Bcl2-interacting Mediator Research Articles

In silico design of potential Mcl-1 peptide-based inhibitors.

BIM (Bcl-2 interacting mediator of apoptosis)-derived peptides that specifically target over-expressed Mcl-1 (myeloid cell leukemia-1) protein and induce apoptosis are potentially anti-cancer agents. Since the helicity of BIM-derived peptides has a crucial role in their functionality, a range of strategies have been used to increase the helicity including the introduction of unnatural residues and stapling methods that have some drawbacks such as the accumulation in the liver. To avoid these drawbacks, this study aimed to design a more helical peptide by utilizing bioinformatics algorithms and molecular dynamics simulations without exploiting unnatural residues and stapling methods. MM-PBSA results showed that the mutations of A4fE and A2eE in analogue 5 demonstrate a preference towards binding with Mcl-1. As evidenced by Circular dichroism results, the helicity increases from 18 to 34%, these findings could enhance the potential of analogue 5 as an anti-cancer agent targeting Mcl-1. The applied strategies in this research could shed light on the in silico peptide design. Moreover, analogue 5 as a drug candidate can be evaluated in vitro and in vivo studies. The sequence of the lead peptide was determined using the ApInAPDB database and PRALINE program. Contact finder and PDBsum web server softwares were used to determine the contact involved amino acids in complex with Mcl-1. All identified salt bridge contributing residues were unaltered to preserve the binding affinity. After proposing novel analogues, their secondary structures were predicted by Cham finder web server software and GOR, Neural Network, and Chou-Fasman algorithms. Finally, molecular dynamics simulations run for 100 ns were done using the GROMACS, version 5.0.7, with the CHARMM36 force field. MM-PBSA was used to assess binding affinity specificity in targeting Mcl-1 and Bcl-xL (B-cell lymphoma extra-large).

Vitamin D deficiency triggers intrinsic apoptosis by impairing SPP1-dependent antiapoptotic signaling in chronic hematogenous osteomyelitis

Chronic hematogenous osteomyelitis (CHOM) is a common bone disease characterized by the development of sequestra after bacterial infection. Emerging evidence has shown that vitamin D (VD) deficiency raises the risk of osteomyelitis, but the underlying mechanisms remain obscure. Here, we establish a CHOM model in VD diet-deficient mice by intravenous inoculation of Staphylococcus aureus. Whole-genome microarray analyses using osteoblast cells isolated from sequestra reveal significant downregulation of SPP1 (secreted phosphoprotein 1). Molecular basis investigations show that VD sufficiency activates the VDR/RXR (VD receptor/retinoid X receptor) heterodimer to recruit NCOA1 (nuclear receptor coactivator 1) and transactivate SPP1 in healthy osteoblast cells. Secreted SPP1 binds to the cell surface molecule CD40 to activate serine/threonine-protein kinase Akt1, which then phosphorylates forkhead box O3a (FOXO3a), blocking FOXO3a-mediated transcription. By contrast, VD deficiency impairs the NCOA1-VDR/RXR-mediated overexpression of SPP1, leading to the inactivation of Akt1 and the accumulation of FOXO3a. FOXO3a then upregulates the expression of the apoptotic genes BAX (Bcl2-associated X-protein), BID (BH3 interacting death domain), and BIM (Bcl2-interacting mediator of cell death), to induce apoptosis. Administration of the NCOA1 inhibitor gossypol to the CHOM mice also promotes the occurrence of sequestra. VD supplementation can reactivate the SPP1-dependent antiapoptotic signaling and improve the outcomes of CHOM. Collectively, our data reveal that VD deficiency promotes bone destruction in CHOM by the removal of SPP1-dependent antiapoptotic signaling.

Abstract 2530: DELE1 loss and dysfunctional integrated stress signaling in TP53 mutated AML is a novel pathway for venetoclax resistance

Abstract Background: Venetoclax (ven) in combination with hypomethylating agents or low dose cytarabine leads to rapid and durable remission in patients (pts) with acute myeloid leukemia (AML) unfit for intensive chemotherapy (IC), however, pts with TP53 mutations (TP53mut) exhibit adverse prognosis. Here, we identify dysregulation of the integrated stress response (ISR) pathway in pts with TP53mut, specifically through the actions of DAP3 binding cell death enhancer 1 (DELE1) and its activating protease OMA1. Methods: RNA sequencing was performed on pre-treatment bone marrow-derived mononuclear cells (BMMCs) from pts with AML ineligible for IC across four clinical trials (NCT02993523, NCT02203773, NCT03069352, and NCT02287233). CRISPR-Cas9 editing of TP53-intact AML cell lines was used to generate TP53-/-, TP53mut, DELE1-/-, OMA1-/- and TP53/DELE1 double deficient cell lines. Quantification of BCL2 and Myeloid cell leukemia 1 (MCL1) complexes with BCL2 interacting mediator (BIM) was performed using chemiluminescence assays. A drug screen to assess viability and gene expression was performed in cells treated with ven in combination with 63 drugs. Results: In total 401 pts were included in the analyses, of which 17% harbored TP53mut and 83% were TP53wt. Analysis of genes differentially expressed (DE) between pts with TP53mut and TP53wt AML revealed 19 DE genes shared across all trials. DELE1, an ISR adaptor, was associated with genes mapping to the ISR-related eukaryotic initiation factor-α (eIF2α) in TP53mut AML BMMCs. Deletion of DELE1 or OMA1 in AML cell lines blocked eIF2α activation and induction of the transcription factor ATF4, a critical ISR effector, in response to the mitochondrial stressor FCCP, ven, and azacitidine, and resulted in ven resistance similar to that of TP53 deficient cells. All modified AML cell lines exhibited a concomitant decrease in the pro-apoptotic regulator PMAIP1, encoding NOXA, and a 14-18-fold increase in MCL1-BIM complexes following ven treatment, as well as increased MCL1 expression, compared to parental lines. Further screening of these cell lines for ven sensitizing activity revealed the BH3 mimetic S63845, which targets MCL1, as the top hit, suggesting that combined BH3 mimetics may overcome ven resistance during ISR pathway defects. As DELE1 is located on chromosome 5 (CH); the frequent loss of CH 5q among TP53mut AML may contribute to low DELE1 expression. Conclusions: These data suggest that defective ISR signaling may be a factor in TP53mut AML treatment outcome and point to DELE1 dysregulation as a driver of ISR inactivation in pts with TP53mut AML. The ISR induces the expression of NOXA, which displaces MCL1 from BIM and lowers the apoptotic threshold. Our data identify p53, DELE1, and OMA1 as regulators of NOXA expression post ven treatment and indicate that co-targeting MCL1 in pts with TP53mut AML may be beneficial to overcome ven resistance. Citation Format: David Sharon, Paul Jung, Yan Sun, Weiguo Feng, Ziping Yang, Valerie Robinson, Diya Mitra, Wei Liu, Pingping Zheng, Tamar Uziel, Lloyd Lam, Mark D. Minden, Jeremy Ross, Wellington Mendes, Jalaja Potluri, Andrew H. Wei, Marina Konopleva, Monique Dail, Brenda Chyla, PK Epling-Burnette. DELE1 loss and dysfunctional integrated stress signaling in TP53 mutated AML is a novel pathway for venetoclax resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2530.

Chemotherapy-Free Targeted Anti-BCR-ABL+ Acute Lymphoblastic Leukemia Therapy May Benefit the Heart.

Simple SummaryRisk-adapted multiagent chemotherapy has led to a remarkable improvement in the life expectancy of patients with acute lymphoblastic leukemia (ALL). Nevertheless, in high-risk subgroups such as BCR-ABL+ ALL, relapse rates remain high without allogeneic hematopoietic stem cell transplantation, and the adverse effects of chemotherapy may cause acute and chronic cardiac complications or dysfunction. Here, we demonstrated that chemotherapy-free targeted therapies designed to optimize apoptosis induction in BCR-ABL+ ALL may circumvent cardiac on-target side effects and may even activate cardiac recovery.Targeted therapies are currently considered the best cost–benefit anti-cancer treatment. In hematological malignancies, however, relapse rates and non-hematopoietic side effects including cardiotoxicity remain high. Here, we describe significant heart damage due to advanced acute lymphoblastic leukemia (ALL) with t(9;22) encoding the bcr-abl oncogene (BCR-ABL+ ALL) in murine xenotransplantation models. Echocardiography reveals severe cardiac dysfunction with impaired left ventricular function and reduced heart and cardiomyocyte dimensions associated with increased apoptosis. This cardiac damage is fully reversible, but cardiac recovery depends on the therapy used to induce ALL remission. Chemotherapy-free combination therapy with dasatinib (DAS), venetoclax (VEN) (targeting the BCR-ABL oncoprotein and mitochondrial B-cell CLL/Lymphoma 2 (BCL2), respectively), and dexamethasone (DEX) can fully revert cardiac defects, whereas the depletion of otherwise identical ALL in a genetic model using herpes simplex virus type 1 thymidine kinase (HSV-TK) cannot. Mechanistically, dexamethasone induces a pro-apoptotic BCL2-interacting mediator of cell death (BIM) expression and apoptosis in ALL cells but enhances pro-survival B-cell lymphoma extra-large (BCLXL) expression in cardiomyocytes and clinical recovery with the reversion of cardiac atrophy. These data demonstrate that therapies designed to optimize apoptosis induction in ALL may circumvent cardiac on-target side effects and may even activate cardiac recovery. In the future, combining the careful clinical monitoring of cardiotoxicity in leukemic patients with the further characterization of organ-specific side effects and signaling pathways activated by malignancy and/or anti-tumor therapies seems reasonable.

Overexpression of HIPK2 removes the transrepression of proapoptotic genes mediated by the CtBP1-p300-FOXO3a complex and increases the chemosensitivity in osteosarcoma cells.

Decreased expression of proapoptotic genes can lead to the chemoresistenance in cancer therapy. Carboxyl-terminal binding protein 1 (CtBP1), a transcriptional corepressor with multiple oncogenic effects, has been previously identified to suppress the expression of two proapoptotic genes [BAX (BCL2 associated X) and BIM (Bcl-2 interacting mediator of cell death)] by assembling a complex with the Forkhead box O3 (FOXO3a) transcription factor and the p300 histone acetyltransferase. However, the upstream regulatory signaling of the CtBP1-p300-FOXO3a complex is obscure, and the effects of changing this signaling on chemosensitivity in osteosarcoma are unknown. Herein, we discovered that the downregulation of HIPK2 (Homeodomain-interacting protein kinase 2) was essential for the function of the CtBP1-p300-FOXO3a complex. Downregulation of HIPK2 prevented the phosphorylation and subsequent degradation of CtBP1, thereby allowing the assembly of the CtBP1-p300-FOXO3a complex and suppression of the expression of proapoptotic genes, such as BAX, BIM, BIK (Bcl-2 interacting killer) and NOXA/PMAIP1 (Phorbol-12-myristate-13-acetate-induced protein 1). Overexpression of HIPK2 promoted the phosphorylation of CtBP1 and the degradation of CtBP1 by proteasomes, thereby preventing the formation of the CtBP1-p300-FOXO3a complex. The abolition of CtBP1 transrepression increased the expression of proapoptotic genes to induce apoptosis and increase chemosensitivity in osteosarcoma cells. Taken together, our in vitro and in vivo results revealed that overexpression of HIPK2 could remove the CtBP1-mediated transrepression of proapoptotic genes, indicating a new therapeutic option for the treatment of osteosarcoma.

IGF-1 Inhibits Apoptosis of Porcine Primary Granulosa Cell by Targeting Degradation of BimEL.

Insulin-like growth factor-1 (IGF-1) is an intra-ovarian growth factor that plays important endocrine or paracrine roles during ovarian development. IGF-1 affects ovarian function and female fertility through reducing apoptosis of granulosa cells, yet the underlying mechanism remains poorly characterized. Here, we aimed to address these knowledge gaps using porcine primary granulosa cells and examining the anti-apoptotic mechanisms of IGF-1. IGF-1 prevented the granulosa cell from apoptosis, as shown by TUNEL and Annexin V/PI detection, and gained the anti-apoptotic index, the ratio of Bcl-2/Bax. This process was partly mediated by reducing the pro-apoptotic BimEL (Bcl-2 Interacting Mediator of Cell Death-Extra Long) protein level. Western blotting showed that IGF-1 promoted BimEL phosphorylation through activating p-ERK1/2, and that the proteasome system was responsible for degradation of phosphorylated BimEL. Meanwhile, IGF-1 enhanced the Beclin1 level and the rate of LC3 II/LC3 I, indicating that autophagy was induced by IGF-1. By blocking the proteolysis processes of both proteasome and autophagy flux with MG132 and chloroquine, respectively, the BimEL did not reduce and the phosphorylated BimEL protein accumulated, thereby indicating that both proteasome and autophagy pathways were involved in the degradation of BimEL stimulated by IGF-1. In conclusion, IGF-1 inhibited porcine primary granulosa cell apoptosis via degradation of pro-apoptotic BimEL. This study is critical for us to further understand the mechanisms of follicular survival and atresia regulated by IGF-1. Moreover, it provides a direction for the treatment of infertility caused by ovarian dysplasia, such as polycystic ovary syndrome and the improvement of assisted reproductive technology.

Re-inforcing the cell death army in the fight against breast cancer.

Metastatic breast cancer is responsible for most breast cancer-related deaths. Disseminated cancer cells have developed an intrinsic ability to resist anchorage-dependent apoptosis (anoikis). Anoikis is caused by the absence of cellular adhesion, a process that underpins lumen formation and maintenance during mammary gland development and homeostasis. In healthy cells, anoikis is mostly governed by B-cell lymphoma-2 (BCL2) protein family members. Metastatic cancer cells, however, have often developed autocrine BCL2-dependent resistance mechanisms to counteract anoikis. In this Review, we discuss how a pro-apoptotic subgroup of the BCL2 protein family, known as the BH3-only proteins, controls apoptosis and anoikis during mammary gland homeostasis and to what extent their inhibition confers tumor suppressive functions in metastatic breast cancer. Specifically, the role of the two pro-apoptotic BH3-only proteins BCL2-modifying factor (BMF) and BCL2-interacting mediator of cell death (BIM) will be discussed here. We assess current developments in treatment that focus on mimicking the function of the BH3-only proteins to induce apoptosis, and consider their applicability to restore normal apoptotic responses in anchorage-independent disseminating tumor cells.

Pancreatic β-cell protection from inflammatory stress by the endoplasmic reticulum proteins thrombospondin 1 and mesencephalic astrocyte-derived neutrotrophic factor (MANF)

Cytokine-induced endoplasmic reticulum (ER) stress is one of the molecular mechanisms underlying pancreatic β-cell demise in type 1 diabetes. Thrombospondin 1 (THBS1) was recently shown to promote β-cell survival during lipotoxic stress. Here we show that ER-localized THBS1 is cytoprotective to rat, mouse, and human β-cells exposed to cytokines or thapsigargin-induced ER stress. THBS1 confers cytoprotection by maintaining expression of mesencephalic astrocyte-derived neutrotrophic factor (MANF) in β-cells and thereby prevents the BH3-only protein BIM (BCL2-interacting mediator of cell death)-dependent triggering of the mitochondrial pathway of apoptosis. Prolonged exposure of β-cells to cytokines or thapsigargin leads to THBS1 and MANF degradation and loss of this prosurvival mechanism. Approaches that sustain intracellular THBS1 and MANF expression in β-cells should be explored as a cytoprotective strategy in type 1 diabetes.

Genetic Modifiers of Progression-Free Survival in Never-Smoking Lung Adenocarcinoma Patients Treated with First-Line Tyrosine Kinase Inhibitors.

Patients with non-small cell lung cancer (NSCLC) with mutated epidermal growth factor receptor (EGFR) are relatively sensitive to EGFR-tyrosine kinase inhibitor (TKI) treatment and have longer progression-free survival (PFS) when treated with EGFR-TKI compared with platinum-based chemotherapy. However, many patients with advanced NSCLC who have mutated EGFR do not respond to first-line EGFR-TKI treatment and still have shorter PFS. The aim of this study was to identify genetic variants associated with PFS among patients with lung adenocarcinoma who were treated with first-line EGFR-TKIs. A genome-wide association study on PFS was performed in never-smoking women diagnosed with lung adenocarcinoma and who were treated with first-line EGFR-TKIs (n = 128). Significant single-nucleotide polymorphisms (SNPs) were selected for follow-up association analysis (n = 198) and for replication assay in another independent cohort (n = 153). We identified SNPs at 4q12 associated with PFS at genome-wide significance (P < 10-8) and with an estimated hazard ratio of more than 4. This association was also replicated in a larger but similar cohort and in an independent NSCLC cohort. Follow-up functional analyses showed that these SNPs were associated with the expression of EGFR, which encodes the TKI target, and with a nearby gene neuromedin-U, which encodes a G protein-coupled receptor ligand known to be involved in the progression of NSCLC. Considering these as possible prognostic biomarkers for the treatment of patients with late-stage lung cancer, we found that these SNPs were not associated with EGFR mutation status or with polymorphism of the Bcl2-interacting mediator of cell death gene. Genetic variants in 4q12 merit further investigation to assess their potential as pharmacogenomic predictors for and to understand the biology underlying its influence on PFS in patients treated with TKI therapy.

BIM (BCL-2 interacting mediator of cell death) SAHB (stabilized α helix of BCL2) not always convinces BAX (BCL-2-associated X protein) for apoptosis

The interaction of BAX (BCL-2-associated X protein) with BIM (BCL-2 interacting mediator of cell death) SAHB (stabilized α helix of BCL2) directly initiates BAX-mediated mitochondrial apoptosis. This molecular dynamics study reveals that BIM SAHB forms a stable complex with BAX but it remains in a non-functional conformation. N terminal of BAX folds towards the core which has been reported exposed in the functional monomer. The α1-α2 loop, which has been reported in open conformation in functional BAX, acquires a closed conformation during the simulation. BH3/α2 remains less exposed as compared to initial structure. The hydrophobic residues of BIM accommodates in the rear pocket of BAX during the simulation. A steep decrease in radius of gyration and solvent accessible surface area (SASA) indicates the complex folding to acquire a more stable but inactive conformation. Further the covariance matrix reveals that the backbone atoms’ motions favour the inactive conformation of the complex. This is the first report on the non-functional BAX-BIM SAHB complex by molecular dynamics simulation in the best of our knowledge.

Molecular mechanisms of lipotoxicity and glucotoxicity in nonalcoholic fatty liver disease

The exposure of hepatocytes to high concentrations of lipids and carbohydrates and the ensuing hepatocellular injury are termed lipotoxicity and glucotoxicity, respectively. A common denominator is metabolic derangement, especially in regards to intracellular energy homeostasis, which is brought on by glucose intolerance and insulin resistance in tissues. In this review, we highlight the lipids and carbohydrates that provoke hepatocyte injury and the mechanisms involved in lipotoxicity and glucotoxicity, including endoplasmic reticulum stress, oxidative stress and mitochondrial impairment. Through upregulation of proteins involved in various pathways including PKR-like ER kinase (PERK), CCAAT/enhancer-binding homologous protein (CHOP), c-Jun NH2-terminal kinase-1 (JNK), Bcl-2 interacting mediator (BIM), p53 upregulated modulator of apoptosis (PUMA), and eventually caspases, hepatocytes in lipotoxic states ultimately undergo apoptosis. The protective role of certain lipids and possible targets for pharmacological therapy are explored. Finally, we discuss the role of high fructose and glucose diets in contributing to organelle impairment and poor glucose transport mechanisms, which perpetuate hyperglycemia and hyperlipidemia by shunting of excess carbohydrates into lipogenesis.

Knockdown of miR-221 promotes the cisplatin-inducing apoptosis by targeting the BIM-Bax/Bak axis in breast cancer.

Accumulating evidence shows that microRNAs (miRNAs) have a critical role in the initiation and progression of types of human cancer, including breast cancer. Recent research indicated that miRNAs are also related with the chemotherapy on cancers. In this study, the expression of miR-221 in breast cancer (BC) patients' serum and cancer tissues was found to be significantly up-regulated. The results of in vitro MTT assay indicated that although the anti-miR-221 oligonucleotide alone did not influence the viability of BC cell lines markedly, it significantly promoted the cytotoxicity of cisplatin (DDP) to BC cells. Mechanistic studies demonstrated that the gene of BIM (Bcl-2 interacting mediator of cell death), a pro-apoptotic Bcl-2 family protein, was up-regulated by the knockdown of miR-221. We found that the synergetic effect of anti-miR-221 on increasing the sensitivity of MDA-MB-231 was BIM dependant. Furthermore, results of immunoprecipitation showed the up-regulated BIM directly combined with the Bax and Bak, leading to mitochondrial dysfunction. Our results suggest the anti-miR-221 could promote the cisplatin-inducing apoptosis by targeting the Bim-Bax/Bak axis in breast cancer.

Genetic targeting of protease activated receptor 2 reduces inflammatory astrogliosis and improves recovery of function after spinal cord injury

Inflammatory-astrogliosis exacerbates damage in the injured spinal cord and limits repair. Here we identify Protease Activated Receptor 2 (PAR2) as an essential regulator of these events with mice lacking the PAR2 gene showing greater improvements in motor coordination and strength after compression-spinal cord injury (SCI) compared to wild type littermates. Molecular profiling of the injury epicenter, and spinal segments above and below, demonstrated that mice lacking PAR2 had significantly attenuated elevations in key hallmarks of astrogliosis (glial fibrillary acidic protein (GFAP), vimentin and neurocan) and in expression of pro-inflammatory cytokines (interleukin-6 (IL-6), tumor necrosis factor (TNF) and interleukin-1 beta (IL-1β)). SCI in PAR2−/− mice was also accompanied by improved preservation of protein kinase C gamma (PKCγ)-immunopositive corticospinal axons and reductions in GFAP-immunoreactivity, expression of the pro-apoptotic marker BCL2-interacting mediator of cell death (BIM), and in signal transducer and activator of transcription 3 (STAT3). The potential mechanistic link between PAR2, STAT3 and astrogliosis was further investigated in primary astrocytes to reveal that the SCI-related serine protease, neurosin (kallikrein 6) promotes IL-6 secretion in a PAR2 and STAT3-dependent manner. Data point to a signaling circuit in primary astrocytes in which neurosin signaling at PAR2 promotes IL-6 secretion and canonical STAT3 signaling. IL-6 promotes expression of GFAP, vimentin, additional IL-6 and robust increases in both neurosin and PAR2, thereby driving the PAR2-signaling circuit forward. Given the significant reductions in astrogliosis and inflammation as well as superior neuromotor recovery observed in PAR2 knockout mice after SCI, we suggest that this receptor and its agonists represent new drug targets to foster neuromotor recovery.

Intracellular Delivery System for Antibody–Peptide Drug Conjugates

Antibodies armed with biologic drugs could greatly expand the therapeutic potential of antibody-drug conjugates for cancer therapy, broadening their application to disease targets currently limited by intracellular delivery barriers. Additional selectivity and new therapeutic approaches could be realized with intracellular protein drugs that more specifically target dysregulated pathways in hematologic cancers and other malignancies. A multifunctional polymeric delivery system for enhanced cytosolic delivery of protein drugs has been developed that incorporates endosomal-releasing activity, antibody targeting, and a biocompatible long-chain ethylene glycol component for optimized safety, pharmacokinetics, and tumor biodistribution. The pH-responsive polymeric micelle carrier, with an internalizing anti-CD22 monoclonal targeting antibody, effectively delivered a proapoptotic Bcl-2 interacting mediator (BIM) peptide drug that suppressed tumor growth for the duration of treatment and prolonged survival in a xenograft mouse model of human B-cell lymphoma. Antitumor drug activity was correlated with a mechanistic induction of the Bcl-2 pathway biomarker cleaved caspase-3 and a marked decrease in the Ki-67 proliferation biomarker. Broadening the intracellular target space by more effective delivery of protein/peptide drugs could expand the repertoire of antibody-drug conjugates to currently undruggable disease-specific targets and permit tailored drug strategies to stratified subpopulations and personalized medicines.

Paxillin confers resistance to tyrosine kinase inhibitors in EGFR-mutant lung cancers via modulating BIM and Mcl-1 protein stability.

Tyrosine kinase inhibitors (TKIs) have been documented to have substantial clinical benefits to non-small cell lung cancer with epidermal growth factor receptor (EGFR) mutation. TKI resistance occurs in nearly all patients who receive TKI-targeting therapy, resulting in a modest overall survival benefit. Therefore, establishing a biomarker for early prediction and exploring the mechanism of primary TKI resistance is essential for improving the therapeutic efficacy in non-small cell lung cancer patients. In this study, we provide evidence indicating that paxillin (PXN) overexpression may confer TKI resistance in EGFR-mutant lung cancer cells. Mechanistically, PXN-mediated extracellular signal-regulated kinases (ERK) activation is responsible for TKI resistance via decreased Bcl2-interacting mediator of cell death (BIM) and increased Mcl-1 expression due to modulating their protein stabilities by phosphorylation of BIM at serine 69 and Mcl-1 at threonine 163. The mechanistic action in the cell model was further confirmed by the observation of xenograft tumors in nude mice, revealing that the PXN-mediated TKI resistance was conquered by ERK inhibitor (AZD6244) and Bcl-2 family inhibitor (obatoclax), but the TKI resistance overcome by AZD6244 is more effective than that of obatoclax. Therefore, we suggest that PXN expression may be useful in predicting primary TKI resistance, and combining TKI with ERK inhibitors may clinically benefit EGFR-mutant non-small cell lung cancer patients whose tumors exhibit high PXN expression.

Assessment of ABT-263 activity across a cancer cell line collection leads to a potent combination therapy for small-cell lung cancer

BH3 mimetics such as ABT-263 induce apoptosis in a subset of cancer models. However, these drugs have shown limited clinical efficacy as single agents in small-cell lung cancer (SCLC) and other solid tumor malignancies, and rational combination strategies remain underexplored. To develop a novel therapeutic approach, we examined the efficacy of ABT-263 across >500 cancer cell lines, including 311 for which we had matched expression data for select genes. We found that high expression of the proapoptotic gene Bcl2-interacting mediator of cell death (BIM) predicts sensitivity to ABT-263. In particular, SCLC cell lines possessed greater BIM transcript levels than most other solid tumors and are among the most sensitive to ABT-263. However, a subset of relatively resistant SCLC cell lines has concomitant high expression of the antiapoptotic myeloid cell leukemia 1 (MCL-1). Whereas ABT-263 released BIM from complexes with BCL-2 and BCL-XL, high expression of MCL-1 sequestered BIM released from BCL-2 and BCL-XL, thereby abrogating apoptosis. We found that SCLCs were sensitized to ABT-263 via TORC1/2 inhibition, which led to reduced MCL-1 protein levels, thereby facilitating BIM-mediated apoptosis. AZD8055 and ABT-263 together induced marked apoptosis in vitro, as well as tumor regressions in multiple SCLC xenograft models. In a Tp53; Rb1 deletion genetically engineered mouse model of SCLC, the combination of ABT-263 and AZD8055 significantly repressed tumor growth and induced tumor regressions compared with either drug alone. Furthermore, in a SCLC patient-derived xenograft model that was resistant to ABT-263 alone, the addition of AZD8055 induced potent tumor regression. Therefore, addition of a TORC1/2 inhibitor offers a therapeutic strategy to markedly improve ABT-263 activity in SCLC.

Hepatocyte autophagy is linked to C/EBP-homologous protein, Bcl2-interacting mediator of cell death, and BH3-interacting domain death agonist gene expression

BackgroundEndoplasmic reticulum (ER) stress and autophagy each play important roles in hepatocyte cell injury. We hypothesized that gene expression of C/EBP-homologous protein (CHOP) and the BH3 proteins Bcl2-interacting mediator of cell death (BIM) and BH3-interacting domain death agonist (BID) are involved in a complex interplay that regulates ER stress-induced autophagy and cell death. Materials and methodsHepatocytes were cultured from lean Zucker rats. Confluent hepatocytes were incubated with single or combined small interfering RNA for CHOP, BIM, and/or BID for 24 h providing gene inhibition. Incubation with tunicamycin (TM) for another 24 h stimulated ER stress. Quantitative real-time polymerase chain reaction determined the expression levels of CHOP, BIM, and BID. Immunostaining with microtubule-associated protein 1 light chain 3 measured autophagy activity. Trypan blue exclusion determined the cell viability. ResultsTM treatment increased the messenger RNA levels of CHOP and BIM but decreased the messenger RNA levels of BID. TM increased autophagy and decreased cell viability. Individual inhibition of CHOP, BIM, or BID protected against autophagy and cell death. However, simultaneous treatment with any combination of CHOP, BIM, and BID small interfering RNAs reduced autophagy activity but increased cell death independent of ER stress induction. ConclusionsAutophagy in hepatocytes results from acute ER stress and involves interplay, at the gene expression level, of CHOP, BIM, and BID. Inhibition of any one of these individual genes during acute ER stress is protective against cell death. Conversely, inhibition of any two of the three genes results in increased nonautophagic cell death independent of ER stress induction. This study suggests interplay between CHOP, BIM, and BID expression that can be leveraged for protection against ER stress-related cell death. However, disruption of the CHOP/BH3 gene expression homeostasis is detrimental to cell survival independent of other cellular stress.

KEAP the balance between life and death

Hepatocyte apoptosis in association with oxidative stress represent key pathogenic factors involved in tumor development in patients with non-alcoholic fatty liver disease (NAFLD). In our recent study, we established that cellular degradation of Kelch-like ECH-associated protein 1 (KEAP1) through sequestrosome (SQSTM)1/p62-dependent autophagy activates c-Jun NH2 terminal kinase (JNK), upregulates expression of Bcl-2–interacting mediator (BIM) and p53 upregulated modulator of apoptosis (PUMA), and contributes to hepatocyte apoptosis induced by saturated free fatty acids. These findings raise the possibility that dysregulation of KEAP1 may contribute to liver cell death and tumorigenesis during chronic inflammatory liver disease.

The increase in BIK expression following ERK1/2 pathway inhibition is a consequence of G1 cell-cycle arrest and not a direct effect on BIK protein stability

BIK (BCL2-interacting killer) is a pro-apoptotic BH3 (BCL2 homology domain 3)-only protein and a member of the BCL2 protein family. It was proposed recently that BIK abundance is controlled by ERK1/2 (extracellular-signal-regulated kinase 1/2)-catalysed phosphorylation, which targets the protein for proteasome-dependent destruction. In the present study, we examined ERK1/2-dependent regulation of BIK, drawing comparisons with BIM(EL) (BCL2-interacting mediator of cell death; extra long), a well-known target of ERK1/2. In many ERK1/2-dependent tumour cell lines, inhibition of BRAF(V600E) (v-raf murine sarcoma viral oncogene homologue B1, V600E mutation) or MEK1/2 (mitogen-activated protein kinase/ERK kinase 1/2) had very little effect on BIK expression, whereas BIM(EL) was strongly up-regulated. In some cell lines we observed a modest increase in BIK expression; however, this was not apparent until ~16 h or later, whereas BIM(EL) expression increased rapidly within a few hours. Although BIK was degraded by the proteasome, we found no evidence that this was regulated by ERK1/2 signalling. Rather, the delayed increase in BIK expression was prevented by actinomycin D, and was accompanied by increases in BIK mRNA. Finally, the delayed increase in BIK expression following ERK1/2 inhibition was phenocopied by a highly selective CDK4/6 (cyclin-dependent kinases 4 and 6) inhibitor, which caused a strong G₁ cell-cycle arrest without inhibiting ERK1/2 signalling. In contrast, BIM(EL) expression was induced by ERK1/2 inhibition, but not by CDK4/6 inhibition. We conclude that BIK expression is not subject to direct regulation by the ERK1/2 pathway; rather, we propose that BIK expression is cell-cycle-dependent and increases as a consequence of the G₁ cell-cycle arrest which results from inhibition of ERK1/2 signalling.

Targeting Small Cell Lung Cancer Harboring PIK3CA Mutation with a Selective Oral PI3K Inhibitor PF-4989216

Constitutive activation of phosphoinositide 3-kinase (PI3K) occurs frequently in many human tumors via either gene mutation in the p110α catalytic subunit of PI3K or functional loss of tumor suppressor PTEN. Patients with small-cell lung cancer (SCLC) have very poor prognosis and survival rates such that an effective targeted therapy is in strong demand for these patients. In this study, we characterized the highly selective oral PI3K inhibitor, PF-4989216, in preclinical SCLC models to investigate whether targeting the PI3K pathway is an effective targeted therapy option for SCLCs that harbor a PIK3CA mutation. A panel of SCLC cell lines with PIK3CA mutation or PTEN loss were treated with PF-4989216 in several in vitro assays, including PI3K pathway signaling, cell viability, apoptosis, cell-cycle progression, and cell transformation. SCLC cell lines that were sensitive in vitro to PF-4989216 were further evaluated by in vivo animal studies to determine the pharmacokinetic/pharmacodynamic relationship and tumor growth inhibition (TGI) by PF-4989216 treatment. PF-4989216 inhibited PI3K downstream signaling and subsequently led to apoptosis induction, and inhibition in cell viability, transformation, and xenograft tumor growth in SCLCs harboring PIK3CA mutation. In SCLCs with PTEN loss, PF-4989216 also inhibited PI3K signaling but did not induce BCL2-interacting mediator (BIM)-mediated apoptosis nor was there any effect on cell viability or transformation. These results implicate differential tumorigenesis and apoptosis mechanisms in SCLCs harboring PIK3CA mutation versus PTEN loss. Our results suggest that PF-4989216 is a potential cancer drug candidate for patients with SCLC with PIK3CA mutation but not PTEN loss.