C/EBPα-p30 confers AML cell susceptibility to the terminal unfolded protein response and resistance to Venetoclax by activating DDIT3 transcription.

BACKGROUND: Acute myeloid leukemia (AML) with biallelic (CEBPAbi) as well as single mutations located in the bZIP region is associated with a favorable prognosis, but the underlying mechanisms are still unclear. Here, we propose that two isoforms of C/EBPα regulate DNA damage-inducible transcript 3 (DDIT3) transcription in AML cells corporately, leading to altered susceptibility to endoplasmic reticulum (ER) stress and related drugs. METHODS: Human AML cell lines and murine myeloid precursor cell line 32Dcl3 cells were infected with recombinant lentiviruses to knock down CEBPA expression or over-express the two isoforms of C/EBPα. Quantitative real-time PCR and western immunoblotting were employed to determine gene expression levels. Cell apoptosis rates were assessed by flow cytometry. CFU assays were utilized to evaluate the differentiation potential of 32Dcl3 cells. Luciferase reporter analysis, ChIP-seq and ChIP-qPCR were used to validate the transcriptional regulatory ability and affinity of each C/EBPα isoform to specific sites at DDIT3 promoter. Finally, an AML xenograft model was generated to evaluate the in vivo therapeutic effect of agents. RESULTS: We found a negative correlation between CEBPA expression and DDIT3 levels in AML cells. After knockdown of CEBPA, DDIT3 expression was upregulated, resulting in increased apoptotic rate of AML cells induced by ER stress. Cebpa knockdown in mouse 32Dcl3 cells also led to impaired cell viability due to upregulation of Ddit3, thereby preventing leukemogenesis since their differentiation was blocked. Then we discovered that the two isoforms of C/EBPα regulate DDIT3 transcription in the opposite way. C/EBPα-p30 upregulated DDIT3 transcription when C/EBPα-p42 downregulated it instead. Both isoforms directly bound to the promoter region of DDIT3. However, C/EBPα-p30 has a unique binding site with stronger affinity than C/EBPα-p42. These findings indicated that balance of two isoforms of C/EBPα maintains protein homeostasis and surveil leukemia, and at least partially explained why AML cells with disrupted C/EBPα-p42 and/or overexpressed C/EBPα-p30 exhibit better response to chemotherapy stress. Additionally, we found that a low C/EBPα p42/p30 ratio induces resistance in AML cells to the BCL2 inhibitor venetoclax since BCL2 is a major target of DDIT3. This resistance can be overcome by combining ER stress inducers, such as tunicamycin and sorafenib in vitro and in vivo. CONCLUSION: Our results indicate that AML patients with a low C/EBPα p42/p30 ratio (e.g., CEBPAbi) may not benefit from monotherapy with BCL2 inhibitors. However, this issue can be resolved by combining ER stress inducers.

Antioxidant and Anti-Obesity Effects of Juglans mandshurica in 3T3-L1 Cells and High-Fat Diet Obese Rats.

Juglans mandshurica Maxim. walnut (JMW) is well-known for the treatment of dermatosis, cancer, gastritis, diarrhea, and leukorrhea in Korea. However, the molecular mechanism underlying its anti-obesity activity remains unknown. In the current study, we aimed to determine whether JMW can influence adipogenesis in 3T3-L1 preadipocytes and high-fat diet rats and determine the antioxidant activity. The 20% ethanol extract of JMW (JMWE) had a total polyphenol content of 133.33 ± 2.60 mg GAE/g. Considering the antioxidant capacity, the ABTS and DPPH values of 200 µg/ml of JMWE were 95.69 ± 0.94 and 79.38 ± 1.55%, respectively. To assess the anti-obesity activity of JMWE, we analyzed the cell viability, fat accumulation, and adipogenesis-related factors, including CCAAT-enhancer-binding protein alpha (C/EBPα), sterol regulatory element-binding protein-1c (SREBP1c), peroxisome proliferator-activated receptor-gamma (PPARγ), fatty acid synthase (FAS), and acetyl-CoA carboxylase (ACC). We found that total lipid accumulation and triglyceride levels were reduced, and the fat accumulation rate decreased in a dose-dependent manner. Furthermore, JMWE suppressed adipogenesis-related factors C/EBPα, PPARγ, and SREBP1c, as well as FAS and ACC, both related to lipogenesis. Moreover, animal experiments revealed that JMWE could be employed to prevent and treat obesity-related diseases. Hence, JMWE could be developed as a healthy functional food and further explored as an anti-obesity drug.

Xiao-Gao-Jiang-Zhuo-containing serum inhibits adipogenesis through SIRT1-IGF-1 crosstalk in 3T3-L1 preadipocytes.

PPARγ, CEBP/α, and SREBP1C are the major transcriptional factors participating in adipogenesis and lipogenesis. SIRT1 and IGF-1 signaling pathways are important pathways involved in body endocrine and metabolism. Our unique Chinese herbal medicine Xiao-Gao-Jiang-Zhuo (XGJZ) has a remarkable clinical effect on obesity. However, the molecular basis remains unknown. XGJZ-containing serum was treated in the incubation of 3T3-L1 preadipocytes to observe its function in the 3T3-L1 cell differentiation. Oil Red O staining was used to monitor the lipid droplets accumulated after 8 days of incubation. RT-qPCR and western blotting were used to investigate the regulatory effects of XGJZ-containing serum on adipogenesis-related factors. The protein levels of main molecules in SIRT1 and IGF-1 signaling pathways were also detected by western blotting. XGJZ-containing serum notably suppressed the lipid accumulation in differentiated adipocytes through SIRT1/IGF-1 pathway. XGJZ-containing serum activated the SIRT1/IGF-1 pathway and reduced the expression levels of PPARγ, CEBP/α, and SREBP1C through this pathway. Additionally, XGJZ-containing serum enhanced the phosphorylation of ATGL and HSL and then induced lipolysis. XGJZ-containing serum has inhibitory effects on adipogenesis in 3T3-L1 preadipocytes through SIRT1/IGF-1 signaling pathway. Our study affirmed the effect of XGJZ-containing serum in the treatment of obesity. It provides a basis for the mechanism of obesity.

Serine Threonine-Protein Kinase-Derived IW13 Improves Lipid Metabolism via C/EBP-α/SREBP1/FAS Signaling Pathways in HFD-Induced Zebrafish In Vivo Larval Model.

Obesity is linked to the development of major metabolic disorders such as type 2 diabetes, cardiovascular disease, and cancer. Recent research has focused on the molecular link between obesity and oxidative stress. Obesity impairs antioxidant function, resulting in dramatically increased reactive oxygen levels and apoptosis. In this study, we investigated the effect of IW13 peptide on inhibiting lipid accumulation and regulating the antioxidant mechanism to normalize the lipid metabolism in HFD induced zebrafish larvae. Our results showed that co-treatment with IW13 peptide showed a protective effect in HFD zebra fish larvae by increasing the survival and heart rate. However, IW13 peptide co-treatment reduced triglycerides and cholesterol levels while also restoring the SOD and CAT antioxidant enzymes. In addition, IW13 co-treatment inhibited the formation of lipid peroxidation and superoxide anion by regulating the glutathione level. Also, the results showed that IW13 specifically downregulated the expression of the lipogenic-specific genes (C/EBP-α, SREBP1, and FAS). The findings exhibited that the IW13 peptide with effective antioxidant and anti-obesity activity could act as a futuristic drug to treat obesity and oxidative stress-related diseases.

Development and validation of a prognostic scoring model to risk stratify childhood acute myeloid leukaemia.

To create a personal prognostic model and modify the risk stratification of paediatric acute myeloid leukaemia, we downloaded the clinical data of 597 patients from the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) database as a training set and included 189 patients from our centre as a validation set. In the training set, age at diagnosis, -7/del(7q) or -5/del(5q), core binding factor fusion genes, FMS-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD)/nucleophosmin 1 (NPM1) status, Wilms tumour 1 (WT1) mutation, biallelic CCAAT enhancer binding protein alpha (CEBPA) mutation were strongly correlated with overall survival and included to construct the model. The prognostic model demonstrated excellent discriminative ability with the Harrell's concordance index of 0.68, 3- and 5-year area under the receiver operating characteristic curve of 0.71 and 0.72 respectively. The model was validated in the validation set and outperformed existing prognostic systems. Additionally, patients were stratified into three risk groups (low, intermediate and high risk) with significantly distinct prognosis, and the model successfully identified candidates for haematopoietic stem cell transplantation. The newly developed prognostic model showed robust ability and utility in survival prediction and risk stratification, which could be helpful in modifying treatment selection in clinical routine.

Sinomenine alleviates glomerular endothelial permeability by activating the C/EBP-α/claudin-5 signaling pathway.

Diabetic nephropathy (DN) is one of the main complications of diabetes. It is closely associated with the dysfunction of glomerular endothelial cells (GECs) under hyperglycemia. Severe inflammation is an important inducer for the development of GECs dysfunction, and it contributes to the disruption of tight junctions in GECs and the increased endothelial permeability. Sinomenine, an alkaloid monomer extracted from the rhizome of Sinomenium acutum, is recognized for its multiple pharmacological functions, including an anti-DN property. The present study aimed to explore the potential functional mechanism of Sinomenine against DN. Animals were randomly divided into Sham, DN, DN + Sinomenine (20 mg/kg), and DN + Sinomenine (40 mg/kg) groups. The Sinomenine or vehicle was administered every day for 6 weeks, followed by collecting renal tissues for further detection. Increased body weights, elevated blood glucose levels and UAE values, aggravated renal tissue pathology, higher concentrations of IL-18 and IL-1ß in renal tissues, and reduced claudin-5 expression were observed in DN rats. However, the administration of Sinomenine significantly alleviated all these DN-related changes. Furthermore, human renal glomerular endothelial cells (HrGECs) were treated with high glucose (HG, 30 mM) with or without Sinomenine (50, 100 µM) for 24 h. We found that Sinomenine treatment ameliorated the elevated production of IL-18 and IL-1ß, increased fluorescence intensity of FITC-dextran, declined trans-endothelial electrical resistance (TEER) value, and reduction of claudin-5 and C/EBP-α in HG-treated HrGECs. Moreover, the regulatory effect of Sinomenine on endothelial monolayer permeability in HG-treated HrGECs was abolished by the knockdown of C/EBP-α, indicating C/EBP-α is required for the effect of Sinomenine. We concluded that Sinomenine alleviated diabetic nephropathy-induced renal glomerular endothelial dysfunction via activating the C/EBP-α/claudin-5 axis.

Treatment of Pancreatic Cancer by Aptamer Conjugated C/EBPα-saRNA.

Pancreatic cancer is estimated to become the second-leading cause of cancer-related mortality by 2020. While the death rates of most other cancers continue to decline recently, the death rates of pancreatic cancer are still increasing, with less than 5% of patients achieving 5-year survival. Despite great efforts to improve treatment with combinational therapies in pancreatic cancer patients, limited progress has been made. V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) has been depicted as a therapeutic target in pancreatic cancer for many years. However, the clinical outcome of KRAS-directed therapies has not been successful, suggesting that KRAS is an undruggable target. For the new druggable target, epigenetically silenced transcriptional factor C/EBPα (CCAAT/enhancer-binding protein α), upregulator of a strong inhibitor of cell proliferation (p21), is upregulated by small activating RNA (saRNA) in pancreatic cancer. For the cell type-specific delivery, pancreatic cancer-specific 2'-Fluoropyrimidine RNA-aptamers (2'F-RNAs) are conjugated with C/EBPα-saRNA via sticky bridge sequences. The conjugates of aptamer-C/EBPα-saRNA upregulate the expression of C/EBPα in vitro and inhibit the tumor growth in vivo. It suggests that aptamer-mediated targeted delivery of therapeutic C/EBPα-saRNA might be the effective therapeutics under the current therapeutic modality failure in pancreatic cancer.