SF3B1 is a core component of splicing machinery. Mutations in SF3B1 are frequently found in myelodysplastic syndromes (MDS), particularly in patients with refractory anemia with ringed sideroblasts (RARS), characterized by isolated anemia. SF3B1 mutations have been implicated in the pathophysiology of RARS; however, the physiological function of SF3B1 in erythropoiesis remains unknown. shRNA-mediated approach was used to knockdown SF3B1 in human CD34+ cells. The effects of SF3B1 knockdown on human erythroid cell differentiation, cell cycle, and apoptosis were assessed by flow cytometry. RNA-seq, qRT-PCR, and western blot analyses were used to define the mechanisms of phenotypes following knockdown of SF3B1. We document that SF3B1 knockdown in human CD34+ cells leads to increased apoptosis and cell cycle arrest of early-stage erythroid cells and generation of abnormally nucleated late-stage erythroblasts. RNA-seq analysis of SF3B1-knockdown erythroid progenitor CFU-E cells revealed altered splicing of an E3 ligase Makorin Ring Finger Protein 1 (MKRN1) and subsequent activation of p53 pathway. Importantly, ectopic expression of MKRN1 rescued SF3B1-knockdown-induced alterations. Decreased expression of genes involved in mitosis/cytokinesis pathway including polo-like kinase 1 (PLK1) was noted in SF3B1-knockdown polychromatic and orthochromatic erythroblasts comparing to control cells. Pharmacologic inhibition of PLK1 also led to generation of abnormally nucleated erythroblasts. These findings enabled us to identify novel roles for SF3B1 in human erythropoiesis and provided new insights into its role in regulating normal erythropoiesis. Furthermore, these findings have implications for improved understanding of ineffective erythropoiesis in MDS patients with SF3B1 mutations.

Encapsulation of chloroquine and doxorubicin by MPEG-PLA to enhance anticancer effects by lysosomes inhibition in ovarian cancer Ming Shao,1,* Weitao Zhu,2,* Xianping Lv,1 Qiankun Yang,1 Xin Liu,1 Ying Xie,1 Ping Tang,3 Ling Sun3 1Department of Blood Transfusion, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan Province 450052, China; 2Department of Clinical Laboratory, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan Province 450052, China; 3Department of Hematology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan Province 450052, China *These authors contributed equally to this work Purpose: As the deadliest gynecological malignancy, ovarian cancer ranks as a major cause of disease-related deaths to women worldwide and is treated with transurethral resection or systemic chemotherapy. However, traditional chemotherapeutic drug in antitumor therapy has shown unavoidable limitations, such as poor curative effects, systemic toxicity and development of drug resistance, leading to failure of tumor inhibition and recurrence. This study aims to explore an innovative method to enhance the clinical efficiency of ovarian cancer.Materials and methods: Using MTT assay, the cell viability was detected under different culture systems. Western blot was used to examine the expression of P-gp in doxorubicin-resistant and wild-type A2780/SKOV3 cells. We used confocal to examine the drug concentration under different culture conditions. Also, flow cytometry was used to detect the drug absorption at the determined time points under different culture systems. Using nude mice model, we evaluated the killing efficacy of chemotherapeutic drugs with or without nanoparticle encapsulation. ELISA was used to examine the levels of creatinine, alanine aminotransferase and aspartate aminotransferase in plasma.Results: We found that pretreatment of chloroquine (CQ) as chemosensitizer markedly enhanced the anticancer effects in ovarian cancer. We also provided evidence that CQ efficiently increase the pH value of lysosomes in tumor cells, leading to the reverse of drug sequestration induced by lysosomes. To further improve the pharmacokinetics profiles and avoid the systemic toxicity caused by chemotherapeutic agents, we encapsulated CQ and chemotherapeutic drugs by polymeric nanoparticles methoxy poly(ethylene glycol)-poly(l-lactic acid). Codelivery of CQ and chemotherapeutic agents by nanocarrier revealed enhanced anticancer effects compared with the free drug delivery by tail vein injection. More importantly, accumulated drugs, prolonged drug circulation and reduced organic damages were observed in nanoparticles delivery.Conclusion: Codelivery of CQ and chemotherapeutic drugs by methoxy poly(ethylene glycol)-poly(l-lactic acid) could significantly improve the anticancer effects and might have important potency in clinical applications for ovarian cancer therapy. Keywords: ovarian cancer, chloroquine, cisplatin, MPEG-PLA, nanoparticles

Acute myeloid leukemia (AML) is a hematological malignancy derived from immature myeloid cells, which have the characteristics of abnormal proliferation and differentiation. Glycolysis has been a popular topic of research in recent years, with increasing uptake and consumption of glucose. The present study aimed to investigate the glycolysis of tumor cells in patients with AML; in particular, how programmed cell death 1 ligand 1 (PD‑L1) regulates tumor cells glycolysis using real time PCR (RT‑PCR), western blotting and flow cytometry. PD‑L1 high expression predicted poor outcome in patients with AML in the public database Gene Expression Profiling Interactive Analysis. PD‑L1 expression was decreased in the samples from patients with AML with complete remission compared to that in patients with relapsed or refractory AML. In AML cell lines, glycolysis‑associated genes ALDOA, PGK1, LDHA and HK2 were highly expressed in a PD‑L1 high‑expressed cell line. Overexpressed PD‑L1 enhanced glucose consumption and the extracellular acidification rate, accompanied by decreased apoptosis and accumulation of cells in the S phase. In contrast, the apoptosis rate of tumor cells and the percentage of cells in the S phase were significantly increased following PD‑L1 knockdown in the THP1 cell line. HK2 and LDHA expression decreased after AML tumor cells were treated with Akt inhibitor or rapamycin. In addition, the PD‑L1‑overexpressed cell line (PD‑L1‑OV) MOLM‑13 exhibited rapid tumor progression. Glycolysis‑associated genes were highly expressed in tumor tissues of PD‑L1‑OV MOLM‑13, with increased Ki67. Based on these findings, PD‑L1 may be considered as a suitable marker for prognosis and treatment in the clinical setting.

MLN4924 is a potent and selective small-molecule inhibitor of NEDD8-activating enzyme, which showed antitumor effect in several types of malignant tumor types. However, the mechanism of action of MLN4924 in acute myeloid leukemia (AML) requires further investigation. Real-time fluorescent quantitative polymerase chain reaction (RT-qPCR) was conducted to detect the mRNA levels of genes. Gene expression was knocked down by short hairpin RNA (shRNA). Moreover, the protein expression was detected by Western blotting (WB) assay. The proliferation and apoptosis of AML cells were measured by Cell Counting Kit-8 (CCK8) assay and flow cytometry (FCM). In the present study, we observed that the mRNA expression levels of NEDD8, UBA3, UBE2M and RBX1 in AML patients were up-regulated compared with healthy controls, which were correlated with worse overall survival (OS) of patients. Besides, knockdown of UBA3, UBE2M and RBX1 inhibited the NEDDylation of CULs and increased the protein expression of p53 and p21 in MOLM-13 cell line. In AML cells, MLN4924 inhibited cell proliferation, promoted cell apoptosis, and induced cell cycle arrest at the G2/M phase. As revealed by experiments in vivo and in vitro, the NEDDylation of CULs was significantly inhibited and the p53 signaling pathway was activated after MLN4924 treatment. So, we concluded that NEDD8, UBA3, UBE2M and RBX1 may serve as the prognostic biomarkers and novel therapeutic targets for AML. Inhibition of the NEDDylation pathway resulted in an anti-leukemia effect by activating the p53 signaling pathway.

Enucleation is a key event in mammalian erythropoiesis responsible for the generation of enucleated reticulocytes. Although progress is being made in developing mechanistic understanding of enucleation, our understanding of mechanisms for enucleation is still incomplete. The MAPK pathway plays diverse roles in biological processes, but its role in erythropoiesis has yet to be fully defined. Analysis of RNA-sequencing data revealed that the MAPK pathway is significantly upregulated during human terminal erythroid differentiation. The MAPK pathway consists of 3 major signaling cassettes: MEK/ERK, p38, and JNK. In the present study, we show that among these 3 cassettes, only ERK was significantly upregulated in late-stage human erythroblasts. The increased expression of ERK along with its increased phosphorylation suggests a potential role for ERK activation in enucleation. To explore this hypothesis, we treated sorted populations of human orthochromatic erythroblasts with the MEK/ERK inhibitor U0126 and found that U0126 inhibited enucleation. In contrast, inhibitors of either p38 or JNK had no effect on enucleation. Mechanistically, U0126 selectively inhibited formation/accumulation of cytoplasmic vesicles and endocytosis of the transferrin receptor without affecting chromatin condensation, nuclear polarization, or enucleosome formation. Treatment with vacuolin-1 that induces vacuole formation partially rescued the blockage of enucleation by U0126. Moreover, phosphoproteomic analysis revealed that inactivation of the ERK pathway led to downregulation of the endocytic recycling pathway. Collectively, our findings uncovered a novel role of ERK activation in human erythroblast enucleation by modulating vesicle formation and have implications for understanding anemia associated with defective enucleation.