SIRT1 regulates the phosphorylation and degradation of P27 by deacetylating CDK2 to promote T-cell acute lymphoblastic leukemia progression.

BACKGROUND: Despite marked advances in the clinical therapies, clinical outcome of most T-cell acute lymphoblastic leukemia (T-ALL) patients remains poor, due to the high risk of relapse, even after complete remission. Previous studies suggest that the NAD-dependent deacetylase sirtuin 1 (SIRT1) has a dual role in hematologic malignancies, acting as a tumor suppressor or tumor promoter depending on the tumor type. However, little is known about the expression and functions of SIRT1 in T-ALL leukemogenesis. METHODS: Public RNA-seq data, a Notch1 driven T-ALL mouse model and γ-secretase inhibitor were used to identify SIRT1 expression in T-ALL. We knocked down SIRT1 expression with ShRNAs and assessed the impacts of SIRT1 deficiency on cell proliferation, colony formation, the cell cycle and apoptosis. Transgenic SIRT1 knockout mice were used to determine the function of SIRT1 in vivo. RT-PCR, western blot, co-immunoprecipitation and ubiquitination analyses were used to detect SIRT1, p27 and CDK2 expression and their interactions. RESULTS: SIRT1 protein expression was positively correlated with the activation of Notch1. Downregulation of SIRT1 expression suppressed the proliferation and colony formation of T-ALL cell lines, which was reversed by SIRT1 overexpression. SIRT1 silencing prolonged the lifespan of T-ALL model mice. We demonstrated that p27 was involved in the downstream mechanism of cell cycle arrest induced by silencing SIRT1. SIRT1 increased the phosphorylation of p27 on Thr187 by deacetylating CDK2 and enhanced the interaction between p27 and SKP2 leading to the degradation of p27. CONCLUSION: Our findings suggest that SIRT1 is a promising target in T-ALL and offer a mechanistic link between the upregulation of SIRT1 and downregulation of p27.

[Corrigendum] Integrated transcriptomic and epigenetic data analysis identifies aberrant expression of genes in acute myeloid leukemia with MLL­AF9 translocation.

Following the publication of this paper, the authors have realized that the final article did not indicate in the Authors' Contribution section that Fangce Wang and Zheng Li made equal contributions to this work (FW and ZL performed most of the statistical analyses and drafted the initial version of the manuscript). Therefore, the affiliations for this paper should have been written as follows (changes are highlighted in bold): FANGCE WANG1*, ZHENG LI1*, GUANGMING WANG1, XIAOXUE TIAN1, JIE ZHOU1, WENLEI YU1, ZHUOYI FAN1, LIN DONG1, JINYUAN LU1, JUN XU2, WENJUN ZHANG1 and AIBIN LIANG1. 1Department of Hematology, Tongji Hospital, Tongji University School of Medicine, Shanghai 200092; 2Medical Center for Stem Cell Engineering and Transformation, East Hospital, Tongji University School of Medicine, Shanghai 200120, P.R. China. *Contributed equally. The authors confirm that there are no further errors in the paper, and all the authors agree to this correction. The authors and the Editor apologize for any inconvenience caused. [the original article was published in Molecular Medicine Reports 21: 883­893, 2020, DOI: 10.3892/mmr.2019.10849].

HCK maintains the self-renewal of leukaemia stem cells via CDK6 in AML.

BACKGROUND: Leukaemia stem cells (LSCs) are responsible for the initiation, maintenance, and recurrence of acute myeloid leukaemia (AML), an aggressive haematological malignancy associated with drug resistance and relapse. Identifying therapeutic LSC targets is critical to curing AML. METHODS: Bioinformatics databases were used to identify therapeutic LSC targets. The conditional knockout mice were used to analyse the role of HCK in leukaemogenesis or normal haematopoiesis. Colony-forming assays, cell counting, and flow cytometry were used to detect the viability and function of leukaemia cells. RT-PCR, western blotting, and RNA sequencing were used to detect mRNA and protein expression. RESULT: HCK is expressed at higher levels in LSCs than in haematopoietic stem cells (HSCs), and high HCK levels are correlated with reduced survival time in AML patients. Knockdown of HCK leads to cell cycle arrest, which results in a dramatic decrease in the proliferation and colony formation in human AML cell lines. Moreover, HCK is required for leukemogenesis and leukaemia maintenance in vivo and in vitro. HCK is necessary for the self-renewal of LSCs during serial transplantation and limiting dilution assay. The phenotypes resulting from HCK deficiency can be rescued by CDK6 overexpression in the human cell line. RNA sequencing and gene expression have demonstrated that HCK may sustain cell cycle entry and maintain the self-renewal ability of LSCs through activating the ERK1/2-c-Myc-CDK6 signalling axis. In contrast, HCK deletion does not affect normal haematopoiesis or haematopoietic reconstruction in mice. CONCLUSIONS: HCK maintains the self-renewal of leukaemia stem cells via CDK6 in AML and may be an ideal therapeutic target for eradicating LSCs without influencing normal haematopoiesis.

Whole Transcriptome Data Analysis Reveals Prognostic Signature Genes for Overall Survival Prediction in Diffuse Large B Cell Lymphoma.

BACKGROUND: With the improvement of clinical treatment outcomes in diffuse large B cell lymphoma (DLBCL), the high rate of relapse in DLBCL patients is still an established barrier, as the therapeutic strategy selection based on potential targets remains unsatisfactory. Therefore, there is an urgent need in further exploration of prognostic biomarkers so as to improve the prognosis of DLBCL. METHODS: The univariable and multivariable Cox regression models were employed to screen out gene signatures for DLBCL overall survival (OS) prediction. The differential expression analysis was used to identify representative genes in high-risk and low-risk groups, respectively, where student t test and fold change were implemented. The functional difference between the high-risk and low-risk groups was identified by the gene set enrichment analysis. RESULTS: We conducted a systematic data analysis to screen the candidate genes significantly associated with OS of DLBCL in three NCBI Gene Expression Omnibus (GEO) datasets. To construct a prognostic model, five genes (CEBPA, CYP27A1, LST1, MREG, and TARP) were then screened and tested using the multivariable Cox model and the stepwise regression method. Kaplan-Meier curve confirmed the good predictive performance of this five-gene Cox model. Thereafter, the prognostic model and the expression levels of the five genes were validated by means of an independent dataset. High expression levels of these five genes were significantly associated with favorable prognosis in DLBCL, both in training and validation datasets. Additionally, further analysis revealed the independent value and superiority of this prognostic model in risk prediction. Functional enrichment analysis revealed some vital pathways responsible for unfavorable outcome and potential therapeutic targets in DLBCL. CONCLUSION: We developed a five-gene Cox model for the clinical outcome prediction of DLBCL patients. Meanwhile, potential drug selection using this model can help clinicians to improve the clinical practice for the benefit of patients.

Periostin Secreted by Carcinoma-Associated Fibroblasts Promotes Ovarian Cancer Cell Platinum Resistance Through the PI3K/Akt Signaling Pathway.

Periostin (POSTN) is a protein secreted by mesenchymal cells. Periostin is upregulated in several cancer types and overexpression is associated with poor prognosis. However, the functional role and molecular underpinnings of periostin in epithelial ovarian cancer (EOC) is unknown. In the present study, periostin was found to be significantly upregulated in EOC stroma. Functional studies revealed that periostin could decrease cisplatin (DDP)-induced apoptosis in EOC. Periostin led to DDP resistance in EOC cells, potentially through the PI3K/Akt signaling pathway. We generated periostin-overexpressing fibroblasts and found that EOC cells were resistant to DDP when co-cultured with periostin-overexpressing fibroblasts. The findings of the present study indicated that periostin secreted by cancer-associated stromal cells may be a potential therapeutic target for EOC.

Checkpoint kinase­1 inhibition and etoposide exhibit a strong synergistic anticancer effect on chronic myeloid leukemia cell line K562 by impairing homologous recombination DNA damage repair.

Leukemia, a malignant hematological disease, has poor therapeutic outcomes due to chemotherapeutic resistance. Increasing evidence has confirmed that the elevated capacity for DNA damage repair in cancer cells is a major mechanism of acquired chemotherapeutic resistance. Thus, combining chemotherapy with inhibitors of DNA damage repair pathways is potentially an ideal strategy for treating leukemia. Checkpoint kinase 1 (CHK1) is an important component of the DNA damage response (DDR) and is involved in the G2/M DNA damage checkpoint. In the present study, we demonstrated that shRNA­mediated CHK1 silencing suppressed cell proliferation and enhanced the cytotoxic effects of etoposide (VP16) in the chronic myeloid leukemia (CML) cell line K562 through the results of CCK­8, and comet assay. The results demonstrated that shRNA­induced CHK1 silencing can override G2/M arrest and impair homologous recombination (HR) repair by reducing breast cancer susceptibility gene 1 (BRCA1) expression. Cells had no time, and thus limited ability, to repair the damage and were thus more sensitive to chemotherapy after CHK1 downregulation. Second, we tested the therapeutic effect of VP16 combined with CCT245737, an orally bioavailable CHK1 inhibitor, and observed strong synergistic anticancer effects in K562 cells. Moreover, we discovered that CCT245737 significantly prevented the G2/M arrest caused by acute exposure to VP16. Interestingly, CCT245737 inhibited both BRCA1 and Rad51, the most important component of the HR repair pathway. In conclusion, these results revealed that CHK1 is potentially an ideal therapeutic target for the treatment of CML and that CCT245737 should be considered a candidate drug.

Integrated transcriptomic and epigenetic data analysis identifiesaberrant expression of genes in acute myeloid leukemia with MLL­AF9 translocation.

Rearrangement of the mixed lineage leukemia (MLL; also known as lysine methyltransferase 2A) gene is a recurrent genomic aberration in acute myeloid leukemia (AML). MLLT3, super elongation complex subunit (AF9) is one of the most common MLL fusion partners in AML. The present study aimed to explore the aberrant expression of genes associated with the MLL­AF9 translocation and identified potential new targets for the therapy of AML with MLL­AF9 translocation. The transcriptomic and epigenetic datasets were downloaded from National Center of Biotechnology Information Gene Expression Omnibus (GEO) database. Differentially expressed genes were obtained from two independent datasets (GSE68643 and GSE73457). Gene Ontology biological process and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis was performed using the Database for Annotation, Visualization and Integrated Discovery. MLL­AF9­associated chromatin immunoprecipitation sequencing (ChIP­Seq) data was analyzed and identified binding sites for MLL­AF9 and wild type MLL (MLL WT). The ChIP­Seq of histone modification data was downloaded from the GEO database, including histone 3 lysine 4 trimethylation (H3K4me3), histone 3 lysine 79 dimethylation (H3K79me2) and histone 3 lysine 27 acetylation (H3K27ac), was used for comparing histone modification marks between the MLL­AF9 leukemia cells and normal hematopoietic cells at MLL­AF9 and MLL WT binding sites. The differentially expressed genes with the same trend in H3K79me2, H3K27ac and H3K4me3 alteration were identified as potential MLL­AF9 direct target genes. Upon validation using RNA­Seq data from the Therapeutically Applicable Research to Generate Effective Treatments AML project, eight potential direct target genes of MLL­AF9 were identified and further confirmed in MLL­AF9 mouse model using reverse transcription­quantitative polymerase chain reaction. These genes may have a critical role in AML with MLL­AF9 translocation.

SIRT1 Involved in the Regulation of Alternative Splicing Affects the DNA Damage Response in Neural Stem Cells.

BACKGROUND/AIMS: Alternative splicing and DNA damage exhibit cross-regulation, with not only DNA damage inducing changes in alternative splicing, but alternative splicing itself possibly modulating the DNA damage response (DDR). Sirt1, a prominent anti-aging player, plays pivotal roles in the DDR. However, few studies have examined alternative splicing with DNA damage in neural stem cells (NSCs) and, in essence, nothing is known about whether SIRT1 regulates alternative splicing. Hence, we investigated the potential involvement of Sirt1-mediated alternative splicing in the NSC DDR. METHODS: Genome-wide alternative splicing profiling was performed upon DNA damage induction and SIRT1 deletion. RESULTS: DNA damage caused genome-wide changes in alternative splicing in adult NSCs and Sirt1 deficiency dramatically altered DDR-related alternative splicing. In particular, extensive alternative splicing changes in DDR-related processes such as cell cycle control and DNA damage repair were observed; these processes were dramatically influenced by Sirt1 deficiency. Phenotypically, Sirt1 deficiency altered the proliferation and DNA repair of adult NSCs, possibly by regulating alternative splicing. CONCLUSION: SIRT1 helps to regulate alternative splicing, which itself affects the DDR of NSCs. Our findings provide novel insight into the mechanisms underlying the DDR in stem cells.

A three­lncRNA signature for prognosis prediction of acute myeloid leukemia in patients.

Long non-coding RNAs (lncRNAs) are transcripts characterized by >200 nucleotides, without validated protein production. Previous studies have demonstrated that certain lncRNAs have a critical role in the initiation and development of acute myeloid leukemia (AML). In the present study, the subtype­specific lncRNAs in AML was identified. Following the exclusion of the subtype­specific lncRNAs, the prognostic value of lncRNAs was investigated and a three­lncRNA expression­based risk score [long intergenic non­protein coding RNA 926, family with sequence similarity 30 member A and LRRC75A antisense RNA 1 (LRRC75A­AS1)] was developed for AML patient prognosis prediction by analyzing the RNA­seq data of AML patients from Therapeutically Available Research to Generate Effective Treatments (TARGET) and The Cancer Genome Atlas (TCGA) projects. In the training set obtained from TARGET, patients were divided into poor and favorable prognosis groups by the median risk score. The prognostic effectiveness of this lncRNA risk score was confirmed in the validation set obtained from TCGA by the same cut­off. Furthermore, the lncRNA risk score was identified as an independent prognostic factor in the multivariate analysis. As further verification of the independent prognostic power of the lncRNA risk score, stratified analysis was performed by a cytogenetics risk group and revealed a consistent result. The prognostic predictive ability of the risk score was compared with the cytogenetics risk group by time­dependent receiver operating characteristic curves analysis. It was revealed that the combination of the lncRNA risk score and cytogenetics risk group provided a higher prognostic value than a single prognostic factor. The present study also performed co­expression analysis to predict the potential regulatory mechanisms of these lncRNAs in a cis/trans/competing endogenous RNA manner. The results suggested that LRRC75A­AS1 was highly associated with the target genes of transcription factors tumor protein 53 and ETS variant 6. Overall, these results highlighted the use of the three­lncRNA expression­based risk score as a potential molecular biomarker to predict the prognosis in AML patients.