not non-vascular related (NVR) Ki-67. Additionally, MTX reduced SOX2, nestin, postsynaptic density protein 95 (PSD-95) and B-cell lymphoma-2 family of proteins (Bcl-2), whereas Bax and caspase-3 were enhanced in the hippocampal tissues. Interestingly, co-treatment with Hsd and MTX revealed upregulation of SOX2, nestin and VR Ki-67 positive cells as well as elevated SOX2, nestin, PSD-95 and Bcl-2 proteins. Moreover, receiving both Hsd and MTX significantly suppressed increased Bax and caspase-3. These results confirm that Hsd can ameliorate MTX-induced impairments of hippocampal NSC proliferation and neuronal apoptosis.

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.

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.

Novel Compounds Synergize With Venetoclax to Target KMT2A-Rearranged Pediatric Acute Myeloid Leukemia.

In pediatric acute myeloid leukemia (AML), fusions involving lysine methyltransferase 2A (KMT2A) are considered hallmarks of aggressive AML, for whom the development of targeted specific therapeutic agents to ameliorate classic chemotherapy and obtain a complete eradication of disease is urgent. In this study, we investigated the antiapoptotic proteins in a cohort of 66 pediatric AML patients, finding that 75% of the KMT2A-r are distributed in Q3 + Q4 quartiles of BCL-2 expression, and KMT2A-r have statistically significant high levels of BCL-2, phospho-BCL-2 S70, and MCL-1, indicating a high anti-apoptotic pathway activation. In an attempt to target it, we tested novel drug combinations of venetoclax, a B-cell lymphoma-2 (BCL-2) inhibitor, in KMT2A-MLLT3, for being the most recurrent, and KMT2A-AFDN, for mediating the worst prognosis, rearranged AML cell lines. Our screening revealed that both the bromodomain and extra-terminal domain (BET) inhibitor, I-BET151, and kinase inhibitor, sunitinib, decreased the BCL-2 family protein expression and significantly synergized with venetoclax, enhancing KMT2A-r AML cell line death. Blasts t (6; 11) KMT2A-AFDN rearranged, both from cell lines and primary samples, were shown to be significantly highly responsive to the combination of venetoclax and thioridazine, with the synergy being induced by a dramatic increase of mitochondrial depolarization that triggered blast apoptosis. Finally, the efficacy of novel combined drug treatments was confirmed in KMT2A-r AML cell lines or ex vivo primary KMT2A-r AML samples cultured in a three-dimensional system which mimics the bone marrow niche. Overall, this study identified that, by high-throughput screening, the most KMT2A-selective drugs converged in different but all mitochondrial apoptotic network activation, supporting the use of venetoclax in this AML setting. The novel drug combinations here unveiled provide a rationale for evaluating these combinations in preclinical studies to accelerate the introduction of targeted therapies for the life-threatening KMT2A-AML subgroup of pediatric AML.

The Effect of NeuroAid (MLC901) on Cholestasis-Induced Spatial Memory Impairment with Respect to the Expression of BAX, BCL-2, BAD, PGC-1α and TFAM Genes in the Hippocampus of Male Wistar Rats.

Cholestasis is a bile flow reduction that is induced following Bile Duct Ligation (BDL). Cholestasis impairs memory and induces apoptosis. Apoptosis consists of two pathways: intrinsic and extrinsic. The intrinsic pathway is modulated by BCL-2 (B cell lymphoma-2) family proteins. BCL-2 (a pro-survival BCL-2 protein) has anti-apoptotic effect, while BAD (BCL-2-associated death) and BAX (BCL-2-associated X), the other members of BCL-2 family have pro-apoptotic effect. Furthermore, TFAM (mitochondrial transcriptional factor A) is involved in transcription and maintenance of mitochondrial DNA and PGC-1α (peroxisome proliferator-activated receptor γ coactivator-1α) is a master regulator of mitochondrial biogenesis. On the other hand, NeuroAid is a Traditional Chinese Medicine with neuroprotective and anti-apoptosis effects. In this study, we evaluated the effect of cholestasis on spatial memory and expression of BCL-2, BAD, BAX, TFAM, and PGC-1α in the hippocampus of rats. Additionally, we assessed the effect of NeuroAid on cholestasis-induced cognitive and genetic alterations. Cholestasis was induced by BDL surgery and NeuroAid was injected intraperitoneal at the dose of 0.4mg/kg. Furthermore, spatial memory was evaluated using Morris Water Maze (MWM) apparatus. The results showed cholestasis impaired spatial memory, increased the expression of BAD and BAX, decreased the expression of TFAM and PGC-1α, and did not alter the expression of BCL-2. Also, NeuroAid decreased the expression of BAD and BAX and increased the expression of TFAM, PGC-1α, and BCL-2. In conclusion, cholestasis impaired spatial memory and increased the expression of pro-apoptotic genes. Also, cholestasis decreased the expression of TFAM and PGC-1α. Interestingly, NeuroAid restored the effects of cholestasis.

Mitochondrial-nuclear p53 trafficking controls neuronal susceptibility in stroke.

Stroke is a major cause of death and long‐term disability in the adult. Neuronal apoptosis plays an essential role in the pathophysiology of ischemic brain damage and impaired functional recovery after stroke. The tumor suppressor protein p53 regulates key cellular processes, including cell cycle arrest, DNA repair, senescence, and apoptosis. Under cellular stress conditions, p53 undergoes post‐translational modifications, which control protein localization, stability, and proapoptotic activity. After stroke, p53 rapidly accumulates in the ischemic brain, where it activates neuronal apoptosis through both transcriptional‐dependent and ‐independent programs. Over the last years, subcellular localization of p53 has emerged as an important regulator of ischemia‐induced neuronal apoptosis. Upon an ischemic insult, p53 rapidly translocates to the mitochondria and interacts with B‐cell lymphoma‐2 family proteins, which activate the mitochondrial apoptotic program, with higher efficacy than through its activity as a transcription factor. Moreover, the identification of a human single nucleotide polymorphism at codon 72 of the Tp53 gene that controls p53 mitochondrial localization and cell susceptibility to apoptosis supports the important role of the p53 mitochondrial program in neuronal survival and functional recovery after stroke. In this article, we review the relevance of mitochondrial and nuclear localization of p53 on neuronal susceptibility to cerebral ischemia and its impact on functional outcome of stroke patients.

Anti-tumor activity and immunogenicity of a succinoglycan riclin

Natural polysaccharides have attracted considerable interests due to diverse biological activities. Succinoglycan is an extracellular polysaccharide produced by most Agrobacterium strains. Here, we confirmed riclin was a typical succinoglycan by NMR and methylation analysis, and investigated the antitumor effects of riclin in sarcoma 180 tumor-bearing mice. The results showed that riclin inhibited the tumor growth significantly as well as cyclophosphamide (CTX). While CTX caused serious damage to spleen structure, riclin increased the spleen index and promoted lymphocytes proliferation in peripheral blood, spleen and lymph nodes. Riclin decreased splenocytes apoptosis as evidenced by alterations of B-cell lymphoma-2 family proteins and Cleaved Caspase-3 protein. Moreover, 1H nuclear magnetic resonance (NMR)-based metabolomics analysis revealed that riclin partially altered the metabolic profiles of splenocytes. In conclusion, riclin is a succinoglycan that performed strong immunogenicity and suppressed sarcoma growth in mice. Succinoglycan riclin could be a potential antitumor agent for functional food and pharmaceutical purpose.

Targeting Bcl-2 Proteins in Acute Myeloid Leukemia.

B cell lymphoma 2 (BCL-2) family proteins play an important role in intrinsic apoptosis. Overexpression of BCL-2 proteins in acute myeloid leukemia can circumvent resistance to apoptosis and chemotherapy. Considering this effect, the exploration of anti-apoptotic BCL-2 inhibitors is considered to have tremendous potential for the discovery of novel pharmacological modulators in cancer. This review outlines the impact of BCL-2 family proteins on intrinsic apoptosis and the development of acute myeloid leukemia (AML). Furthermore, we will also review the new combination therapy with venetoclax that overcomes resistance to venetoclax and discuss biomarkers of treatment response identified in early-phase clinical trials.

Selective Covalent Targeting of Anti-apoptotic BFL-1 by a Sulfonium-Tethered Peptide.

Anti-apoptotic B cell lymphoma 2 (BCL-2) family proteins are proven targets for human cancers. Targeting the BH3-binding pockets of these anti-apoptotic proteins could reactivate apoptosis in BCL-2-depedent cancers. BFL-1 is a BCL-2 family protein overexpressed in various chemoresistant cancers. A unique cysteine at the binding interface of the BH3 and BFL-1 was previously proven to be an intriguing targeting site to irreversibly inhibit BFL-1 functions with stabilized cyclic peptide bearing a covalent warhead. Recently, we developed a sulfonium-tethered peptide cyclization strategy to construct peptide ligands that could selectively and efficiently react with the cysteine(s) of target proteins near the interacting interface. Using this method, we constructed a BFL-1 peptide inhibitor, B4-MC, that could selectively conjugate with BFL-1 both in vitro and in cell. B4-MC showed good cellular uptake, colocalized with BFL-1 on mitochondria, and showed obvious growth inhibition of BFL-1 over-expressed cancer cell lines.

Heterogeneous Pattern of Dependence on Anti-Apoptotic BCL-2 Family Proteins upon CHOP Treatment in Diffuse Large B-Cell Lymphoma.

Expression of the anti-apoptotic B-cell lymphoma 2 (BCL-2) protein in patients with diffuse large B-cell lymphoma (DLBCL) strongly correlates with resistance to standard therapy with cyclophosphamide, vincristine, doxorubicin, prednisolone, and rituximab (R-CHOP). Although studies focus mainly on the contribution of BCL-2, here we also investigate the contribution of other anti-apoptotic proteins to CHOP-therapy resistance in DLBCL. Functional dynamic BCL-2 homology (BH)3 profiling was applied to DLBCL cell lines upon CHOP treatment or single CHOP compounds. Cell-specific anti-apoptotic dependencies were validated with corresponding BH3-mimetics. We found high expression of anti-apoptotic BCL-2, MCL-1, and BCL-XL in DLBCL cell lines and patients. CHOP treatment resulted in both enhanced and altered anti-apoptotic dependency. Enhanced sensitivity to different BH3-mimetics after CHOP treatment was confirmed in specific cell lines, indicating heterogeneity of CHOP-induced resistance in DLBCL. Analysis of single CHOP compounds demonstrated that similar changes could also be induced by doxorubicin or vincristine, providing evidence for clinical combination therapies of doxorubicin or vincristine with BH3-mimetics in DLBCL. In conclusion, we show for the first time that CHOP treatment induces increased anti-apoptotic dependency on MCL-1 and BCL-XL, and not just BCL-2. These results provide new perspectives for the treatment of CHOP-resistant DLBCL and underline the potential of BH3 profiling in predicting therapy outcomes.

Updated Results from the Venetoclax (Ven) in Combination with Idasanutlin (Idasa) Arm of a Phase 1b Trial in Elderly Patients (Pts) with Relapsed or Refractory (R/R) AML Ineligible for Cytotoxic Chemotherapy

Updated Results from the Venetoclax (Ven) in Combination with Idasanutlin (Idasa) Arm of a Phase 1b Trial in Elderly Patients (Pts) with Relapsed or Refractory (R/R) AML Ineligible for Cytotoxic Chemotherapy

Autophagy and apoptosis are regulated by stress on Bcl2 by AMBRA1 in the endoplasmic reticulum and mitochondria

BackgroundAutophagy and apoptosis are two important physiological processes that determine cell survival or death in response to different stress signals. The regulatory mechanisms of these two processes share B-cell lymphoma-2 family proteins and AMBRA1, which are present in both the endoplasmic reticulum and mitochondria. B-cell lymphoma-2 family proteins sense different stresses and interact with AMBRA1 to regulate autophagy and apoptosis, which are respectively mediated by Beclin1 and Caspases. Therefore, we investigated how different levels of stress on B-cell lymphoma-2 family proteins that bind to AMBRA1 in the endoplasmic reticulum and mitochondria regulate the switch from autophagy to apoptosis.MethodsIn this paper, we considered the responses of B-cell lymphoma-2 family proteins, which bind to AMBRA1 in both the endoplasmic reticulum and mitochondria, to two different levels of stress in a model originally proposed by Kapuy et al. We investigated how these two stress levels affect the transition from autophagy to apoptosis and their effects on apoptosis activation over time. Additionally, we analyzed how the feedback regulation in this model affects the bifurcation diagrams of two levels of stress and cell fate decisions between autophagy and apoptosis.ResultsAutophagy is activated for minor stress in mitochondria regardless of endoplasmic reticulum stress, while apoptosis is activated for only significant stress in mitochondria. Apoptosis is only sensitive to mitochondria stress. The time duration before apoptosis activation is longer in the presence of high AMBRA1 levels with high endoplasmic reticulum and mitochondria stress. AMBRA1 can compete with B-cell lymphoma-2 family proteins to bind and activate Beclin1 and thus promote the autophagy process for a long time before apoptosis. Furthermore, apoptosis is prone to occur with increasing activation of Caspases, inactivation of Beclin1-A and the Michaelis constant of Caspases.ConclusionA novel mathematical model has been developed to understand the complex regulatory mechanisms of autophagy and apoptosis. Our model may be applied to further autophagy-apoptosis dynamic modeling experiments and simulations.

Designing anticancer copper(II) complexes by optimizing 2-pyridine-thiosemicarbazone ligands

To develop potential next-generation metal anticancer agents, we designed and synthesised five Cu(II) 2-pyridine-thiosemicarbazone complexes by modifying the hydrogen atom at the N-4 position of ligands, and then investigated their structure-activity relationships and anticancer mechanisms. Modification of the N-4 position with different groups caused significant differences in cellular uptake and produced superior antitumor activity. Cu complexes arrested the cell cycle at S phase, leading to down-regulation of levels of cyclin and cyclin-dependent kinases and up-regulation of expression of cyclin-dependent kinase inhibitors. Cu complexes exerted chemotherapeutic effects via activating p53 and inducing production of reactive oxygen species to regulate expression of the B-cell lymphoma-2 family of proteins, causing a change in the mitochondrial membrane potential and release of cytochrome c to form a dimer with apoptosis protease activating factor-1, resulting in activation of caspase-9/3 to induce apoptosis. In addition, Cu complexes inhibited telomerase by down-regulating the c-myc regulator gene and expression of the human telomerase reverse transcriptase.

Shutting Down Acute Myeloid Leukemia and Myelodysplastic Syndrome with BCL-2 Family Protein Inhibition.

Apoptosis results from the interaction between pro- and anti-apoptotic proteins, mediated by BCL-2 homology 3 (BH3) proteins. B cell lymphoma-2 (BCL-2) is an inhibitor of apoptosis which stabilizes the mitochondria, resulting in the prevention of activation of the pro-apoptotic proteins. In addition, BCL-2 is overexpressed in the leukemic stem cell (LSC) population, and its inhibition may lead to selective LSC eradication. Herein, we will discuss the mechanism and rationale of BCL-2 inhibition in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) with an overview of the selective BCL-2 inhibitor venetoclax. Venetoclax has activity against AML and has displayed synergistic activity with hypomethylating agents in the preclinical setting. In the clinical setting, although it has only modest activity as a single agent in relapsed and refractory AML, in the older, treatment-naïve population, in combination with either a hypomethylator or low-dose cytarabine, it is well tolerated with impressive efficacy. In addition, BCL-2 inhibition may also have activity in MDS, and although clinical trials are in their early phases, this may be an effective strategy in both the up-front and relapsed setting. BCL-2 inhibition with venetoclax is well tolerated and active in older patients with newly diagnosed AML and in the relapsed setting has activity that may be improved in combination with other therapies. It may prove to be effective in MDS and is an exciting treatment strategy for myeloid malignancies.

The small molecule AU14022 promotes colorectal cancer cell death via p53-mediated G2/M-phase arrest and mitochondria-mediated apoptosis.

The p53 tumor suppressor plays critical roles in cell cycle regulation and apoptotic cell death, with its activation capable of sensitizing cancer cells to radiotherapy or chemotherapy. To identify small molecules that induce apoptosis via increased p53 transcriptional activity, we used a novel in-house library containing 96 small-molecule compounds. Using a cell-based screening method with a p53-responsive luciferase-reporter assay system involving benzoxazole derivatives, we found that AU14022 administration significantly increased p53 transcriptional activity in a concentration-dependent manner. Treatment with AU14022 increased p53 protein expression, p53 Ser15 phosphorylation, p53-mediated expression of downstream target genes, and apoptosis in p53-wild-type HCT116 human colon cancer cells, but not in p53-knockout HCT116 cells. Additionally, p53-wild-type HCT116 cells treated with AU14022 exhibited mitochondrial dysfunction, including modulated expression of B-cell lymphoma-2 family proteins and cytochrome c release. Combination treatment with AU14022 and ionizing radiation (IR) synergistically induced apoptosis as compared with IR or AU14022 treatment alone, with further investigation demonstrating that cell cycle progression was significantly arrested at the G2/M phase following AU14022 treatment. Furthermore, in a mouse p53-wild-type HCT116 colon cancer xenograft model, combined treatment with AU14022 and IR inhibited tumor growth more effectively than radiation alone. Therefore, AU14022 treatment induced apoptosis through p53-mediated cell cycle arrest involving mitochondrial dysfunction, leading to enhanced radiosensitivity in colon cancer cells. These results provide a basis for further assessments of AU14022 as a promising anticancer agent.

Fas- and Mitochondria-Mediated Signaling Pathway Involved in Osteoblast Apoptosis Induced by AlCl3.

Aluminum (Al) is known to induce apoptosis of osteoblasts (OBs). However, the mechanism is not yet established. To investigate the apoptotic mechanism of OBs induced by aluminum trichloride (AlCl3), the primary OBs from the craniums of fetal Wistar rats were exposed to 0mg/mL (control group, CG), 0.06mg/mL (low-dose group, LG), 0.12mg/mL (mid-dose group, MG), and 0.24mg/mL (high-dose group, HG) AlCl3 for 24h, respectively. We observed that AlCl3 induced OB apoptosis with the appearance of apoptotic morphology and increase of apoptosis rate. Additionally, AlCl3 treatment activated mitochondrial-mediated signaling pathway, accompanied by mitochondrial membrane potential (ΔΨm) depolarization, release of cytochrome c from the mitochondria to the cytoplasm, as well as survival signal-related factor caspase-9 and caspase-3 activation. AlCl3 exposure also activated Fas/Fas ligand signaling pathway, presented as Fas, Fas ligand, and Fas-associated death domain expression enhancement and caspase-8 activation, as well as the hydrolysis of Bid to truncated Bid, suggesting that the Fas-mediated signaling pathway might aggravate mitochondria-mediated OB apoptosis through hydrolyzing Bid. Furthermore, AlCl3 exposure inhibited Bcl-2 protein expression and increased the expressions of Bax, Bak, and Bim in varying degrees. These results indicated that AlCl3 exposure induced OB apoptosis through activating Fas- and mitochondria-mediated signaling pathway and disrupted B-cell lymphoma-2 family proteins.