Alternative splicing of HOXB-AS3 underlie the promoting effect of nuclear m6A reader YTHDC1 on the self-renewal of leukemic stem cells in acute myeloid leukemia.

This research sought to elucidate the mechanism underlying the self-renewal capacity of leukemic stem cells (LSCs) to offer new insights into the treatment of acute myeloid leukemia (AML). The expression of HOXB-AS3 and YTHDC1 in the AML samples was screened and verified in THP-1 cells and LSCs. The relationship between HOXB-AS3 and YTHDC1 was determined. HOXB-AS3 and YTHDC1 were knocked down through cell transduction to examine the effect of HOXB-AS3 and YTHDC1 on LSCs isolated from THP-1 cells. Tumor formation in mice was used to verify fore experiments. HOXB-AS3 and YTHDC1 were robustly induced in AML, in correlation with adverse prognosis in patients with AML. We found YTHDC1 bound HOXB-AS3 and regulated its expression. Overexpression of YTHDC1 or HOXB-AS3 promoted the proliferation of THP-1 cells and LSCs and impaired their apoptosis, increasing the number of LSCs in the blood and bone marrow of AML mice. YTHDC1 could upregulate the expression of HOXB-AS3 spliceosome NR_033205.1 via the m6A modification of HOXB-AS3 precursor RNA. By this mechanism, YTHDC1 accelerated the self-renewal of LSCs and the subsequent AML progression. This study identifies a crucial role for YTHDC1 in the regulation of LSC self-renewal in AML and suggests a new perspective for AML treatment.

Acetylshikonin suppresses diffuse large B-Cell Lymphoma cell growth by targeting the T-lymphokine-activated killer cell-originated protein kinase signalling pathway.

Diffuse large B-cell lymphoma (DLBCL) is one of the most common causes of cancer death worldwide, and responds poorly to the existing treatments. Thus, identifying novel therapeutic targets of DLBCL is urgently needed. In this study, we found that T-lymphokine-activated killer cell-originated protein kinase (TOPK) was highly expressed in DLBCL cells and tissues. Data from the GEPIA database also indicated that TOPK was highly expressed in DLBCL tissues. The high expression levels of proteins were identified via Western blots and immunohistochemistry (IHC). TOPK knockdown inhibited cell growth and induced apoptosis of DLBCL cells with 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 H-tetrazolium (MTS) and flow cytometry. Further experiments demonstrated that acetylshikonin, a compound that targeted TOPK, could attenuate cell growth and aggravate cell apoptosis through TOPK/extracellular signal-regulated kinase (ERK)-1/2 signaling, as shown by MTS, flow cytometry and Western blots. In addition, we demonstrated that TOPK modulated the effect of acetylshikonin on cell proliferation and apoptosis in U2932 and OCI-LY8 cells using MTS, flow cytometry and Western blots. Taken together, the present study suggests that acetylshikonin suppresses the growth of DLBCL cells by attenuating TOPK signaling, and the targeted inhibition of TOPK by acetylshikonin may be a promising approach for the treatment of DLBCL.

The expression of STAT3 inhibited the NF-ΚB signalling pathway and reduced inflammatory responses in mice with viral myocarditis.

BACKGROUND: The aim of this study was to investigate the mechanism of STAT3 in reducing the inflammatory responses in mice with viral myocarditis (VMC). METHODS: Induce and generate viral myocarditis by using coxsackievirus B3 (CVB3) infected cardiomyocyte-specific STAT3 conditional knockout (STAT3cKO) mice and BALB/c mice. Use RT-PCR and western blot techniques to detect the expression of related cytokines in the uninfected wild-type mice group (Control group), myocarditis wild-type mice group (Model group) and STAT3cKO group, as well as the differentiation of spleen T cells in each group. Eukaryotic expression plasmid pcDNA3-STAT3 can reduce the expression of inflammatory factors the in vitro cultured cardiomyocytes of the STAT3cKO group. RESULTS: RT-PCR showed that compared with the Control group, the expression levels of VMC-related genes (NF-κB, TNF­α, IL-1ß and IL-1) and anti-inflammation-related cytokines (IL-10 and TGF-ß) in the Model group went up (*p < 0.05, **p < 0.01, ***p < 0.001); and also compared with the Control group, the rise in the expression levels of the above VMC-related genes in the STAT3cKO group was particularly significant (***p < 0.001, ****p < 0.0001) but there was no significant difference in the expression of IL-10 and TGF-ß. After 4 weeks, a second RT-PCR showed that the expression of inflammation-related genes in the STAT3cKO group continued to be activated (***p < 0.001, ****p < 0.0001). Western blotting was performed to detect the expression of p65, a key protein of the NF-κB signalling pathway. The results showed that the p65 protein content was increased and the IL-10 protein content was decreased in the STAT3cKO group; the results of the T cell differentiation test showed that the T cell differentiation rate increased in the STAT3cKO group (**p < 0.01). Eukaryotic expression plasmid pcDNA3-STAT3 could reduce the expression of NF-κB, TNF-α, IL-1ß and IL-17 (**p < 0.01). CONCLUSION: The expression of STAT3 gene in VMC could to a certain extent inhibit the NF-κB signalling pathway and reduce the inflammatory responses of VMC.

ORP8 induces apoptosis by releasing cytochrome c from mitochondria in non­small cell lung cancer.

Non­small cell lung cancer (NSCLC) is the most common type of lung cancer, and numerous oncogenes are associated with this disease. Oxysterol­binding protein­related protein 8 (ORP8) is essential for cell growth, migration and the modulation of mitochondrial respiration and morphology. However, the underlying role of ORP8 in NSCLC remains unclear. In the present study, it was reported that the expression of ORP8 was low in NSCLC cells and tissues. The ORP8 expression levels were analyzed by immunohistochemistry (IHC), quantitative real­time PCR (qPCR) and western blot analysis. ORP8 overexpression inhibited cell growth and induced apoptosis in NSCLC cells with MTS, anchorage­independent growth and Hoechst 33342 staining assay. Further experiments demonstrated that ORP8 overexpression induced the apoptosis of NSCLC cells via the release of cytochrome c from mitochondria into the cytoplasm with western blot analysis and confocal microscopy results. In addition, qPCR analysis showed that miR­421 was upregulated in NSCLC cell lines, with the bioinformatics analysis, western blot analysis and Dual­Luciferase reporter assay, it was determined that miR­421 could target ORP8. The inhibition of cell proliferation via ORP8 overexpression was rescued by a miR­421 mimic, which aided in maintaining the proliferative potential of the cells. Overall, the present study revealed that ORP8 may be a candidate target in the prevention and treatment of NSCLC.

The interaction of tumor cells and myeloid-derived suppressor cells in chronic myelogenous leukemia.

Myeloid-derived suppressor cells (MDSCs) are considered to be a strong contributor to the immunosuppressive tumor microenvironment. In our study, the counts of MDSCs were correlated with the remission status of CML patients, especially the M-MDSCs. M-MDSCs promoted the proliferation of K562 cells or CD34+ cells from newly diagnosed CML patients, no matter in cells or mice experiments. We also established a TKI discontinuation model using the K562 cell line for examining the effect of microvesicles (MVs) derived from K562 cells before and after TKI discontinuation on MDSCs. We found a mutual promotion of proliferation of tumor cells and MDSCs. Moreover, MVs derived from K562 cells after TKI discontinuation significantly improved the proliferation of MDSCs compared with MVs from before TKI discontinuation. The bidirectional interaction results in a vicious cycle, by providing a protective niche against immune attacks. Therapeutic interventions modulating this interaction might accelerate the success of TFR.

Tumor necrosis factor α knockout impaired tumorigenesis in chronic myeloid leukemia cells partly by metabolism modification and miRNA regulation.

PURPOSE: Half of the chronic myeloid leukemia (CML) patients with sustained deep molecular response suffer from relapse after discontinuation mainly because tyrosine kinase inhibitors (TKIs) cannot eradicate leukemia stem cells (LSCs). In addition, tumor necrosis factor α (TNF-α) is highly detected in CML patients. Our aim was to explore whether TNF-α is a potential target for LSC elimination. MATERIALS AND METHODS: We applied a CRISPR/Cas9 gene editing technique, colony-forming cell assay, subcutaneous tumor models, miRNA-seq and liquid chromatography-mass spectroscopy (LC-MS) on metabonomics to explore the feasibility and mechanism of TNF-α as a new therapeutic target for CML. RESULTS: We demonstrated that TNF-α knockout remarkably decreased the proliferative, colony-forming and in vivo tumorigenesis capacities of the CML K562 cell line. The apoptosis was increased when TNF-α knockout cells were cultured with imatinib. The mechanisms involved in the abovementioned phenomena were that TNF-α knockout inhibited the citrate cycle and increased starch, sucrose, amino sugar and nucleotide sugar metabolism. In addition, differentially expressed miRNAs between TNF-α knockout and control cells were involved in the cell cycle, CML, P13K-Akt and pathways in cancer. CONCLUSION: We identified that TNF-α may serve as a new target therapy for CML and described the metabolic pathways associated with TNF-α in CML cells for the first time.

Leukemia cell-derived microvesicles induce T cell exhaustion via miRNA delivery.

T cell function in cancer patients is usually impaired due to the constitutive activation of immune checkpoint inhibitors. This state is known as 'exhaustion' and is often associated with the inefficient control of tumors or persistent infections. In this work, we investigated the role of leukemia cell-derived microvesicles (MVs) in T cell exhaustion. Following incubation with MVs from various sources, all T cell subtypes exhibited the exhaustion phonotype and impaired cytokine secretion in vitro. Mice models also showed the connection between immune checkpoint inhibitors and MV injection. Sequencing and bioinformatics analyses indicated that a number of transcription factors and microRNAs (miRNAs) were attributable to the dysregulation of pathways and exhaustion in T cells. Further work revealed that functional miR-92a-3p, miR-21-5p, miR-16-5p, miR-126 and miR-182-5p in MVs could be delivered into T cells to induce the exhaustion phenotype. SerpinB2, IL-1ß and CXCL5, which are mediators of the NF-κB pathway, were identified as the targets of the miRNAs mentioned above. We demonstrated that leukemia-derived MVs could initiate T cell exhaustion via the progressive temporal delivery of multiple exogenous miRNAs into T cells and the subsequent interaction of these miRNAs with their targets. Therefore, MVs can be expected not only to become new indicators of the T cell status in patients but also to be used as novel targets for personalized patient treatment.

The epigenetic effect of microRNA in BCR-ABL1­positive microvesicles during the transformation of normal hematopoietic transplants.

Epigenetics have been demonstrated to play a pivotal role in the progression of multiple cancers. Our previous study has demonstrated that microvesicles (MVs) derived from K562 cells could malignantly transform normal hematopoietic cells. The aim of this section was to elucidate the epigenetic effects of RNA in K562-MVs. We altered some epigenetic RNAs (miR-106a-5p, miR-106b-5p and lincPOU3F3) in K562-MVs and followed the process of transformation. Global DNA methylation and DNA methyltransferase (DNMT) levels were observed respectively. Our findings revealed that increased miR-106a/b in K562-MVs accelerated the transformation process (8.33±0.94 vs. 13.29±1.28 days; P<0.01) whereas decreased lincPOU3F3 delayed the transformation (17.83±0.29 days; P<0.05). The targets of miR-106a/b and lincPOU3F3 in the recipient cells were DNMT3a and DNMT3b. We found that lincPOU3F3 directly increased the DNMT3a/b while miR-106a/b only in part by targeting RB. However, global DNA methylation and special gene methylation was altered due to the concurrent regulation of DNMT3a and DNMT3b. Consequently, we demonstrated that tumor-derived MVs represent a notable intercellular epigenetic communication between cancer cells and recipient cells.

Decitabine for relapsed acute lymphoblastic leukemia after allogeneic hematopoietic stem cell transplantation.

Relapse after allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains a main question on treatment failure. Current strategies for management that usually include salvage chemotherapy, donor lymphocytic infusion and second transplantation. Our study assessed the efficacy of decitabine (DAC) for treating patients with acute lymphoblastic leukemia (ALL) who relapsed after allogeneic hematopoietic stem cell transplantation (allo-HSCT). We retrospectively analyzed the outcomes of 12 patients with relapsed ALL after allo-HSCT who received DAC therapy. Nine patients received DAC combined with chemotherapy and donor stem cell infusion, and 3 patients received single- agent DAC. Ten of the 12 patients achieved complete remission (CR), 1 achieved a partial remission (PR), and 1 had no response (NR) after treatment at the latest follow-up (LFU), the median survival was 11.2 months (range, 3.8-34, 7 months). The 1- and 2-year overall survival (OS) rates were 50% (6/12) and 25% (3/12), respectively. Five patients were still alive; 4 had maintained CR and 1 was alive with disease. Patients with Philadelphia chromosome-positive ALL had higher survival rate than patients with Philadelphia chromosome-negative ALL (57.1% vs. 20%). No aggravated flares of graft-versus-host disease (GVHD) were observed during DAC treatment. Therefore, DAC may be a promising therapeutic agent for ALL recurrence after allo-HSCT.

Decitabine for Relapsed Acute Lymphoblastic Leukemia after Allogeneic Hematopoietic Stem Cell Transplantation / 华中科技大学学报（医学）（英德文版）

Relapse after allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains a main question on treatment failure.Current strategies for management that usually include salvage chemotherapy,donor lymphocytic infusion and second transplantation.Our study assessed the efficacy of decitabine (DAC) for treating patients with acute lymphoblastic leukemia (ALL) who relapsed after allogeneic hematopoietic stem cell transplantation (allo-HSCT).We retrospectively analyzed the outcomes of 12 patients with relapsed ALL after allo-HSCT who received DAC therapy.Nine patients received DAC combined with chemotherapy and donor stem cell infusion,and 3 patients received single-agent DAC.Ten of the 12 patients achieved complete remission (CR),1 achieved a partial remission (PR),and 1 had no response (NR) after treatment at the latest follow-up (LFU),the median survival was 11.2 months (range,3.8-34,7 months).The 1-and 2-year overall survival (OS) rates were 50％ (6/12) and 25％ (3/12),respectively.Five patients were still alive;4 had maintained CR and 1 was alive with disease.Patients with Philadelphia chromosome-positive ALL had higher survival rate than patients with Philadelphia chromosome-negative ALL (57.1％ vs.20％).No aggravated flares of graft-versus-host disease (GVHD) were observed during DAC treatment.Therefore,DAC may be a promising therapeutic agent for ALL recurrence after allo-HSCT.