Mcl-1 Research Articles

Long noncoding RNA XIST regulates cardiomyocyte apoptosis by targeting miR-873-5p/MCL1 axis.

The purpose of this study was to investigate the expression of miR-873-5p and long non-coding RNA X-inactive specific transcript (lncRNA-XIST) in myocardial infarction (MI), the interaction mechanism and the effect of target gene MCL1 on apoptosis in H9c2 cells. quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) was used to detect and compare the expressions of miR-873-5p and lncRNA XIST in 8 myocardial infarction rats and 8 normal rats tissues, respectively, and the correlation between the expressions of miR-873-5p and lncRNA XIST in the myocardial tissues was explored. Next, qRT-PCR and Western blot were used to detect the effects of upregulation of miR-873-5p and downregulation of lncRNA XIST, as well as the impacts of their interactions on the expression level of MCL1 in H9c2 cells and the apoptosis of cells. It was found that the downregulation of miR-873-5p protected the heart against apoptosis after AMI, and lncRNA XIST inhibited apoptosis in H9c2 cells after hypoxia. Besides, inhibiting lncRNA XIST could upregulate miR-873-5p and downregulate MCL1, thus increasing apoptosis in the H9c2 cells after hypoxia. LncRNA XIST can regulate cardiomyocyte apoptosis by targeting miR-873-5p.

Trial in Progress: A Phase I Trial of BTX-A51 in Patients with Relapsed or Refractory AML or High-Risk MDS

Background: For patients with relapsed or refractory (R/R) acute myeloid leukemia (AML) or high-risk myelodysplastic syndrome (MDS), low response rates and poor overall survival remain unmet clinical needs. AML cells evade apoptosis through overexpression of antiapoptotic genes and inactivation of p53. The antiapoptotic gene Mcl1 is overexpressed in AML cell lines resistant to venetoclax. Similarly, the sensitivity of AML patients' samples to venetoclax inversely correlates with the presence of a TP53 mutation or low expression of p53. In AML, p53 inactivation more commonly results from overexpression of its negative regulators, Mdmx and Mdm2. BTX-A51 is a novel, oral, direct inhibitor of Casein kinase 1α (CK1α), cyclin dependent kinase 7 (CDK7), and CDK9. CK1α phosphorylates Mdmx and Mdm2 leading to enhanced binding and degradation of p53. CDK7 and CDK9 phosphorylate RNA polymerase II (Pol II) to enable transcriptional initiation and elongation, particularly at large clusters of transcriptional enhancers termed super-enhancers (SE). Preclinical studies have demonstrated that BTX-A51 robustly increased p53 protein levels via CK1a inhibition and Mdm2 downregulation while preferentially decreasing SE transcription of key oncogenes such as Myc andMcl1, enabling selective apoptosis of leukemia cells. The combination of CK1α, CDK7, and CDK9 inhibition was synergistic and prolonged survival in multiple genetic and patient-derived xenograft AML models. Study Design and Methods: This is an open-label, multi-center, first-in-human Phase 1 study evaluating the safety of BTX-A51 in patients with R/R AML or high-risk MDS. The trial will be performed in two phases, a dose escalation (phase 1a) and dose expansion (phase 1b). Phase 1a utilizes a hybrid accelerated titration with single patient cohorts and a Bayesian optimal interval (BOIN) design to assess 8 potential dosing cohorts. The maximum tolerated dose (MTD) will be the dose for which the isotonic estimate of the toxicity rate is closest to the target toxicity rate of 0.3. Up to a maximum of 35 patients will be enrolled in the dose escalation phase of the study at Memorial Sloan Kettering Cancer Center, MD Anderson Cancer Center, and City of Hope Cancer Center. Following determination of the MTD, 15 patients will be enrolled in the dose expansion phase for further evaluation of dose-limiting toxicities (DLTs) and for preliminary evidence of efficacy. BTX-A51 will be dosed 3 weeks on drug, followed by 1 week off drug over a 28-day cycle. For the first cycle, patients will receive tumor lysis syndrome prophylaxis with allopurinol and intravenous fluids and be closely monitored. Key inclusion criteria are age ³ 18 years, R/R AML or R/R high-risk MDS, Eastern Cooperative Oncology Group (ECOG) £ 2 and life expectancy of ³ 6 weeks, and adequate kidney and liver function. Key exclusion criteria are receipt of cancer chemotherapy (other than hydroxyurea) within 2 weeks prior to the start of study drug, transplantation within 3 months prior to screening, active graft-versus-host disease requiring systemic immunosuppressive medications, and a white blood cell count > 20 × 109/L. The primary objective for the Phase 1 study is to determine the MTD and recommended Phase 2 dose (RP2D) of BTX-A51. Secondary objectives include evaluating overall response (complete remission, complete remission with incomplete blood count recovery, and partial remission) according to the European LeukemiaNet (ELN) 2017 criteria (Döhner et al. Blood. 2017), survival (overall survival and event-free survival) and pharmacokinetics. Correlative objectives include determining the changes in SEs and SE-driven expression of antiapoptotic genes by chromatin immunoprecipitation and RNA-sequencing. Recruitment is ongoing and this trial is registered on clinicaltrials.gov: NCT04243785 Disclosures Stein: Amgen: Consultancy, Speakers Bureau; Stemline: Consultancy, Speakers Bureau. Borthakur:BioLine Rx: Consultancy; Argenx: Consultancy; PTC Therapeutics: Consultancy; FTC Therapeutics: Consultancy; BioTherix: Consultancy; Curio Science LLC: Consultancy; Xbiotech USA: Research Funding; Oncoceutics: Research Funding; Polaris: Research Funding; PTC Therapeutics: Research Funding; Cyclacel: Research Funding; GSK: Research Funding; Jannsen: Research Funding; Abbvie: Research Funding; Novartis: Research Funding; Incyte: Research Funding; BioLine Rx: Research Funding; BMS: Research Funding; AstraZeneca: Research Funding; Nkarta Therapeutics: Consultancy; Treadwell Therapeutics: Consultancy. Murray:Salamandra: Current Employment. Kook:Salamandra: Current Employment. Chan:BioTheryx: Current Employment. Stein:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Syros: Membership on an entity's Board of Directors or advisory committees; Syndax: Consultancy, Research Funding; Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bayer: Research Funding; Biotheryx: Consultancy; Agios Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy; PTC Therapeutics: Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Consultancy; Amgen: Consultancy; Celgene Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Astellas Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

MiR-935/HIF1α Feedback Loop Inhibits the Proliferation and Invasiveness of Glioma.

ObjectiveThe biological functions and molecular mechanisms of miR-935 have been widely investigated in various types of cancer. The aim of the present study was to explore the function of miR-935 in glioma.MethodsBioinformatic analysis and quantitative real-time fluorescent PCR (qRT-PCR) were used to determine the expression of miR-935 in glioma tissues and glioma cell lines. Chi-square test was performed to analyze the relationship between the expression of miR-935 and clinical traits. CCK-8 assay, colony formation assay, cell cycle analysis and subcutaneous tumorigenesis model in nude mice were conducted to determine the effects of miR-935 on the proliferation of glioma cells both in vitro and in vivo. Wound healing and transwell assays were used to detect the effects of miR-935 on the migration and invasion of glioma cells in vitro. The relationship between miR-935 and HIF1α was analyzed using bioinformatics, luciferase reporter assay and Western blotting.ResultsThe expression of miR-935 was lower in glioma tissues than in the adjacent tissues, and in cell lines than in the normal human astrocytes (NHAs), and the low expression levels of miR-935 predicted a poor outcome. Exogenous overexpression of miR-935 inhibited the proliferation of glioma cells both in vitro and in vivo, and suppressed the migration and invasion of glioma cells in vitro. HIF1α was identified as the target of miR-935, whereas miR-935 overexpression decreased the expression of HIF1α and its target genes VEGF, MCL1 and GLUT1. Strikingly, overexpression of HIF1α significantly decreased the expression of miR-935, whereas silencing HIF1α increased the expression of miR-935. Similarly, HIF1α overexpression remarkably reversed the inhibitory effects of miR-935 on the proliferation, migration and invasion of glioma cells.ConclusionOverall, present study reveals the presence of miR-935/HIF1α feedback loop in glioma, which inhibits the development of glioma. This feedback loop may be a potential target for the treatment of glioma.

MicroRNAs as Biomarkers in Canine Osteosarcoma: A New Future?

Sarcomas are frequent in dogs and canine species are excellent animal models for studying the human counterpart. However, osteosarcomas are a rare form of sarcoma with high death rates in humans and dogs. miRNAs are small endogenous RNAs that regulate gene expression post-transcriptionally. The discovery of miRNAs could give a contribute in the diagnosis and prognosis of different types of tumors in animal species, as already in humans. The differentiated expression of miRNAs is a frequent finding in cancers and is related to their pathogenesis in many cases. Most canine and human sarcomas show similar miRNA aberrations. Lower levels of miR-1 and miR-133b in canine osteosarcoma tissues were found to increase tumorigenesis through a higher expression of their target genes MET and MCL1. The overexpression of miR-9 promotes a metastatic phenotype in canine osteosarcomas and its capacity as a prognostic biomarker for the disease is currently being evaluated. MicroRNAs at the 14q32 locus could be used as prognostic biomarkers, since their decreased expression has been associated with poor prognosis in canine and human osteosarcomas. Furthermore, a decreased expression of miR-34a in osteosarcoma tumour cells has been associated with shorter disease-free survival times and its reintroduction as a synthetic prodrug shows good potential as a novel therapeutic target to fight the disease. Circulating miR-214 and miR-126 are significantly increased in a broad-spectrum cancer and have the ability to successfully predict the prognosis of dogs. However, further studies are needed to make the use of miRNAs as biomarkers a common practice.

Expression differences of miR-142-5p between treatment-na\xefve chronic myeloid leukemia patients responding and non-responding to imatinib therapy suggest a link to oncogenic ABL2, SRI, cKIT and MCL1 signaling pathways critical for development of therapy resistance

BackgroundChronic myeloid leukemia (CML) is a myeloproliferative neoplasm characterized by constitutive activity of the tyrosine kinase BCR-ABL1. Although the introduction of tyrosine kinase inhibitors (TKIs) has substantially improved patients’ prognosis, drug resistance remains one of the major challenges in CML therapy. MicroRNAs (miRNAs), a class of short non-coding RNAs acting as post-transcriptional regulators, are implicated in CML progression and drug resistance. The aim of the present study was to analyze the miRNA expression profiles of 45 treatment-naïve CML patients in chronic phase (28 peripheral blood and 17 bone marrow samples) with respect to future response to imatinib therapy.MethodsTaqMan low density arrays were used to analyze the miRNA expression pattern of the patient samples. For selected microRNAs, reporter gene assays were performed to study their ability to regulate CML associated target genes.ResultsSignificant lower expression levels of miR-142-5p were identified in both, peripheral blood and bone marrow samples of future non-responders suggesting a potential tumor suppressor role of this miRNA. This was supported by reporter gene assays that identified the survival, proliferation and invasion promoting CML related genes ABL2, cKIT, MCL1 and SRI as targets of miR-142-5p and miR-365a-3p, the latter identified as potential biomarker in peripheral blood samples.ConclusionMiR-142-5p and to a certain extend also miR-365a-3p were able to discriminate treatment-naïve CML patients not responding to imatinib in the course of their treatment from patients, who responded to therapy. However, further large-scale studies should clarify if the identified miRNAs have the potential as predictive biomarkers for TKI resistance.

Discovery of novel indazole-acylsulfonamide hybrids as selective Mcl-1 inhibitors

Overexpressing myeloid cell leukemia sequence 1 (Mcl-1) protein is an important way to confer the resistance of cancer cells to conventional anti-cancer treatments. Therefore, developing Mcl-1 inhibitors has become an attractive strategy for cancer therapy. In the studies, a series of new indazole-acylsulfonamide hybrids were designed, synthesized and evaluated as potent Mcl-1 inhibitors. Among them, the most potent compound 17 (Ki = 0.43 μM) showed a little better inhibitory activity against Mcl-1 protein than positive control AT-101 (Ki = 0.45 μM). Pleasingly, it displayed > 40-fold selectivity over Bcl-2 (Ki = 18 μM) and Bcl-xL (no activity). Furthermore, compound 17 had good inhibitory activities against PC-3, MDA-MB-231 and K562 cells (IC50 = 12.3, 10.6 and 6.62 μM, respectively) and could effectively induce apoptosis and the activation of caspase-3 in a dose-dependent manner in K562 cells.

Relationship between Chromosomal Aberrations and Gene Expressions in the p53 Pathway in Chronic Lymphocytic Leukemia.

Chronic lymphocytic leukemia (CLL) is a neoplasm characterized by excessive accumulation of B lymphocytes in the peripheral blood, bone marrow and lymph nodes. We assessed the expressions of 22 genes in the p53 pathway in 30 CLL patients and 15 healthy subjects by a RT2 Profiler PCR (polymerase chain reaction) Array technique and their relation to cytogenetic aberrations detected by fluorescent in situ hybridization (FISH). Our Student’s t-test results indicated that ATM, ATR, BAX, CASP9, CDK4, CDKN2A, CHEK1, CHEK2, E2F3, MCL1, MDM2, MDM4, PCNA, RB1, P53 and BCL2 genes were statistically significant (p <0.001). For six genes (APAF1, CDKN1A, E2F1, GADD45A, PTEN and PTX3) were not statistically significant. The ATM, ATR, BAX, CASP9, CDK4, CDKN1A, CDKN2A, CHEK1, CHEK2, MDM2, MDM4, PCNA, RB1, P53, E2F1, GADD45A and BCL2 genes were found to be upregulated by the 2-ᐃᐃCt (relative fold change in gene expression) method. The highest up-regulation was detected in CDKN2A and BCL2 genes, 10.22- and 8.51-fold, respectively. On the other hand, the PTX3 gene with a fold regulation of 1.84 was found to the highest downregulation. Overall, the CDNK2A BCL2 and PTX3 genes are related to the mechanism of the disease in the p53 pathway and may be an important predictor of the prognosis of the disease. The BCL2 gene may be associated with increased risk of developing CLL. We suggest that the PTX3 gene may be considered as a marker associated with CLL disease. The CDKN2A gene expression seems to play a protective role in CLL.

Ilimaquinone Induces Apoptosis and Autophagy in Human Oral Squamous Cell Carcinoma Cells.

In this study, the anti-tumor activity of ilimaquinone (IQ), a sesquiterpene quinone isolated from marine sponge Halichondria sp., in oral squamous cell carcinoma (OSCC) cells, was investigated. IQ suppressed the viability of the OSCC cell lines SCC4 and SCC2095 with IC50 values of 7.5 and 8.5 μM, respectively. Flow cytometric analysis demonstrated that IQ induced caspase-dependent apoptosis in SCC4 cells and modulated the expression of several cell growth-related gene products, including Akt, p38, Mcl-1, and p53. Notably, p53 knockdown caused higher resistance to IQ’s anti-tumor activity. In addition, IQ increased reactive oxygen species generation, which was partially reversed by the addition of antioxidants. Furthermore, it triggered autophagy, as evidenced by acidic organelle formation and LC3B-II and Atg5 expression in SCC4 cells. Pretreatment with the autophagy inhibitor 3-methyladenine or chloroquine partially decreased IQ-induced apoptosis, suggesting that IQ induced protective autophagy. In summary, IQ has potential to be used in OSCC therapy.

Chloride intracellular channel 4 is dysregulated in endometrium of women with infertility and alters receptivity

The endometrium remodels in each menstrual cycle to become receptive in preparation for embryo implantation which occurs in the mid-secretory phase of the cycle. Failure of blastocyst adhesion and implantation cause infertility. We compared chloride intracellular channel 4 (CLIC4) expression in human endometrium from women with normal fertility and primary unexplained infertility in the mid-secretory/receptive phase of the menstrual cycle. CLIC4 localised to both the epithelial and stromal regions of the endometrium of fertile tissues across the cycle. CLIC4 expression was significantly reduced in the luminal and glandular epithelium and remained unchanged in the stromal region of mid-secretory infertile endometrium compared to fertile endometrium. siRNA knockdown of CLIC4 significantly compromised adhesive capacity of Ishikawa cells (endometrial epithelial cell line). This reduced adhesion and CLIC4 expression was associated with elevated SGK1, p53, SIRT1, BCL2 and MCL1 gene expression in the Ishikawa cells. CLIC4 expression was increased in primary human endometrial stromal cells during decidualization, however, siRNA knockdown of CLIC4 did not affect decidualization. Our data provide evidence that CLIC4 may regulate receptivity and facilitate blastocyst attachment initiating implantation. Reduced CLIC4 levels may be causative of implantation failure in women.

Abstract 73: Development of APG-3526 as a novel and highly efficacious MCL-1 inhibitor

Abstract Background MCL-1 is an important anti-death BCL-2 family protein and plays a key role in blocking apoptosis in cancer cells. MCL-1 gene is located in one of the most frequently amplified locus in various hematologic malignancies and solid tumors, including prostate, lung, pancreatic, breast, ovarian and cervical cancers, as well as melanoma, B-cell chronic lymphocytic leukemia (B-CLL), acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL)); moreover, MCL-1 overexpression is implicated as a resistance factor for multiple therapies including widely prescribed microtubule-targeted agents for breast cancers. Therefore, MCL-1 is an attractive therapeutic target for the treatment of cancers. In this study, we discovered the lead preclinical compound APG-3526, from its chemical synthesis optimization to potent antiproliferative and antitumor activity using multiple in vitro and in vivo xenograft models, respectively. Methods and Experiments APG-3526 was chemically synthesized by using one convergent multistep synthesis in gram scale. The binding affinity of APG-3526 was determined by a FP assay using a fluorescein labelled peptide (FAM-Bid) which binds to the MCL-1 protein leading to an increased anisotropy measured in milli-polarization (mP) values. The effect of APG-3526 on cell viability was determined using CCK-8 assay and its antitumor activities were tested in NCI-H929 and OPM-2 multiple myeloma (MM) xenograft SCID mice model (dosed i.v. at 50 mg/kg and p.o. at 25 mg/kg). Results Our biochemical study has demonstrated that APG-3526 binds to MCL-1 with a nanomolar affinity (IC50 = 7 nM). It is highly potent in inhibition of cell growth in MCL-1-dependent multiple myeloma cell lines (NCI-H929 cells: IC50 = 14 nM; OPM-2 cells: IC50 = 62 nM). Administration of APG-3526 (p.o. at 25 mg/kg, QDx14D, or i.v. at 50 mg/kg, once) achieved complete tumor regression in both NCI-929 and OPM-2 multiple myeloma xenograft models. The pharmacodynamics (PD) study using tumor samples further revealed caspase 3 activation and PARP cleavage triggered by APG-3526. MCL-1 complex MSD assay confirmed that APG-3526 disrupted MCL-1:BIM binding thus freeing BIM to initiate the apoptotic cascade. In conclusion, we have developed a novel and highly potent MCL-1 inhibitor, APG-3526, which displays clinically relevant pharmacokinetic properties and elicits potent antiproliferative and antitumor activities via disrupting MCL-1 complex and triggering caspase activation, especially in MCL-1 driven MM models. These results support APG-3526 as a promising MCL-1inhibitor for further clinical development. Citation Format: Jianyong Chen, Chengzhe Wu, Lingling Jiao, Leilei Zhao, Yunlong Zhou, Dongbo Li, Guozhi Tang, Shoulai Gu, Jing Deng, Guangfeng Wang, Douglas D. Fang, Shaomeng Wang, Dajun Yang, Yifan Zhai. Development of APG-3526 as a novel and highly efficacious MCL-1 inhibitor [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 73.

Abstract 5684: Drug-anchored in vitro and in vivo CRISPR screens to identify targetable vulnerabilities and modifiers of response to MRTX849 in KRASG12C-mutant models

Abstract MRTX849 is a recently described covalent KRASG12C inhibitor in active clinical development in non-small cell lung cancer (NSCLC) and other cancers harboring this mutation. Additional genetic alterations in hallmark pathways are co-incident with KRASG12C mutations in NSCLC and several of these alterations have been shown to contribute to the oncogenic phenotype. The impact of co-occurring genetic alterations on KRAS dependency or response to KRAS targeted therapy is unknown. Evaluation of MRTX849 across a panel of cell and patient-derived xenograft models demonstrated strong single agent activity in the majority of models tested, however, a subset of models were less responsive. A focused sgRNA library targeting ~1,000 genes was tested in MRTX849-anchored CRISPR/Cas9 screens in vitro and in vivo in multiple KRASG12C-mutant cell lines. Several genes that act either upstream or downstream of KRAS were depleted with MRTX849 treatment which illuminated specific targetable vulnerabilities in the context of KRASG12C inhibition. These data provided a catalogue of putative combination targets. sgRNAs targeting EGFR, PTPN11 (SHP2), SOS1, mTOR, CDK1/2/4/6 were all depleted in MRTX849-treated (or vehicle-treated) cell lines or xenograft models. Furthermore, treatment with small molecules targeting selected vulnerabilities confirmed these genetic data. Finally, targeting FGFR1 exhibited context-dependent dropout that may conditionally address receptor tyrosine kinase reactivation following MRTX849 treatment. This observation was further confirmed utilizing the FGFR family inhibitor BGJ398. Further evaluation of dependencies utilizing an integrated analysis of RNAseq data also revealed KRAS regulates and is critically dependent on specific pro-survival genes BCL2L1, MCL1 and BIRC5 (Survivin) for cancer cell viability. On the other hand, loss of tumor suppressors including KEAP1, NF1, CBL, RB1 and PTEN conferred a growth advantage and partial resistance to MRTX849 treatment in selected models. sgRNAs targeting the RAS pathway genes SPRY2 and NRAS were also enriched providing additional insight into the wiring of KRAS mutant tumors and adaptive response to KRASG12C inhibition. These data provide key insight into the genes that mediate the mechanism of action of, as well as confer partial resistance to, MRTX849, and identify combination targets that augment the anti-tumor effect of MRTX849. Citation Format: Lars D. Engstrom, Laura Waters, Sole Gatto, Julio Fernandez-Banet, Ruth Aranda, Adam Pavlicek, James G. Christensen, Peter Olson. Drug-anchored in vitro and in vivo CRISPR screens to identify targetable vulnerabilities and modifiers of response to MRTX849 in KRASG12C-mutant models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5684.

Simultaneously Inhibiting BCL2 and MCL1 Is a Therapeutic Option for Patients with Advanced Melanoma.

There is an urgent need to develop treatments for patients with melanoma who are refractory to or ineligible for immune checkpoint blockade, including patients who lack BRAF-V600E/K mutations. This is often the case in patients diagnosed with rare melanoma subtypes such as mucosal and acral melanoma. Here, we analyzed data from the cutaneous melanoma The Cancer Genome Atlas Network (TCGA) transcriptomic and proteomic databases for differential expression of apoptosis molecules between melanomas with or without BRAF hotspot mutations. Our data indicated higher B-cell CLL/lymphoma 2 (BCL2) expression in melanoma without BRAF hotspot mutations, suggesting that BH3 mimetics, such as ABT-199 (venetoclax, a small molecule against BCL2), may be a potential therapeutic option for these patients. We explored the efficacy of combining two BH3 mimetics, ABT-199 and a myeloid cell leukemia sequence 1 (MCL1) inhibitor (S63845 or S64315/MIK665) in cutaneous, mucosal and acral melanomas, in vitro and in vivo. Our data indicate this combination induced cell death in a broad range of melanoma cell lines, including melanoma initiating cell populations, and was more potent in melanoma cells without BRAF-V600E/K mutations. Our knockdown/knockout experiments suggest that several pro-apoptotic BCL2 family members, BCL2-like 11 (apoptosis facilitator) (BIM), phorbol-12-myristate-13-acetate-induced protein 1 (NOXA) or BID, play a role in the combination-induced effects. Overall, our study supports the rationale for combining an MCL1 inhibitor with a BCL2 inhibitor as a therapeutic option in patients with advanced melanoma.

NCAPH is negatively associated with Mcl‑1 in non‑small cell lung cancer.

Lung cancer has a high mortality rate worldwide. Non-SMC condensin I complex subunit H (NCAPH) has been identified to be one of the regulatory subunits of the condensin I complex, which is essential for the correct packaging and segregation of chromosomes in eukaryotes. NCAPH is abnormally overexpressed in various types of cancer. A pro-survival member of the Bcl-2 family, myeloid cell leukemia sequence 1 (Mcl-1) is also frequently overexpressed in multiple cancers and is associated with poorer clinical outcomes for patients. The association of NCAPH and Mcl-1 proteins with the clinical and pathological features of non-small cell lung cancer (NSCLC) remains to be elucidated. In the current study, the positive percentage of NCAPH in the non-cancerous lung tissues was revealed to be higher compared with that in NSCLC. However, the positive percentage of Mcl-1 in the non-cancerous lung tissues was lower compared with NSCLC. In addition, NCAPH high-expression patients had a higher overall survival rate compared with patients exhibiting low expression, whereas the Mcl-1 high-expression group had a lower survival rate. Pairwise association in 260 cases of NSCLC revealed that overexpression of the NCAPH protein was negatively associated with Mcl-1 expression and vice versa. The results of multivariate Cox proportional hazard regression analysis also indicated that NCAPH and Mcl-1 demonstrated potential as distinct prognostic factors that may be used in NSCLC. The expression of NCAPH and Mcl-1 may be associated with, and act as distinct molecular marks for the prediction of a poor prognosis in patients with NSCLC.

MNDA controls the expression of MCL-1 and BCL-2 in chronic lymphocytic leukemia cells

The myeloid nuclear differentiation antigen (MNDA) is a stress-induced protein that promotes degradation of the anti-apoptotic factor MCL-1 and apoptosis in myeloid cells. MNDA is also expressed in normal lymphoid cells and in B-cell clones isolated from individuals with chronic lymphocytic leukemia (CLL), a disease characterized by abnormal apoptosis control. We found that MNDA expression levels inversely correlate with the amount of the anti-apoptotic proteins MCL-1 and BCL-2 in human CLL samples. We report that in response to chemotherapeutic agents that induce genotoxic stress, MNDA exits its typical nucleolar localization and accumulates in the nucleoplasm of CLL and lymphoid cells. Then, MNDA binds chromatin at Mcl1 and Bcl2 genes and affects the transcriptional competence of RNA polymerase II. Our data also reveal that MNDA specifically associates with Mcl1 and Bcl2 (pre-) mRNAs and favors their rapid turnover as a prompt response to genotoxic stress. We propose that this rapid dynamic tuning of RNA levels, which leads to the destabilization of Mcl1 and Bcl2 transcripts, represents a post-transcriptional mechanism of apoptosis control in CLL cells. These results provide an explanation of previous clinical data and corroborate the finding that higher MNDA expression levels in CLL are associated with a better clinical course.

Notch-1 Signaling Modulates Macrophage Polarization and Immune Defense against Mycobacterium avium paratuberculosis Infection in Inflammatory Diseases.

Despite the extensive research on Notch signaling involvement in inflammation, its specific role in macrophage response in autoimmune disease and defense mechanisms against bacterial infection, such as Mycobacterium avium paratuberculosis (MAP), remains unknown. In this study, we investigated the molecular role of Notch-1 signaling in the macrophage response during MAP infection. In particular, we measured the in vitro effect of MAP on Notch-1 signaling and downstream influence on interleukin (IL)-6 and myeloid cell leukemia sequence-1 (MCL-1) and consequent cellular apoptosis, MAP viability, and macrophage polarization. Overall, the data show significant upregulation in Notch-1, IL-6, and MCL-1 in MAP-infected macrophages, parallel with a decrease in apoptosis and elevated pro-inflammatory response in these infected cells. On the contrary, blocking Notch signaling with γ-secretase inhibitor (DAPT) decreased MAP survival and burden, increased apoptosis, and diminished the pro-inflammatory response. In particular, the treatment of infected macrophages with DAPT shifted macrophage polarization toward M2 anti-inflammatory phenotypic response. The outcome of this study clearly demonstrates the critical role of Notch signaling in macrophage response during infection. We conclude that MAP infection in macrophages activates Notch-1 signaling and downstream influence on IL-6 which hijack MCL-1 dependent inhibition of apoptosis leading to its chronic persistence, and further inflammation. This study supports Notch-1 signaling as a therapeutic target to combat infection in autoimmune diseases such as Crohn’s disease and Rheumatoid Arthritis.

Multimodality of human epidermal growth factor receptor-2 antagonism restores the apoptotic capacity of liver cancer cells

Background: Hepatocellular carcinoma, the most widespread form of liver cancer and one of the most common and lethal malignancies, is characterized by poor prognosis, late onset, and a lack of clear-cut diagnostic markers. Novel therapeutic approaches are desperately required to develop effective treatment regimens. Methods: In this study, we attempted to reverse the proliferative capacity of liver cancer cells through employing a 3 – prong approach. We evaluated the antitumorigenic effects of some medicinal plant extracts that contain bioactive phytochemicals. In addition, we used Imatinib – a tyrosine kinase inhibitor (TKI), with human epidermal growth factor receptor (Her2)-specific small interfering RNA(siRNA) species to counteract the Her2-induced proliferative capacity of cancer cells. In our model, we evaluated the extent of activation of apoptotic mechanisms versus the proliferative and antiapoptotic strategies mounted by cancer cells. Results: Our results showed that HepG2 cells treated with 0.5 mM Imatinib exhibited marked downregulation of Her2 expression, upregulation of the proapoptotic marker, BAX and a downregulation of proliferative markers GPC3 and transforming growth factor (TGF)-β (45, 29, 95, and 115 folds, respectively). In the meantime, there was also significant downregulation of Her2, TGF-β, Mcl1, Spp1, GLUL and GPC3 expression and activation of apoptotic system in the cells treated with a mixture of anti-Her2 siRNA, Imatinib along with some selected extracts where the mixture successfully decreased viability of cancer cells. Conclusion: our study reveals the potential of using TKI along with phytochemical therapy and RNA interference as adjuvant regimen for treatment of liver cancer to augment the efficacy of the current control programs, yet, minimizing the side effects by transition to targeted rather than mass therapies.

Circular RNA circ_0067934 functions as an oncogene in breast cancer by targeting Mcl-1.

Since this article has been suspected of research misconduct and the corresponding authors did not respond to our request to prove originality of data and figures, "Circular RNA circ_0067934 functions as an oncogene in breast cancer by targeting Mcl-1, by J.-M. Wang, X.-J. Li, J. Wang, published in Eur Rev Med Pharmacol Sci 2019; 23 (21): 9499-9505-DOI:10.26355/eurrev_201911_19444-PMID: 31773702" has been withdrawn. The Publisher apologizes for any inconvenience this may cause. https://www.europeanreview.org/article/19444.

Shrimp miR-965 induced the human melanoma stem-like cell apoptosis and inhibited their stemness by disrupting the MCL-1-ER stress-XBP1 feedback loop in a cross-species manner

BackgroundMelanoma is a type of aggressive skin cancer with a poor survival rate. The resistance to conventional therapy of this disease is, at least in part, attributed to its cancer stem cell population. However, the mechanism of survival and stemness maintenance of cancer stem cells remains to be investigated.MethodsTumorsphere formation assay was used to study the stem-like property of melanoma stem-like cells (MSLC). Chromatin immunoprecipitation (ChIP), promoter luciferase reporter assay were included for exploring the role of MCL-1 in MSLC and electrophoretic mobility shift assay were used to evaluate the interaction between shrimp miR-965 and human Ago2 protein. Melanoma xenograft nude mice were used to study the inhibition of tumor development.ResultsIn the present study, our results showed that myeloid cell leukemia sequence 1 (MCL-1) knocking down induced ER stress and apoptosis, and the expression reduction of stemness associated genes in MSLC, which implied a significant role of MCL-1 in MSLC. Further study indicated that ER stress agonist (tunicamycin) treatment in MSLC results in the translocation of XBP1, an ER stress sensor, into the nucleus to induce MCL-1 expression through direct binding to the − 313- to − 308-bp region of MCL-1 promoter. In addition, we found that a shrimp-derived miRNA (shrimp miR-965) could interact with the human Ago2 protein and suppressed the human MCL-1 expression by binding to the 3′ UTR of MCL-1 mRNA, thereby inhibiting the MSLC proliferation and stemness in vitro and in vivo in a cross-species manner.ConclusionIn conclusion, we identified an important role of MCL-1-ER stress-XBP1 feedback loop in the stemness and survival maintenance of MSLC, and shrimp miR-965, a natural food derived miRNA, could regulate MSLC stemness and survival by targeting MCL-1 and disrupting the balance of MCL-1-ER stress-XBP1 feedback loop. In conclusion, this study indicated an important mechanism of the regulation of MSLC stemness and survival, otherwise it also demonstrated the significance of cross-species-derived miRNA as promising natural drugs in melanoma therapy.

Inhibition of the STAT3 Signaling Pathway Contributes to the Anti-Melanoma Activities of Shikonin.

BackgroundMalignant melanoma is an extremely aggressive and metastatic cancer, and highly resistant to conventional therapies. Signal transducer and activator of transcription 3 (STAT3) signaling promotes melanoma development and progression, which has been validated as an effective target in melanoma treatment. Natural naphthoquinone shikonin is reported to exert anti-melanoma effects. However, the underlying mechanisms have not been fully elucidated.PurposeThis study aims to evaluate the anti-melanoma activities of shikonin and explore the involvement of STAT3 signaling in these effects.MethodsZebrafish tumor model was established to evaluate the anti-human melanoma effects of shikonin in vivo. MTT assay and colony formation assay were employed to investigate the anti-proliferative effects of shikonin on human melanoma A375 and A2058 cells. Flow cytometry was used to analyze cell cycle distribution and apoptosis induction. Wound healing assay and Transwell chamber assay were conducted to examine the cell migratory and invasive abilities. Immunofluorescence assay was used to observe F-actin, Tubulin, and STAT3 localization. Western blotting was used to determine the expression levels of proteins associated with apoptosis and key proteins in the STAT3 signaling pathway. Immunoblotting was performed in DSS cross-linked cells to determine the homo-dimerization of STAT3. Gelatin zymography was employed to evaluate the enzymatic activity of MMP-2 and MMP-9. Transient transfection was used to overexpress STAT3 in cell models.ResultsShikonin suppressed melanoma growth in cultured cells and in zebrafish xenograft models. Shikonin induced melanoma cells apoptosis, inhibited cell migration and invasion. Mechanistic study indicated that shikonin inhibited the phosphorylation and homo-dimerization of STAT3, thus reduced its nuclear localization. Further study showed that shikonin decreased the levels of STAT3-targeted genes Mcl-1, Bcl-2, MMP-2, vimentin, and Twist, which are involved in melanoma survival, migration, and invasion. More importantly, overexpression of constitutively active STAT3 partially abolished the anti-proliferative, anti-migratory, and anti-invasive effects of shikonin.ConclusionThe anti-melanoma activity of shikonin is at least partially attributed to the inhibition on STAT3 signaling. These findings provide new insights into the anti-melanoma molecular mechanisms of shikonin, suggesting its potential in melanoma treatment.

Effects of sorafenib on fibroblast-like synoviocyte apoptosis in rats with adjuvant arthritis

Rheumatoid arthritis (RA) is a chronic autoimmune disease that is characterized by synovial inflammation and hyperplasia resulting from an imbalance between the proliferation and apoptosis of fibroblast-like synoviocytes (FLSs). Our previous study found that sorafenib had inhibitory effects in rats with adjuvant arthritis (AA). The present study investigated the role of sorafenib in the induction of AA FLS apoptosis in vitro. FLSs obtained from AA rats were cultured in vitro and identified. Cell apoptosis was detected using terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) and annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) labeling methods. Real-time PCR and Western blotting assays were used to quantify the expression levels of Fas, Caspase-3, Mcl-1, NF-κB and C-jun gene products in AA FLSs. Our data revealed that sorafenib (4 μmol/L) induced apoptosis in AA FLSs, and flow cytometry analysis showed that AA FLSs treated with sorafenib (4 μmol/L) in vitro accumulated in early and late apoptosis. There were significant increases in the expression levels of Fas, Caspase-3 and Mcl-1, and significant decreases in NF-κB and C-jun expression in AA FLSs treated with sorafenib. In summary, these results demonstrate that sorafenib promotes AA FLS apoptosis, which may be related to the upregulation of Fas and Caspase-3 and downregulation of NF-κB and C-jun. All of these findings suggest that sorafenib exerts an inhibitory effect on AA rats in vivo via AA FLS apoptotic induction, which has potential therapeutic implications for RA.