Fbxo45 facilitates the malignant progression of breast cancer by targeting Bim for ubiquitination and degradation.

BACKGROUND: Breast cancer is one of the common malignancies in women. Evidence has demonstrated that FBXO45 plays a pivotal role in oncogenesis and progression. However, the role of FBXO45 in breast tumorigenesis remains elusive. Exploration of the regulatory mechanisms of FBXO45 in breast cancer development is pivotal for potential therapeutic interventions in patients with breast cancer. METHODS: Hence, we used numerous approaches to explore the functions of FBXO45 and its underlaying mechanisms in breast cancer pathogenesis, including CCK-8 assay, EdU assay, colony formation analysis, apoptosis assay, RT-PCR, Western blotting, immunoprecipitation, ubiquitination assay, and cycloheximide chase assay. RESULTS: We found that downregulation of FBXO45 inhibited cell proliferation, while upregulation of FBXO45 elevated cell proliferation in breast cancer. Silencing of FBXO45 induced cell apoptosis, whereas overexpression of FBXO45 inhibited cell apoptosis in breast cancer. Moreover, FBXO45 interacted with BIM and regulated its ubiquitination and degradation. Furthermore, knockdown of FBXO45 inhibited cell proliferation via regulation of BIM pathway. Notably, overexpression of FBXO45 facilitated tumor growth in mice. Strikingly, FBXO45 expression was associated with poor survival of breast cancer patients. CONCLUSION: Our study could provide the rational for targeting FBXO45 to obtain benefit for breast cancer patients. Altogether, modulating FBXO45/Bim axis could be a promising strategy for breast cancer therapy.

Small molecule inhibitor targeting the Hsp70-Bim protein-protein interaction in estrogen receptor-positive breast cancer overcomes tamoxifen resistance.

INTRODUCTION: Estrogen receptor (ER) positive patients compromise about 70% of breast cancers. Tamoxifen, an antagonist of ERα66 (the classic ER), is the most effective and the standard first-line drug. However, its efficacy is limited by the development of acquired resistance. METHODS: A specific inhibitor of Hsp70-Bim protein-protein interaction (PPI), S1g-2, together with an inhibitor of Hsp70-Bag3 PPI, MKT-077 and an ATP-competitive inhibitor VER155008, were used as chemical tools. Cell viability assays, co-immunoprecipitation and gene knockdown were used to investigate the role of Hsp70 in tamoxifen resistance. A xenograft model was established in which tamoxifen-resistant breast cancer (MCF-7/TAM-R) cells maintained in the presence of 5 µM tamoxifen were subcutaneously inoculated. The anti-tumor efficiency of S1g-2 was measured after a daily injection of 0.8 mg/kg for 14 days. RESULTS: It was revealed that Hsp70-Bim PPI protects ERα-positive breast cancer from tamoxifen-induced apoptosis through binding and stabilizing ERα36, rather than ERα66, resulting in sustained EGFR mRNA and protein expression. Disruption of Hsp70-Bim PPI and downregulation of ERα36 expression in tumor samples are consistent with the in vitro functions of S1g-2, resulting in about a three-fold reduction in tumor volume. CONCLUSIONS: The in vivo activity and safety of S1g-2 illustrated that it is a potential strategy for Hsp70-Bim disruption to overcome tamoxifen-resistant ER-positive breast cancer.

Protein interaction networks characterizing the A549 cells Klotho transfected are associated with activated pro-apoptotic Bim and suppressed Wnt/ß-catenin signaling pathway.

Invasive assays and lung tumor-bearing mice models using a human lung adenocarcinoma cell line A549 cells transfected with the Klotho (KL) gene, A549/KL cells, have confirmed that KL suppresses invasive/metastatic potential. This study aimed to identify the co-expression protein networks and proteomic profiles associated with A549/KL cells to understand how Klotho protein expression affects molecular networks associated with lung carcinoma malignancy. A two-step application of a weighted network correlation analysis to the cells' quantitative proteome datasets of a total of 6,994 proteins, identified by mass spectrometry-based proteomic analysis with data-independent acquisition (DIA), identified one network module as most significantly associated with the A549/KL trait. Upstream analyses, confirmed by western blot, implicated the pro-apoptotic Bim (Bcl-2-like protein 11) as a master regulator of molecular networks affected by Klotho. GeneMANIA interaction networks and quantitative proteome data implicated that Klotho interacts with two signaling axes: negatively with the Wnt/ß-catenin axis, and positively by activating Bim. Our findings might contribute to the development of future therapeutic strategies.

Non-canonical BIM-regulated energy metabolism determines drug-induced liver necrosis.

Paracetamol (acetaminophen, APAP) overdose severely damages mitochondria and triggers several apoptotic processes in hepatocytes, but the final outcome is fulminant necrotic cell death, resulting in acute liver failure and mortality. Here, we studied this switch of cell death modes and demonstrate a non-canonical role of the apoptosis-regulating BCL-2 homolog BIM/Bcl2l11 in promoting necrosis by regulating cellular bioenergetics. BIM deficiency enhanced total ATP production and shifted the bioenergetic profile towards glycolysis, resulting in persistent protection from APAP-induced liver injury. Modulation of glucose levels and deletion of Mitofusins confirmed that severe APAP toxicity occurs only in cells dependent on oxidative phosphorylation. Glycolytic hepatocytes maintained elevated ATP levels and reduced ROS, which enabled lysosomal recycling of damaged mitochondria by mitophagy. The present study highlights how metabolism and bioenergetics affect drug-induced liver toxicity, and identifies BIM as important regulator of glycolysis, mitochondrial respiration, and oxidative stress signaling.

BATF represses BIM to sustain tolerant T cells in the periphery.

T cells that encounter self-antigens after exiting the thymus avert autoimmunity through peripheral tolerance. Pathways for this include an unresponsive state known as anergy, clonal deletion, and T regulatory (Treg) cell induction. The transcription factor cues and kinetics that guide distinct peripheral tolerance outcomes remain unclear. Here, we found that anergic T cells are epigenetically primed for regulation by the non-classical AP-1 family member BATF. Tolerized BATF-deficient CD4+ T cells were resistant to anergy induction and instead underwent clonal deletion due to proapoptotic BIM (Bcl2l11) upregulation. During prolonged antigen exposure, BIM derepression resulted in fewer PD-1+ conventional T cells as well as loss of peripherally induced FOXP3+ Treg cells. Simultaneous Batf and Bcl2l11 knockdown meanwhile restored anergic T cell survival and Treg cell maintenance. The data identify the AP-1 nuclear factor BATF as a dominant driver of sustained T cell anergy and illustrate a mechanism for divergent peripheral tolerance fates.

circ_0000018 downregulation peripherally ameliorates neuroprotection against acute ischemic stroke through the miR­871/BCL2L11 axis.

Acute ischemic stroke (AIS) is a common acute cerebrovascular disease. Circular RNAs (circRNAs) have been demonstrated to have critical functions in a wide range of physiological processes and disorders in humans. However, their precise function in ischemic stroke (IS) remains largely unknown. The present study explored the function and potential mechanisms of circ_0000018 in AIS in vivo and in vitro. The cerebral ischemia/reperfusion injury model was established in vivo and in vitro using the oxygen­glucose deprivation (OGD/R) and transient middle cerebral artery occlusion (tMCAO) methods. Subsequently, the impact of circ_0000018 on cerebral ischemia/reperfusion injury was assessed using various techniques, including TTC staining, quantitative PCR, western blotting, cell counting kit­8 assay, Annexin V­FITC Apoptosis Detection Kit, luciferase reporter gene assays, and others. The levels of circ_0000018 were markedly increased in the OGD/R­treated neuronal cells and in a mouse model of tMCAO. The blocking of microRNA (miR)­871 by circ_0000018 promoted Bcl­2­like protein 11 (BCL2L11) expression to increase neuronal cell damage. Furthermore, circ_0000018 knockdown significantly improved neuronal cell viability and attenuated OGD/R­treated neuronal cell death. Meanwhile, circ_0000018 knockdown improved brain infarct volume and neuronal apoptosis in tMCAO mice. The present study found that circ_0000018 knockdown relieved cerebral ischemia­reperfusion injury progression in vitro and in vivo. Mechanistically, circ_0000018 regulated the levels of BCL2L11 by sponging miR­871.

Dynamic BH3 profiling identifies pro-apoptotic drug combinations for the treatment of malignant pleural mesothelioma.

Malignant pleural mesothelioma (MPM) has relatively ineffective first/second-line therapy for advanced disease and only 18% five-year survival for early disease. Drug-induced mitochondrial priming measured by dynamic BH3 profiling identifies efficacious drugs in multiple disease settings. We use high throughput dynamic BH3 profiling (HTDBP) to identify drug combinations that prime primary MPM cells derived from patient tumors, which also prime patient derived xenograft (PDX) models. A navitoclax (BCL-xL/BCL-2/BCL-w antagonist) and AZD8055 (mTORC1/2 inhibitor) combination demonstrates efficacy in vivo in an MPM PDX model, validating HTDBP as an approach to identify efficacious drug combinations. Mechanistic investigation reveals AZD8055 treatment decreases MCL-1 protein levels, increases BIM protein levels, and increases MPM mitochondrial dependence on BCL-xL, which is exploited by navitoclax. Navitoclax treatment increases dependency on MCL-1 and increases BIM protein levels. These findings demonstrate that HTDBP can be used as a functional precision medicine tool to rationally construct combination drug regimens in MPM and other cancers.

The carboxyl-terminal sequence of PUMA binds to both anti-apoptotic proteins and membranes.

Anti-apoptotic proteins such as BCL-XL promote cell survival by sequestering pro-apoptotic BCL-2 family members, an activity that frequently contributes to tumorigenesis. Thus, the development of small-molecule inhibitors for anti-apoptotic proteins, termed BH3-mimetics, is revolutionizing how we treat cancer. BH3 mimetics kill cells by displacing sequestered pro-apoptotic proteins to initiate tumor-cell death. Recent evidence has demonstrated that in live cells the BH3-only proteins PUMA and BIM resist displacement by BH3-mimetics, while others like tBID do not. Analysis of the molecular mechanism by which PUMA resists BH3-mimetic mediated displacement from full-length anti-apoptotic proteins (BCL-XL, BCL-2, BCL-W, and MCL-1) reveals that both the BH3-motif and a novel binding site within the carboxyl-terminal sequence (CTS) of PUMA contribute to binding. Together these sequences bind to anti-apoptotic proteins, which effectively 'double-bolt locks' the proteins to resist BH3-mimetic displacement. The pro-apoptotic protein BIM has also been shown to double-bolt lock to anti-apoptotic proteins however, the novel binding sequence in PUMA is unrelated to that in the CTS of BIM and functions independent of PUMA binding to membranes. Moreover, contrary to previous reports, we find that when exogenously expressed, the CTS of PUMA directs the protein primarily to the endoplasmic reticulum (ER) rather than mitochondria and that residues I175 and P180 within the CTS are required for both ER localization and BH3-mimetic resistance. Understanding how PUMA resists BH3-mimetic displacement will be useful in designing more efficacious small-molecule inhibitors of anti-apoptotic BCL-2 proteins.

MicroRNA-17-5p Protects against Propofol Anesthesia-Induced Neurotoxicity and Autophagy Impairment via Targeting BCL2L11.

Background: Propofol (PPF) has been shown in studies to cause cognitive impairment and neuronal cell death in developing animals. PPF has been demonstrated to decrease the expression of microRNA-17-5p (miR-17-5p) in a recent study. Nonetheless, the function of miR-17-5p in PPF-induced neurotoxicity and related mechanisms is uncharacterized. Methods: After the induction of neurotoxicity by treating the SH-SY5Y cells with PPF, qRT-PCR was conducted to evaluate the level of miR-17-5p. Using MTT and flow cytometry, cell viability and apoptosis rate were assessed, respectively. Interaction between miR-17-5p and BCL2 like 11 was (BCL2L11) studied using a Luciferase reporter assay. With the help of western blot analysis, we determined the level of proteins of apoptosis-related genes and autophagy-related markers. Results: In SH-SY5Y cells, PPF treatment induced neurotoxicity and downregulated miR-17-5p expression. In SH-SY5Y cells post-PPF exposure, overexpression of miR-17-5p increased cell viability and decreased apoptosis. Consistently, miR-17-5p mimics mitigated PPF-generated autophagy via inhibition of Atg5, Beclin1, and LC3II/I level and elevation of p62 protein expression. In addition, BCL2L11, which was highly expressed in PPF-treated SH-SY5Y cells, was directly targeted by miR-17-5p. Further, in PPF-treated SH-SY5Y cells, overexpressed BCL2L11 counteracted the suppressing behavior of miR-17-5p elevation on PPF-induced apoptosis. Conclusion: Overexpressed miR-17-5p alleviates PPF exposure-induced neurotoxicity and autophagy in SH-SY5Y cells via binding to BCL2L11, suggesting the possibility that miR-17-5p can serve as a candidate in the treatment of neurotoxicity (caused by PPF).

Significant role of long non-coding RNA MALAT1 in deep vein thrombosis via the regulation of vascular endothelial cell physiology through the microRNA-383-5p/BCL2L11 axis.

Deep vein thrombosis (DVT) is a vascular disease. The long non-coding RNA (lncRNA), metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), is positively expressed in DVT tissues, and regulates the biological behavior of endothelial progenitor cells. Here, we explored whether MALAT1 affected the physiology of human vascular endothelial cells (HUVECs) and analyzed its underlying mechanism. To overexpress/silence the expression of MALAT1 in HUVECs, MALAT1-plasmid/MALAT1-small interfering RNA (siRNA) was used. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide and flow cytometry analyses were performed to observe the cell viability and apoptosis. Reverse transcription-quantitative polymerase chain reaction and western blotting were used to determine the apoptosis-related protein and gene expression levels. We used Starbase software to predict the associations among MALAT1, microRNA (miR)-383-5p, and BCL2-like 11 (BCL2L11). Luciferase reporter assay was used to validate their relationship. Compared to the control vector group, MALAT1-plasmid suppressed the viability and induced apoptosis of HUVECs, while improving Bcl-2-associated X protein (Bax) expression and decreasing Bcl-2 expression. There was an interaction between MALAT1 and miR-383-5p. Compared to the control siRNA group, MALAT1-siRNA increased the cell viability, reduced cell apoptosis, upregulated Bcl-2 expression, and suppressed Bax expression. These changes were reversed by the miR-383-5p inhibitor. Additionally, we verified that BCL2L11 is a target of miR-383-5p. miR-383-5p improved the cell proliferation, while decreasing cell apoptosis in HUVECs by targeting BCL2L11. Therefore, the lncRNA-MALAT1/miR-383-5p/BCL2L11 axis may be effective for DVT treatment.

Codon Usage Analysis of Pro-Apoptotic Bim Gene Isoforms.

BACKGROUND: Bim is a Bcl-2 homology 3 (BH3)-only proteins, a group of pro-apoptotic proteins involved in physiological and pathological conditions. Both the overexpression and under-expression of Bim protein are associated with the diseased condition, and various isoforms of Bim protein are present with differential apoptotic potential. OBJECTIVE: The present study attempted to envisage the association of various molecular signatures with the codon choices of Bim isoforms. METHODS: Molecular signatures like composition, codon usage, nucleotide skews, the free energy of mRNA transcript, physical properties of proteins, codon adaptation index, relative synonymous codon usage, and dinucleotide odds ratio were determined and analyzed for their associations with codon choices of Bim gene. RESULTS: Skew analysis of the Bim gene indicated the preference of C nucleotide over G, A, and T and preference of G over T and A nucleotides was observed. An increase in C content at the first and third codon position increased gene expression while it decreased at the second codon position. Compositional constraints on nucleotide C at all three codon positions affected gene expression. The analysis revealed an exceptionally high usage of CpC dinucleotide in all the envisaged 31 isoforms of Bim. We correlated it with the requirement of rapid demethylation machinery to fine-tune the Bimgene expression. Also, mutational pressure played a dominant role in shaping codon usage bias in Bim isoforms. CONCLUSION: An exceptionally high usage of CpC dinucleotide in all the envisaged 31 isoforms of Bim indicates a high order selectional force to fine tune Bim gene expression.

Bim Deletion Reduces Functional Deficits Following Ischemic Stroke in Association with Modulation of Apoptosis and Inflammation.

Cellular apoptosis is a key pathological mechanism contributing to neuronal death following ischemic stroke. The pro-apoptotic Bcl-2 family protein, Bim, is an important regulator of apoptosis. In this study we investigated the effect of Bim expression on post-stroke functional outcomes, brain injury and inflammatory mechanisms. Wild type (WT) and Bim-deficient mice underwent 1-h middle cerebral artery occlusion (MCAO) followed by 23 h of reperfusion. At 24-h post-stroke, we assessed functional deficit, infarct volume, immune cell death, as well as the number of infiltrating immune cells in the brain and circulating immune cells. Bim deficiency did not affect infarct volume (P > 0.05), but resulted in less motor impairment (~ threefold greater latency to fall in hanging grip strength test, P < 0.05) and a lower median clinical score than WT mice (P < 0.05). Additionally following MCAO, Bim-deficient mice exhibited fewer myeloid cells (particularly neutrophils) in the ischemic brain hemisphere and less apoptosis of CD3+ T cells in the spleen and thymus compared with WT (all P < 0.05). After MCAO, Bim-deficient mice also tended to have more M2-polarised macrophages in the brain than WT mice. In sham-operated mice, we found that Bim deficiency resulted in greater numbers of circulating total CD45+ leukocytes, Ly6Clo+ monocytes and CD3+ T cells, although MCAO did not affect the number of circulating cells at 24 h in either genotype. Our findings suggest that Bim deficiency modulates post-stroke outcomes, including reductions in motor impairment, brain inflammation and systemic post-stroke leukocyte apoptosis. Bim could therefore serve as a potential therapeutic target for stroke.

Evaluation of microRNA 92a Expression and Its Target Protein Bim in Colorectal Cancer.

BACKGROUND: Colorectal cancer is one of the most commonly diagnosed cancers and leading causes of malignancy-related deaths all over the world. MicroRNAs (miRNAs) can regulate more than 60% of human genes, including tumor-stimulating, and -suppressor genes. Therefore, they can promote cancer development and affect risk of malignancy. miR-92a overexpression in CRC enhances tumor proliferation, invasion, and metastasis through downregulating different pro-apoptosis proteins including Bim. This study aimed to assess the role of plasma miR-92a as non-invasive marker in CRC patients, outline correlation between plasma miR-92a and serum Bim, and determine their correlations with clinicopathological parameters in CRC and adenoma patients. METHODS: A total of 54 newly diagnosed CRC patients, 15 colonic adenoma patients, and 15 age- and sex-matched control subjects were recruited in this study. Plasma miR-92a was assayed by TaqMan qRT-PCR and serum Bim was measured by ELISA. RESULTS: Statistically significant overexpression of serum miR-92a was observed in CRC patients as compared to adenoma and control groups (p<0.001 each) and lower serum Bim in CRC patients as compared to adenoma and control groups (p=0.001, p <0.001 respectively). The ROC curve analysis showed excellent AUC for plasma miR-92a in discriminating CRC from control (AUC=0.994), and adenoma (AUC=0.993) groups with highest diagnostic performance in discriminating CRC from controls (at cutoff 1.43, sensitivity 98.1%, specificity 93.9%), and adenoma patients (at cutoff 1.78, sensitivity 92.6%,  specificity 93.3%). The diagnostic performance in discriminating early from late CRC was good (at cutoff 15, AUC=0.641, sensitivity 61.2%, specificity 80%). A significant negative correlation was evident between plasma miR-92a and serum Bim both in adenoma and CRC groups (P<0.001 for both). Higher plasma miR-92a expression (r=0.275, p=0.044) and lower serum Bim (r=-0.299, p=0.028) were found to be correlated with late CRC stages. CONCLUSION: Circulating miR-92a and Bim could be promising, non-invasive diagnostic and prognostic biomarkers in CRC.
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Circular RNA Rbms1 inhibited the development of myocardial ischemia reperfusion injury by regulating miR-92a/BCL2L11 signaling pathway.

Acute myocardial infarction (AMI) is characterized by high morbidity and mortality rates. Circular RNAs collectively participate in the initiation and development of AMI. The purpose of this study was to investigate the role of circRbms1 in AMI. Ischemia-reperfusion (I/R) was performed to establish an AMI model. RT-qPCR and Western blotting were performed to detect mRNA and analyze protein expression, respectively. The interaction between miR-92a and circRbms1/BCL2L11 was confirmed by luciferase and RNA pull-down assays. circRbms1 is overexpressed in AMI. However, circRbms1 knockdown alleviated H9c2 cell apoptosis and reduced the release of reactive oxygen species. circRbms1 targeted miR-92a, the downregulation of which alleviated the effects of circRbms1 knockdown and increased oxidative stress and H9c2 cell apoptosis. Moreover, circRbms1 sponged miR-92a to upregulate BCL2L11, which modulated the expression of apoptosis-related genes. circRbms1 participated in myocardial I/R injury by regulating the miR-92a/BCL2L11 signaling pathway, which may provide a new strategy for the treatment of AMI.

Inhibition of histone demethylase KDM4 by ML324 induces apoptosis through the unfolded protein response and Bim upregulation in hepatocellular carcinoma cells.

Hepatocellular carcinoma (HCC) is an extremely aggressive malignancy that ranks as the sixth-leading cause of cancer-associated death worldwide. Recently, various epigenetic mechanisms including gene methylation were reported to be potential next era HCC therapeutics and biomarkers. Although inhibition of epigenetic enzymes including histone lysine demethylase 4 (KDM4) enhanced cell death in HCC cells, the detailed mechanism of cell death machinery is poorly understood. In this study, we found that ML324, a small molecule KDM4-specific inhibitor, induced the death of HCC cells in a general cell culture system and 3D spheroid culture with increased cleavage of caspase-3. Mechanistically, we identified that unfolded protein responses (UPR) were involved in ML324-induced HCC cell death. Incubation of HCC cells with ML324 upregulated death receptor 5 (DR5) expression through the activation transcription factor 3 (ATF3)-C/EBP homologous protein (CHOP)-dependent pathway. Moreover, we identified BIM protein as a mediator of ML324-induced apoptosis using CRISPR/Cas9 knockout analysis. We showed that the loss of Bim suppressed ML324-induced apoptosis by flow cytometry analysis, colony formation assay, and caspase-3 activation assay. Interestingly, BIM protein expression by ML324 was regulated by ATF3, CHOP, and DR5 which are factors involved in UPR. Specifically, we confirmed the regulating roles of KDM4E in Bim and CHOP expression using a chromatin immune precipitation (ChIP) assay. Physical binding of KDM4E to Bim and CHOP promoters decreased the response to ML324. Our findings suggest that KDM4 inhibition is a potent anti-tumor therapeutic strategy for human HCC, and further studies of UPR-induced apoptosis and the associated epigenetic functional mechanisms may lead to the discovery of novel target for future cancer therapy.

Melatonin induces apoptotic cell death through Bim stabilization by Sp1-mediated OTUD1 upregulation.

Melatonin, secreted by the pineal gland, regulates the circadian rhythms and also plays an oncostatic role in cancer cells. Previously, we showed that melatonin induces the expression of Bim, a pro-apoptotic Bcl-2 protein, at both the transcriptional and post-translational levels. In the present study, we investigated the molecular mechanisms underlying the melatonin-mediated Bim upregulation through post-translational regulation. We found that ovarian tumor domain-containing protein 1 (OTUD1), a deubiquitinase belonging to the OTU protein family, is upregulated by melatonin at the mRNA and protein levels. OTUD1 knockdown inhibited melatonin-induced Bim upregulation and apoptosis in cancer cells. OTUD1 directly interacted with Bim and inhibited its ubiquitination. Melatonin-induced OTUD1 upregulation caused deubiquitination at the lysine 3 residue of Bim, resulting in its stabilization. In addition, melatonin-induced activation of Sp1 was found to be involved in OTUD1 upregulation at the transcriptional level, and pharmacological inhibition and genetic ablation of Sp1 (siRNA) interrupted melatonin-induced OTUD1-mediated Bim upregulation. Furthermore, melatonin reduced tumor growth and induced upregulation of OTUD1 and Bim in a mouse xenograft model. Notably, Bim expression levels correlated with OTUD1 levels in patients with renal clear cell carcinoma. Thus, our results demonstrated that melatonin induces apoptosis by stabilizing Bim via Sp1-mediated OTUD1 upregulation.

Prognostic Value of BIM Deletion in EGFR-Mutant NSCLC Patients Treated with EGFR-TKIs: A Meta-Analysis.

BACKGROUND: Resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) is inevitable in EGFR-mutant non-small-cell lung cancer (NSCLC) patients. A germline 2903 bp deletion polymorphism of Bcl-2-like protein 11 (BIM) causes reduced expression of proapoptotic BH3-only BIM protein and blocks TKI-induced apoptosis of tumor cells. Yet the association between the deletion polymorphism and response to EGFR-TKI treatment remains inconsistent among clinical observations. Thus, we performed the present meta-analysis. METHODS: Eligible studies were identified by searching PubMed, Embase, and ClinicalTrials.gov databases prior to March 31, 2021. Hazard ratios (HRs) and 95% confidence intervals (CIs) of progression-free survival (PFS) and overall survival (OS) and odds ratios (ORs) and 95% CIs of objective response rate (ORR) and disease control rate (DCR) were calculated by using a random effects model. Sensitivity, metaregression, and publication bias analyses were also performed. RESULTS: A total of 20 datasets (3003 EGFR-mutant NSCLC patients receiving EGFR-TKIs from 18 studies) were included. There were 475 (15.8%) patients having the 2903-bp intron deletion of BIM and 2528 (84.2%) wild-type patients. BIM deletion predicted significantly shorter PFS (HR = 1.35, 95% CI: 1.10-1.64, P = 0.003) and a tendency toward an unfavorable OS (HR = 1.22, 95% CI: 0.99-1.50, P = 0.068). Patients with deletion polymorphism had lower ORR (OR = 0.60, 95% CI: 0.42-0.85, P = 0.004) and DCR (OR = 0.59, 95% CI: 0.38-0.90, P = 0.014) compared with those without deletion. CONCLUSION: BIM deletion polymorphism may confer resistance to EGFR-TKIs and can be used as a biomarker to predict treatment response to EGFR-TKIs in EGFR-mutant NSCLC patients from Asian populations.

Dual Inhibition of Histone Deacetylases and the Mechanistic Target of Rapamycin Promotes Apoptosis in Cell Line Models of Uveal Melanoma.

Purpose: Over 90% of uveal melanomas harbor pathogenic variants of the GNAQ or GNA11 genes that activate survival pathways. As previous studies found that Ras-mutated cell lines were vulnerable to a combination of survival pathway inhibitors and the histone-deacetylase inhibitor romidepsin, we investigated whether this combination would be effective in models of uveal melanoma. Methods: A small-scale screen of inhibitors of bromodomain-containing protein 4 (BRD4; OTX-015), extracellular signal-related kinase (ERK; ulixertinib), mechanistic target of rapamycin (mTOR; AZD-8055), or phosphoinositide 3-kinase (PI3K; GDC-0941) combined with a clinically relevant administration of romidepsin was performed on a panel of uveal melanoma cell lines (92.1, Mel202, MP38, and MP41) and apoptosis was quantified by flow cytometry after 48 hours. RNA sequencing analysis was performed on Mel202 cells treated with romidepsin alone, AZD-8055 alone, or the combination, and protein changes were validated by immunoblot. Results: AZD-8055 with romidepsin was the most effective combination in inducing apoptosis in the cell lines. Increased caspase-3 and PARP cleavage were noted in the cell lines when they were treated with romidepsin and mTOR inhibitors. RNA sequencing analysis of Mel202 cells revealed that apoptosis was the most affected pathway in the romidepsin/AZD-8055-treated cells. Increases in pro-apoptotic BCL2L11 and decreases in anti-apoptotic BIRC5 and BCL2L1 transcripts noted in the sequencing analysis were confirmed at the protein level in Mel202 cells. Conclusions: Our data suggest that romidepsin in combination with mTOR inhibition could be an effective treatment strategy against uveal melanoma due in part to changes in apoptotic proteins.

Mechanisms and management of 3rd­generation EGFR­TKI resistance in advanced non­small cell lung cancer (Review).

Targeted therapy with epidermal growth factor receptor (EGFR)­tyrosine kinase inhibitors (TKIs) is a standard modality of the 1st­line treatments for patients with advanced EGFR­mutated non­small cell lung cancer (NSCLC), and substantially improves their prognosis. However, EGFR T790M mutation is the primary mechanism of 1st­ and 2nd­generation EGFR­TKI resistance. Osimertinib is a representative of the 3rd­generation EGFR­TKIs that target T790M mutation, and has satisfactory efficacy in the treatment of T790M­positive NSCLC with disease progression following use of 1st­ or 2nd­generation EGFR­TKIs. Other 3rd­generation EGFR­TKIs, such as abivertinib, rociletinib, nazartinib, olmutinib and alflutinib, are also at various stages of development. However, the occurrence of acquired resistance is inevitable, and the mechanisms of 3rd­generation EGFR­TKI resistance are complex and incompletely understood. Genomic studies in tissue and liquid biopsies of resistant patients reveal multiple candidate pathways. The present review summarizes the recent findings in mechanisms of resistance to 3rd­generation EGFR­TKIs in advanced NSCLC, and provides possible strategies to overcome this resistance. The mechanisms of acquired resistance mainly include an altered EGFR signaling pathway (EGFR tertiary mutations and amplification), activation of aberrant bypassing pathways (hepatocyte growth factor receptor amplification, human epidermal growth factor receptor 2 amplification and aberrant insulin­like growth factor 1 receptor activation), downstream pathway activation (RAS/RAF/MEK/ERK and PI3K/AKT/mTOR) and histological/phenotypic transformations (SCLC transformation and epithelial­mesenchymal transition). The combination of targeted therapies is a promising strategy to treat osimertinib­resistant patients, and multiple clinical studies on novel combined therapies are ongoing.

SNAI2-Mediated Repression of BIM Protects Rhabdomyosarcoma from Ionizing Radiation.

Ionizing radiation (IR) and chemotherapy are mainstays of treatment for patients with rhabdomyosarcoma, yet the molecular mechanisms that underlie the success or failure of radiotherapy remain unclear. The transcriptional repressor SNAI2 was previously identified as a key regulator of IR sensitivity in normal and malignant stem cells through its repression of the proapoptotic BH3-only gene PUMA/BBC3. Here, we demonstrate a clear correlation between SNAI2 expression levels and radiosensitivity across multiple rhabdomyosarcoma cell lines. Modulating SNAI2 levels in rhabdomyosarcoma cells through its overexpression or knockdown altered radiosensitivity in vitro and in vivo. SNAI2 expression reliably promoted overall cell growth and inhibited mitochondrial apoptosis following exposure to IR, with either variable or minimal effects on differentiation and senescence, respectively. Importantly, SNAI2 knockdown increased expression of the proapoptotic BH3-only gene BIM, and chromatin immunoprecipitation sequencing experiments established that SNAI2 is a direct repressor of BIM/BCL2L11. Because the p53 pathway is nonfunctional in the rhabdomyosarcoma cells used in this study, we have identified a new, p53-independent SNAI2/BIM signaling axis that could potentially predict clinical responses to IR treatment and be exploited to improve rhabdomyosarcoma therapy. SIGNIFICANCE: SNAI2 is identified as a major regulator of radiation-induced apoptosis in rhabdomyosarcoma through previously unknown mechanisms independent of p53.