Inhibited Acute Myeloid Leukemia Cell Research Articles

Rapamycin inhibits the progression of human acute myeloid leukemia by regulating the circ_0094100/miR-217/ATP1B1 axis

Rapamycin has been reported to inhibit the progression of diverse tumor cells. However, little is known about the functions of rapamycin in acute myeloid leukemia (AML). A Cell Counting Kit-8 (CCK-8) assay was conducted to evaluate cell viability. Flow cytometry analysis was employed to analyze cell apoptosis and cell cycle process. Quantitative real-time polymerase chain reaction (qRT-PCR) assay was performed to determine the levels of circRNA_0094100 (circ_0094100) and microRNA-217 (miR-217). Western blot assay was carried out to measure the protein levels of proliferating cell nuclear antigen (PCNA), cyclin D1, B-cell lymphoma-2 (Bcl-2) and ATPase Na+/K+ transporting subunit beta 1 (ATP1B1). Dual-luciferase reporter and RNA immunoprecipitation (RIP) assays were conducted to verify the relationship between miR-217 and circ_0094100 or ATP1B1. Rapamycin treatment suppressed AML cell viability and promoted apoptosis in a dose-dependent way. Circ_0094100 was elevated in AML tissues and cells. Moreover, the circ_0094100 level was reduced in AML cells treated with rapamycin. Circ_0094100 knockdown further inhibited rapamycin-mediated AML cell viability and cell cycle and promoted cell apoptosis. Circ_0094100 silencing reduced the protein levels of PCNA, cyclin D1, and Bcl-2 in rapamycin-treated AML cells. For mechanism analysis, circ_0094100 acted as the sponge for miR-217 and miR-217 inhibition reversed circ_0094100 knockdown-mediated malignant behaviors of rapamycin-treated AML cells. Furthermore, miR-217 overexpression suppressed cell viability and cell cycle and facilitated apoptosis in rapamycin-exposed AML cells, which were abolished by increasing ATP1B1. Rapamycin inhibited AML cell viability and cell cycle process and induced apoptosis through regulation of the circ_0094100/miR-217/ATP1B1 axis.

Homoharringtonine is synergistically lethal with BCL-2 inhibitor APG-2575 in acute myeloid leukemia

BackgroundDespite advances in targeted agent development, effective treatment of acute myeloid leukemia (AML) remains a major clinical challenge. The B-cell lymphoma-2 (BCL-2) inhibitor exhibited promising clinical activity in AML, acute lymphoblastic leukemia (ALL) and diffuse large B-cell lymphoma (DLBCL) treatment. APG-2575 is a novel BCL-2 selective inhibitor, which has demonstrated anti-tumor activity in hematologic malignancies. Homoharringtonine (HHT), an alkaloid, exhibited anti-AML activity.MethodsThe synergistic effects of APG-2575 and HHT were studied in AML cell lines and primary samples. MTS was used to measure the cell viability. Annexin V/propidium iodide staining was used to measure the apoptosis rate by flow cytometry. AML cell xenografted mouse models were established to evaluate the anti-leukemic effect of BCL-2 inhibitor, HHT and their combination in vivo. Western blot was used to determine the expression of related proteins.ResultsAPG-2575 showed comparable anti-leukemic effect to the FDA-approved BCL-2 inhibitor ABT-199 in vitro and in vivo. Combined treatment of HHT with APG-2575 synergistically inhibited AML cell growth and engraftment. Mechanistically, HHT promoted degradation of myeloid cell leukemia-1 (MCL-1), which was reported to induce BCL-2 inhibitor resistant, through the PI3K/AKT/GSK3β signaling pathway.ConclusionOur results provide an effective AML treatment strategy through combination of APG-2575 and HHT, which is worthy of further clinical research.

PD-L1 regulates cell proliferation and apoptosis in acute myeloid leukemia by activating PI3K-AKT signaling pathway

As immune checkpoint inhibitors (ICIs) continue to advance, more evidence has emerged that anti-PD-1/PD-L1 immunotherapy is an effective treatment against cancers. Known as the programmed death ligand-1 (PD-L1), this co-inhibitory ligand contributes to T cell exhaustion by interacting with programmed death-1 (PD-1) receptor. However, cancer-intrinsic signaling pathways of the PD-L1 molecule are not well elucidated. Therefore, the present study aimed to evaluate the regulatory network of PD-L1 and lay the basis of successful use of anti-PD-L1 immunotherapy in acute myeloid leukemia (AML). Data for AML patients were extracted from TCGA and GTEx databases. The downstream signaling pathways of PD-L1 were identified via Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. The key PD-L1 related genes were selected by weighted gene co-expression network analysis (WGCNA), MCC algorithm and Molecular Complex Detection (MCODE). The CCK-8 assay was used to assess cell proliferation. Flow cytometry was used to determine cell apoptosis and cell cycle. Western blotting was used to identify the expression of the PI3K-AKT signaling pathway. PD-L1 was shown to be elevated in AML patients when compared with the control group, and high PD-L1 expression was associated with poor overall survival rate. The ECM-receptor interaction, as well as the PI3K-AKT signaling pathway, were important PD-L1 downstream pathways. All three analyses found eight genes (ITGA2B, ITGB3, COL6A5, COL6A6, PF4, NMU, AGTR1, F2RL3) to be significantly associated with PD-L1. Knockdown of PD-L1 inhibited AML cell proliferation, induced cell apoptosis and G2/M cell cycle arrest. Importantly, PD-L1 knockdown reduced the expression of PI3K and p-AKT, but PD-L1 overexpression increased their expression. The current study elucidates the main regulatory network and downstream targets of PD-L1 in AML, assisting in the understanding of the underlying mechanism of anti-PD-1/PD-L1 immunotherapy and paving the way for clinical application of ICIs in AML.

P490: SNORNA-DERIVED RNAS (SDRNAS) ARE IMPORTANT DRIVERS OF LEUKEMOGENESIS AND LEUKEMIC MAINTENANCE IN AML

Background: Small non-coding RNAs play important roles in leukemogenesis. The canonical function of small nucleolar RNAs (snoRNAs) is to facilitate 2’-O-methylation of rRNA via a target site-specific sequence and thus promote ribosomal biogenesis and function. Recently, we have shown that a subset of small non-coding RNAs, i.e. C/D box snoRNAs are overexpressed in acute myeloid leukemia (AML) and required for AML1/ETO-driven leukemia initiation and maintenance. Depletion of snoRNP protein components or even single snoRNAs can potently inhibit AML cell growth or decrease clonogenicity, and the levels of many snoRNAs positively correlate with leukemic stem cell frequency in AML patients. Over the past years, it has been shown that snoRNAs can be further processed into shorter snoRNA-derived RNA fragments (sdRNAs), but the expression patterns of sdRNAs in AML, as well as mechanistic and functional implications, remain widely elusive. Aims: In this study, we investigated sdRNA expression patterns, functions and mechanisms in AML and healthy hematopoiesis. Methods: We prospectively characterized snoRNA and sdRNA expression in 96 AML patient samples, as well as in healthy hematopoietic stem and progenitor cells and differentiated white blood cells by small RNA sequencing. mRNA sequencing was performed to investigate connections between snoRNAs, sdRNAs and the transcriptome. Further, we depleted AML cells of different snoRNA loci by CRISPR/Cas9-based knock-out and performed lentiviral rescue overexpression of the respective sdRNA or full-length snoRNA. Results: We discovered that more than 120 snoRNAs are further processed into sdRNA fragments in AML. Single snoRNAs often evolve into multiple sdRNA isoforms. Interestingly, sdRNA expression varied significantly between different AML patients. Correlation of sdRNA and full-length snoRNA expression with patient characteristics, clinical outcome and mutational status revealed numerous implications. Those include e.g. a distinct pattern of enriched sdRNAs in NPM1-mutated patients. Expression of several sdRNAs was associated with poor overall, event-free and relapse-free survival in AML. We identified sdRNAs that were overexpressed in AML as compared to HSCs and found characteristic expression patterns among differentiated white blood cells. Notably, ratios of sdRNAs and their host snoRNA were not stable across different samples, suggesting an active mechanism regulating sdRNA processing and thus their downstream pathways. Gene set enrichment analyses based on mRNA and sdRNA correlations suggested distinct cell type specific properties. Forced expression of several sdRNAs rescued the reduced clonogenic potential observed upon depletion of the respective host snoRNA locus. This effect was often identical or even more pronounced than the rescue with the respective snoRNA precursor. Of note, overexpression of distinct sdRNAs in AML cell lines enhanced clonogenic potential. Summary/Conclusion: SnoRNA-derived sdRNAs are a common feature of AML with characteristic expression patterns determined by mutation status and clinical features. Targeting certain sdRNAs or factors involved in sdRNA processing or function might constitute promising novel therapeutic leverage points in AML.

MiR-338-3p Plays a Significant Role in Casticin-Induced Suppression of Acute Myeloid Leukemia via Targeting PI3K/Akt Pathway.

Objective Casticin is generally used in traditional herbal medicine for its anti-inflammatory and anticarcinogenic pharmacological properties. Also, microRNAs are indispensable oncogenes or cancer suppressors being dysregulated in various diseases. In this study, we aimed to elucidate the mechanisms underlying effects of casticin on the progression of acute myeloid leukemia (AML). Methods CCK-8 and flow cytometry were utilized to measure the proliferation and apoptosis of AML cell lines, respectively, after treatment with different concentrations of casticin. The alteration of several microRNA expressions in response to casticin treatment was detected by performing qRT-PCR, and the activity of PI3K/Akt pathways was evaluated through immunoblotting. Afterwards, the potential target gene of miR-338-3p was investigated by dual-luciferase reporter assay. In order to evaluate the role of miR-338-3p in the casticin-induced cellular phenotype changes, AML cells were transfected with miR-338-3p mimics or inhibitor and then subjected to proliferation and apoptosis analysis. Finally, a mouse xenograft model system was employed to investigate the role of casticin in AML progression in vivo. Results Suppressed cellular proliferation and enhanced apoptosis were observed in HL-60 and THP-1 cells after exposure to casticin, accompanied by remarkable upregulation of the miR-338-3p expression as well as a decline in the phosphorylation of PI3K and Akt proteins. RUNX2 was identified as a direct target molecular of miR-338-3p, which might account for the findings that miR-338-3p knockdown enhanced the PI3K/Akt pathway activity, whereas the miR-338-3p overexpression inactivated this signaling pathway. In addition, the inhibition of the miR-338-3p expression attenuated severe cell apoptosis and suppressions of PI3K/Akt pathway induced by casticin. Furthermore, casticin treatment retarded tumor growth rate in mouse models, whilst elevating miR-338 expression and repressing the activity of PI3K/Akt pathway in vivo. However, miR-338-3p depletion could also abolish the phenotypic alterations caused by casticin treatment. Conclusion Casticin promotes AML cell apoptosis but inhibits AML cell proliferation in vitro and tumor growth in vivo by upregulating miR-338-3p, which targets RUNX2 and thereafter inactivates PI3K-Akt signaling pathway. Our results provide insights into the mechanisms underlying the action of casticin in the control of AML progression.

Abstract 3617: METTL16 drives leukemogenesis and maintains leukemia stem cell self-renewal via reprogramming BCAA metabolism

Abstract Background: As the most prevalent internal decorations in mammalian mRNA, N6-methyladenosine (m6A) has been reported to be involved in many physiological and pathological processes, including acute myeloid leukemia (AML). METTL3 and METTL14, the well-recognized m6A methyltransferase complex, contribute to AML. METTL16 is a recently identified m6A methyltransferase that has been reported to deposit m6A in a few targets. While, unlike METTL3/14, the biological functions of METTL16 are largely unknown. Here, we explored the function and mechanism of METTL16 in AML pathogenesis and evaluated its therapeutic potential for AML treatment. Methods: We performed CRISPR-Cas9 screen to evaluate the dependency of METTL16 in AML cells. We created METTL16 knockout (KO) cells and conditional KO mice to evaluate its role in leukemogenesis and normal hematopoiesis. We employed bone marrow transplantation (BMT), xenograft, and AML patient-derived xenograft (PDX) models to determine its role in AML development and progression. To identify the targets of METTL16, we performed m6A-seq and RNA-seq, followed m6A-qPCR, CLIP-qPCR, in vitro methyltransferase assays and RNA stability assays. To examine the effect of METTL16 on branched chain amino acid (BCAA) metabolism, we performed metabolic profiling with 13C, 15N-leucine. Results: CRISPR-Cas9 screen showed METTL16 is one of the most essential genes for the survival of AMLs. The AML cells display more robust dependency on METTL16 than METTL3/14. We found METTL16 is highly expressed in AML patients compared to healthy controls. METTL16 KO significantly inhibited AML cell proliferation, promoted cell apoptosis and myeloid differentiation in vitro, which could be totally reversed by forced expression of wild-type METTL16, but not catalytic-dead mutant. METTL16 depletion dramatically inhibited AML progression and prolonged survival of recipient mice in the BMT, xenograft and PDX models. In addition, METTL16 is highly expressed in LSCs contrast to leukemic bulk cells and METTL16 KO significantly attenuates LSC self-renewal in vitro and in vivo. By contrast, the role of METLL16 is largely spared in normal hematopoietic cells. Via integrated analysis of m6A-seq data and RNA-seq data, we identified two bona fide targets of METTL16, BCAT1 and BCAT2, which encode two critical BCAA transaminases in BCAA biosynthesis pathway. METTL16 promotes the expression of BCAT1 and BCAT2 via an m6A dependent manner. Metabolomics with 13C, 15N-leucine tracing showed that METTL16 KO results in suppressed pools of TCA cycle intermediates, some non-essential amino acids and nucleotides. Conclusion: We uncover a tumor-promoting role of METTL16 in AML and LSC self-renewal via reprogramming BCAA metabolism, in which METTL16 functions as an m6A methyltransferase to regulate expression of BCAT1 and BCAT2. Our data suggest that METTL16 is an attractive target for AML therapy. Citation Format: Li Han, Lei Dong, Keith Leung, Zhicong Zhao, Yangchan Li, Ying Qing, Jianhuang Xue, Chao Shen, Zhenhua Chen, Lei Gao, Kitty Wang, Keren Zhou, Wei Li, Brandon Tan, Zheng Zhang, Xi Qin, Rui Su, Xiaolan Deng, Jianjun Chen. METTL16 drives leukemogenesis and maintains leukemia stem cell self-renewal via reprogramming BCAA metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3617.

Bioactive peptide inhibits acute myeloid leukemia cell proliferation by downregulating ALKBH5-mediated m6A demethylation of EIF4EBP1 and MLST8 mRNA

PurposeN6-methyladenosine (m6A), the most prevalent mRNA modification, plays an essential role in tumorigenesis. Notably, increasing interest has been directed to bioactive peptides (BPs) with antitumor activities. Here, we set out to investigate the potential of the BP-regulated ALKBH5/MLST8/EIF4EBP1 axis on prevention and treatment of acute myeloid leukemia (AML).MethodsThe biological effects of BP on AML cells were detected by MTT and ApoLive-Glo™ multiplex assays. The role of BP in tumor growth was determined by a subcutaneous xenograft model. The ALKBH5/MLST8/EIF4EBP1 axis was identified as a potential BP target in AML via methylated RNA immunoprecipitation sequencing (MeRIP-seq) combined with RNA sequencing (RNA-seq). Western blot, RT-qPCR, MeRIP-qPCR, dual-luciferase reporter and RNA stability assays were performed to validate the function and mode of action of the BP-regulated ALKBH5/MLST8/EIF4EBP1 axis. The clinical relevance of the BP-regulated ALKBH5/MLST8/EIF4EBP1 axis in AML was confirmed by TCGA data analysis.ResultsWe found that BP can inhibit AML cell proliferation and promote apoptosis in vitro, and repress AML tumor growth in vivo. Mechanistically, we found that BP downregulated ALKBH5 expression, which in turn repressed m6A demethylation of MLST8 and EIF4EBP1 mRNAs. Reduction of the m6A levels of MLST8 and EIF4EBP1 facilitated MLST8 and EIF4EBP1 mRNA decay, resulting in inhibition of AML cell proliferation. Furthermore, we found that the BP-regulated ALKBH5/MLST8/EIF4EBP1 axis closely correlates with AML patient prognosis.ConclusionsOur data indicate that BP can inhibit acute myeloid leukemia cell proliferation by downregulating ALKBH5-mediated m6A demethylation of EIF4EBP1 and MLST8 mRNAs, which may have potential to prevent and treat this disease.

Circ_KCNQ5 participates in the progression of childhood acute myeloid leukemia by enhancing the expression of RAB10 via binding to miR-622

ABSTRACT Background : Acute myeloid leukemia (AML) is regarded as a haematological malignancy and seriously threatens the public’s health. Circular RNA (circRNA) is gradually confirmed to be involved in the development of AML. The purpose of this study was to disclose the role of circRNA Potassium Voltage-Gated Channel Subfamily Q Member 5 (circ_KCNQ5) in AML. Methods : Quantitative real-time PCR (qPCR) and western blot were used for expression analysis. Colony formation assay, EdU assay and MTT assay were performed to determine cell proliferation. Flow cytometry assay was conducted to determine cell apoptosis. The predicted binding relationship between miR-622 and circ_KCNQ5 or RAS oncogene family member 10 (RAB10) was verified by dual-luciferase reporter assay. Results : The expression of circ_KCNQ5 was increased in bone marrow samples of childhood AML patients and AML cell lines. The knockdown of circ_KCNQ5 largely suppressed AML cell proliferation and promoted cell apoptosis. Circ_KCNQ5 directly bound to miR-622 and inhibited miR-622 expression. The cotransfection of miR-622 inhibitor reversed the effects of circ_KCNQ5 knockdown and thus recovered cell proliferation and depleted cell apoptosis. RAB10 was a target of miR-622, and circ_KCNQ5 bound to miR-622 to increase the expression of RAB10. MiR-622 restoration inhibited AML cell proliferation and induced cell apoptosis, while RAB10 overexpression abolished these effects. Conclusion : Circ_KCNQ5 high expression was associated with childhood AML malignant development, and circ_KCNQ5 participated in AML progression by regulating the miR-622/RAB10 pathway.

The new mitochondrial uncoupler BAM15 induces ROS production for treatment of acute myeloid leukemia

Acute myeloid leukemia (AML) is a malignant proliferative disease of myeloid hematopoietic origin and cannot be treated appropriately at present. This is due to the fact that leukemia cells are not sensitive to some of the traditional chemotherapy drugs. Or some chemotherapeutic drugs are too toxic to normal cells, affecting their wide clinical application. In this study, we identified BAM15 as a novel mitochondrial uncoupling agent by screening a library of small molecule compounds that inhibit AML cell activity. BAM15 significantly inhibited proliferation and promoted apoptosis in AML cells while at the same time being less cytotoxic to normal cells. The mechanism may be related to the disturbance of the ROS production balance. In vivo investigations revealed that BAM15 effectively suppressed AML progression and prolonged the survival time of mice. In addition, we found that BAM15 can be used in combination with cytarabine to enhance its anti-cancer activity and inhibit the activity of primary cells in AML. Therefore, we identified BAM15 as a potential drug candidate for the treatment of AML.

Circular RNA circ_0040823 inhibits the proliferation of acute myeloid leukemia cells and induces apoptosis by regulating miR-516b/PTEN.

Endogenous circular RNAs (circRNAs) and microRNAs (miRNAs) have been shown to regulate the pathogenesis of acute myeloid leukemia (AML). The current study aimed to identify the role of circRNA 0040823 (circ_0040823) in AML. Microarray datasets were analyzed to identify differentially expressed circRNAs in AML patients. Peripheral blood samples were obtained from healthy volunteers and AML patients for the measurement of circ_0040823 and miR-516b levels. The overexpression or knockdown of a target gene in AML cells was achieved by the transfection with lentiviral vectors or small interfering RNAs. BALB/c nude mice were inoculated with AML cells and monitored for tumor growth. Dual-luciferase reporter assay, RNA immunoprecipitation, and RNA pull-down assay were used to determine the binding relationship between circRNA and miRNA. circ_0040823 was significantly downregulated in AML patients and leukemia cells. Overexpression of circ_0040823 inhibited AML cell proliferation, and induced apoptosis and cell cycle arrest. Upregulation of circ_0040823 also repressed the growth of xenograft tumors in vivo. circ_0040823 acted as a miR-516b sponge and regulated key cellular events in leukemia cells via downregulating miR-516b. Moreover, tumor suppressor phosphatase and tensin homolog (PTEN) was a downstream target of miR-516b. The inhibition of miR-516b impaired the proliferation capacity of leukemia cells and induced apoptosis, while PTEN deficiency attenuated these effects. This study showed that circ_0040823 inhibited proliferation and induced apoptosis of AML cells by sponging miR-516b, thereby diminishing the regulatory effect of miR-516b on PTEN. These findings identified circ_0040823/miR-516b/PTEN as a new therapeutic target for AML.

AKAP9 Upregulation Predicts Unfavorable Prognosis in Pediatric Acute Myeloid Leukemia and Promotes Stemness Properties via the Wnt/β-Catenin Pathway

BackgroundPRKA kinase anchor protein 9 (AKAP9) is a scaffold protein involved in various cellular processes, including cell adhesion, proliferation, differentiation, and apoptosis. Although the oncogenic role of AKAP9 in solid tumors is well elucidated, the functions and mechanisms of AKAP9 in acute myeloid leukemia (AML) are still not understood.MethodsWe used the gene expression omnibus (GEO) database (GSE2191) to determine the mRNA expression of AKAP9 in the bone marrow of pediatric AML and healthy patients. We further used the therapeutically available research to generate effective treatments (TARGET) database to elucidate the relationship between AKAP9 expression and clinical outcomes in pediatric patients with AML. In addition, cell proliferation, cell cycle, apoptosis, RT-PCR, and Western blotting assays were applied to reveal the functions of AKAP9 and the underlying mechanisms of AKAP9 silencing in THP1 and HL60 cell lines.ResultsAKAP9 is overexpressed in the bone marrow of pediatric AML patients as compared with that of healthy patients. High expression of AKAP9 was found to be a predictor of poor overall survival (OS) and event-free survival (EFS). Using univariate and multivariate survival analyses, we found that high AKAP9 expression is an independent predictor of a worse OS and EFS. Functionally, AKAP9 silencing significantly inhibited AML cell proliferation, and cell cycle progression and promoted apoptosis. Moreover, AKAP9 silencing significantly downregulated the expression of stemness markers and β-catenin.ConclusionAKAP9 upregulation is a predictor of unfavorable prognosis, promotes stemness, and activates the Wnt/β-catenin pathway in AML patients. AKAP9 may act as a prognostic biomarker of AML in pediatric patients and a future therapeutic target.

LINC00998 functions as a novel tumor suppressor in acute myeloid leukemia via regulating the ZFP36 ring finger protein/mammalian target of rapamycin complex 2 axis

ABSTRACT Acute myeloid leukemia (AML) is a severe hematologic malignancy that threatens human health. Long non-coding RNA (lncRNA) is emerged as a key player in human cancer. Herein, we explored the role of LINC00998 in human AML. LINC00998 was significantly decreased in human AML, which was linked to relapse and poor prognosis. Stable overexpression of LINC00998 inhibited AML cell viability, colony ability, DNA synthesis rate and increased apoptosis. LINC00998 was mainly located in the cytoplasm, in which interacted with ZFP36 ring finger protein (ZFP36), a mRNA destabilizing factor, resulting in increased decay of mammalian target of rapamycin complex 2 (mTORC2), a well-known proto-oncogene in AML. Overexpression of mTORC2 partly blocked the tumor suppressive effects of LINC00998. Importantly, LINC00998 shortened in vivo AML cell survival in xenograft tumor model. Taken together, we found that LINC00998 is a novel tumor-inhibiting lncRNA in human AML. The dysregulation of LINC00998/ZFP36/mTORC2 axis is linked to leukemogenesis and progression.

Upregulation of O-GlcNAc transferase is involved in the pathogenesis of acute myeloid leukemia.

Acute myeloid leukemia (AML) is the most common acute leukemia in adults. Patients have a low survival rate and a high recurrence rate, and AML is a highly heterogeneous disease without an effective and specific targeted therapy. Therefore, it is urgent to explore new AML markers to enable early diagnosis and find drug targets for individualized treatment. Herein, we demonstrate that O-linked-N-acetylglucosamine transferase (OGT) is significantly upregulated in AML tissues compared with normal tissues. The high level of OGT expression is significantly related to poor overall survival (OS) in AML. Inhibition of OGT can inhibit AML cell proliferation and promote AML cell apoptosis. These results suggest that OGT plays an important role in the pathogenesis of AML, and may become a potential biomarker and molecular drug target for precision therapy for AML.

MicroRNA‑93 knockdown inhibits acute myeloid leukemia cell growth via inactivating thePI3K/AKT pathway by upregulating DAB2.

Acute myeloid leukemia (AML) is associated with a poor prognosis in elderly adults and currently lacks optimal treatment strategies. MicroRNAs (miRNAs or miRs) have increasingly been reported to be associated with AML progression; however, the mechanisms of action of miR-93 in AML with the involvement of disabled 2 (DAB2) are currently unknown. In the present study, miR-93 expression was assessed in patients with AML and in AML cell lines. The association between miR-93 expression and the pathological characteristics of patients with AML was analyzed. AML cells were then transfected to knockdown or overexpress miR-93 in order to elucidate its function in AML progression. The target gene of miR-93 was assessed using a dual-luciferase reporter gene assay. The expression levels of miR-93, DAB2 and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) pathway-related proteins were measured and in vivo experiments were conducted to confirm the results. It was observed that miR-93 was highly expressed in patients with AML and in AML cells. The knockdown of miR-93 in HL-60 cells inhibited AML cell proliferation and resistance to apoptosis, while the overexpression of miR-93 in THP-1 cells led to contrasting results. Moreover, miR-93 targeted DAB2 to inactivate the PI3K/AKT pathway, and the overexpression of DAB2 reversed the effects of miR-93 on THP-1 cell growth. Tumor volume, tumor weight, and the positive expression of Ki67, survivin and p53 were increased in THP-1 cells overexpressing miR-93. On the whole, the present study demonstrates that miR-93 is highly expressed in AML cells, and that the suppression of miR-93 inhibits AML cell growth by targeting DAB2 and inhibiting the PI3K/AKT pathway.

LINC00987 knockdown inhibits the progression of acute myeloid leukemia by suppressing IGF2BP2-mediated PA2G4 expression.

This study aimed to investigate the role and potential mechanisms of LINC00987 in acute myeloid leukemia (AML) progression. The expression of LINC00987 in bone marrow specimens of AML patients and cell lines was measured by quantitative reverse transcription PCR (RT-qPCR). Small interfering RNA targeting LINC00987 (si-LINC00987) was transfected into AML cell lines HL-60 and KG-1, and the proliferation, invasion and apoptosis were detected with Cell Counting Kit-8 (CCK-8), Transwell and flow cytometry, respectively. Moreover, the binding between LINC00987 and insulin like growth factor 2 mRNA binding protein 2 (IGF2BP2) was validated with an RNA pull-down assay. Co-immunoprecipitation assay was used to verify the binding between IGF2BP2 and proliferation-associated 2G4 (PA2G4). Then rescue experiments were performed to explore the effects of LINC00987/IGF2BP2/PA2G4 axis on HL-60 and KG-1 cell functions. Additionally, HL-60 cells transfected with si-LINC00987 were injected into mice, followed by the evaluation of xenograft tumor growth. LINC00987 was upregulated in AML patient specimens and cell lines. LINC00987 knockdown inhibited proliferation and invasion and promoted apoptosis in AML cells. LINC00987 could bind with IGF2BP2 and promote its expression, and IGF2BP2 overexpression reversed the effects of LINC00987 knockdown on the proliferation, invasion and apoptosis in AML cells. Besides, IGF2BP2 could bind with PA2G4. IGF2BP2 knockdown inhibited proliferation and invasion, and promoted apoptosis in AML cells, whereas PA2G4 overexpression reversed these effects. Additionally, the LINC00987 knockdown inhibited the xenograft tumor growth of AML in vivo. Knockdown of LINC00987 inhibits AML cell proliferation and invasion, and promotes apoptosis in vitro and reduces tumor growth in vivo by suppressing IGF2BP2-mediated PA2G4 expression.

WT1 inhibits AML cell proliferation in a p53-dependent manner

ABSTRACT WT1 has been reported to function as an oncogene and a tumor suppressor in acute myeloid leukemia (AML). The molecular mechanisms have not yet been fully elucidated. Here, we report that p53, served as a tumor suppressor, plays a critical role in regulating the function of WT1 in AML. For details, we performed a meta-analysis on 1131 AML cases, showing that WT1 gene mutation and TP53 gene exhibited a mutually exclusive predisposition in AML. p53 can be recruited to the promoter region of WT1’s target genes to modulate their expression by physically interacting with WT1. The AML-derived p53 mutation (p53R248Q) can disrupt the interaction between WT1 and p53, resulting in the loss of modulation of WT1’s target genes. Furthermore, wild-type p53 maintained the anti-proliferation activity of WT1 in AML cells. In contrast, WT1 promoted AML cell proliferation in the absence of p53 (or mutated p53). In conclusion, we demonstrated a novel explanation of the controversial function of WT1 in AML. These results provided a mechanism by which WT1 inhibited AML cell proliferation in a p53-dependent manner.

Long non-coding RNA LINC01018 inhibits the progression of acute myeloid leukemia by targeting miR-499a-5p to regulate PDCD4.

Acute myeloid leukemia (AML) is a highly heterogeneous disease with a very high mortality rate. In recent years, an increasing number of studies have proven that long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) may serve as useful biomarkers in various cancer types. However, the mechanism of LINC01018 and miR-499a-5p in AML requires further investigation. The mRNA expression of LINC01018, miR-499a-5p and PDCD4 in AML tissues and cells was detected using reverse transcription-quantitative polymerase chain reaction. Cell proliferation was measured using Cell Counting kit-8 and EdU assays. Cell apoptosis was monitored via a TUNEL staining assay. Protein expression of PDCD4, Bax and Bcl-2 was measured using western blot analysis. The interaction between PDCD4 and LINC01018 or miR-499a-5p was verified by RNA pull-down, RIP and dual-luciferase reporter assays. LINC01018 and PDCD4 were downregulated in AML, while miR-499a-5p was upregulated. LINC01018-overexpression suppressed AML cell proliferation and induced AML cell apoptosis, while miR-499a-5p transfection reversed these effects. LINC01018 acted as a sponge of miR-499a-5p, and PDCD4 was demonstrated to be targeted by miR-499a-5p. Knockdown of miR-499a-5p suppressed AML cell proliferation and promoted AML cell apoptosis, but silencing PDCD4 abolished this effect. LINC01018 inhibited AML cell growth by modulating PDCD4 through suppression of miR-499a-5p, providing a feasible theoretical basis for the treatment of AML.

Identification and functional study of novel oligonucleotides: CpG Seq 13 and CpG Seq 19.

Aim: This study explored new immunoadjuvants with stronger immune activity to enhance therapeutic effects against leukemia. Materials & methods: Whole blood and bone marrow of acute myeloid leukemia (AML) patients and healthy volunteers were collected. Isolated mononuclear cells were treated with two newly designed CpG oligodeoxynucleotides, CpG sequence 13 and 19, and known CpG oligodeoxynucleotides and analyzed via flow cytometry. Results: CpG Seq 13 and 19 possess strong immune activation and enhance the proliferation, degranulationand cytotoxicity of T cells. They also inhibit AML cell proliferation. When CpG Seq 13/19 are combined with anti-OX40 antibodies, the cytotoxicity of T cells on AML cells are further enhanced. Conclusion: CpG Seq 13 and 19 are strong immune adjuvant candidates for AML treatment.

Circular RNA circCRKL inhibits the proliferation of acute myeloid leukemia cells via the miR-196a-5p/miR-196b-5p/p27 axis

ABSTRACT As a new type of non-coding RNA, the role of circular RNA (circRNA) in various diseases and tumors has received considerable attention. Studies have shown that circRNAs play an important role in the progression of acute myeloid leukemia (AML) via different mechanisms. However, the specific underlying molecular mechanism of circRNAs in the proliferation of AML cells remians unclear. This study aimed to clarify the biological role and mechanism of circCRKL in AML. The results indicated low circCRKL expression in AML cell lines and samples. Moreover, the overexpression of circCRKL inhibited the proliferation and colony-forming ability of AML cells, while its silencing promoted them. In addition, bioinformatics tools and luciferase assays revealed that circCRKL could sponge miR-196a-5p and miR-196b-5p to promote the expression of p27. Furthermore, circCRKL inhibited AML cell proliferation via the miR-196a-5p/miR-196b-5p/p27 axis, suggesting a potential new target for AML therapy.

Essential role for the p110δ isoform in phosphoinositide 3-kinase activation and cell proliferation in acute myeloid leukemia

AbstractThe phosphoinositide 3-kinase (PI3K)/Akt signaling pathway has been shown to be frequently activated in blast cells from patients with acute myeloid leukemia (AML) and to contribute to survival and proliferation of these cells. Of the 8 distinct mammalian isoforms of PI3K, it is the class I PI3Ks (p110α, p110β, p110γ, and p110δ) that are responsible for Akt activation. It is not known which PI3K isoform is critical in AML. Here we show that the p110δ isoform of PI3K is consistently expressed at a high level in blast cells from AML, in contrast to the other class I isoforms, the expression of which was very variable among patients. IC87114, a p110δ-selective inhibitor, suppressed both constitutive and Flt-3–stimulated Akt activation in blasts to the same extent as Ly294002, an inhibitor of all PI3K isoforms. Moreover, IC87114 inhibited AML cell proliferation without affecting the proliferation of normal hematopoietic progenitor cells. These observations identify p110δ as a potential therapeutic target in AML.