Proliferation Of Acute Promyelocytic Leukemia Cells Research Articles

Phase Separation of PML/RARα Microspeckles Governs Transcriptional Dysregulation through Genomic Rewiring of BRD4 in Acute Promyelocytic Leukemia

Introduction: Abnormal nuclear morphologies are frequently observed in cancer cells, which have been implicated in biological reactions such as gene regulation and DNA damage repair. However, the nature of altered nuclear structures, particularly molecular components determining the specificity, regulatory functions and oncogenic activity of nuclear compartments, has remained largely enigmatic and awaits characterization. In acute promyelocytic leukemia (APL), the PML/RARα fusion oncoprotein, which is produced by t(15;17), has been long observed under the microscope for its ability in destroying PML nuclear bodies (NBs) and subsequently forming microspeckles. Restoration of PML NBs has been reported during the treatment with retinoic acid (RA) or arsenic trioxide (ATO). Despite being long observed, how PML/RARα-mediated microspeckles are formed and linked to APL leukemogenesis remains a mystery. To address this knowledge gap, we present a comprehensive analysis of the driving forces and constituent components of PML/RARα-assembled microspeckles and their functional implications in transcriptional dysregulation and leukemogenesis. Methods: High-resolution imaging techniques, multi-omics strategies, integrative bioinformatics analysis, and in-depth functional validation are used in this study. We employ Immunofluorescence, Fluorescence recovery after photobleaching and in vitro droplet formation assays to determine the chemical and physical properties and constituent components of PML/RARα microspeckles. Co-IP and mass spectrometry are performed to identify interactome of PML/RARα. ChIP-seq is utilized to determine the exact genomic regions where PML/RARα microspeckles occur and to evaluate the impact on chromatin occupancy of PML/RARα and its co-factors upon genetic or pharmacological perturbation. RNA-seq is performed to evaluate the transcriptional output of repression of condensates mediated co-factor activity. Functional experiments, including CCK-8 assay and Flow cytometry, are used to assess the pathological functions of PML/RARα assembled and BRD4 recruited microspeckles. Results: We uncover the biophysical mechanism of liquid-liquid phase separation (LLPS) underlying the assembly of PML/RARα microspeckles and elucidate their role in APL leukemogenesis. Our findings reveal that PML/RARα co-assembles with BRD4 to form de novo nuclear phase-separated condensates, which distinguish them from PML nuclear bodies. PML/RARα and BRD4 co-assembled condensates exhibit preferential occupancy on super-enhancers and broad-promoters, targeting genes essential for APL leukemogenesis. Mechanically, PML/RARα incorporates BRD4 into nuclear condensates, thereby facilitating its chromatin binding and redistribution. Importantly, blockage of the condensate-mediated co-activator BRD4 activity suppresses APL cell proliferation and induces apoptosis, thereby impairing PML/RARα-driven leukemogenesis. Finally, perturbation of LLPS depletes the chromatin co-occupancy of PML/RARα and BRD4 and attenuates their target gene activation, reinforcing the importance of LLPS in transcriptional dysregulation. Conclusions: In this study, we have provided a comprehensive analysis of PML/RARα-assembled microspeckles, shedding light on their driving forces, constituent components, and regulatory function. Our findings have resolved the long-standing cognitive stagnation regarding the morphological characteristics of PML/RARα microspeckles. We have demonstrated that PML/RARα forms de novo phase-separated condensates, which selectively recruit the co-activator BRD4 to gene loci with both super enhancers and broad promoters. This aberrant recruitment leads to dysregulated transcriptional programs and the development of malignant phenotypes. Importantly, our present study is the first to reveal the biophysical nature of PML/RARα-mediated microspeckles and elucidate the fundamental regulatory mechanism underlying their formation. We have also highlighted the role of PML/RARα phase separation in the pathogenesis of APL leukemogenesis. Therefore, our study provides valuable biophysical insights into the molecular basis for PML/RARα to exert its oncogenic activity.

Realgar (As4S4), a traditional Chinese medicine, induces acute promyelocytic leukemia cell death via the Bcl-2/Bax/Cyt-C/AIF signaling pathway in vitro.

Acute promyelocytic leukemia (APL) is a specific subtype of acute myelogenous leukemia (AML) characterized by the proliferation of abnormal promyelocytes. Realgar, a Chinese medicine containing arsenic, can be taken orally. Traditional Chinese medicine physicians have employed realgar to treat APL for over a thousand years. Therefore, realgar may be a promising candidate for the treatment of APL. Nevertheless, the underlying mechanism behind realgar therapy is largely unclear. The present study aimed to investigate the effect of realgar on cell death in the APL cell line (NB4) in vitro and to elucidate the underlying mechanism. In this study, after APL cells were treated with different concentrations of realgar, the cell survival rate, apoptotic assay, morphological changes, ATP levels and cell cycle arrest were assessed. The expression of Bcl-2, Bax, Cytochrome C (Cyt-C) and apoptosis-inducing factor (AIF) at the mRNA and protein levels were also measured by immunofluorescence, quantitative PCR (qPCR) and Western blotting. We found that realgar could significantly inhibit APL cell proliferation and cell death in a time- and dose-dependent manner. Realgar effectively decreased the ATP levels in APL cells. Realgar also induced APL cell cycle arrest at the S and G2/M phases. Following realgar treatment, the mRNA and protein levels of Bcl-2 were significantly downregulated, whereas the levels of Bax, Cyt-C, and AIF were significantly upregulated. In summary, realgar can induce APL cell death via the Bcl-2/Bax/Cyt-C/AIF signaling pathway, suggesting that realgar may be an effective therapeutic for APL.

Interplay between hypertriglyceridemia and acute promyelocytic leukemia mediated by the cooperation of peroxisome proliferator-activated receptor-α with the PML/RAR α fusion protein on super-enhancers.

Patients with newly diagnosed acute promyelocytic leukemia (APL) are often obese or overweight, accompanied by metabolic disorders, such as dyslipidemia. However, the link between dyslipidemia and leukemia is obscure. Here, we conducted a retrospective study containing 1,412 cases (319 newly diagnosed APL patients, 393 newly diagnosed non-APL acute myeloid leukemia patients, and 700 non-tumor controls) and found that APL patients had higher triglyceride levels than non- APL and control groups. Using clinical data, we revealed that hypertriglyceridemia served as a risk factor for early death in APL patients, and there was a positive correlation between triglyceride levels and leukocyte counts. RNA sequencing analysis of APL patients having high or normal triglyceride levels highlighted the contribution of peroxisome proliferatoractivated receptor-α (PPARα), a crucial regulator of cell metabolism and a transcription factor involved in cancer development. The genome-wide chromatin occupancy of PPARα revealed that PPARα co-existed with PML/RARα within the super-enhancer regions to promote cell proliferation. PPARα knockdown affected the expression of target genes responsible for APL proliferation, including FLT3, and functionally inhibited the proliferation of APL cells. Moreover, in vivo results in mice having high fat diet-induced high triglyceride levels supported the connection between high triglyceride levels and the leukemic burden, as well as the involvement of PPARα-mediated-FLT3 activation in the proliferation of APL cells. Our findings shed light on the association between APL proliferation and high triglyceride levels and provide a genetic link to PPARα-mediated hyperlipidemia in APL.

Safety and efficacy of Compound Huangdai Tablets combined with all-trans retinoic acid for treatment of acute promyelocytic leukemia: Clinical evidence and potential mechanisms

ObjectiveTo evaluate the safety and efficacy of Compound Huangdai Tablets (Realgar-Indigo Naturalis formula, RIF) combined with all-trans retinoic acid (ATRA) to treat acute promyelocytic leukemia (APL). MethodsThis study was registered in PROSPERO (CRD42018108118). The relevant literatures on RIF treatment of APL were systematically searched in the following databases: China National Knowledge Infrastructure, Wanfang, VIP Medical Information System, Chinese Biomedical Database, EMBASE, Cochrane Library, and PubMed. The quality of the included studies was evaluated and Review Manager 5.3 software and Stata 13.0 software were used to perform the Meta-analysis. In addition, this study used the method of network pharmacology to conduct a preliminary exploration of the mechanism of RIF on APL. ResultsThe study included 12 studies involving 775 APL patients. The Meta-analysis showed that there was no significant difference (P ＞ 0.05) between the RIF group and the arsenic trioxide (ATO) group for primary outcomes, secondary outcomes apart from liver dysfunction. The incidence of liver dysfunction (P = 0.006) in the RIF group were significantly lower than those in the ATO group. In addition, the cost of maintenance therapy in the RIF group was significantly lower (P ＜ 0.05) than the ATO group. Besides, the active ingredients in RIF mainly act on targets proteins such as ACHE, NCOA2, RXRA, and then play a role in the treatment of APL through regulating multiple molecular mechanisms, such as TP53 regulates transcription of cell cycle genes, nuclear receptor transcription pathway. ConclusionThere was no significant difference in efficacy of oral RIF combined with ATRA compared with intravenous ATO combined with ATRA for the treatment of APL. The oral RIF exposed patients to less risk, offered more convenience and had lower prices. RIF can treat APL by multi-target and multi-pathway interventions that inducing apoptosis of APL cells and inhibiting the proliferation of APL cells, and so on. Therefore, oral RIF in the treatment of APL is worthy of further research and development.

LncRNA KCNQ1OT1 activated by c-Myc promotes cell proliferation via interacting with FUS to stabilize MAP3K1 in acute promyelocytic leukemia

Uncontrolled proliferation is the hallmark of cancer cells. Previous studies mainly focused on the role of protein-coding genes in cancer cell proliferation. Emerging evidence showed that long non-coding RNAs (lncRNAs) also play critical roles in cancer cell proliferation and growth. LncRNA KCNQ1OT1 is found to contribute to carcinogenesis, but its role in acute promyelocytic leukemia (APL) is unclear. In this study, by analyzing data from Gene Expression Omnibus, The Cancer Genome Atlas database and our clinical samples, we found that KCNQ1OT1 was selectively highly expressed in APL. Functional assays demonstrated that knockdown of KCNQ1OT1 reduced APL cell proliferation and increased apoptosis. Further evidence showed that KCNQ1OT1 was mainly located in the cytoplasm of APL patient-derived NB4 cells and APL patient bone marrow samples. Mechanistically, KCNQ1OT1 bound to RNA binding protein FUS, and silencing either KCNQ1OT1 or FUS reduced the expression level and stability of MAP3K1 mRNA. Whereas KCNQ1OT1 and FUS did not affect each other. Importantly, knockdown of MAP3K1 impaired APL cell proliferation. Finally, c-Myc transactivated KCNQ1OT1 in APL cells through binding to its promoter while knockdown of c-Myc decreased KCNQ1OT1 expression. Our results not only revealed that c-Myc transactivated KCNQ1OT1 and upregulated KCNQ1OT1 promoted APL cell proliferation, but also demonstrated that KCNQ1OT1 bound to FUS to synergistically stabilize MAP3K1 mRNA, thus facilitating APL cell proliferation. This study established a previously unidentified role of KCNQ1OT1 in the development of APL, and KCNQ1OT1 may serve as a potential therapeutic target for APL.

Abstract 1017: Arsenic trioxide reduces the expression of E2F1, cyclin E, and phosphorylation of PI3K signaling molecules in acute leukemia cells

Abstract Arsenic trioxide (ATO) has been used for the treatment of acute promyelocytic leukemia (APL). Although ATO modulates cell cycle progression and apoptosis in APL cells, its exact mechanism of action remains elusive. In this research, we investigated its effects on E2F1, cyclin E, p53, pRb and PI3K signaling molecules by western blotting, immunocytochemistry and/or confocal imaging. We found that ATO inhibited the proliferation of APL cells through down-regulation of E2F1 and cyclin E expression, and stimulation of pRb. It also reduced the interaction of pRb and E2F1with binding to the E2F1 promoter, by stimulating pRb association. ATO also effected the phosphorylation of pRb at S608 and T373 residues and association of E2F1, pRb, and p53, simultaneously. However, in p53-knockdown NB4 cells, ATO did not significantly reduce E2F1 and cyclin E expression. Our findings demonstrate that ATO inhibits APL cell growth through reduced expression of E2F1, cyclin E, and stimulation of pRb. It also effected both interaction and association of E2F1, pRb and p53 by phosphorylation of pRb at T373 and S608 residues and reduced phosphorylation of PI3K signaling molecules. This novel mode of action of ATO in APL cells may be useful for designing new APL drugs. Citation Format: Paul B. Tchounwou, Sanjay Kumar. Arsenic trioxide reduces the expression of E2F1, cyclin E, and phosphorylation of PI3K signaling molecules in acute leukemia cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1017.

Arsenic trioxide reduces the expression of E2F1, cyclin E, and phosphorylation of PI3K signaling molecules in acute leukemia cells.

Arsenic trioxide (ATO) has been used for the treatment of acute promyelocytic leukemia (APL). Although ATO modulates cell cycle progression and apoptosis in APL cells, its exact mechanism of action remains elusive. In this research, we investigated its effects on E2F1, cyclin E, p53, pRb, and PI3K signaling molecules by western blotting, immunocytochemistry and/or confocal imaging. We found that ATO inhibited the proliferation of APL cells through down‐regulation of E2F1 and cyclin E expression, and stimulation of pRb. It also reduced the interaction of pRb and E2F1with binding to the E2F1 promoter, by stimulating pRb association. ATO also effected the phosphorylation of pRb at S608 and T373 residues and association of E2F1, pRb, and p53, simultaneously. However, in p53‐knockdown NB4 cells, ATO did not significantly reduce E2F1 and cyclin E expression. Our findings demonstrate that ATO inhibits APL cell growth through reduced expression of E2F1, cyclin E, and stimulation of pRb. It also effected both interaction and association of E2F1, pRb, and p53 by phosphorylation of pRb at T373 and S608 residues and reduced phosphorylation of PI3K signaling molecules. This novel mode of action of ATO in APL cells may be useful for designing new APL drugs.

Oncogenic role of lncRNA CRNDE in acute promyelocytic leukemia and NPM1-mutant acute myeloid leukemia

The PML/RARα fusion protein acts in concert with cooperative genetic events in the development of acute promyelocytic leukemia (APL). However, oncogenic long non-coding RNAs (lncRNAs) cooperating with PML/RARα remain under-explored. Here, we first identified a set of pathogenesis-related lncRNAs, aberrantly expressed in APL using RNA-seq data from a large cohort of acute myeloid leukemia (AML) patients and normal counterparts. Among the pathogenesis-related lncRNAs, one of the evolutionarily conservative lncRNAs CRNDE (Colorectal Neoplasia Differentially Expressed) drew our attention. We found that CRNDE was highly expressed in the disease state but not in the preleukemic stage of APL, suggesting that CRNDE might be a secondary event coordinating with PML/RARα to promote APL development. Functional analysis showed that CRNDE knockdown induced differentiation and inhibited proliferation of APL cells, and prolonged survival of APL mice. Further mechanistic studies showed that CRNDE elicited its oncogenic effects through binding the miR-181 family and thereby regulating NOTCH2. Finally, we found that high CRNDE expression was also significantly correlated with NPM1 mutations and contributed to the differentiation block in NPM1-mutant AML. Collectively, our findings shed light on the importance of oncogenic lncRNAs in the development of AML and provide a promising target for AML therapy.

DDX5-targeting fully human monoclonal autoantibody inhibits proliferation and promotes differentiation of acute promyelocytic leukemia cells by increasing ROS production

Acute promyelocytic leukemia (APL) therapy involves the compounds cytotoxic to both malignant tumor and normal cells. Relapsed APL is resistant to subsequent chemotherapy. Novel agents are in need to kill APL cells selectively with minimal toxicity. DDX5 has been recognized to be a novel target to suppress acute myeloid leukemia (AML). However, the role of DDX5 remains elusive in APL. Here a DDX5-targeting fully human monoclonal autoantibody named after 2F5 was prepared. It is demonstrated that 2F5 selectively inhibited APL cell proliferation without toxicity to normal neutrophil and tissues. Moreover, 2F5 was confirmed to induce G0/G1 phase arrest in APL cells, and promote APL cell differentiation combined with decreased DDX5 expression and increased reactive oxygen species (ROS) production. Knockdown of DDX5 by siRNA also inhibited proliferation, promoted cell differentiation and enhanced ROS production in APL cells. However, the ROS inhibitor reversed the effects of 2F5 on DDX5 and ROS in APL cells. Thus, we conclude that DDX5-targeting 2F5 inhibits APL cell proliferation, and promotes cell differentiation via induction of ROS. 2F5 showed the therapeutic value of fully human monoclonal autoantibody in APL, which provides a novel and valid approach for treatment of relapse/refractory APL.

MiR-218 Targeting Bmi-1 Inhibits Proliferation of Acute Promyelocytic Leukemia Cells

To explore the molecular mechanism by which miR-218 targeting Bmi-1 inhibits the proliferation of acute promyelocytic leukemia (APL) cells. APL cell line HL-60 was transfected by miR-218 and RNA-negative control sequences, respectively. The expression of miR-218 in cells was detected by real-time fluorescence quantitative PCR. The effect of transfected miR-218 on the proliferation of APL cells was detected by MTT assay. Cell apoptosis was detected by flow cytometry. The regulation effect of miR-218 on Bmi-1 expression was determined by Western blot. The correlation of miR-218 expressions with Bmi-1 was analyzed by Spearman test. The targeted relationship between miR-218 and Bmi-1 was verified by luciferase assay. MTT assay showed that the proliferation of HL-60 cells in vitro was inhibited by high expression miR-218 significantly. Flow cytometry showed that the G1 and G2 phase cells increased while the S phase cells decreased after transfected by miR-218. Western blot showed that the level of Bmi-1 protein in HL-60 cells decreased significantly after transfection of miR-218 (P＜0.05). Spearman correlation analysis showed that the mRNA level of miR-218 negatively correlated with the protein content of Bmi-1 (r=-0.326, P＜0.01). Luciferase assay indicated that Bmi-1 could targeted on miR-218 directly. miR-218 can inhibit the proliferation, metastasis and invasion of APL cells, which can be related with the down-regulated of Bmi-1.

ATPR induces acute promyelocytic leukemia cells differentiation and growth arrest by blockade of SHP2/Rho/ROCK1 pathway

Acute promyelocytic leukemia (APL) is a form of acute myeloid leukemia with a unique chromosome translocation t (15;17), commonly complicated by a complex coagulopathy. 4-Amino-2-trifuoromethyl-phenyl retinate (ATPR), a novel all-trans retinoic acid (ATRA) derivative, was synthesized by our group and known to possess obvious biological anti-tumor activities. It has previously been shown that ATPR could induce differentiation and inhibit proliferation of APL cells, although the mechanism responsible for this effect was not well understood. In this study, we demonstrated that ATPR remarkably inhibited the expression and activity of SHP2. Further experiments showed silencing SHP2 or using SHP2 inhibition (SHP099) enhanced the effect of ATPR on cell proliferation and maturation. In addition, we also demonstrated that Rho/ROCK1 might be regulated by SHP2. Using Y-27632, a ROCK inhibitor, further proved that ROCK1 played an important role in ATPR-induced differentiation and proliferation suppression. In conclusion, the results from this study revealed that ATPR induced APL cells terminal differentiation and growth arrest by blockade of SHP2/Rho/ ROCK1 pathway.

THE EFFECT OF INHIBITION OF LNCRNA MIR100HG ON THE PROLIFERATION OF HUMAN PROMYELOCYTIC LEUKEMIA CELLS

lncRNAs are one of the main classes of non-coding RNAs, responsible for RNA regulation in a variety of cellular processes, and their effect on human cancer remains largely unexplored; Abnormal expression of lncRNAs has been shown to be associated with many human diseases and cancers, such as leukemia. Acute promyelocytic leukemia (APL) is the M3 subtype of acute myeloid leukemia (AML), with the aberrant accumulation of promyelocytes. One of the lncRNAs that showed upregulation in AML is lncRNA MIR100HG, involved in different cancers. The aim of our study is to investigate the functional role of MIR100HG antisense LNA GapmeRs, on APL cells. In this experimental study, we have used an Antisense LNA GapmeRs, in order to block MIR100HG in APL Cells. HL60 (APL cell line) cells were transfected with MIR100HG antisense LNA GapmeRs and at three different time points (24, 48 and 72 h) and were investigated apoptosis, necrosis, MIR100HG and TGFβ expression. MIR100HG inhibition could reduce the viability of HL- 60 cells, through induction of apoptosis; because of the TGFβ upregulation. qRT-PCR was performed to determine the MIR100HG expression by antisense LNA GapmeRs. Our results suggest that degradation of MIR100HG could serve as a novel approach for controlling the proliferation of APL cells and therefore, can be used in translational medicine for targeted therapy in APL

Abstract 1503: Arsenic trioxide inhibits proliferation of acute leukemia cells through reduced expression of E2F1, cyclin E, and activation of retinoblastoma protein

Abstract Arsenic trioxide (ATO) is has been used successfully in the treatment of all age group of acute promyelocytic leukemia (APL) patients in both induction and consolidation therapy. It inhibits growth APL cells through various mechanism including oxidative stress, cell cycle regulation, and apoptosis. However, detail molecular mechanism ATO-induced APL cell proliferation inhibition mostly poorly known. E2F1, a transcriptional factor widely involved proliferation and differention of many types of cancer cells through providing signal to accumulation p53, cell cycle regulation, and apoptosis. We hypothesized that ATO inhibits proliferation and differentiation of APL cells through reduced expression of E2 F1, cyclin E and stimulation of retinoblastoma and AMPKα2 expressions in APL cells. To test hypothesis, we used APL cell lines and after treatment with different concentration (2,4,6, and 8μg/ml) ATO, checked expression level of E2F1, cyclin A, cyclin E, Akt, retinoblastoma (Rb D20 & H1) and AMPKα2 by western blotting, immunocytochemistry, and confocal imaging. We found that it reduced expression significantly of E2F1, cyclin E by modulating expression profile of retinoblastoma (Rb D20 & H1), Akt and AMPKα2 proteins in APL cells concentration dependent manner. Our data showed that E2F1 associated with p53 and its association may involve ATO -induced cell cycle regulation and apoptosis. On the basis of these findings, we conclude that ATO inhibits APL cells proliferation by reducing E2F1, cyclin E expression through stimulation of retinoblastoma (Rb D20 & H1), Akt , and AMPKα2 proteins. It is novel mode of action of ATO in APL cells and may help in the designing of new anti-leukemic drugs for treatment of APL patients more efficiently and quickly. Keywords: Arsenic trioxide, E2F1, cyclin E, Rb proteins, AMPKα2, and APL cells. Acknowledgements: This research was financially supported by National Institutes of Health NCRR Grant No. 5G12RR013459 and MIMHD Grant No. 8G12MD007581, through the RCMI-Center for Environmental Health at Jackson State University. Citation Format: Sanjay Kumar, Paul Tchounwou. Arsenic trioxide inhibits proliferation of acute leukemia cells through reduced expression of E2F1, cyclin E, and activation of retinoblastoma protein [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1503.

Deubiquitinase USP48 promotes ATRA-induced granulocytic differentiation of acute promyelocytic leukemia cells.

All-trans retinoic acid (ATRA) has been used for the treatment of acute promyelocytic leukemia (APL). However, its molecular mechanisms of action are unclear. Ubiquitin-specific protease48 (USP48) is a deubiquitinase enzyme that can post-translationally remove ubiquitin molecules from substrates. In the present study, the role of USP48 in ATRA-induced differentiation of APL cells was studied. The expression of USP48 decreased following ATRA treatment. Functionally, overexpression of USP48 using electroporation-mediated delivery inhibited the proliferation of APL cells and promoted ATRA-mediated differentiation. The inverse observations were made upon siRNA-mediated knockdown of USP48. Furthermore, the expression of USP48 was increased in the nucleus upon ATRA exposure for ≤24h, suggesting that USP48 was translocated into the nucleus. Interestingly, regulation of p65, a substrate of USP48, did not contribute to the downstream mechanism of ATRA-induced differentiation of APL cells. In addition, upstream mechanistic studies demonstrated that the expression of USP48 was regulated by microRNA-301a-3p. In conclusion, the present study highlights the function of USP48 in the ATRA-induced granulocytic differentiation of APL cells and provides a theoretical basis for identifying novel targets for differentiation therapy of APL.

Arsenic trioxide nanoparticles inhibit acute promyelocytic leukemia cell proliferation and induce apoptosis via PTEN/AKT signalling pathway

Background: Arsenic Trioxide (As2O3) is a FDA-approved agent for the treatment of Acute Promyelocytic Leukemia (APL). But the high-toxicity is a bottleneck of the effect of As2O3. Methods: In our previous work, we made a novel nanoparticle formulation of As2O3. The aim of the present study was to preliminary study the possible mechanisms of the antitumor effect of As2O3 nanoparticles on NB4 cells. We examined the proliferation and apoptosis of NB4 cells incubated with the As2O3 or As2O3 nanoparticles. Protein levels of p-PTEN, p-AKT, Bax, caspase-3, caspase-9 and AIF of NB4 cells after using As2O3 nanoparticles and traditional As2O3 were determined by Western blotting analysis. Results: In vitro cytotoxicity test showed that the inhibition rate and apoptosis level of NB4 cells treated with As2O3 nanoparticles was much higher than that of traditional As2O3. Moreover, As2O3 nanoparticles resulted in a more significant increase in p-PTEN expression and a greater reduction in p- Akt expression compared with traditional As2O3. Conclusions: Our findings indicated the obvious anticancer effect of As2O3 nanoparticles and demonstrate the possible mechanism of its therapeutic potential. The results provide a foundation for the future clinical studies of As2O3 nanoparticles in APL patients.

Vernodalol mediates antitumor effects in acute promyelocytic leukemia cells.

Acute promyelocytic leukemia (APL) remains a challenge to cure due to the side effects of cytotoxic chemotherapy and drug resistance. The present study demonstrated that vernodalol, an active compound isolated from Centratherum anthelminticum, suppresses APL cell proliferation and induces cell cycle arrest in the G2/M phase through the upregulation of p21 and cell division cycle 25. In addition, vernodalol induced cellular apoptosis via the mitochondrial pathway as observed by the cleavage of caspase-9 as well as the release of cytochrome c and Smac/DIABLO into the cytosol. A mechanistic study revealed that vernodalol may exert its antitumor activity through the suppression of phosphoinositide 3-kinase/protein kinase B/mechanistic target of rapamycin signaling. In conclusion, vernodalol may be developed as a potential therapeutic compound for the treatment of APL.

Inducing cell proliferative prevention in human acute promyelocytic leukemia by miR-182 inhibition through modulation of CASP9 expression.

MicroRNAs (miRNAs) are one class of endogenous non-coding RNAs that involved in post-transcriptional regulation of the gene. MiRNAs through interaction with messenger RNA (mRNA) involved in several biological processes such as cell cycle, differentiation, growth, metabolism, aging and apoptosis. MiRNAs may act as an oncogene or a tumor suppressor via up or down regulation in cancerous cells. MiR-182 located in a miR-183/-96/-182 cluster, this is the highly conserved cluster to have an important role in cancer development and tumorigenesis. Abnormal expression of miR-182 in a variety of human cancers has reported. Oncogenic features of miR-182 confirmed through negative regulation of various tumor suppressor genes. In this study, miR-182 inhibition in acute promyelocytic leukemia (APL) cell line (HL60) was performed by locked nucleic acid (LNA) anti-miR. MTT assay in three-time points 24, 48 and 72h after LNA-anti-miR-182 transfection was performed. Our study demonstrated inhibition of miR-182 can expansively decrease cell proliferation of APL cells. The Western blotting analysis presents that CASP9 expression associated with inhibition of miR-182. CASP9 protein has an important role in the mitochondrial cell death pathway as the initiator of apoptosis. These results can offer a way for inhibition of APL cells proliferates and produce translational medicine based on microgenomics and antisense therapy.

PML-Rara/Nrf2-Regulated Mir-125b Targets CEBPA and Influences Acute Promyelocytic Leukemia Cell Proliferation

Acute promyelocytic leukemia (APL) is characterized by the presence of PML-RARA fusion protein. The PML-RARA oncoprotein is known to be the initiating factor for APL development. We have previously found that miR-125b is highly expressed in APL and upregulated by PML-RARA. However, the molecular mechanisms by which PML-RARA controls miR-125b expression had been uncharacterized. In the present study, we further confirmed that expression of PML-RARA resulted in a significant increase in primary miR-125b expression, and we found that the nuclear transcription factor E2-related factor 2 (Nrf2) targeting genes (HO1) was also upregulated (Figure 1A). Consistently, HO1 and miR-125b are transcriptionally upregulated by proteasome inhibitor (MG132) which has been showed to activate Nrf2 (Figure 1B). Knockdown of Nrf2 can inhibit the effect of PML-RARA expression and MG132 (Figure 1C). Thus, these results indicated that Nrf2 may mediate PML-RARA-induced miR-125b expression. Furthermore, we demonstrated the importance of miR-125b in APL cell proliferation (Figure 1D), we showed that promoting leukemic cell grow is associated with AKT pathway and MAPK signaling (Figure 1E). Subsequent bioinformatic and RNA-seq data analysis (Figure 1F and G) identified CEBPA as a putative target of miR-125b, and we further experimentally verified using luciferase reporter constructs (Figure H). In conclusion, we identified the mechanisms responsible for the high expression and the function of miR-125b in APL, suggesting that the expression of miR-125b could be a primary oncogenic event in APL leukemogenesis.This study was supported by grants from the National Natural Science Foundation of China (No.81400102), the China Postdoctoral Science Foundation (No.2015M570751), and the Medical Scientific Research Foundation of Guangdong Province, China (A2015420). [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Identification of miR-125b targets involved in acute promyelocytic leukemia cell proliferation

Acute promyelocytic leukemia (APL) is characterized by the presence of the PML-RARα fusion protein. We have previously found that PML-RARα-regulated miR-125b is highly expressed in APL; however, the characteristics of the regulatory effects and mechanisms of miR-125b involved in APL proliferation have yet to be clarified. In this study, we demonstrate that miR-125b promotes the proliferation of APL cells with the involvement of the PI3K/Akt and MAPK signaling pathways. Furthermore, we identified BTG2, MAP3K11, RPS6KA1 and PRDM1 as putative targets of miR-125b, which we verified using luciferase reporter constructs. Moreover, we demonstrate that the expression of miR-125b targets is downregulated in leukemic cells in patients with APL. Thus, our results provide evidence that miR-125b can modulate multiple oncogenic cell proliferation pathways and may be a novel therapeutic target for APL.

Overexpression of the long non-coding RNA PVT1 is correlated with leukemic cell proliferation in acute promyelocytic leukemia

BackgroundAcute promyelocytic leukemia (APL) is associated with chromosomal translocation t(15;17), which results in the proliferation of morphologically abnormal promyelocytes. Gain of supernumerary copies of the 8q24 chromosomal region, which harbors MYC and PVT1, has been shown to be the most common secondary alteration in human APL. Increased MYC can accelerate the development of myeloid leukemia in APL. However, the role that the expression of the long non-coding RNA (lncRNA) PVT1 plays in the pathogenesis of APL remains largely unknown.FindingsIn this study, we first analyzed the lncRNA PVT1 expression level in peripheral blood cells from 28 patients with de novo APL, and significantly upregulated PVT1 was found in APL patients compared with healthy donors. We then observed significantly lower MYC and PVT1 expression during all-trans retinoic acid (ATRA)-induced differentiation and cell cycle arrest in the APL cell line. MYC knockdown in NB4 cells led to PVT1 downregulation. Moreover, PVT1 knockdown by RNA interference led to suppression of the MYC protein level, and cell proliferation was inhibited.ConclusionOur findings reveal that the lncRNA PVT1 may play an important role in the proliferation of APL cells and may be useful for future therapeutic management.Electronic supplementary materialThe online version of this article (doi:10.1186/s13045-015-0223-4) contains supplementary material, which is available to authorized users.