Leukemogenesis Of Acute Promyelocytic Leukemia Research Articles

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

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

Recurrent noncoding somatic and germline WT1 variants converge to disrupt MYB binding in acute promyelocytic leukemia

AbstractGenetic alternations can occur at noncoding regions, but how they contribute to cancer pathogenesis is poorly understood. Here, we established a mutational landscape of cis-regulatory regions (CREs) in acute promyelocytic leukemia (APL) based on whole-genome sequencing analysis of paired tumor and germline samples from 24 patients and epigenetic profiling of 16 patients. Mutations occurring in CREs occur preferentially in active enhancers bound by the complex of master transcription factors in APL. Among significantly enriched mutated CREs, we found a recurrently mutated region located within the third intron of WT1, an essential regulator of normal and malignant hematopoiesis. Focusing on noncoding mutations within this WT1 intron, an analysis on 169 APL patients revealed that somatic mutations were clustered into a focal hotspot region, including one site identified as a germline polymorphism contributing to APL risk. Significantly decreased WT1 expression was observed in APL patients bearing somatic and/or germline noncoding WT1 variants. Furthermore, biallelic WT1 inactivation was recurrently found in APL patients with noncoding WT1 variants, which resulted in the complete loss of WT1. The high incidence of biallelic inactivation suggested the tumor suppressor activity of WT1 in APL. Mechanistically, noncoding WT1 variants disrupted MYB binding on chromatin and suppressed the enhancer activity and WT1 expression through destroying the chromatin looping formation. Our study highlights the important role of noncoding variants in the leukemogenesis of APL.

Obesity in children with acute promyelocytic leukemia: What is its prevalence and prognostic significance?

To compare outcomes of obese and nonobese pediatric patients with acute promyelocytic leukemia (APL) from the Cancer and Leukemia Group B trial (CALGB) 9710 and the Children's Oncology Group trial AAML0631. Data including demographics, adverse events, overall and event-free survival (EFS) were analyzed. The prevalence of obesity was 34% on C9710 and 35% on AAML0631. There was significantly lower overall and EFS in the obese population on multivariable analysis on AAML0631 but not on CALGB 9710. Eleven patients died during therapy or in follow-up. The prevalence of obesity is higher in pediatric patients with APL compared to the general population. The decreased EFS and OS in obese patients on AAML0631 suggest that the presence of obesity can influence outcomes using the most current treatment. These findings support the need for further research on the potential role of obesity in pediatric APL leukemogenesis.

Long Noncoding RNA Profiling Reveals an Abundant Crnde That Inhibits Granulocytic Differentiation in APL

Long noncoding RNAs (lncRNAs) emerge as important factors involved in the regulation of gene expression during normal tissue development. Abnormally expressed lncRNAs lead to the initiation of cancers, and also promote tumor cell proliferation and metastasis in various kinds of cancers. In acute promyelocytic leukemia (APL), it is general accepted that PML/RARα fusion protein acts as a transcriptional repressor, which disrupts the differentiation program, and arrests cells at the promyelocytic stage of myeloid differentiation. However, a significant amount of evidence has demonstrated that PML/RARα is not sufficient to cause APL and additional cooperating events are required for the development of APL. As the key regulators of cancers, the specific expression patterns and functions of lncRNAs involved in APL leukemogenesis are not clear. In our study, we aimed to identify APL-specific lncRNAs and explore the important biological roles of these dysregulated lncRNAs in APL leukemogenesis. To discover the unique dysregulated lncRNAs in APL, we first established a lncRNA identification pipeline by analyzing RNA-seq data of 171 AML patient samples from the TCGA database and totally obtained 3,892 lncRNAs. We further analyzed the differentially expressed lncRNAs in 16 APL patient samples compared with 155 non-APL AML patient samples and identified a total of 414 APL-specific lncRNAs. These unique lncRNAs in APL contained 219 APL-specific upregulated lncRNAs, such as CRNDE, PVT1, MEG3, etc., and 195 APL-specific downregulated lncRNAs, such as HOTAIRM1, NEAT1, HOXA-AS2, etc., which may serve as novel biomarkers for APL. We then evaluated the expression of these dysregulated lncRNAs in response to all- trans retinoic acid (ATRA), which is the first-line drug for APL therapy that efficiently induces APL cells differentiation. We analyzed our RNA-seq data of APL cell line NB4 treated with or without ATRA and performed gene set enrichment analysis (GSEA) to evaluate the potential association of APL-specific lncRNAs with the induction of ATRA treatment. We noticed that highly expressed lncRNAs in APL were significantly enriched in ATRA-downregulated lncRNAs dataset, and the APL-specific downregulated lncRNAs were enriched in ATRA-upregulated lncRNAs dataset. These results suggested that APL-dysregulated lncRNAs could also participate in ATRA-induced granulocytic differentiation. To further identify the possible functional roles of APL-specific dysregulated lncRNAs, we selected one of the APL-specific overexpressed lncRNA, CRNDE, which is involved in the pathogenesis of some solid tumors. We found that the highly expressed CRNDE could be repressed by ATRA in a time dependent manner. Furthermore, functional analysis was performed to identify the potential roles of CRNDE in the leukemogenesis and the differentiation therapy of APL. We designed CRNDE-specific shRNA to downregulate its expression in NB4 cells, and found that silencing of CRNDE could promote differentiation of NB4 cells and enhance ATRA-induced differentiation. Further RNA-seq analysis to explore the mechanism revealed that knockdown of CRNDE could affect the expression of a number of myeloid differentiation-associated genes and signaling pathways. Collectively, our studies have systematically identified a distinct set of lncRNAs in APL, which may serve as novel biomarkers for APL. The further functional analysis of CRNDE reveals its essential roles involved in the leukemogenesis and ATRA-induced differentiation of APL, which provides new insights into the underlying mechanisms of dysregulated lncRNAs in APL. DisclosuresNo relevant conflicts of interest to declare.

Crosstalk between hnRNP K and SET in ATRA-induced differentiation in acute promyelocytic leukemia.

HnRNP K protein is a heterogeneous nuclear ribonucleoprotein which has been proposed to be involved in the leukemogenesis of acute promyelocytic leukemia (APL), as well as in differentiation induced by all‐trans retinoic acid (ATRA). We previously demonstrated a connection between SET and hnRNP K function in head and neck squamous cell carcinoma (HNSCC) cells related to splicing processing. The objective of this study was to characterize the participation of hnRNP K and SET proteins in ATRA‐induced differentiation in APL. We observed higher (5‐ to 40‐fold) levels of hnRNP K and SET mRNA in APL patients at the diagnosis phase compared with induction and maintenance phases. hnRNP K knockdown using short‐hairpin RNA led to cell death in ATRA‐sensitive NB4 and resistant NB4‐R2 cells by apoptosis with SET cleavage. In addition, hnRNP K knockdown increased granulocytic differentiation in APL cells, mainly in NB4‐R2 with ATRA. hnRNP K knockdown had an effect similar to that of treatment with U0126 (an meiosis‐specific serine/threonine protein kinase/ERK inhibitor), mainly in NB4‐R2 cells. SET knockdown in APL cells revealed that apoptosis induction in cells with hnRNP K knockdown occurred by SET cleavage rather than by reduction in SET protein. Transplantation of NB4‐R2 cells into nude mice confirmed that arsenic trioxide (ATO) combined with U0126 has higher potential against tumor progression when compared to ATO. Therefore, hnRNP K/SET and ERK are potential therapeutic targets for both antineoplastic leukemia therapy and relapsed APL patients with ATRA resistance.

A PML/RARα direct target atlas redefines transcriptional deregulation in acute promyelocytic leukemia

Transcriptional deregulation initiated by oncogenic fusion proteins plays a vital role in leukemia. The prevailing view is that the oncogenic fusion protein promyelocytic leukemia/retinoic acid receptor-α (PML/RARα), generated by the chromosome translocation t(15;17), functions as a transcriptional repressor in acute promyelocytic leukemia (APL). Here, we provide rich evidence of how PML/RARα drives oncogenesis through both repressive and activating functions, particularly the importance of the newly identified activation role for the leukemogenesis of APL. The activating function of PML/RARα is achieved by recruiting both abundant P300 and HDAC1 and by the formation of super-enhancers. All-trans retinoic acid and arsenic trioxide, 2 widely used drugs in APL therapy, exert synergistic effects on controlling super-enhancer-associated PML/RARα-regulated targets in APL cells. We use a series of in vitro and in vivo experiments to demonstrate that PML/RARα-activated target gene GFI1 is necessary for the maintenance of APL cells and that PML/RARα, likely oligomerized, transactivates GFI1 through chromatin conformation at the super-enhancer region. Finally, we profile GFI1 targets and reveal the interplay between GFI1 and PML/RARα on chromatin in coregulating target genes. Our study provides genomic insight into the dual role of fusion transcription factors in transcriptional deregulation to drive leukemia development, highlighting the importance of globally dissecting regulatory circuits.

Arsenic induced epigenetic changes and relevance to treatment of acute promyelocytic leukemia and beyond

Epigenetic alterations regulate gene expression without changes in the DNA sequence. It is well-demonstrated that aberrant epigenetic changes contribute to the leukemogenesis of acute promyelocytic leukemia (APL). Arsenic trioxide (ATO) is one of the most common drugs used in the frontline treatment of APL that act through targeting and destabilizing the PML/RARα oncofusion protein. ATO together with all-trans retinoic acid (ATRA) lead to durable remission of more than 90% non-high-risk APL patients, turning APL treatment into a paradigm of oncoprotein targeted cure. Although relapse and drug resistance in APL are yet to be resolved in the clinic, epigenetic machineries might hold the key to address this issue. Further, ATO also showed promising anticancer activities against a variety of malignancies, but its application is particularly restricted due to limited understanding of the mechanism. Thus, a thorough understanding of epigenetic mechanism behind anti-leukemic effects of ATO would benefit the development of ATO-based anticancer strategy. Role of ATRA on APL associated epigenetic alterations has been extensively studied and reviewed. Recently, accumulating evidence suggest that ATO also induces some epigenetic changes that might favor APL eradication. In this article, we comprehensively discuss arsenic induced epigenetic changes and its relevance in APL treatment and beyond, so as to provide novel insights into overcoming arsenic resistance in APL and promote application of this drug to other malignancies.

TRIB3 Stabilizes High TWIST1 Expression to Promote Rapid APL Progression and ATRA Resistance.

The resistance to differentiation therapy and early death caused by fatal bleeding endangers the health of a significant proportion of patients with acute promyelocytic leukemia (APL). This study aims to investigate the molecular mechanisms of all-trans retinoic acid (ATRA) resistance and uncover new potential therapeutic strategies to block the rapid progression of early death. The important role of TWIST1 in APL leukemogenesis was first determined by gain- and loss-of-function assays. We then performed in vivo and in vitro experiments to explore the interaction of TWIST1 and TRIB3 and develop a potential peptide-initiated therapeutic opportunity to protect against early death and induction therapy resistance in patients with APL. We found that the epithelial-mesenchymal transition (EMT)-inducing transcription factor TWIST1 is highly expressed in APL cells and is critical for leukemic cell survival. TWIST1 and TRIB3 were highly coexpressed in APL cells compared with other subtypes of acute myeloid leukemia cells. We subsequently demonstrated that TRIB3 could bind to the WR domain of TWIST1 and contribute to its stabilization by inhibiting its ubiquitination. TRIB3 depletion promoting TWIST1 degradation reverses resistance to induction therapy and improves sensitivity to ATRA. On the basis of a detailed functional screen of synthetic peptides, we discovered a peptide analogous to the TWIST1 WR domain that specifically represses APL cell survival by disrupting the TRIB3/TWIST1 interaction. Our data not only define the essential role of TWIST1 as an EMT-TF in patients with APL but also suggest that disrupting the TRIB3/TWIST1 interaction reverses induction therapy resistance and blocks rapid progression of APL early death.See related commentary by Peeke and Gritsman, p. 6018.

VITAMIN A, RETINOIC ACID AND TAMIBAROTENE, A FRONT TOWARD ITS ADVANCES: A REVIEW

Vitamin A and its derivative retinoic acid (13-cis RA, 9-cis RA, all-trans RA) and recent tamibarotene have been shown a broad variety of biological actives in human, such as vision, embryonic development, cell growth and cellular differentiation and immune function. These precise functions of RA are mediated by their retinoic acid receptors (RARs). In the past five decades, retinoic acid (RA) proved therapeutic benefits in cancer prevention, in skin diseases and in acute promyelocytic leukemia (APL).The elucidation of the molecular basis of vitamin A acid and its retinoid pharmacology in APL has been illustrated in several publications, the detail molecular model of gene regulation had also been proposed by Zhu in earlier 90s. A molecular model is further revised. As an approach to APL treatment, one possible the action of retinoic acid (RA), A consensus sequence (TCAGGTCA motif ) has been postulated for thyroid hormone (TRE) and retinoic acid responsive element (RARE)-containing in the promoter region of target genes. High dose of RA-RARE-PML/RARa complexes in intracellular localization appears to relieve repressors from DNA-bound receptor, including the dissociation of co-re pressor complexes N-CoR, SMRT and HDACs from PML-RARa or PML-RARa/RXR. Also release PML/RARa -mediated transcription repression. This transcriptional derepression occurs at RARa target gene promoter. Consequentially, PML-RARa chimera converted receptor from a repressor to a RA-dependent activator of transcription. The resulting pml-RARA oncoprotein proteolytic degradation occurs through the autophagy-lysosome pathway and the ubiquitin SUMO-proteasome system(UPS) as well as caspase 3, or lysosomal protease (cathepsin D) enzyme or/and EI-like ubiquitin-activating enzyme(UBEIL) induction. Accordingly the expression level of PML-RARa downregulated. PML protein relocalizes into the wild-type nuclear body (PML-NB) configuration or a truncated PML-RARa fusion fragment detected or/and the wild-type RAR upregulated. An effect is to relieve the blockade of pml/RARa-mediated RA dependent promyelocytic differentiation, and retinoic acid (9-cid RA,ATRA, Am80) in APL therapy. Here, oncogenic pml/RARa as constitutive transcriptional repressor that block myeloid differentiation at promyelocytic phenotype. RA can overcome the transcrptional repressor activity of pml/RARa. The oncogenic pml/RARa uncovers a pathogenic role in leukemogenesis of APL through blocking promyelocytic differentiation. This oncogenic receptor derivative pml/ RARa chimera is locked in their "off" regular mode thereby constitutively repressing transcription of target genes or key enzymes (such as AP-1, PTEN, DAPK2,UP.1, p21WAF/CCKN1A）that are critical for differentiation of hematopoietic cells. This is first described in eukaryotes.

PML/RARa blocks the differentiation and promotes the proliferation of acute promyelocytic leukemia through activating MYB expression by transcriptional and epigenetic regulation mechanisms.

The promyelocytic leukemia (PML)/retinoic acid receptor-alpha (RARα) onco-fusion protein that is generated from t(15;17) chromosome translocation is crucial for the leukemogenesis of acute promyelocytic leukemia (APL) and is well documented as a transcriptional repressor. To understand the relationship between PML/RARα and the oncogene in the development of APL, we investigate the regulation mechanism of PML/RARα to MYB proto-oncogene and the role of this regulation on the proliferation and differentiation of APL cells. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assays show that MYB expression was significantly higher in PML/RARα positive cell lines. Microarray data verify that the MYB expression was significantly higher in APL patient samples than in normal promyelocyte samples. Further expression analysis from RT-qPCR and microarray data verifies that the expression of MYB is upregulated by PML/RARα. Transcriptional factor binding analysis shows that MYB is directly bound by PML/RARα and its cofactors. Luciferase assays show that PML/RARα transactivated MYB promoter activity through the RARα binding site and the coexistence of CCAAT enhancer binding protein ε. We also find that PML/RARα increases the acetylation level of the promoter region of MYB. Further evidence demonstrates that PML/RARα regulates MYB expression through long-range interaction. Functionally, PML/RARα increases the cell proliferation and blocks the differentiation through activating MYB expression. Collectively, this study uncovers a novel mechanism of PML/RARα-mediated transcriptional activation and enriches our knowledge of the onco-fusion protein-mediated transcription activation.

Repression of CDKN2C caused by PML/RARα binding promotes the proliferation and differentiation block in acute promyelocytic leukemia.

Inappropriate cell proliferation during oncogenesis is often accompanied by inactivation of components involved in the cell cycle machinery. Here, we report that cyclin-dependent kinase inhibitor 2C (CDKN2C) as a member of the cyclin-dependent kinase inhibitors is a target of the PML/RARα oncofusion protein in leukemogenesis of acute promyelocytic leukemia (APL).We found that CDKN2C was markedly downregulated in APL blasts compared with normal promyelocytes. Chromatin immunoprecipitation combined with quantitative polymerase chain reaction demonstrated that PML/RARα directly bound to the CDKN2C promoter in the APL patient-derived cell line NB4. Luciferase assays indicated that PML/RARα inhibited the CDKN2C promoter activity in a dose-dependent manner. Furthermore, all-trans retinoic acid treatment induced CDKN2C expression by releasing the PML/RARα binding on chromatin in NB4 cells. Functional studies showed that ectopic expression of CDKN2C induced a cell cycle arrest at the G0/G1 phase and a partial differentiation in NB4 cells. Finally, the transcriptional regulation of CDKN2C was validated in primary APL patient samples. Collectively, this study highlights the importance of CDKN2C inactivation in the abnormal cell cycle progression and differentiation block of APL cells and may provide new insights into the study of pathogenesis and targeted therapy of APL.

PML nuclear body disruption impairs DNA double-strand break sensing and repair in APL

Proteins involved in DNA double-strand break (DSB) repair localize within the promyelocytic leukemia nuclear bodies (PML-NBs), whose disruption is at the root of the acute promyelocytic leukemia (APL) pathogenesis. All-trans-retinoic acid (RA) treatment induces PML-RARα degradation, restores PML-NB functions, and causes terminal cell differentiation of APL blasts. However, the precise role of the APL-associated PML-RARα oncoprotein and PML-NB integrity in the DSB response in APL leukemogenesis and tumor suppression is still lacking. Primary leukemia blasts isolated from APL patients showed high phosphorylation levels of H2AX (γ-H2AX), an initial DSBs sensor. By addressing the consequences of ionizing radiation (IR)-induced DSB response in primary APL blasts and RA-responsive and -resistant myeloid cell lines carrying endogenous or ectopically expressed PML-RARα, before and after treatment with RA, we found that the disruption of PML-NBs is associated with delayed DSB response, as revealed by the impaired kinetic of disappearance of γ-H2AX and 53BP1 foci and activation of ATM and of its substrates H2AX, NBN, and CHK2. The disruption of PML-NB integrity by PML-RARα also affects the IR-induced DSB response in a preleukemic mouse model of APL in vivo. We propose the oncoprotein-dependent PML-NB disruption and DDR impairment as relevant early events in APL tumorigenesis.

In Vivo Analysis of PML-RARA in a Humanized Mouse Model

Acute promyelocytic leukemia (APL) is a subtype of acute myeloid leukemia (AML) characterized by the formation of a PML-RARa fusion protein, which leads to the accumulation of abnormal promyelocytes. Xenograft mouse models with human leukemic cells have advantages for analyzing the human leukemias in vivo, especially for genetic analyses. However, human primary APL cells are difficult to engraft even in very severely immunodeficient mice, such as NOD/shi-SCID IL2Rg-/- (NOG) mice. In order to understand the mechanisms involved in human APL leukemogenesis, we established a humanized in vivo APL model using the transplantation of PML-RARA-transduced CD34+ cells from human cord blood into NOG mice. The expression of PML-RARa in the CD34+ cells disrupted the nuclear bodies in vitro. The clonogenic assay showed that PML-RARa inhibited the total colony formation, but favored the growth of myeloid colonies. When CD34+ cells with PML-RARA were transplanted, they proliferated in the NOG mice for more than three to four months after transplantation (in 24 out of the 34 mice). All 16 mice with more than 3,000 PML-RARA-transduced CD34+ cells were engrafted, while the engraftment was only detected in eight out of 18 mice when the cell density used for transplantation was less than 3,000 cells. These cells possessed abundant azurophilic abnormal granules in the cytoplasm, and some of them had bundles of Auer rods. They expressed CD13, CD33 and CD117, but not HLA-DR or CD34. In addition, the gene expression analysis revealed that these cells and human primary APL were clustered together among various types of AML, suggesting that these induced APL cells well recapitulated human primary APL. Similar to human primary APL, the induced APL cells possessed the ability for myeloid differentiation after treatment with all-trans retinoic acid in vitro and in vivo, and a very low potential for re-transplantation, which was similarly observed in both unsorted induced APL cells and the CD34- fraction. When human cord blood was fractionated before the PML-RARA transduction, the CD34+/CD38+ cells and common myeloid progenitors (CMP) in the CD34+/CD38+ cells led to the efficient development of APL in vivo. These findings demonstrate that CMP is a target for PML-RARA in APL, whereas the resultant CD34- APL cells may share the ability to maintain the leukemia. DisclosuresNo relevant conflicts of interest to declare.

Establishment of a humanized APL model via the transplantation of PML-RARA-transduced human common myeloid progenitors into immunodeficient mice.

Recent advances in cancer biology have revealed that many malignancies possess a hierarchal system, and leukemic stem cells (LSC) or leukemia-initiating cells (LIC) appear to be obligatory for disease progression. Acute promyelocytic leukemia (APL), a subtype of acute myeloid leukemia characterized by the formation of a PML-RARα fusion protein, leads to the accumulation of abnormal promyelocytes. In order to understand the precise mechanisms involved in human APL leukemogenesis, we established a humanized in vivo APL model involving retroviral transduction of PML-RARA into CD34+ hematopoietic cells from human cord blood and transplantation of these cells into immunodeficient mice. The leukemia well recapitulated human APL, consisting of leukemic cells with abundant azurophilic abnormal granules in the cytoplasm, which expressed CD13, CD33 and CD117, but not HLA-DR and CD34, were clustered in the same category as human APL samples in the gene expression analysis, and demonstrated sensitivity to ATRA. As seen in human APL, the induced APL cells showed a low transplantation efficiency in the secondary recipients, which was also exhibited in the transplantations that were carried out using the sorted CD34− fraction. In order to analyze the mechanisms underlying APL initiation and development, fractionated human cord blood was transduced with PML-RARA. Common myeloid progenitors (CMP) from CD34+/CD38+ cells developed APL. These findings demonstrate that CMP are a target fraction for PML-RARA in APL, whereas the resultant CD34− APL cells may share the ability to maintain the tumor.

MicroRNA-181a-mediated downregulation of AC9 protein decreases intracellular cAMP level and inhibits ATRA-induced APL cell differentiation.

AC9 is one of the adenylate cyclase (AC) isoforms, which catalyze the conversion of ATP to cAMP, an important second messenger. We previously found that the integration of cAMP/PKA pathway with nuclear receptor-mediated signaling was required during all-trans retinoic acid (ATRA)-induced maturation of acute promyelocytic leukemia (APL) cells. Here we showed that AC9 could affect intracellular cAMP level and enhance the trans-activity of retinoic acid receptor. Knockdown of AC9 in APL cell line NB4 could obviously inhibit ATRA-induced differentiation. We also demonstrated that miR-181a could decrease AC9 expression by targeting 3′UTR of AC9 mRNA, finally controlling the production of intracellular cAMP. The expression of miR-181a itself could be inhibited by CEBPα, probably accounting for the differential expression of miR-181a in NB4 and ATRA-resistant NB4-R1 cells. Moreover, we found that AC9 expression was relatively lower in newly diagnosed or relapsed APL patients than in both complete remission and non-leukemia cases, closely correlating with the leukemogenesis of APL. Taken together, our studies revealed for the first time the importance of miR-181a-mediated AC9 downregulation in APL. We also suggested the potential value of AC9 as a biomarker in the clinical diagnosis and treatment of leukemia.

Novel Treatment of Acute Promyelocytic Leukemia: As<sub>2</sub>O<sub>3</sub>, Retinoic Acid and Retinoid Pharmacology

Acute promyelocytic leukemia(APL), a specific characteristic of t(15;17) chromosome translocation, represents 5% to 15% of cases of acute nonlymphocytic leukemia. An alternative approach is to consider retinoic acid(all-trans RA, ATRA or 13-cis RA or 9-cis RA) plus chemotherapy or RA plus As₂O₃ regimens as now novel therapy. Molecular gene analyses are conclusive in vivo evidence that oncogenic PML/RARa plays a crucial role in APL leukemogenesis. As a novel approach to APL treatment, one possible the action of RA, A consense sequence (5'-TCAGGTCATGACCTGA-3') has been postulated for the thyroid hormone (TRE) and retinoic acid responsive element (RARE) containing half palindromes, which located in the promoter region of target genes. High dose (100-fold) of RA-RARE-PML/RARa complex in intracellular localization appears to relieve repressor from DNA binding, including corepressors N-CoR, SMRT and HDACs, release PML/RARa- mediated transcriptional repression, and release histone deacetylase activity from PMLRARa. The resulting PML/RARa oncoprotein proteolytic degradation through the autophagy-lysosome pathway and the ubiquitin SUMO-proteasome system (UPS), as well as caspase 3 (cleavage site Asp522 within a-helics region of PML component of the fusion protein) or neutrophil elastase, or lysosomal protease enzyme induction. PML protein relocalizes into the wild-type nuclear body (PML-NB) configuration or/and wild-type RARa upregulated. An effect to relieve the blockade (inhibition) of PML/RARA-mediated RA dependent promyelocytic differentiation, and retinoic acid in APL therapy (see Figure in the full text, George Zhu, 1991). Here, like v-erbA, PML/RARa is a (strong) transcriptional repressor of the RA receptor (RAR) complex, and PML/RARa fusion receptor gene act as conditional oncogenic receptor (translocated chimeric retinoic acid a signaling) or oncogenic PML/RARa may participate in leukemogenesis of APL through blocking RA-mediated promyelocytic differentiation. This is first described in eukaryotes.

Transcriptional Repression Of CDKN2D by PML/RARα Contributes To The Altered Proliferation and Differentiation Block Of Acute Promyelocytic Leukemia Cells

Acute promyelocytic leukemia (APL), a distinct subtype of acute myeloid leukemia, is characterized by an oncogenic fusion protein of a translocation between chromosomes 15 and 17, promyelocytic leukemia/retinoic acid receptor a (PML/RARα). PML/RARα plays an essential role in the leukemogenesis of APL by interfering with its target genes, eventually leading to a differentiation block at the promyelocytic stage and a hyperproliferation of blocked promyelocytes; both are the hallmarks of APL. It has been well-documented that cell differentiation and proliferation are two fundamental but distinct, even mutually exclusive, processes. In APL, the leukemic differentiation block is generally attributed to deregulated transcription of hematopoietic differentiation related transcription factors, RARα and PU.1, which is disrupted by PML/RARα. On the other hand, altered proliferation is usually caused by the impairment of the formation of functional PML nuclear bodies that act as a growth suppressor. There is emerging evidence to suggest that cell differentiation and proliferation can be controlled by the same regulator or pathway. Several studies have demonstrated that cell cycle regulators, especially CDK inhibitors (CKIs), are involved in the regulation of differentiation in addition to their well-documented function of controlling the cell cycle. Based on the exploration of verified PML/RARα binding sites derived from several genome-wide screening, of particular interest is CDKN2D, one of the INK4 family CKIs encoding p19INK4D. To understand the mechanisms coordinating these two processes in APL, we investigated the regulatory mechanism of the negative cell cycle regulator CDKN2D by the PML/RARα fusion protein and its role in differentiation and proliferation. We found that CDKN2D expression in APL cells was significantly lower than that in normal promyelocytes. Chromatin immunoprecipitation and luciferase reporter assays showed that PML/RARα directly bound to and inhibited the transactivation of the CDKN2D promoter. Further evidence by the truncated CDKN2D promoters revealed the localization of PML/RARa binding, which resided on the ER8 sites of the promoter. Moreover, ectopic expression of CDKN2D induced G0/G1 phase arrest and a partial granulocytic differentiation in APL-derived NB4 cells, suggesting its role in regulating both cell proliferation and granulocytic differentiation. Finally, we found that ATRA could significantly induce CDKN2D expression in APL cells. Knockdown of CDKN2D expression during ATRA treatment partially blocked the ATRA induced-differentiation and cell cycle arrest, further supporting our findings that CDKN2D expression was required for these two processes. Collectively, our data indicate that CDKN2D repression by PML/RARα disrupts both proliferation and differentiation to facilitate the pathogenesis of APL, and induced-expression of CDKN2D by ATRA alleviates the disruption of both processes to ensure treatment efficiency. This study suggests a mechanism for coupling proliferation and differentiation in leukemic cells through the action of CDKN2D. Disclosures:No relevant conflicts of interest to declare.

Expression Profiling Of Selected Polycomb Complex Genes In Childhood Acute Myeloid Leukemia Revealed An Overexpression Of EZH2 In Acute Promyelocytic Leukemia

Acute promyelocytic leukemia (APL) in children and adolescents accounts for about 20% of cases of acute myeloid leukemia (AML) in Brazil. The reasons for the relatively high incidence of APL among children and adolescents in Brazil and other Latin countries remain elusive. Epigenetic constitutional and/or environmental factors might be implicated in the mechanism of APL. The Polycomb group (PcG) genes are critical for differentiation and cell-cycle regulation and maintenance of epigenetic memory of living organisms. Aberrant expression of PcG genes has been observed in human tumors, including AML. In this study, we sought to determine the expression levels of 4 genes from the PcG repressive complexes EZH2, YY1, BMI1 and SUZ12 in acohort of 52 children with AML or APL (male, 32; female, 20; median age 7.8 years, range 4 months-18 years).Cells from healthy children (male, 2; female, 2; median age, 10.7 years, range 6-15 years) were used as the control group. Quantitative determination of mRNA levels was performed using Power SYBR Green PCR Master Mix® (Applied Biosystems, Foster City, CA, USA) in a Rotor Gene® thermocycler (QIAGEN). Expression levels were estimated in triplicate, and ß-actin was used as an internal control. All statistical analyses were performed using the GraphPad Prim 5.0 System. Multiple pairwise comparisons were made using a one-way analysis of variance (ANOVA) test; P<0.05 was considered statistically significant.Despite showing broad variation among patients and controls, the expression levels of YY1 (controls, 1.16 ±SE 0.73; M1/M2, 0.63±1.07; M4/M5 0.217±0.71; APL, 0.62±3.24; p=0.19); SUZ12 (control, 1.11 ±0.93; M1/M2, 0.12±1.65; APL 2.4±3.42; M4/M5, 0.34 ± 10.30; p=0.18), and BMI1 (control, 0.041 ± 0.77; M1/M2: 0.043 ±0.10; M4/M5, 0.016± 0.23; APL, 0.12 ± 0.59; p=0.37)did not differsignificantly between controls and patients grouped according to the major AML subtypes (M1/M2, 15 cases, median age 10.8 years, range 4-18 years; M4/M5, 21 cases,median 4.5 years, range 5 months-18 years; APL 16 cases, median 9.8 years, range, 1-17 years). However, the EZH2 expression levels (control, 0.0008818±0.03675; M1/M2, 0.001495±0.03296; M4/M5 0.008215±0.09313; APL, 0.07180± 0.3402; p=0.0092) were significantly higher in APL. Among patients with APL, the expression levels of EZH2 did not differ significantly according to age (EZH2 expression, 0.23±0.44, age 0-8 years; 0.05±0.46, age 9-18 years; p=0.112), white blood cell count (EZH2 expression 0.07±0.65, WBC < 10 x 109/L; 0.10±0.38, WBC ≥ 10 x 109/L; p=0.148) or platelet count (EZH2 expression 0.20±0.49, platelet count < 40 x109/L; 0.06 ±0.04, platelet count ≥ 40 x 109/L; p=0.13).Several studies have shown that EZH2 is deregulated in several human cancers. Here we extend this data by informing that EZH2 is highly overexpressed in pediatric APL. APL in children less than 8 years of age tends to be associated with higher EZH2 gene expression levels, suggesting that constitutional epigenetic factors may play a driver role in pediatric APL leukemogenesis. Disclosures:No relevant conflicts of interest to declare.

Characterization of arsenic-induced cytogenetic alterations in acute promyelocytic leukemia cell line, NB4

Gain or loss of genes plays important roles in leukemogenesis of APL via cooperation with PML-RARA. Fluorescence in situ hybridization (FISH) was applied to investigate the DNA copy number changes of hTERT, ERG, CDKN1B (P27), CDKN2A (P16), and TP53 genes in an acute promyelocytic leukemia (APL) cell line (NB4). Five bacterial artificial chromosome probes (BAC) for 9p21.3, 17p13.1, 12p13.2, 5p15.33, 21q22.2 regions were prepared using sequence independent amplification (SIA) and were hybridized to NB4 cells treated with different doses of arsenic trioxide (As(2)O(3); ATO) at various time intervals. NB4 cells were also karyotyped by G-banded chromosome analysis 24 h after culture initiation. FISH analysis prior to treatment showed CDKN1B, CDKN2A, and TP53 gene deletion but ERG and hTERT gene amplification. After treatment with ATO, the number of the NB4 cells with deleted CDKN1B and CDKN2A as well as the counts of the cells with hTERT amplification was significantly reduced in time- and does-dependent manners. In addition, we observed expressive increase in signal patterns of CDKN1B and CDKN2A along with significant decline in hTERT signal patterns in ATO-treated cells as compared with the control group (in time- and dose-dependent manners). On the other hand, no difference in signal patterns for Erg and p53 was observed in response to ATO exposure. The results of the present study show the cytogenetic alteration in hTERT, CDKN1B, and CDKN2A in NB4 cells after treatment with ATO might introduce a new mechanism of antitumor activities of ATO in APL cell line, NB4.

Positive evidence for a role of the PML pathway in leukemogenesis of acute promyelocytic leukemia

Acute Promyelocytic Leukemia is characterized by the production of oncogenic Retinoic acid receptor alpha (RARα) fusion proteins. The most frequently observed fusion protein is PML-RAR (PR). All fusion partners of RARα contribute an oligomerization motif. Previously, we have shown that replacement of PML with an unrelated oligomerization domain renders RARα oncogenic, albeit also reduces its potency. Here we have, conversely, attempted to create a more oncogenic RARα fusion protein. To do so, we fused the corresponding PML domain of murine PML to RARα. As PML, different from RARα, is only moderately conserved between mouse and man, we reasoned, that the biological potency might be suboptimal when human PR is used in the mouse. This prompted us to design a RAR fusion protein with the corresponding mouse PML homologue (mPR). Indeed, in the mouse system, mPR proved more potent than the human PR. We observed this both in in-vitro studies as well as in bone marrow transduction/transplantation experiments in mice.