Tanshinone IIa Induces Autophagy and Apoptosis via PI3K/Akt/mTOR Axis in Acute Promyelocytic Leukemia NB4 Cells

Tanshinone IIa (TanIIa), an ingredient of Radix Salviae Miltiorrhizae, has an anticancer effect on various solid tumors with high efficiency and low toxicity. Nonetheless, the underlying role of TanIIa in acute promyelocytic leukemia (APL) remains unclear. Here, we revealed that TanIIa drastically inhibited NB4 cell viability with an IC50 value of 31.25 μmol/L. Using flow cytometry apoptosis assay, we identified that TanIIa dose-dependently exacerbated NB4 cell apoptosis. Mechanistically, TanIIa upregulated apoptotic factor levels, namely, cleaved-caspase 9, cleaved-caspase 3, and cleaved-PARP-1. Moreover, we noticed that TanIIa dose-dependently suppressed the PI3K/Akt/mTOR axis. This axis not only functions as an essential antiapoptotic modulator but also serves as a suppressant regulator of autophagy. Correspondingly, we detected the levels of autophagic marker, namely, LC3B, which were increased after the TanIIa treatment. Furthermore, the autophagy inhibitor Baf-A1 could effectively reverse the TanIIa-induced apoptosis, manifesting that TanIIa eliminated NB4 cells in an autophagy-dependent manner. In conclusion, tanshinone IIa exerts anti-APL effects through triggering autophagy and apoptosis in NB4 cells.

KDM5A suppresses PML-RARα target gene expression and APL differentiation through repressing H3K4me2

Epigenetic abnormalities are frequently involved in the initiation and progression of cancers, including acute myeloid leukemia (AML). A subtype of AML, acute promyelocytic leukemia (APL), is mainly driven by a specific oncogenic fusion event of promyelocytic leukemia–RA receptor fusion oncoprotein (PML-RARα). PML-RARα was reported as a transcription repressor through the interaction with nuclear receptor corepressor and histone deacetylase complexes leading to the mis-suppression of its target genes and differentiation blockage. Although previous studies were mainly focused on the connection of histone acetylation, it is still largely unknown whether alternative epigenetics mechanisms are involved in APL progression. KDM5A is a demethylase of histone H3 lysine 4 di- and tri-methylations (H3K4me2/3) and a transcription corepressor. Here, we found that the loss of KDM5A led to APL NB4 cell differentiation and retarded growth. Mechanistically, through epigenomics and transcriptomics analyses, KDM5A binding was detected in 1889 genes, with the majority of the binding events at promoter regions. KDM5A suppressed the expression of 621 genes, including 42 PML-RARα target genes, primarily by controlling the H3K4me2 in the promoters and 5′ end intragenic regions. In addition, a recently reported pan-KDM5 inhibitor, CPI-455, on its own could phenocopy the differentiation effects as KDM5A loss in NB4 cells. CPI-455 treatment or KDM5A knockout could greatly sensitize NB4 cells to all-trans retinoic acid–induced differentiation. Our findings indicate that KDM5A contributed to the differentiation blockage in the APL cell line NB4, and inhibition of KDM5A could greatly potentiate NB4 differentiation.

(–)-Epigallocatechin-3-gallate induces apoptosis and differentiation in leukaemia by targeting reactive oxygen species and PIN1

Abstract(–)-Epigallocatechin-3-gallate (EGCG), the major active polyphenol extracted from green tea, has been shown to induce apoptosis and inhibit cell proliferation, cell invasion, angiogenesis and metastasis. Herein, we evaluated the in vivo effects of EGCG in acute myeloid leukaemia (AML) using an acute promyelocytic leukaemia (APL) experimental model (PML/RARα). Haematological analysis revealed that EGCG treatment reversed leucocytosis, anaemia and thrombocytopenia, and prolonged survival of PML/RARα mice. Notably, EGCG reduced leukaemia immature cells and promyelocytes in the bone marrow while increasing mature myeloid cells, possibly due to apoptosis increase and cell differentiation. The reduction of promyelocytes and neutrophils/monocytes increase detected in the peripheral blood, in addition to the increased percentage of bone marrow cells with aggregated promyelocytic leukaemia (PML) bodies staining and decreased expression of PML-RAR oncoprotein corroborates our results. In addition, EGCG increased expression of neutrophil differentiation markers such as CD11b, CD14, CD15 and CD66 in NB4 cells; and the combination of all-trans retinoic acid (ATRA) plus EGCG yield higher increase the expression of CD15 marker. These findings could be explained by a decrease of peptidyl-prolyl isomerase NIMA-interacting 1 (PIN1) expression and reactive oxygen species (ROS) increase. EGCG also decreased expression of substrate oncoproteins for PIN1 (including cyclin D1, NF-κB p65, c-MYC, and AKT) and 67 kDa laminin receptor (67LR) in the bone marrow cells. Moreover, EGCG showed inhibition of ROS production in NB4 cells in the presence of N-acetyl-L-cysteine (NAC), as well as a partial blockage of neutrophil differentiation and apoptosis, indicating that EGCG-activities involve/or are in response of oxidative stress. Furthermore, apoptosis of spleen cells was supported by increasing expression of BAD and BAX, parallel to BCL-2 and c-MYC decrease. The reduction of spleen weights of PML/RARα mice, as well as apoptosis induced by EGCG in NB4 cells in a dose-dependent manner confirms this assumption. Our results support further evaluation of EGCG in clinical trials for AML, since EGCG could represent a promising option for AML patient ineligible for current mainstay treatments.

miR-139-5p Regulates the Proliferation of Acute Promyelocytic Leukemia Cells by Targeting MNT

Acute promyelocytic leukemia (APL) patients with progressive leukocytosis are more likely to have various complications and poor outcomes. However, the regulatory roles of microRNAs in the leukocytosis of APL have not been clarified. Our study aims to evaluate the effects of miRNAs on leukocytosis during induction therapy of APL patients and explore its potential mechanisms. During induction treatment, patients with white blood cell count higher than 10 × 109/L were divided into leukocytosis group and others were nonleukocytosis group. Using microarray assays, we found that miR-139-5p was significantly downregulated in the leukocytosis group. Elevated expression of miR-139-5p inhibited the proliferation of NB4 cells by arresting the cell cycle and inducing apoptosis. We further identified that MNT was a target of miR-139-5p. miR-139-5p significantly inhibited the proliferation, invasion, and migration function of NB4 cells through targeting MNT. Strategies for regulating miR-139-5p or MNT expression might provide new therapeutic approaches for progressive leukocytosis in APL.

Effect of Cassiopea andromeda Venom on P15INK4b, P21 WAF1/CIP1, P53, DNA methyltransferase 1, and Bcl-2 Genes Expression, Apoptosis Induction, and Cell Growth Inhibition in Acute Promyelocytic Leukemia NB4 Cell line

Background: One of the acute hematologic malignancies is acute promyelocytic leukemia (APL) that resulted in translocation of chromosomes 15 and 17, t (15; 17), and cessation in the maturation of myeloid cell line, and ultimate aggregation of neoplastic promyelocytes. Regarding that appetence of using herbal and marine medicine studies is increasing, and on the other hand, the features of Cassiopea andromeda Venom remained unclear; this study was conducted to determine its effects on NB4 cells as a model for APL. Materials and Methods: In this experimental study, the cells were treated with C. andromeda Venom concentrations at different periods and times. Growth inhibition and toxic effects of C. andromeda Venom were evaluated through methyl thiazole tetrazolium salt reduction (MTT test). The flow cytometry analysis was carried out using 7AAD and Annexin V stains for evaluating this venom’s effect on apoptotic pathways. Besides, Real-Time polymerase chain reaction was performed to evaluate the relative gene expression. Results: C. andromeda Venom inhibited the growth of NB4 cells as correlated with concentration and time. Cell growth was inhibited by 49.1%, after 24 hours of treating NB4 cells with 1000µg/mL C. andromeda Venom. This venom increased the apoptotic process, which was then verified by 7AAD/AnnexinV staining. The fold change of p15INK4b, p21 WAF1/CIP1, P53, DNMT1, and Bcl-2 genes in the NB4 cell line were 144, 2.78, 1.75, 15.24, and 0.33, respectively, which meant that the expression level of p15INK4b, p21 WAF1/CIP1, P53, and DNMT1 were increased by 14400%, 278%, 175%, and 1524%, respectively and the expression of Bcl-2 was decreased by 67%. Conclusion: Considering the inhibitory property of C. andromeda Venom, the authors recommended it as a part of combinational medication for treating APL in animal trials and for other leukemias’ in vitro studies.

Honokiol induces paraptosis-like cell death of acute promyelocytic leukemia via mTOR & MAPK signaling pathways activation

Acute promyelocytic leukemia (APL) is a blood system disease caused by the accumulation of a large number of immature blood cells in bone marrow. Although the introduction of all-trans retinoic acid (ATRA) and arsenic has reached a high level of complete remission rate and 5-year disease-free survival rate, the occurrence of various adverse reactions still severely affects the quality of life of patients. As a natural product, honokiol (HNK) has the advantages of low toxicity and high efficiency, and it is a potential drug for the treatment of cancer. Since cancer cells can escape apoptotic cell death through multiple adaptive mechanisms, HNK, a drug that induces cancer cell death in a nonapoptotic way, has attracted much interest. We found that HNK reduced the viability of human APL cell line (NB4 cells) by inducing paraptosis-like cell death. The process was accompanied by excessive reactive oxygen species (ROS), mitochondrial damage, endoplasmic reticulum stress, and increased microtubule-associated protein 1 light chain 3 (LC3) processing. The inactivation of proteasome activity was the main cause of misfolded and unfolded protein accumulation in endoplasmic reticulum, such as LC3II/I and p62. This phenomenon could be alleviated by adding cycloheximide (CHX), a protein synthesis inhibitor. We found that mTOR signaling pathway participated in paraptosis-like cell death induced by HNK in an autophagy-independent process. Moreover, the mitogen-activated protein kinase (MAPK) signaling pathway induced paraptosis of NB4 cells by promoting endoplasmic reticulum stress. In summary, these findings indicate that paraptosis may be a new way to treat APL, and provide novel insights into the potential mechanism of paraptosis-like cell death.

Stimulation of Peroxisome Proliferator-Activated Receptor-Gamma (PPARγ) using Pioglitazone Decreases the Survival of Acute Promyelocytic Leukemia Cells through Up-Regulation of PTEN Expression

Background: The intertwining between cancer pathogenesis and aberrant expression of either oncogenes or tumor suppressor proteins ushered the cancer therapeutic approaches into a limitless road of modern therapies. For the nonce and among the plethora of promising anticancer agents, intense interest has focused on pioglitazone, a first in-class of Thiazolidinedione (TZD) drugs that is currently used to treat patients with diabetes. Objective: Intrigued by the overexpression of PPARγ in Acute Promylocytic Leukemia (APL), this study was designed to investigate the effects of pioglitazone in APL-derived NB4 cells. Methods: To assess the anti-leukemic effect of pioglitazone on myeloid leukemia cell lines, we used MTT and trypan blue assays. Given the higher expression level of PPARγ in NB4 cells, we then expanded our experiments on this cell line. To ascertain the molecular mechanism action of pioglitazone in APL-derived NB4 cells, we evaluated the expression levels of a large cohort of target genes responsible for the regulation of apoptosis, autophagy and cell proliferation. Afterward, to examine whether there is a correlation between PPARγ and the PI3K signaling pathway, the amount of Akt phosphorylation was evaluated using western blot analysis. Results: Our results showed that pioglitazone exerted its cytotoxic effect in wild-type PTEN-expressing NB4 cells, but not in leukemic K562 cells harboring mutant PTEN; suggesting that probably this member of TZD drugs induced its anti-leukemic effects through a PTEN-mediated manner. Moreover, we found that not only pioglitazone reduced the survival rate of NB4 through the induction of p21-mediated G1 arrest, also elevated the intracellular level of Reactive Oxygen Species (ROS) which was coupled with upregulated FOXO3a. Notably, this study proposed for the first time that the stimulation of autophagy as a result of the compensatory activation of PI3K pathway may act as a plausible mechanism through which the anti-leukemic effect of pioglitazone may be attenuated; suggestive of the application of either PI3K or autophagy inhibitors along with pioglitazone in APL. Conclusion: By suggesting a mechanistic pathway, the results of the present study shed more light on the favorable anti-leukemic effect of pioglitazone and suggest it as a promising drug that should be clinically investigated in APL patients.

Crispra: A Promising Tool for β-Thalassemia Treatment

Induction of hemoglobin γ expression is a reliable strategy to treat β-thalassemia. Gene editing using CRISPR/Cas9 technology has been widely used. However, application in vivo is limited due to the uncertainty on genomic cleavages of Cas9. In contrast, CRISPR/Cas9-based gene activation (CRISPRa) can only locate genomic locus but not interrupt sequence. Here, we use SAM system of CRISPRa to locate and activate HBG1 and HBG2, exploring the great potential of CRISPRa for β-thalassemia treatment. WWe designed 8 single-guide RNAs (sgRNAs) online and cloned into vector SAM V2, which fused dCas9 and VP64. To test the over-expression efficiency, vector containing sgRNA and MPH (fused HSF1, p65 and MS2) were transfected into 293T cell. After 72h transfection, 293T cells were collected. Q-PCR data showed that two sgRNAs were excellent on activating HBG expression with over 1000-fold increase. WTo test the activating function in hematological cell and the persistence of hemoglobin γexpression, two screened sgRNA were transfected into NB4 cells using lentivirus system. We harvested NB4 cells at different time-point (3 day, 1 week and 2 weeks), and implemented q-PCR assay. HBG expression were increased 50-hold and 1000-hold, respectively. However, the expression were reducing over time and the intrinsic mechanism is unknown. WThis study set out to increase HBG without interrupt genome using CRISPRa system. This study has found two sgRNA to activate the expression of HBG in 293T cell and NB4 cell. Further research is required to vertify the efficiency of sgRNA in hematopoietic stem cells and prolong the expression time. Figure Disclosures No relevant conflicts of interest to declare.

PML-RAR Binds to the +7kb Enhancer of CEBPE and Inhibits Its Expression

Acute promyelocytic leukemia (APL) is a unique subtype of acute myeloid leukemia (AML). The disease is identified by distinctive morphology and is distinguished by a balanced reciprocal translocation between chromosomes 15 and 17. This aberration leads to the fusion between promyelocytic leukemia (PML) gene located on chromosome 15q21, and retinoic acid receptor α (RARA) gene from chromosome 17q21, leading to the resultant chimeric onco-fusion protein PML-RARA, which is detectable in more than 95% patients and disturbs proper promyelocytic differentiation. All-trans retinoic acid (ATRA) can induce granulocytic differentiation in APL and is used to treat APL patients. Genes containing PML-RARA-targeted promoters are transcriptionally suppressed in APL and most likely constitute a major mechanism of transcriptional repression occurring in APL. A growing body of evidence points to the role of distal regulatory elements, including enhancers, in the control of gene expression. In order to understand the unique sets of enhancers that might be under the control of PML-RAR and crucial for granulocytic differentiation of NB4 cells, we analysed the enhancer landscape of control and ATRA treated NB4 cells. H3K9Ac mapping identified a repertoire of enhancers that were gained in NB4 cells treated with ATRA. Closer investigation of these enhancer elements revealed enrichment of H3K9Ac signals around major drivers of myeloid differentiation. Of note, we identified a gain in enhancer signature for a region about 7kb downstream of the CEBPE gene. Our previous studies identified a novel enhancer for CEBPE in murine hematopoietic cells, which was 6 downstream of CEBPE core promoter. It appears that the +7kb region we identified in human APL cells may be analogous to the murine enhancer. We also observed that PML-RAR binds this +7kb region and ATRA treatment of NB4 cells displaced binding of PML-RAR from the + 7kb region, suggestive of a transcriptional repressive effect of PML-RAR at such enhancer elements. To test the transcription regulating potential of this +7kb region, we used catalytically inactive Cas9 fused to Krüppel associated box (KRAB) domain (dCas9-KRAB). We designed three guide RNAs covering this regulatory region. The sgRNAs effectively repressed expression of CEBPE accompanied by lowered granulocytic differentiation of these guide RNA targeted NB4 cells after ATRA treatment. To explore transcription factor (TF) occupancy at this +7 kb region, we analysed public available ChIP-seq datasets for hematopoiesis-specific factors. Analysis revealed that the +7kb region was marked by an open chromatin signature, accompanied by binding of a majority of hematopoietic TFs around this putative regulatory element with concurrent binding of EP300. Strikingly we noticed binding of CEBPA, CEBPB and CEBPE at this regulatory element. To assess whether binding of these members of the CEBP family of TFs is functionally relevant, luciferase reporter and electrophoretic mobility shift assays (EMSA) were performed. Co expression of the CEBP TFs led to significant induction of luciferase expression, and this data was further confirmed using EMSA assays. Based on these observations, we propose that PML-RAR blocks granulocytic differentiation by occupying this +7kb enhancer of CEBPE, hinders binding of other cell type/lineage specific TFs, and blocks CEBPE expression. When cells are stimulated with ATRA, PML-RAR is displaced from the CEBPE enhancer, allowing for efficient binding of myeloid-specific TFs. This results in increased CEBPE expression, which in turn promotes efficient granulocytic differentiation. The findings from our study expands our current understanding of the mechanism of differentiation therapy, the role of onco-fusion proteins in inhibiting myeloid differentiation, and may provide new therapeutic approaches to many acute myeloid leukemias. Disclosures Ong: National University of Singapore: Other: Royalties.