NB4 Acute Promyelocytic Leukemia Cells Research Articles

Clinical and Functional Studies Reveal That TP73 Isoforms Levels Are Associated with Prognosis and RA-Resistance in Acute Promyelocytic Leukemia

Clinical and Functional Studies Reveal That TP73 Isoforms Levels Are Associated with Prognosis and RA-Resistance in Acute Promyelocytic Leukemia

Reduced SLIT2 Are Associated with Increased Cell Proliferation and Arsenic Trioxide Resistance in APL Cells

Background: Recently, the SLIT/ROBO axis became a therapeutic target for a variety of non-cerebral solid tumors because of its relevance in the regulation of angiogenesis, inflammatory cell chemotaxis, tumor cell migration and metastasis. In acute myeloid leukemia (AML), lower levels of SLIT2 expression were associated with poorer prognosis in studies that excluded patients with acute promyelocytic leukemia (APL) of the analysis (Golos et al., Arch Immunol 2019). Our group previously reported that higher SLIT2 expression was associated with improved overall survival, disease-free survival and decreased cumulative incidence of relapse. Subsequently, our in vitro functional studies indicated anti-proliferative effects exercised by exogenous SLIT2 peptide treatment in APL (Weinhauser et al., Blood 2018). Although, the expression of SLIT/ROBO was detected on leukemic cells, the fact that in healthy subjects the main source of SLIT2 derives from bone marrow stromal cells (BMSC) (Smith-Berdan et al., Cell Cycle 2012) was so far neglected in the context of hematological malignancies. Aims: Here, we expanded our data on the role of SLIT2 using lentivirally knocked down APL cells, and addressed the issue of the importance of BMSC-derived SLIT2 on APL development. Methods: NB4 and NB4R2 (RA-resistant) and primary APL cells (age, 25-47y; n=4) were transduced with shRNA-SLIT2 or shCT (control). After synchronization using double-thymidine block, transduced cells were submitted to proliferation and cell cycle assays. For the apoptosis analysis, cells were treated with ATO (1 µM) alone or in combination with RA (1 µM) for 24, 48 and 72 hours. The granulocytic differentiation in response to RA treatment alone (1 µM) or in combination with ATO (1 µM) for 48 and 72 hours, was evaluated based on the CD11b and CD11c levels. To assess the effect of SLIT2 silencing in BMSCs, HS5 and HS27A cell lines were transduced with shSLIT2 or shCT. Non-transduced NB4, NB4R2 and primary APL cells were co-cultured with engineered BMSCs and proliferation, apoptosis and granulocytic differentiation was performed in response to ATO alone or in combination with RA for 48 and 72 hours. In vivo assessment was performed using four 8-12 week-old C57/BL6PepBoy (CD45.1) lethally irradiated, transplanted with 5 x 106 hCG-PMLRARa blasts (CD45.2) transduced with shSlit2-murine and shCT. Results: Silencing SLIT2 in primary APL samples resulted in a significant increase in cell growth at the sixth day of culture compared to shCT samples (P<.001). Accordingly, enhanced staining of Ki67 was detected in NB4 and NB4R2-shSLIT2 transduced compared to shCT cells. Moreover, NB4-shSLIT2 cells exhibited lower ATO-induced apoptosis either when ATO was used alone or in combination with RA. Nevertheless, SLIT2 knockdown had no effect on NB4R2 cells suggesting that RARA moiety of PML/RARA may be important to the observed effect. Concomitantly, NB4 cell differentiation rate, was greatly increased in NB4-shSLIT2 in the presence of RA after 48 and 72h (P<.05) and 48h when RA was combined with ATO (P<.05). In vivo transplantation of primary murine shSLIT2 hCG-PMLRARa blasts, resulted in significant increase of white blood cell count during the course of APL emergence and increased extramedullary hematopoiesis in the spleen, yet with no difference in the BM at the time of sacrifice. Next, we evaluated the number of transcripts and the function of SLIT2 in AML derived MSC cells. SLIT2 transcript levels were significantly reduced in primary AML-derived BMSC compared to BMSCs obtained from healthy donors (P<.05). In vitro assays co-culturing NB4 and NB4R2 cells on engineered HS5 or HS27A BMSC cells showed no difference in drug-induced apoptosis or differentiation (P>.05). In contrast to shSLIT2 APL induced cell growth, co-culture of primary APL blast cells with transduced HS27A cells had no impact on proliferation, while the expression of the neutrophilic differentiation gene ELANE2 was significantly decreased in APL cells co-cultured with HS27A-shSLIT2.ConclusionSLIT2 is expressed in APL cells and exerts an anti-proliferative effect. On the other hand, SLIT2 produced by BMSC seemed to promote APL cellular stemness. Our results reinforce that SLIT2 function is cell type dependent and may be involved in the cross-talk between microenvironment and leukemic cells. Disclosures Figueiredo-Pontes: Novartis: Honoraria.

KDM3B suppresses APL progression by restricting chromatin accessibility and facilitating the ATRA-mediated degradation of PML/RAR\u03b1

BackgroundA hallmark of acute promyelocytic leukemia (APL) is the expression of PML/RARα fusion protein. Treatment with all-trans retinoic acid (ATRA) results in the terminal differentiation of neutrophil granulocytes. However, the underlying mechanisms remain largely unknown. Here, we identify and elucidate a novel differentiation-suppressive model of APL involving the histone demethylase KDM3B, which has been identified as a suppressor of the tumor genes involved in hematopoietic malignancies.MethodsFirst, we established a KDM3B knockdown NB4 cell model to determine the functional characteristics of KDM3B by cell proliferation assay and flow cytometry. Then, we performed ChIP-seq and ATAC-seq to search for potential relationships among KDM3B, histone modification (H3K9me1/me2) and the chromatin state. Finally, molecular biological techniques and a multi-omics analysis were used to explore the role of KDM3B in differentiation of the leukemia cells after ATRA treatment.ResultsWe found that knocking down KDM3B contributed to the growth of NB4 APL cells via the promotion of cell-cycle progression and blocked granulocytic differentiation. Through global and molecular approaches, we provided futher evidence that knocking down KDM3B altered the global distribution of H3K9me1/me2 and increased the chromatin accessibility. Moreover, knocking down KDM3B inhibited the ATRA-induced degradation of the PML/RARα oncoprotein.ConclusionOur study suggested that KDM3B was able to inhibit APL progression by maintaining chromatin in a compact state and facilitating the ATRA-mediated degradation of PML/RARα. Taken together, the results show that KDM3B may be an alternative target for the treatment regimens and the targeted therapy for APL by sustaining the function of PML/RARα fusion protein.

Genome-wide miRNA profiling and pivotal roles of miRs 125a-5p and 17-92 cluster in human neutrophil maturation and differentiation of acute myeloid leukemia cells.

MicroRNAs (miRNAs, miRs) are short non-coding post-transcriptional regulators of gene expression in normal physiology and disease. Acute myeloid leukemia is characterized by accumulation of malignantly transformed immature myeloid precursors, and differentiation therapy, used to overcome this differentiation blockage, has become a successful therapeutic option. The human HL-60 acute leukemia cell line serves as a cell culture model for granulocytic maturation, and dimethyl sulfoxide (DMSO) incubation leads to its differentiation towards neutrophil-like cells, as assessed by biochemical, functional and morphological parameters. DMSO-induced HL-60 cell differentiation constitutes an excellent model to examine molecular processes that turn a proliferating immortal leukemic cell line into mature non-proliferating and apoptosis-prone neutrophil-like end cells. By performing genome-wide miRNA profiling and functional assays, we have identified a signature of 86 differentially expressed canonical miRNAs (51 upregulated; 35 downregulated) during DMSO-induced granulocytic differentiation of HL-60 cells. Quantitative real-time PCR was used to validate miRNA expression. Among these differentially expressed canonical miRNAs, we found miR-125a-5p upregulation and miR-17-92 cluster downregulation acted as major regulators of granulocytic differentiation in HL-60 cells. Enforced expression of miR-125a-5p promoted granulocytic differentiation in HL-60 cells, whereas miR-17-92 ectopic expression inhibited DMSO-induced HL-60 granulocytic differentiation. Ectopic expression of miR-125a-5p also promoted granulocytic differentiation in human acute promyelocytic leukemia NB4 cells, as well as in naïve human primary CD34+-hematopoietic progenitor/stem cells. These findings provide novel molecular insights into the identification of miRNAs regulating granulocytic differentiation of human leukemia cells and normal CD34+-hematopoietic progenitor/stem cells, and may assist in the development of novel miRNA-targeted therapies for leukemia.

Ferritin nanovehicle for targeted delivery of cytochrome C to cancer cells

In this work, we have exploited the unique properties of a chimeric archaeal-human ferritin to encapsulate, deliver and release cytochrome c and induce apoptosis in a myeloid leukemia cell line. The chimeric protein combines the versatility in 24-meric assembly and cargo incorporation capability of Archaeglobus fulgidus ferritin with specific binding of human H ferritin to CD71, the “heavy duty” carrier responsible for transferrin-iron uptake. Delivery of ferritin-encapsulated cytochrome C to the Acute Promyelocytic Leukemia (APL) NB4 cell line, highly resistant to transfection by conventional methods, was successfully achieved in vitro. The effective liberation of cytochrome C within the cytosolic environment, demonstrated by double fluorescent labelling, induced apoptosis in the cancer cells.

Retinoic acid and arsenic trioxide induce lasting differentiation and demethylation of target genes in APL cells

Acute promyelocytic leukemia (APL) is characterized by arrested differentiation of promyelocytes. Patients treated with all-trans retinoic acid (ATRA) alone experience relapse, while patients treated with ATRA and arsenic trioxide (ATO) are often relapse-free. This suggests sustained changes have been elicited by the combination therapy. To understand the lasting effects of the combination therapy, we compared the effects of ATRA and ATO on NB4 and ATRA-resistant NB4-MR2 APL cells during treatment versus post treatment termination. After treatment termination, NB4 cells treated with ATRA or ATO reverted to non-differentiated cells, while combination-treated cells remained terminally differentiated. This effect was diminished in NB4-MR2 cells. This suggests combination treatment induced more permanent changes. Combination treatment induced higher expression of target genes (e.g., transglutaminase 2 and retinoic acid receptor beta), which in NB4 cells was sustained post treatment termination. To determine whether sustained epigenetic changes were responsible, we quantified the enrichment of histone modifications by chromatin immunoprecipitation, and CpG methylation by bisulfite-pyrosequencing. While ATRA and combination treatment induced similar histone acetylation enrichment, combination treatment induced greater demethylation of target genes, which was sustained. Therefore, sustained demethylation of target genes by ATRA and ATO combination treatment is associated with lasting differentiation and gene expression changes.

Indole itself and its novel derivative affect PML cells proliferation via controlling the expression of cell cycle genes

Recently the role of indole and pyran rings in carcinogenesis has been well studied. Here we studied the effects and the possible mechanisms of the action of basal indole (I3A) and its novel indole derivative (C19H15F3N2O) on inhibition of proliferation cells in acute promyelocytic leukemia NB4 cell line by examining the expression of cell cycle genes. We treated NB4 cells with concentration of C19H15F3N2O for 24-72 h. The MTT and PI/Annexin V examinations were employed for assessment of the proliferation and apoptosis of NB4 cells. Both of Cyclin D and P21 were detected by the Real-time PCR. The western blotting analysis was also performed to show the protein levels for P21. A difference was regarded significant if p-value was less than 0.05. MTT assay showed that 15.12-1000 µg/mL C19H15F3N2O caused a time and concentration-dependent inhibition of NB4 cell proliferation. Exposure to higher concentrations of C19H15F3N2O resulted in significantly increased apoptosis rate in NB4 cells. RT PCR showed that C19H15F3N2O has up-regulated the expression of P21 and down-regulated the expression of Cyclin D. Western blotting experiments also demonstrated that the P21 expression in C19H15F3N2O treated cells has significantly increased, where compared with either untreated control cells or I3A treated cells. This newly (C19H15F3N2O) was able to inhibit NB4 cells proliferation and causes apoptosis of these cells more than I3A, and these effects are probably facilitated via cell cycle arrest. C19H15F3N2O might probably be introduced as a promising organic therapeutic reagent against APL.

Autophagy is Required to Regulate Mitochondria Renewal, Cell Attachment, and All-trans–Retinoic Acid–Induced Differentiation in NB4 Acute Promyelocytic Leukemia Cells

All-trans-retinoic acid (ATRA) is a potent inducer of cellular differentiation, growth arrest, and apoptosis as well as a front-line therapy for acute promyelocytic leukemia (APL). The present study provides evidence that induction of autophagy is required for ATRA to induce differentiation of APL (NB4) cells into granulocytes. ATRA treatment causes ~12-fold increase in the number of acidic vesicular organelles and induces marked up-regulation of LC3-II, autophagy-related 5 (ATG5), and Beclin-1. Transmission electron microscopy (TEM) revealed a decrease in mitochondria and ATRA-induced differentiation. To determine the role of autophagy in the differentiation of APL, we knocked down ATG5 in NB4 cells to find that ATRA-induced differentiation is significantly inhibited during ATG5 knock down in cells, indicating the role of autophagy in differentiation of APL. Further experiments revealed restriction of autophagy during ATRA-induced differentiation and inhibition of tissue transglutaminase 2 (TG2) and phospho-focal adhesion kinase (p-FAK), which are known to have roles in differentiation and cell attachment. We examinedexpression of Beclin-1 and B-cell lymphoma-2 (Bcl-2) and levels of mechanistic target of rapamycin (mTOR) after ATRA treatment. ATRA inhibits Bcl-2, up-regulates Beclin-1 expression, and reduces induction of mTOR activation/phosphorylation in NB4 cells. Our results reveal that autophagy has roles in regulation of differentiation, mitochondria elimination, and cell attachment during ATRA-induced APL differentiation.

Hexokinase Proteins Impart Distinct Functions in Myeloid Development and Cell Death

Many key oncogenic pathways converge to adapt tumor cell metabolism to requirements of cancer cells. Aberrant proliferation that is frequently associated with cancer cells is also linked to an adjustment of metabolism in order to fuel cell growth and division. Cancer cells prefer utilizing glycolysis for energy production and providing essential building blocks for a variety of macromolecules. Hexokinases (HKs) are rate-limiting enzymes that catalyze the first and irreversible step of glycolysis, the ATP-dependent phosphorylation of glucose to glucose-6-phosphate. Four HK isoforms are expressed in mammalian cells, HK1, HK2, HK3 and HK4 (also known as glucokinase). HKs promote and sustain a concentration gradient that facilitates glucose entry, which ensures the initiation of glucose dependent pathways. In general, HKs have a cytoprotective role that was highlighted by enhanced sensitivity of cancer cells to drugs when HKs were inhibited. Previously we have reported that HK3 was transcriptionally regulated by PU.1 (SPI-1) in myeloid cells. Further, HK3 expression was significantly reduced in patient acute myeloid leukemia (AML) cells, particularly in acute promyelocytic leukemia (APL) cells expressing the PML-RARA oncofusion protein.We now report on the expression and regulatory function of HKs, particularly HK3, during myeloid differentiation and granulocyte associated cell death. First, we analyzed mRNA HK levels in human CD34+ hematopoietic progenitors cells differentiated towards granulocytes or macrophages by qPCR. Interestingly, while HK1 and HK2 levels remain stable during all stages of myeloid differentiation, HK3 mRNA levels significantly increased. The same pattern of HK mRNA expression was seen in NB4 APL and HL60 AML cell lines differentiated towards granulocytes and monocytes using all-trans retinoic acid (ATRA) and vitamin D3, respectively.To determine a specific role for HK1-3 function in myeloid cells, HK1-3 knockdowns (KD) and knockouts (KO) in NB4 and HL60 cell lines, using shRNA or gRNAs (Cas9/CRISPR technology), respectively, were generated. NB4 HK KD AML cells were tested for their differentiation upon ATRA treatment. Knockdown of HKs generally resulted in a decreased differentiation response of about 20% as assessed by the differentiation marker CD11b. We next determined energy metabolism of HK altered KD and KO cells, relative to parental cells, using a seahorse analyzer. While lowering HK1 or HK3 levels in NB4 and HL60 AML cells did not affect glycolytic capacity at steady state, HK2 inhibition significantly reduced steady state glycolytic capacity. In contrast, knocking down or knocking out HK3 resulted in a higher sensitivity to ATRA-induced cell death during differentiation, which was coupled with higher glycolytic capacity.Together, our findings suggest that HK2 has an important role in steady state metabolism of AML cells while HK3 appears to be a metabolic switch for cell survival during myeloid differentiation. DisclosuresNo relevant conflicts of interest to declare.

Elucidating the Non-Catalytic Function of Fatty Acid Synthase and Its Autophagy-Dependent Degradation in Acute Myelocytic Leukemia Differentiation Therapy

Acute myeloid leukemia (AML) is characterized by a block in myeloid differentiation and an aberrant cell proliferation of leukemia blasts. While other AML therapies cure only 30% of the patients, acute promyelocytic leukemia (APL) is successfully treated by differentiation therapy using all-trans retinoic acid (ATRA) in combination with chemotherapy or arsenic trioxide. A better understanding of the molecular mechanisms underlying ATRA therapy in APL may offer new perspectives in the treatment of additional AML subtypes.Fatty acid synthase (FASN) is the only human lipogenic enzyme able of de novo fatty acid synthesis. Its expression is very low in healthy adult tissues, whereas increased expression is commonly found in a variety of cancerous tissues. Therefore, FASN represents an attractive potential drug target.To investigate if a similar expression pattern is found in normal versus malignant myeloid cells, we determined FASN expression in fresh leukemic blast cells from untreated AML patients obtained at the Inselspital Bern (Switzerland) (n=68) and expression data available online from the Bloodspot server (n=203). In both AML cohorts, we found that FASN expression is significantly higher in AML patients than in healthy granulocytes (p<0.05). Accordingly, FASN protein levels are reduced in NB4 and HT93 APL cells upon ATRA-induced granulocyte differentiation. Additionally, silencing FASN in NB4 cells led to an acceleration of differentiation upon ATRA treatment. Surprisingly, solely inhibiting the catalytic function of FASN using C75 or Orlistat did not affect granulocyte differentiation nor FASN expression. To validate that FASN levels and not activity attenuate granulocyte differentiation, we took advantage of the green tea polyphenol epigallocatechin-3-gallate (EGCG) that reduces FASN protein expression. Indeed, similar to knocking down FASN, EGCG treatment of NB4 cells resulted in increased differentiation paralleled by decreased FASN protein levels.We and others demonstrated the importance of autophagy for successful ATRA-induced APL differentiation. Autophagy is an intracellular degradation system that ensures a dynamic recycling of cytoplasmic contents. This process is regulated by the so-called autophagy related genes or ATGs. ATG1, or ULK1, is a key autophagy gene of the initiation complex that is inhibited by mTOR-mediated phosphorylation leading to reduced autophagy activity.FASN expression correlates with mTOR activity in several solid cancers, therefore we hypothesized that FASN activates mTOR resulting in decreased autophagy in AML cells. Indeed, lowering FASN protein expression by either shRNA or EGCG resulted in increased autophagy together with a decreased mTOR-mediated phosphorylation of ULK1.Next, we aimed at understanding how FASN expression is regulated during ATRA treatment. We found a negative correlation between KLF5 and FASN expression in both AML patient cohorts (r=-0.3) and 4 potential KLF binding sites on the FASN promoter, suggesting that KLF5 inhibits FASN transcription.However, since the protein half-life of FASN is around 24 hours, transcriptional inhibition is not sufficient to fully explain the reduced protein levels upon ATRA treatment. We therefore tested if FASN protein itself is degraded via autophagy. Co-treatment of NB4 cells with ATRA and the lysosome inhibitor Bafilomycin A1 led to a dose dependent accumulation of FASN protein suggesting that FASN is an autophagy substrate during ATRA treatment. Furthermore, using mCherry-LC3B expressing NB4 cells, we were able to show FASN and LC3B co-localization by fluorescence microscopy.Finally, we asked whether inhibiting FASN expression levels would increase ATRA sensitivity in non-APL AML cells. To this end, we treated KG-1, OCI-AML2 and MOLM-13 AML cells with EGCG in combination with ATRA. Importantly, combining ATRA with EGCG resulted in increased differentiation of these non-APL cells paralleled by reduced FASN levels, decreased mTOR activity and ULK1 phosphorylation.Together, our data suggest that EGCG treatment is beneficial for differentiation therapy of non-APL AML cells by activating autophagy via degradation of FASN and thereby inactivating mTOR. Furthermore, high FASN expression in AML is likely due to low expression of its positive transcriptional regulator KLF5 and low autophagy activity. DisclosuresNo relevant conflicts of interest to declare.

The Genes for Tissue Factor F3 and Nuclear Receptor 4A Are Down-Regulated in Early Death Acute Promyelocytic Leukemia Patients

Introduction: Acute promyelocytic leukemia (APL) patients are successfully treated via differentiation therapy with all-trans retinoic acid (ATRA) and arsenic trioxide (ATO). Attempts to apply ATRA-based differentiation therapy to non-APL acute myeloid leukemia (AML) patients have not been effective so far (Johnson & Redner, 2015). Furthermore, 10-30% of the APL patients suffer an early death (ED) within 30 days, due to hemorrhages, infections, differentiation syndrome and thrombosis. Different risk factors have been identified, but the underlying mechanisms and successful treatment of ED in APL patients still have not been established (Kwaan et al., 2014, Lehmann et al., 2017). Hence, it is highly important to identify novel risk factors to understand the mechanistic processes in APL ED patients.Methods: To identify target genes, 50 bp single-read RNA sequencing (RNAseq) of 6 ED and 9 APL controls (cohort #1; n=15) and 4 ED and 5 APL controls (cohort #2; n=9) was executed. Samples were taken from patients with confirmed diagnosis of APL before beginning of treatment. Cohort #1 consisted of 15 patients: median age 60 years (range 37-73 years), 10 high-risk patients and 5 low/intermediate-risk patients according to Sanz score. Patients of cohort #1 were treated according to the AIDA2000 protocol of the Study Alliance Leukemia (SAL) study group. Cohort #2 consisted of 9 patients: median age 55 years (range 36-79 years), 4 high-risk patients and 5 low/intermediate-risk patients. Most patients of cohort 2 were treated according to the AIDA2000 (SAL) or APL0406 protocol. Gene validation was performed via RT-qPCR. PML-RARα variants were determined by Mentype AMLplexQS (Biotype). Survival and RT-qPCR analyses were performed with 64 non-APL AML patients´ samples treated within the clinical trial of the AMLSG HD98B study (Schlenk et al., 2004). In this trial, all patients received ICE (idarubicin, cytarabine, etoposide) chemotherapy. The patients were randomly assigned to ATRA or no ATRA. In our cohort, 26 patients had received ICE-ATRA, whereas 38 only ICE. Amaxa nucleofector technology was used for overexpression of PML-RARα bcr1 or bcr3 variant in U937 cells. NB4 or AML cells treated with 1 µM ATRA up to 72 h or 1-2 µM ATO up to 48 h were analyzed via flow cytometry, CellTiter-Glo viability assay, RT-qPCR or Western blot.Results: RNAseq of cohorts 1 and 2 showed F3 (Tissue Factor) and members of nuclear receptor 4A family, NR4A1/2/3, significantly downregulated in ED compared to control APL cases. GSEA analysis further identified a gene family including F3 and members of nuclear receptor 4A family, NR4A1/2/3, co-regulated upon leukotriene and thrombin treatment. Downregulation of F3 was further validated by RT-qPCR. Analysis of PML-RARα variants in APL control and ED cases (n=38, Chi-square p < 0.041) showed a significant enrichment of the short variant bcr3 in ED APL. Artificial overexpression of the short bcr3 and long bcr1 PML/RARα variant in U937 further revealed a correlation between bcr3 and downregulation of NR4A2/3. Moreover, treatment of the APL NB4 cell line with ATRA or ATO induced further downregulation of F3 but upregulation of NR4A2/3 transcripts. ATRA treatment induced the same effects on F3 and NR4A3 protein levels, while ATO led not only to a decrease of F3 but also NR4A3 protein levels.We next sought to address the role of F3 and NR4A in non-APL AML. In 5 non-APL AML cell lines high F3 transcript and protein levels were positively correlated with a better response to ATRA treatment in vitro. Consistently, analyzing samples taken from the AMLSG HD98B trial, AML patients with high F3 but also NR4A3 transcript levels treated with ICE chemotherapy showed a significantly prolonged overall survival upon additional ATRA treatment in vivo (Log-rank p < 0.008).Conclusions: Expression of F3 and NR4A1/2/3 is downregulated in APL ED and decreased expression of NR4A2/3 is associated with short PML-RARα variant bcr3, which is significantly enriched in ED APL. Since NR4A members are associated with coagulation and inflammation, they may be important F3-related factors, contributing to the bcr3 ED APL phenotype. As ATRA and ATO treatment is known to further inhibit F3 expression, alternative therapies not inhibiting F3 expression could be worthwhile in APL ED patients. Moreover, F3 and NR4A3 expression levels seem to be highly relevant as marker for the prediction of ATRA response in non-APL AML patients. [Display omitted] DisclosuresPlatzbecker:Celgene: Research Funding. Thiede:Novartis: Honoraria, Research Funding; AgenDix: Other: Ownership. Schlenk:Pfizer: Research Funding, Speakers Bureau.

Effects of EGCG on Proliferation, Cell Cycle and DAPK1 Gene Methylation of Acute Promyelocytic Leukemia NB4 Cell Line

To investigate the effects of epigallocatechin-3-gallate (EGCG) on proliferation and cell cycle of acute promyelocytic leukemia NB4 cell line and to clarify the molecular mechanism. NB4 cells were treated with 0,50,75,100 and 125µmol/L of EGCG for 24, 48, 72 and 96 h, respectively. The proliferation level of NB4 cells was measured by CCK-8 assay. The cell cycle progression of NB4 cells was assayed by flow cytometry. The mRNA expression levels of DNMT1, DNMT3a and DAPK1 were detected by RT-PCR. The methylation status of gene was tested by methylation specific PCR, and the expression level of DAPK1 protein was detected by Western blot. The proliferation and cell cycle progression of NB4 cells treated with EGCG were inhibited and showed the characteristic of time-dependent and dose-dependent manner. The expression level of DAPK1 and DNMT3a decreased in NB4 cells treated with EGCG. The expression level of DAPK increased in NB4 cells treated with EGCG, while the methylation of DAPK1 gene decreased. EGCG inhibits the proliferation and cell cycle progression of NB4 cells by inhibiting the expression of DNMT1 and DNMT3a and down-regulating the methylation status of DAPK1 gene.

Study of the mechanisms of crocetin-induced differentiation and apoptosis in human acute promyelocytic leukemia cells.

Crocetin, the major carotenoid in saffron, exhibits potent anticancer effects. However, the antileukemic effects of crocetin are still unclear, especially in primary acute promyelocytic leukemia (APL) cells. In the current study, the potential antipromyelocytic leukemia activity of crocetin and the underlying molecular mechanisms were investigated. Crocetin (100 µM), like standard anti-APL drugs, all-trans retinoic acid (ATRA, 10 µM) and As2 O 3 (arsenic trioxide, 50 µM), significantly inhibited proliferation and induced apoptosis in primary APL cells, as well as NB4 and HL60 cells. The effect was associated with the decreased expressions of prosurvival genes Akt and BCL2, the multidrug resistance (MDR) proteins, ABCB1 and ABCC1 and the inhibition of tyrosyl-DNA phosphodiesterase 1 (TDP1), while the expressions of proapoptotic genes CASP3, CASP9, and BAX/BCL2 ratio were significantly increased. In contrast, crocetin at relatively low concentration (10 µM), like ATRA (1 µM) and As 2 O 3 (0.5 µM), induced differentiation of leukemic cells toward granulocytic pattern, and increased the number of differentiated cells expressing CD11b and CD14, while the number of the immature cells expressing CD34 or CD33 was decreased. Furthermore, crocetin suppressed the expression of clinical marker promyelocytic leukemia/retinoic acid receptor-α ( PML/RARα) in NB4 and primary APL cells, and reduced the expression of histone deacetylase 1 ( HDAC1) in all leukemic cells. The results suggested that crocetin can be considered as a candidate for future preclinical and clinical trials of complementary APL treatment.

Optimization of 5-arylidene barbiturates as potent, selective, reversible LSD1 inhibitors for the treatment of acute promyelocytic leukemia

Histone lysine specific demethylase 1 (LSD1) is overexpressed in diverse hematologic disorders and recognized as a promising target for blood medicines. In this study, molecular docking-based virtual screening united with bioevaluation was utilized to identify novel skeleton of 5-arylidene barbiturate as small-molecule inhibitors of LSD1. Among the synthesized derivatives, 12a exhibited reversible and potent inhibition (IC50 = 0.41 μM) and high selectivity over the MAO-A and MAO-B. Notably, 12a strongly induced differentiation effect on acute promyelocytic leukemia NB4 cell line and distinctly escalated the methylation level on histone 3 lysine 4 (H3K4). Our findings indicate that 5-arylidene barbiturate may represent a new skeleton of LSD1 inhibitors and 12a deserve as a promising agent for the further research.

RACK1 deficiency synergizes with all-trans retinoic acid to induce apoptosis in human acute promyelocytic leukemia cells.

As a classic differentiation agent, all-trans retinoic acid (ATRA) has been widely used in the treatment of acute promyelocytic leukemia (APL). However, the clinical application of ATRA has strict limitations, for its severe side effects due to the accumulation of peripheral blood leukocytes. The scaffold protein RACK1 (Receptor for activated C kinase 1), which regulates multiple signaling pathways, has been proposed to contribute to the survival of leukemic progenitors. But it remains unclear whether it is also involved in the oncogenic growth of APL. In the present study, we demonstrate that silencing of endogenous RACK1 expression synergized with ATRA to promote the death of NB4 and HL-60 APL cells without effect on cell differentiation induced by ATRA. Interestingly, RACK1 knockdown combined with ATRA treatment mainly induces apoptosis. It is distinct to the necrotic cell death induced by idarubicin in combination with ATRA, a regimen extensively used in the clinic to prevent neutrophil accumulation. Further exploration revealed that the lysosome-autophagy pathway is likely to be responsible for the anti-apoptotic role of RACK1. Taken together, our findings indicate that RACK1 is essential in maintaining the malignant features of APL, and targeting RACK1 may have promising therapeutic implications in the treatment of APL.

Arsenic trioxide promoting ETosis in acute promyelocytic leukemia through mTOR-regulated autophagy

Despite the high efficacy and safety of arsenic trioxide (ATO) in treating acute promyelocytic leukemia (APL) and eradicating APL leukemia-initiating cells (LICs), the mechanism underlying its selective cytotoxicity remains elusive. We have recently demonstrated that APL cells undergo a novel cell death program, termed ETosis, through autophagy. However, the role of ETosis in ATO-induced APL LIC eradication remains unclear. For this study, we evaluated the effects of ATO on ETosis and the contributions of drug-induced ETosis to APL LIC eradication. In NB4 cells, ATO primarily increased ETosis at moderate concentrations (0.5–0.75 μM) and stimulated apoptosis at higher doses (1.0–2.0 μM). Furthermore, ATO induced ETosis through mammalian target of rapamycin (mTOR)-dependent autophagy, which was partially regulated by reactive oxygen species. Additionally, rapamycin-enhanced ATO-induced ETosis in NB4 cells and APL cells from newly diagnosed and relapsed patients. In contrast, rapamycin had no effect on apoptosis in these cells. We also noted that PML/RARA oncoprotein was effectively cleared with this combination. Intriguingly, activation of autophagy with rapamycin-enhanced APL LIC eradication clearance by ATO in vitro and in a xenograft APL model, while inhibition of autophagy spared clonogenic cells. Our current results show that ATO exerts antileukemic effects at least partially through ETosis and targets LICs primarily through ETosis. Addition of drugs that target the ETotic pathway could be a promising therapeutic strategy to further eradicate LICs and reduce relapse.

Overexpression of MiR-138 Inhibits Cell Growth and Induces Caspase-mediated Apoptosis in Acute Promyelocytic Leukemia Cell Line.

Dysregulated expression of miRNAs can play a vital role in pathogenesis of leukemia. The shortened telomere length, and elevated telomerase activity in acute promyelocytic leukemia cells are mainly indicative of extensive proliferative activity. This study aimed to investigate the effect of overexpression of miR-138 on telomerase activity, and cell proliferation of acute promyelocytic leukemia NB4 cells. MiR-138 was overexpressed in NB4 cells using GFP hsa-miR-138-expressing lentiviruses. hTERT mRNA and protein expression levels were assessed by qRT-PCR and western blot analysis. For evaluation of apoptosis, annexin-V staining and activation of caspases were assessed using flow cytometry and western blot analysis, respectively. Our data demonstrate that overexpression of miR-138 attenuated the hTERT mRNA and protein expression levels. In addition, cell growth was inhibited, and malignant cells underwent caspase mediated-apoptosis in response to miR-138 overexpression. These findings suggest that loss of miR-138 expression may be associated with increased telomerase activity in NB4 cells. Therefore, strategies for up-regulation of miR-138 may result in inhibition of malignant cell growth, and provide a promising therapeutic approach for acute promyelocytic leukemia.

Effects of Fifty-Hertz Electromagnetic Fields on Granulocytic Differentiation of ATRA-Treated Acute Promyelocytic Leukemia NB4 Cells

Background/Aims: Life on Earth is constantly exposed to electromagnetic fields (EMFs) and the effects induced by EMFs on biological systems have been extensively studied producing different and sometimes contradictory results. Extremely low-frequency electromagnetic fields (ELF-EMFs) have shown to play a role in regulating cell proliferation and differentiation, although how EMFs influence these processes remains unclear. Human acute promyelocytic leukemia (APL) cells are characterized by the arrest of differentiation at the promyelocytic stage due to epigenetic perturbations induced by PML/RARα fusion protein (Promyelocytic Leukemia protein - PML/Retinoic Acid Receptor alpha - RARα). Therapeutic administration of all-trans retinoic acid (ATRA) re-establishes the leukemogenic mechanism re-inducing the normal differentiation processes. Methods: We studied the effects of ELF-EMFs (50 Hz, 2 mT) on the ATRA-mediated granulocytic differentiation process of APL NB4 cells (a cell line established from the bone marrow of a patient affected by the acute promyelocytic leukemia) by monitoring cellular proliferation and morphology, nitrob lue tetrazolium (NBT) reduction and the expression of differentiation surface markers. Finally, we investigated mechanisms focusing on reactive oxygen species (ROS) generation and related molecular pathways. Results: ELF-EMF exposure decreases cellular proliferation potential and helps ATRA-treated NB4 cells to mature. Furthermore, the analysis of ROS production and the consequent extracellular signal regulated kinases (ERK1/2) phosphorylation suggest that a changed intracellular oxidative balance may influence the biological effects of ELF-EMFs. Conclusions: These results indicate that the exposure to ELF-EMF promotes ATRA-induced granulocytic differentiation of APL cells.

Dihydromyricetin sensitizes human acute myeloid leukemia cells to retinoic acid-induced myeloid differentiation by activating STAT1

The success of all-trans retinoic acid (ATRA) in differentiation therapy for patients with acute promyelocytic leukemia (APL) highly encourages researches to apply a new combination therapy based on ATRA. Therefore, research strategies to further sensitize cells to retinoids are urgently needed. In this study, we showed that Dihydromyricetin (DMY), a 2,3-dihydroflavonol compound, exhibited a strong synergy with ATRA to promote APL NB4 cell differentiation. We observed that DMY sensitized the NB4 cells to ATRA-induced cell growth inhibition, CD11b expression, NBT reduction and myeloid regulator expression. PML-RARα might not be essential for DMY-enhanced differentiation when combined with ATRA, while the enhanced differentiation was dependent on the activation of p38-STAT1 signaling pathway. Taken together, our study is the first to evaluate the synergy of DMY and ATRA in NB4 cell differentiation and to assess new opportunities for the combination of DMY and ATRA as a promising approach for future differentiation therapy.

Chloroquine exerts antitumor effects on NB4 acute promyelocytic leukemia cells and functions synergistically with arsenic trioxide.

Chloroquine (CQ) has been confirmed to exhibit antitumor effects on different types of cancer cell, but whether it exerts the same effect on acute promyelocytic leukemia (APL) cells remains to be confirmed. In the present study, the effects of various concentrations of CQ on the growth, apoptosis and cell cycle distribution of NB4 cells, as well as the potential mechanisms underlying these effects, were examined. The combined effect of CQ and arsenic trioxide (ATO) on the growth of NB4 cells was also determined. The results of the present study demonstrated that CQ treatment inhibited cell proliferation, and induced mitochondrial pathway apoptosis and S phase arrest in a dose-dependent manner by regulating apoptosis- and cell cycle-related proteins. CQ and ATO had a synergistic effect on the growth inhibition of NB4 cells, which may have been induced through the inhibition of autophagy. In conclusion, the results of the present study indicated that CQ exhibits a cytotoxic effect on NB4 cells and has a synergistic effect when combined with ATO, which thereby improves the curative effect of ATO on APL.