ATRA-Induced Activation of the Autophagy Regulator Tfeb Regulates Myeloid Differentiation in Acute Promyelocytic Leukemia

Abstract Introduction Acute promyelocytic leukemia (APL) is characterized by gene fusions involving RARα, which disrupt retinoid signaling and lead to the clonal expansion of myeloid precursors halted at the promyelocyte stage of maturation. Treatment with all-trans-retinoic acid (ATRA) can overcome this differentiation block, restore granulocytic differentiation in APL cells and improve APL patient outcome. ATRA treatment of APL cells induces autophagy, a catabolic process whereby redundant proteins and organelles are degraded using lysosomal machinery. ATRA-induced autophagy is important for successful granulocytic differentiation of APL cells through the degradation of fusion oncoproteins and executing the structural protein remodeling necessary for cellular differentiation. The molecular mechanisms linking retinoid signaling and autophagy pathways are poorly understood and are the focus of our study. By integrating available chromatin immunoprecipitation, coupled with next-generation sequencing (ChIPseq) datasets and transcriptomic approaches, we examined the transcriptional effects of ATRA treatment on autophagy-related genes during APL cell differentiation. We identified transcription factor EB (TFEB), a master transcriptional regulator of autophagy, as a retinoid target and have evaluated the consequences of TFEB knockdown on both ATRA-induced autophagy and APL cell differentiation. Methods & Results A list of 557 autophagy-related genes was generated from 3 public autophagy databases. Cluster analysis of expression of these 557 genes in the APL TCGA RNAseq dataset, suggest autophagy is associated with patient survival (Cluster 1 = 26.4 months / Cluster 2 = 60 months). We therefore examined whether the 557 autophagy related genes may be direct ATRA targets. Accessing a public ChipSeq database (GSE18886), increased RARα binding was detected within the TFEB gene in NB4 cells (APL cell line, t15:17) treated with ATRA for 24 hours. Increased RNAPolII binding and transcriptionally active histone markers were also detected, consistent with a direct transcriptionally activating retinoid effect. To test the effects of retinoid therapy on APL gene expression, we treated NB4 cells with ATRA or ethanol control for 72 hours. Isolated RNAs were sequenced using the Illumina HiSeq 2000 platform. Reads were aligned and annotated to the hg19 genome using Tophat and subsequent differential expression analysis performed using Cufflinks/Cuffdiff and Cluster/Treeview. We observed changes in autophagy gene expression upon ATRA treatment and recorded a significant >2 fold increase in expression in >50 autophagy-related genes including TFEB, p62 and DRAM1. We then carried out lentivirally-mediated shRNA knockdown of TFEB in NB4 cells and treated both knockdown and scrambled control cells with ATRA for 72 hours. Transcriptomic analysis demonstrated that TFEB knockdown blocked ATRA-induced TFEB induction, affected the regulation of key autophagy-related genes and impaired CD11b expression. These results were validated by qRT-PCR (n=6)(P<0.001). We confirmed TFEB upregulation by qRT-PCR in primary human APL samples treated with ATRA ex vivo (n=1) and in primary peripheral blood mononuclear cells isolated from tumour patients treated with ATRA as part of clinical trial NCT00195156 (n=3). We also found that TFEB was only minimally induced by ATRA in the differentiation-resistant NB4R cell line (qRT-PCR n=6), suggesting an important role for TFEB in ATRA-induced APL differentiation. We evaluated the ability of two clinically used differentiating agents - valproic acid (VPA) and arsenic trioxide (ATO), alone and in combination with ATRA, to induce TFEB expression in NB4 cells. While VPA alone induced TFEB (5 fold), combined therapy with ATRA and VPA resulted in a >20 fold induction of TFEB expression, which correlated with enhanced differentiation (n=6)(P<0.001). ATO alone did not induce TFEB nor did it enhance the induction seen with ATRA. Notably, combined therapy with ATRA and ATO did not significantly enhance differentiation over levels seen with ATRA alone. Conclusion Retinoid signaling promotes APL differentiation in part through transcriptionally activating autophagy. Novel or existing therapies, which enhance autophagy signaling, may promote retinoid-induced differentiation and circumvent resistance to retinoid therapy in acute myeloid leukemia. Disclosures Orfali: MSD: Unrestricted educational support Other; Amgen: Unrestricted educational support, Unrestricted educational support Other; BMS: Unrestricted educational support, Unrestricted educational support Other; Pfizer: Unrestricted educational support, Unrestricted educational support Other; Novartis: Unrestricted educational support Other.

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

Blood ◽

10.1182/blood-2020-136230 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 43-43

Author(s):

Pavithra Shyamsunder ◽

Shree Pooja Sridharan ◽

Pushkar Dakle ◽

Zeya Cao ◽

Vikas Madan ◽

...

Keyword(s):

Retinoic Acid ◽

Regulatory Element ◽

Promyelocytic Leukemia ◽

Luciferase Reporter ◽

Myeloid Differentiation ◽

Atra Treatment ◽

Granulocytic Differentiation ◽

Nb4 Cells ◽

Acute Myeloid

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.

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CD14 Mediates Phagocytic Activity during the Granulocytic Differentiation Process in Acute Promyelocytic Leukemia Cells

Blood ◽

10.1182/blood.v124.21.4955.4955 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4955-4955 ◽

Cited By ~ 1

Author(s):

Hui-Chi Hsu ◽

Wen-Hui Tsai ◽

Yu-Chieh Lin

Keyword(s):

Phagocytic Activity ◽

Promyelocytic Leukemia ◽

Time Dependent ◽

Apoptotic Cells ◽

Dependent Manner ◽

Atra Treatment ◽

Granulocytic Differentiation ◽

Nb4 Cells ◽

Pre Treatment

Abstract All-trans retinoic acid (ATRA) can induce acute promyelocytic leukemia (APL) cells differentiation into mature granulocytes. CD14 and Toll-like receptor 4 (TLR-4) play an important role in the phagocytic activity of macrophage, however, their role during granulopoiesis is still unclear. In this study, we determined the role of CD14/TLR-4 in the development of phagocytic activity in NB4 APL cells after induction into the process of granulocytic differentiation by ATRA. Flow cytometry analysis demonstrate that, during ATRA treatment for 6 days, the phagocytic activity of NB4 cells in engulfing either fluorescein-latex beads or idarubicin-induced apoptotic cells increased in a time-dependent manner, and the level of CD14 expression on NB4 cells was also significantly increased in a time dependent manner, though its level was only minimally expressed in ATRA-untreated NB4 cells. However, TLR-4 was constitutionally expressed in ATRA-untreated cells and its level did not changed significantly during the first 5 days of ATRA treatment. Further study demonstrates that the phagocytic activity of ATRA-NB4 cells was significantly inhibited by pre-treating cells with antibodies specific to either CD14 or TLR-4 before phagocytosis assay. In exploring the role of CD14/TLR4 associated signal transduction mediators, NF-κB and IRF-3, we further demonstrate that the phagocytic activity of ATRA-NB4 cells in engulfing beads was significantly inhibited when cells were pretreated with either a NF-κB inhibitor (BAY 11-7082) or an IRF-3 inhibitor (SP600125). However, this activity in engulfing apoptotic cells was only significantly inhibited by pretreatment with BAY11-7082, but not by pre-treatment with SP600125. Finally, our results indicate that the level of CD14(+) microparticles (MPs) released by ATRA-NB4 cells was significantly enhanced when those cells were induced into the process of apoptosis by pre-treatment with idarubicin. Moreover, by incubation with MPs derived from apoptotic ATRA-NB4 cells, the phagocytic activity of living ATRA-NB4 cells in engulfing apoptotic cells was significantly enhanced, and this phagocytic activity was also significantly inhibited by pre-treating MPs with antibody specific to CD14 before phagocytic assay. We conclude that CD14 contributes to the phagocytic activity of APL cells during the process of granulocytic differentiation. Disclosures No relevant conflicts of interest to declare.

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PML/RARa Represses Transactivation of PSMB10 Via a PU.1-Dependent Manner In Acute Promyelocytic Leukemia

Blood ◽

10.1182/blood.v116.21.3866.3866 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3866-3866

Author(s):

Xianwen Yang ◽

Ping Wang ◽

Xujie Zhao ◽

Huahua Zhu ◽

Sai-Juan Chen ◽

...

Keyword(s):

Transcriptional Regulation ◽

Retinoic Acid ◽

Immune System ◽

Acute Promyelocytic Leukemia ◽

Target Genes ◽

Promyelocytic Leukemia ◽

Class I ◽

Luciferase Reporter ◽

Atra Treatment ◽

Nb4 Cells

Abstract Abstract 3866 Immunoproteasome is a special form of proteasome which contains three unique interferonγ (IFNγ) induced catalytic subunits, i.e. PSMB8, PSMB9 and PSMB10. Immunoproteasome plays a pivotal role in generating certain peptide antigens for MHC class I presentation. Dysregulation of the immunoproteasome system may contribute to the pathogenesis of certain types of malignancies, including leukemia. Our previous study has identified the target genes of PML/RARa, the initiating factor of acute promyelocytic leukemia (APL) on the genome-wide scale, and demonstrated that PML/RARa could selectively target PU.1-regulated genes, which is a critical mechanism for the pathogenesis of APL. PSMB10, encoding an important composition of immunoproteasome, is one of the identified target genes which are regulated by PML/RARa in this manner. Here we revealed the detailed transcriptional regulation mechanism of PSMB10 in APL. Chromatin immunoprecipitation (ChIP)-PCR assay showed that PML/RARa and PU.1 could bind to the PSMB10 promoter in APL cells, including patient derived NB4 cells and Zn-treated PR9 cells. Re-ChIP assay further demonstrated that PML/RARa and PU.1 co-existed on the same DNA fragment of the PSMB10 promoter, which provided the possibility that PML/RARa and PU.1 could co-regulate the PSMB10 promoter. Using a transient luciferase reporter system, we found that PU.1 transactivated the PSMB10 promoter and PML/RARa repressed the PU.1-dependent transactivation. All-trans retinoic acid (ATRA) could relief the repression caused by PML/RARa. To further demonstrate that the PU.1 site (-37bp∼-29bp) and related retinoic acid response elements (RAREs) (-555bp∼-549bp, -258bp∼-252bp) were essential for PML/RARa to function as an effective repressor, we prepared a series of mutant constructs, including the PU.1-site mutant, the construct mutated on both RARE half (RAREh) sites and two constructs respectively mutated on one of the two RAREh sites, and then transfected them into myeloid U937 cells. From the results of luciferase reporter assays, we found that both PU.1 site and RAREh sites played important roles in PML/RARa-mediated transcriptional repression, moreover, the second RAREh site (-258bp∼-252bp) contributed more than the first one (-555bp∼-549bp). Through electrophoretic mobility shift assay (EMSA), we further determined that PML/RARa could interact with PU.1 through protein-protein interaction, and then bind to the PU.1 site on the PSMB10 promoter. Recent study has shown that ATRA treatment could induce the production of anti-PML/RARa in APL mouse, which implicates that ATRA plays an important role in activating immune system. As the essential elements for immune response, HLA class I antigens (A, B & C) present peptides, which are produced from digested proteins degraded by immunoproteasome, to the surface of antigen-presenting cells. We thus utilized real time RT-PCR to measure the expression of PSMB10 and HLA-A/B/C during ATRA-induced NB4 cells differentiation. We found the levels of PSMB10 and HLA-A/B/C expression were up-regulated in ATRA-treated NB4 cells. These results suggested that the enhanced expression of PSMB10 availed immunoproteasome restoration, which benefited the reactivation of immune system during ATRA treatment therapy. Our results not only demonstrate the detailed transcriptional regulation of PSMB10 in APL but imply the potential function of PSMB10 during ATRA treatment as well. Disclosures: No relevant conflicts of interest to declare.

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Retinoid-induced differentiation of acute promyelocytic leukemia involves PML-RARalpha-mediated increase of type II transglutaminase

Blood ◽

10.1182/blood.v87.5.1939.bloodjournal8751939 ◽

1996 ◽

Vol 87 (5) ◽

pp. 1939-1950 ◽

Cited By ~ 7

Author(s):

L Benedetti ◽

F Grignani ◽

BM Scicchitano ◽

AM Jetten ◽

D Diverio ◽

...

Keyword(s):

Cell Line ◽

Signaling Pathway ◽

Acute Promyelocytic Leukemia ◽

Molecular Mechanisms ◽

Growth Arrest ◽

Promyelocytic Leukemia ◽

Leukemic Cells ◽

Type Ii ◽

Granulocytic Differentiation ◽

Nb4 Cells

All-trans retinoic acid (t-RA) administration leads to complete remission in acute promyelocytic leukemia (APL) patients by inducing growth arrest and differentiation of the leukemic clone. In the present study, we show that t-RA treatment dramatically induced type II transglutaminase (type II TGase) expression in cells carrying the t(15;17) translocation and expressing the PML-RARalpha product such as the APL-derived NB4 cell line and fresh leukemic cells from APL patients. This induction correlated with t-RA-induced growth arrest, granulocytic differentiation, and upregulation of the leukocyte adherence receptor beta subunit (CD18) gene expression. The increase in type II TGase was not abolished by cycloheximide treatment, suggesting that synthesis of a protein intermediate was not required for the induction. t-RA did not significantly alter the rate of growth arrest and did not stimulate differentiation and type II TGase activity in NB4.306 cells, a t-RA-resistant subclone of the NB4 cell line, or in leukemic cells derived from two patients morphologically defined as APL but lacking the t(15;17). However, in NB4.306 cells, t-RA treatment was able to increase CD18 mRNA expression in a manner similar to NB4 cells. The molecular mechanisms involved in the induction of these genes were investigated. In NB4 cells, using novel receptor-selective ligands such as 9-cis-RA, TTNPB, AM580, and SR11217, we found that RAR- and RARalpha- selective retinoids were able to induce growth arrest, granulocytic differentiation, and type II TGase, whereas the RXR-selective retinoid SR11217 was inactive. Moreover, an RAR alpha-antagonist completely inhibited the expression of type II TGase and CD18 induced by these selective retinoids in NB4 cells. In NB4.306 cells, an RARalpha- dependent signaling pathway was found involved in the modulation of CD18 expression. In addition, expression of the PML-RARalpha gene in myeloid U937 precursor cells resulted in the ability of these cells to induce type II TGase in response to t-RA. On the basis of these results we hypothesize a specific involvement of a signaling pathway involving PML-RAR alpha for the induction of growth arrest, granulocytic differentiation, and type II TGase by retinoids in APL cells.

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Retinoic acid is required for and potentiates differentiation of acute promyelocytic leukemia cells by nonretinoid agents

Blood ◽

10.1182/blood.v84.7.2122.2122 ◽

1994 ◽

Vol 84 (7) ◽

pp. 2122-2129 ◽

Cited By ~ 33

Author(s):

A Chen ◽

JD Licht ◽

Y Wu ◽

N Hellinger ◽

W Scher ◽

...

Keyword(s):

Retinoic Acid ◽

Cell Line ◽

Acute Promyelocytic Leukemia ◽

Drug Exposure ◽

Primary Cultures ◽

Leukemia Cell ◽

Promyelocytic Leukemia ◽

Leukemia Cell Line ◽

Granulocytic Differentiation ◽

Nb4 Cells

Abstract Patients with acute promyelocytic leukemia (APL) associated with the t(15;17) translocation and fusion of the promyelocytic leukemia (PML) and retinoic acid receptor-alpha (RAR-alpha) genes achieve complete remission but not cure with all-trans retinoic acid (RA), NB4, a cell line derived from a patient with t(15;17) APL that undergoes granulocytic differentiation when treated with pharmacologic doses of RA, was used as a model for differentiation therapy of APL. We found that NB4 cells are resistant to differentiation by nonretinoid inducers such as hexamethylene bisacetamide (HMBA), butyrates, vitamin D3, or hypoxanthine, all of which can induce differentiation in the commonly used HL60 leukemia cell line. Preexposure of NB4 cells to low concentrations of RA for a period as short as 30 minutes abolished resistance to nonretinoids and potentiated differentiation. Sequential RA and HMBA treatment yielded maximal differentiation by 3 days of drug exposure, whereas the effect of RA alone peaked after 6 days and yielded a smaller percentage of differentiated cells. RA also reversed NB4 cell resistance to butyrates and allowed for synergistic differentiation by these agents. Pretreatment with HMBA before exposure to RA failed to stimulate differentiation. Sequential RA/HMBA treatment also markedly increased the extent of differentiation of primary cultures of bone marrow and peripheral blood mononuclear cells from three APL patients. In one case RA/HMBA treatment overcame resistance to RA in vitro. Together, these results suggest that intermittent low doses of RA followed by either HMBA or butyrates may be a useful combination in the treatment of APL. This clinical strategy may help prevent or overcome RA resistance in APL.

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Enhancing Neutrophil Differentiation - A Novel Role for the Death-Associated Protein Kinase 2 (DAPK2).

Blood ◽

10.1182/blood.v106.11.1348.1348 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1348-1348

Author(s):

Mattia Rizzi ◽

Christian Britschgi ◽

Mario P. Tschan ◽

Tobias J. Grob ◽

Barbara Huegli ◽

...

Keyword(s):

Gene Expression ◽

Progenitor Cells ◽

Expression Profiles ◽

Chronic Phase ◽

Close Relative ◽

Mrna Levels ◽

Granulocytic Differentiation ◽

Expression Levels ◽

Nb4 Cells ◽

Neutrophil Differentiation

Abstract DAPK2 is a 42-kDa Ca2+/Calmodulin-regulated serine/threonine kinase involved in apoptosis. In gene expression profiles derived from in vitro differentiated myeloid leukemic NB4 cells treated with all-trans retinoid acid (ATRA), we found that DAPK2 was decisively induced during differentiation towards neutrophils. DAPK1, a close relative of DAPK2, is inactivated in a number of hematopoietic malignancies (AML, lymphoma, myeloma), and it may play a role during normal and leukemic myeloid cell differentiation. We therefore investigated DAPK2 for its possible role in both normal and leukemic myelopoiesis. Real time quantitative RT-PCR (RQ-PCR) and Western blot analysis of DAPK2 gene expression in primary myeloid cells revealed significantly higher DAPK2 expression in granulocytes (G; n=9) compared with monocytes/macrophages (M; n=8) and CD34+ progenitor cells (CD34+; n=6) (Δ, p< 0.001; figure, left panel). Moreover, significantly increased DAPK2 mRNA levels were also seen when cord blood CD34+ progenitor cells were induced to differentiate towards neutrophils with human recombinant G-CSF (hrG-CSF). In addition, ATRA-induced neutrophil differentiation of two leukemic cell lines, NB4 and U937, showed significantly higher DAPK2 mRNA expression paralleled by DAPK2 protein induction. However, during differentiation of CD34+ cells (with hrM-CSF) and U937 cells (with PMA) towards monocytes/macrophages, DAPK2 mRNA levels remained low. DAPK2 expression in primary leukemic cells revealed significantly lower DAPK2 expression levels in AML blasts (AML; n=100) than in samples from chronic myeloid leukemia patients in chronic phase (CML-CP; n=9) (ΔΔ, p< 0.001; figure, right panel). Figure Figure Stable lentiviral-mediated expression of wild-type DAPK2 enhanced ATRA-induced granulocytic differentiation of NB4 cells as shown by morphology and by increased CD11b expression. Furthermore, upregulation of mRNA levels of key regulator genes for terminal differentiation, such as C/EBPe, the G-CSF receptor and the secondary granule protein lactoferrin, was also enhanced. Expression of a kinase-inactive DAPK2 mutant did not show these effects, a finding consistent with a role of DAPK2 in granulopoiesis. Conclusion: we demonstrate for the first time, that DAPK2 expression levels correlate with the degree of granulocytic differentiation, and that DAPK2 upregulation is restricted to granulopoiesis. Furthermore lentiviral-mediated DAPK2 expression enhances granulocytic differentiation. The finding that DAPK2 expression is low in AML and high in CML-CP patients suggests that suppressed DAPK2 expression may contribute to the differentiation block in AML.

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Death Associated Protein 5 (DAP5/p97/NAT1) Contributes to Retinoic Acid-Induced Granulocytic Differentiation and Arsenic Trioxide Induced Apotosis through Inhibition of PI3K/Akt/mTOR Pathway in Acute Promyelocytic Leukemia Cells: A Novel Mechanism.

Blood ◽

10.1182/blood.v110.11.2836.2836 ◽

2007 ◽

Vol 110 (11) ◽

pp. 2836-2836

Author(s):

Bulent Ozpolat ◽

Ugur Akar ◽

Isabel Zorilla-Calancha ◽

Pablo Vivas-Mejia ◽

Gabriel Lopez-Berestein

Keyword(s):

Retinoic Acid ◽

Arsenic Trioxide ◽

Acute Promyelocytic Leukemia ◽

Translation Initiation ◽

Promyelocytic Leukemia ◽

Mtor Pathway ◽

Granulocytic Differentiation ◽

Induced Expression ◽

Nb4 Cells ◽

Induced Apoptosis

Abstract All-trans Retinoic Acid (ATRA) is a naturally occurring metabolite of retinol (vitamin A)and acts as a potent inducer of cellular differentiation and growth arrest in acute promyelocytic leukemia (APL), a type of acute myeloid leukemia (M3-AML). APL is characterized by translocation t(15;17), fusing PML (promyelocytic leukemia) and RARα (retinoic acid receptor) genes, leding to expression of PML/RARα receptor protein and differentiation block. Arsenic trioxide (ATO) induces (<0.5 μM) differentiation at low doses and apoptosis at high doses (>1 μM) in APL cells. Currently, both ATRA and ATO are successfully used in the treatment of APL in the clinic. However, the molecular mechanisms of myeloid differentiation and apoptosis induced by these agents are not fully understood. We previously reported that ATRA inhibits the translation initiation through multiple mechanisms, including upregulation of translation initiation inhibitors, DAP5/p97 and PDCD4 tumor suppressor protein. Here we investigated the role and regulation of death associated protein-5 (DAP5/p97/NAT1), a novel inhibitor of translational initiation, in myeloid (granulocytic and monocytic) cell differentiation and apoptosis. We found that ATRA (1 μM) induced a marked DAP5/p97 protein and mRNA expression during granulocytic differentiation of NB4 and HL60 cells but not in differentiation-resistant cells, which express very low levels of DAP5/p97. DAP5/p97 was translocated into nucleus during the differentiation of NB4 cells induced ATRA. At differentiation inducing doses, ATO, dimethysulfoxide, 1,25-dihydroxy-vitamin-D3, and phorbol-12-myristate-13-acetate also induced a significant DAP5/p97 expression in NB4 cells. However, ATO at apoptotic doses, but not ATRA, induced DAP5/p86, a proapoptotic form of DAP5/p97. ATRA and ATO -induced expression of DAP5/p97 was associated with inhibition of phosphaditylinositol 3-kinase (PI3K)/Akt pathway, which is known to stimulate cap-dependent translation of mRNAs. To show direct link between PI3K/Akt/mTOR pathway and DAP5 expression, we treated cell with PI3K and mTOR inhibitors LY294002 and by rapamycin, respectively. We found that inhibition of PI3K/Akt/mTOR pathway upregulated DAP5/p97 expression in NB4 cells. Finally, knockdown of DAP5/p97 expression by small interfering RNA significantly inhibited ATRA-induced granulocytic differentiation detected by expression of CD11b and ATO-induced apoptosis in NB4 cells detected by Annexin V assay (p<0.05). In conclusion, our data suggest that DAP5/p97 plays a role in ATRA-induced differentiation and ATO-induced apoptosis in APL cells. Our data demonstrated for the first time that DAP5/p97 is constitutively suppressed by of PI3K/Akt/mTOR pathway, and ATRA and ATO-induced expression of DAP5 is mediated by the inhibition of this survival pathway, suggesting a novel mechanism of DAP5 regulation and a role of translational control in induction of differentiation and apoptosis. Figure Figure

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Gene expression networks underlying retinoic acid–induced differentiation of acute promyelocytic leukemia cells

Blood ◽

10.1182/blood.v96.4.1496 ◽

2000 ◽

Vol 96 (4) ◽

pp. 1496-1504 ◽

Cited By ~ 160

Author(s):

Ting-Xi Liu ◽

Ji-Wang Zhang ◽

Jiong Tao ◽

Ruo-Bo Zhang ◽

Qing-Hua Zhang ◽

...

Keyword(s):

Gene Expression ◽

Retinoic Acid ◽

Acute Promyelocytic Leukemia ◽

Cellular Proliferation ◽

Expression Profiles ◽

Expression Patterns ◽

Gene Expression Profiles ◽

Promyelocytic Leukemia ◽

Atra Treatment ◽

Induced Differentiation

Abstract To elucidate the molecular mechanism of all-trans-retinoic acid (ATRA)–induced differentiation of acute promyelocytic leukemia (APL) cells, the gene expression patterns in the APL cell line NB4 before and after ATRA treatment were analyzed using complementary DNA array, suppression-subtractive hybridization, and differential-display–polymerase chain reaction. A total of 169 genes, including 8 novel ones, were modulated by ATRA. The ATRA-induced gene expression profiles were in high accord with the differentiation and proliferation status of the NB4 cells. The time courses of their modulation were interesting. Among the 100 up-regulated genes, the induction of expression occurred most frequently 12-48 hours after ATRA treatment, while 59 of 69 down-regulated genes found their expression suppressed within 8 hours. The transcriptional regulation of 8 induced and 24 repressed genes was not blocked by cycloheximide, which suggests that these genes may be direct targets of the ATRA signaling pathway. A balanced functional network seemed to emerge, and it formed the foundation of decreased cellular proliferation, maintenance of cell viability, increased protein modulation, and promotion of granulocytic maturation. Several cytosolic signaling pathways, including JAKs/STAT and MAPK, may also be implicated in the symphony of differentiation.

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The PML/RARα-Regulated MiR-181a/b-Cluster Targets the Tumor Suppressor RASSF1A in Acute Promyelocytic Leukemia

Blood ◽

10.1182/blood.v124.21.2195.2195 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2195-2195

Author(s):

Daniela Braeuer-Hartmann ◽

Jens-Uwe Hartmann ◽

Dennis Gerloff ◽

Christiane Katzerke ◽

Alexander Arthur Wurm ◽

...

Keyword(s):

Bone Marrow ◽

Tumor Suppressor ◽

Acute Promyelocytic Leukemia ◽

Conflicts Of Interest ◽

Fusion Gene ◽

Promyelocytic Leukemia ◽

Granulocytic Differentiation ◽

Nb4 Cells ◽

Knock Down ◽

Functional Studies

Abstract In acute promyelocytic leukemia (APL) bearing the translocation t(15;17), all-trans-retinoic acid (ATRA) treatment induces granulocytic maturation and complete remission of leukemia. Several factors are involved in the formation of the leukemic phenotype. Latest studies identified microRNAs as critical players in this network. In a micro array based microRNA screen we could identify the genomically clustered miR-181a and miR-181b as downregulated in the APL cell line NB4 by treatment with pharmacological doses of ATRA. In addition, the expression of the miR-181a/b-cluster was strongly reduced in bone marrow samples of APL patient while ATRA-based therapy. Furthermore, we showed the transcriptional induction of miR-181a and miR-181b by the APL-associated PML-RARα oncogene in vitro and in vivo. In PR9 cells, carrying a zinc-driven PML/RARα construct, and in PML/RARα-knock in mice the expression of the fusion gene leads to upregulation of the microRNA-cluster expression. Analysis of bone marrow samples of APL patients showed an enhanced expression of miR-181a and miR-181b in comparison to AML patient samples with normal karyotype, whereas other AML subgroups show no significant regulation. Based on siRNA experiments we could propose AP-1 and GATA-2 as potential co-activators for the PML/RARα-dependent regulation of the miR-181a/b-cluster. In functional studies in NB4 cells we observed after lentiviral knock down of miR-181a and miR-181b a significant reduction of colony size and number as well as proliferation rate. In contrast, transient overexpression of miR-181a and miR-181b led to an inhibition of ATRA-induced expression of the differentiation marker CD11b. In a microRNA target search we identified the novel ATRA regulated tumor suppressor RASSF1A as a putative target of miR-181a and miR-181b. In functional studies we showed that enforced expression of miR-181a and miR-181b reduces the protein level of RASSF1A by binding to the 3´UTR of RASSF1A mRNA. Accordingly, RASSF1A protein was enriched after knock down of miR-181b. The role of RASSF1A in ATRA induced differentiation was verified by knock down of RASSF1A protein by specific siRNA: Here we could show the reduction of ATRA induced CD11b expression. Overexpression of RASSF1A in NB4 cells strongly induced apoptosis. Additional, we could show by western blot that the miR-181a/b-cluster and RASSF1A modulate cell cycle via regulation of cyclin D1. In conclusion, we identified the miR-181a/b-cluster as an important player in the PML/RARα associated APL. Moreover, we firstly described the miR-181a/b target RASSF1A as a crucial factor in the ATRA activated granulocytic differentiation program in APL. Our data reveal the importance of deregulated microRNA biogenesis in cancer and may provide novel biomarkers and therapeutic targets in myeloid leukemia. Disclosures No relevant conflicts of interest to declare.

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Retinoic acid is required for and potentiates differentiation of acute promyelocytic leukemia cells by nonretinoid agents

Blood ◽

10.1182/blood.v84.7.2122.bloodjournal8472122 ◽

1994 ◽

Vol 84 (7) ◽

pp. 2122-2129 ◽

Cited By ~ 3

Author(s):

A Chen ◽

JD Licht ◽

Y Wu ◽

N Hellinger ◽

W Scher ◽

...

Keyword(s):

Retinoic Acid ◽

Cell Line ◽

Acute Promyelocytic Leukemia ◽

Drug Exposure ◽

Primary Cultures ◽

Leukemia Cell ◽

Promyelocytic Leukemia ◽

Leukemia Cell Line ◽

Granulocytic Differentiation ◽

Nb4 Cells

Patients with acute promyelocytic leukemia (APL) associated with the t(15;17) translocation and fusion of the promyelocytic leukemia (PML) and retinoic acid receptor-alpha (RAR-alpha) genes achieve complete remission but not cure with all-trans retinoic acid (RA), NB4, a cell line derived from a patient with t(15;17) APL that undergoes granulocytic differentiation when treated with pharmacologic doses of RA, was used as a model for differentiation therapy of APL. We found that NB4 cells are resistant to differentiation by nonretinoid inducers such as hexamethylene bisacetamide (HMBA), butyrates, vitamin D3, or hypoxanthine, all of which can induce differentiation in the commonly used HL60 leukemia cell line. Preexposure of NB4 cells to low concentrations of RA for a period as short as 30 minutes abolished resistance to nonretinoids and potentiated differentiation. Sequential RA and HMBA treatment yielded maximal differentiation by 3 days of drug exposure, whereas the effect of RA alone peaked after 6 days and yielded a smaller percentage of differentiated cells. RA also reversed NB4 cell resistance to butyrates and allowed for synergistic differentiation by these agents. Pretreatment with HMBA before exposure to RA failed to stimulate differentiation. Sequential RA/HMBA treatment also markedly increased the extent of differentiation of primary cultures of bone marrow and peripheral blood mononuclear cells from three APL patients. In one case RA/HMBA treatment overcame resistance to RA in vitro. Together, these results suggest that intermittent low doses of RA followed by either HMBA or butyrates may be a useful combination in the treatment of APL. This clinical strategy may help prevent or overcome RA resistance in APL.

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