Acute Promyelocytic Leukemia Differentiation Research Articles

HSPA8 Chaperone Complex Drives Chaperone-Mediated Autophagy Regulation in Acute Promyelocytic Leukemia Cell Differentiation

Introduction: Acute myeloid leukemia (AML) is a cancer of the hematopoietic system characterized by hyperproliferation of undifferentiated cells of the myeloid lineage. While most of AML therapies are focused toward tumor debulking, all-trans retinoic acid (ATRA) induces neutrophil differentiation in the AML subtype acute promyelocytic leukemia (APL). Macroautophagy has been extensively investigated in the context of various cancers and is often dysregulated in AML where it can have context-dependent pro- or anti-leukemogenic effects. On the contrary, the implications of chaperone-mediated autophagy (CMA) on the pathophysiology of diseases are still being explored and its role in AML remains elusive. Methods: We took advantage of human AML primary samples and databases to analyze CMA gene expression and activity. Furthermore, we used ATRA-sensitive (NB4) and -resistant (NB4-R1) APL cells to further dissect a potential function for CMA in ATRA-mediated neutrophil differentiation. NB4-R1 cells are unique in that they do respond to retinoic acid transcriptionally but do not mature in response to retinoid signaling alone unless maturation is triggered by adding cyclic adenosine monophosphate. Results: Here, we report that CMA-related mRNA transcripts are significantly higher expressed in immature hematopoietic cells as compared to neutrophils, contrasting the macroautophagy gene expression patterns. Accordingly, lysosomal degradation of an mCherry-KFERQ CMA reporter decreases during ATRA-induced differentiation of APL cells. On the other hand, using NB4-R1 cells we found that macroautophagy flux primed ATRA-resistant NB4-R1 cells to differentiate upon ATRA treatment but reduced the association of lysosome-associated membrane protein type 2A (LAMP-2A) and heat shock protein family A (Hsp70) member 8 (HSPA8), necessary for complete neutrophil maturation. Accordingly, depletion of HSPA8 attenuated CMA activity and facilitated APL cell differentiation. In contrast, maintaining high CMA activity by ectopic expression of LAMP-2A impeded APL differentiation. Conclusion: Overall, our findings suggest that APL neutrophil differentiation requires CMA inactivation and that this pathway predominantly depends on HSPA8 and is possibly assisted by other co-chaperones.

USP22 regulates APL differentiation via PML-RARα stabilization and IFN repression

Ubiquitin-specific peptidase 22 (USP22) is a deubiquitinating enzyme (DUB) that underlies tumorigenicity, proliferation, cell death and differentiation through deubiquitination of histone and non-histone targets. Ubiquitination determines stability, localization and functions of cell fate proteins and controls cell-protective signaling pathways to surveil cell cycle progression. In a variety of carcinomas, lymphomas and leukemias, ubiquitination regulates the tumor-suppressive functions of the promyelocytic leukemia protein (PML), but PML-specific DUBs, DUB-controlled PML ubiquitin sites and the functional consequences of PML (de)ubiquitination remain unclear. Here, we identify USP22 as regulator of PML and the oncogenic acute promyelocytic leukemia (APL) fusion PML-RARα protein stability and identify a destabilizing role of PML residue K394. Additionally, loss of USP22 upregulates interferon (IFN) and IFN-stimulated gene (ISG) expression in APL and induces PML-RARα stabilization and a potentiation of the cell-autonomous sensitivity towards all-trans retinoic acid (ATRA)-mediated differentiation. Our findings imply USP22-dependent surveillance of PML-RARα stability and IFN signaling as important regulator of APL pathogenesis, with implications for viral mimicry, differentiation and cell fate regulation in other leukemia subtypes.

NLRP3 participates in the differentiation and apoptosis of PMA‑treated leukemia cells.

Acute myeloid leukemia (AML) is a clonal malignant proliferative disease. In recent years, with the use of all‑trans retinoic acid to induce cancer cell differentiation in acute promyelocytic leukemia, and its advantages of high efficacy and low toxic side effects, tumor differentiation therapy has become a research hotspot; however, the mechanisms underlying its role remain to be fully established. Nod‑like receptor family pyridine domain containing 3 (NLRP3) is the most extensively studied and well‑characterized inflammasome, is involved in a variety of inflammation‑related diseases, including cancer, and is a very attractive potential target for the study of novel therapeutic agents. Activation of the NLRP3 inflammasome is a double‑edged sword in tumor therapy, with evidence of protective anti‑tumor and pro‑tumor effects in different types of cancer. Whether the NLRP3 inflammasome promotes disease progression or exerts a protective anti‑tumor effect in hematological malignancies remains contested. In the present study, the protective anti‑tumor effects of NLRP3 on leukemia cells during their differentiation and maturation were investigated. It was found that the upregulation of NLRP3 expression induced using Phorbol 12‑Myristate 13‑Acetate played a role in promoting the differentiation and maturation of leukemia cells into monocytes/macrophages, and it was directly involved in the apoptosis of leukemia cells and the differentiation and maturation of CD11b+ cells. These results provide novel theoretical evidence for exploring the mechanism of differentiation therapy in leukemia and improves our understanding of the role of NLRP3 in hematologic tumors.

ATRA treatment slowed P-selectin-mediated rolling of flowing HL60 cells in a mechano-chemical-dependent manner.

All-trans retinoic acid (ATRA)-induced differentiation of acute promyelocytic leukemia (APL) toward granulocytes may trigger APL differentiation syndrome (DS), but there is less knowledge about the mechano-chemical regulation mechanism of APL DS under the mechano-microenvironment. We found that ATRA-induced changes in proliferation, morphology, and adhesive molecule expression levels were either dose or stimulus time dependent. An optimal ATRA stimulus condition for differentiating HL60 cells toward neutrophils consisted of 1 × 10-6 M dose and 120h of stimulus time. Under wall shear stresses, catch-slip bond transition governs P-selectin-mediated rolling for neutrophils and untreated or ATRA-treated (1 × 10-6 M, 120h) HL60 cells. The ATRA stimuli slowed down the rolling of HL60 cells on immobilized P-selectin no matter whether ICAM-1 was engaged. The β2 integrin near the PSGL-1/P-selectin axis would be activated within sub-seconds for each cell group mentioned above, thus contributing to slow rolling. A faster β2 integrin activation rate and the higher expression levels of PSGL-1 and LFA-1 were assigned to induce the over-enhancement of ATRA-treated HL60 adhesion in flow, causing APL DS development. These findings provided an insight into the mechanical-chemical regulation for APL DS development via ATRA treatment of leukemia and a novel therapeutic strategy for APL DS through targeting the relevant adhesion molecules.

Safety and Efficacy of Arsenic Trioxide in the Treatment of Newly Diagnosed Pediatric Acute Promyelocytic Leukemia: Results from the JPLSG AML-P13 Study

Aims: Arsenic trioxide (ATO) has shown to be effective in patients with acute promyelocytic leukemia (APL), which has led to a dose reduction of cytotoxic agents in the treatment of APL. To investigate safety and efficacy of ATO in Japanese children with APL, we conducted a nationwide prospective study, JPLSG AML-P13 (jRCTs051180191). Patients & Methods: Between December 2014 and November 2018, children (0-18 years old) with newly diagnosed APL were enrolled in the AML-P13 study. All patients received two courses of induction therapy consisting of ATRA, cytarabine, and daunorubicin. Patients who achieved hematological complete remission (HCR) were allocated to the standard risk (SR) group and received three intensification cycles consisting of ATRA and ATO, followed by intermittent ATRA maintenance therapy. Patients who did not obtain HCR after induction courses were allocated to the high risk (HR) group and received intensified schedule of ATO, followed by gemtuzumab ozogamicin monotherapy and maintenance therapy consisting of 6-mercaptopurine and methotrexate. Results & Discussions: Among the 32 patients registered, 5 patients were excluded due to a lack of PML::RARA fusion or other reasons, and the remaining 27 patients received the protocol therapy (full analysis set). During induction courses, grade 3 disseminated intravascular coagulation was observed in 9 patients (33.3%). APL differentiation syndrome and induction death were not reported. Two patients were taken off protocol therapy due to a progressive disease (regarded as an event) and a safety issue, respectively. Among the 25 patients who completed induction therapy, 23 patients obtained HCR. During intensification cycles of ATO, grade 3 or higher non-hematological adverse events (AEs) were observed in 3 patients: prolonged QTc interval in 2 and dysesthesia in one. Regarding hematological AEs, 10 to 30% patients exhibited grade 4 neutropenia during each intensification cycles, but no patients experienced severe infectious complications. Two patients who did not obtain HCR received HR regimen without any apparent AEs. All 25 patients including 2 HR patients achieved molecular remission at the end of intensification and molecular remission rate was 100.0% [95% C.I. 86.3-100.0%]. No patients suffered from relapse, and 3-year EFS rate and 3-year OS rate in full analysis set were 96.3% [76.5-99.5%] (Figure) and 100.0% [87.2-100.0%], respectively. We previously reported an inferior outcome of younger children (less than 5 years old) treated with conventional ATRA and chemotherapy in the JPLSG AML-P05 study (Takahashi H. Br J Haematol 2016). However, 4 children under 5 years of age at diagnosis in the AML-P13 study remains in molecular remission, indicating efficacy of ATO in this age group of children. Interpretations: The AML-P13 study demonstrated an excellent outcome in children with APL and showed that ATO could be administered safely in children with APL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A scientometric and visualization analysis of studies on arsenic for acute promyelocytic leukemia

The use of arsenic for acute promyelocytic leukemia (APL) has a long history. However, a systematic scientometric study to describe these studies and to explore their research status and development trends has not been carried out. CNKI and Web of Science databases were searched from their inception to June 13, 2021 to identify suitable articles. Knowledge network maps of identified studies focusing on authors, institutions, keywords and their references were summmarised. 3095 articles published between 1996 and 2021 showed an overall fluctuating trend in growth of articles. Main institutions conducting research were Harbin Medical University and Shanghai Jiaotong University School of Medicine. The main research team working outside of China was located at the Sloan Kettering Cancer Center. Research hotspots included efficacy evaluation of arsenic alone or in combination with retinoic acid, mechanism study of apoptosis, RARα, activation, growth inhibition, expression etc. The current research hot topics were APL differentiation syndrome and drug resistance. Chinese patent medicine compound Huangdai tablets, arsenous acid and all-trans retinoic acid were the main drugs. The types of highly cited and burst studies were landmark case series, well-designed RCTs, comprehensive reviews, and authoritative APL guidelines. Articles on the use of arsenic for APL have fluctuated in growth since 1996. The themes have focused on clinical efficacy and mechanism of arsenic and its action. The APL differentiation syndrome and drug resistance are disease research hotspots. The compound Huangdai tablets, arsenious acid and all-trans retinoic acid are potentially the main focus for drugs now and in the future.

Acute Myeloid Leukemia - Update 2022

The suspicion of acute myeloid leukemia (AML) is a haematological emergency that requires a rapid diagnostic workup. Symptoms are usually caused by cytopenias of all blood cell lines. The differentiation of acute promyelocytic leukemia (APL) is important because of the early death rate caused by thrombembolic and bleeding events. Rapid immunophenotypic and genetic characterization is necessary for risk stratification and therapy selection. For this purpose, a center with appropriate expertise should be contacted. Therapy has become more complex due to numerous new approvals. For certain patients, the established intensive induction therapy with cytarabine and anthracycline is now combined with targeted agents, like the antibody conjugate Gemtuzumab-Ozogamicin or the FLT3 inhibitor Midostaurin. Patients with secondary AML benefit from the liposomal chemotherapy combination CPX-351. Therapy with the hypomethylating agent Azacitidine and the BCL2-inhibitor Venetoclax (Aza/Ven) represents the standard for patients who are not fit for intensive therapy. Here, it is important to consider interactions with CYP3A4-effective drugs.In most cases, APL is treated "chemotherapy-free" with all-trans retinoic acid (ATRA) and arsenic trioxide (ATO). In high-risk patients, the combination of chemotherapy and ATRA is still standard.Moreover, maintenance therapies were (re)established as an important therapeutic element of post-remission therapy. For example, Midostaurin is used in patients with FLT3 mutations, as is the multikinase inhibitor sorafenib after allogeneic stem cell transplantation. In addition, oral azacitidine is available for non-allogeneic transplant eligible patients in first complete remission. These new drugs have improved prognosis and resulted in a more individualized therapy mostly driven by genetic aberrations. This development will continue in the next years and will significantly improve treatment options, especially for older patients.

Co-targeting leukemia-initiating cells and leukemia bulk leads to disease eradication.

According to a hierarchical model, targeting leukemia-initiating cells (LICs) was speculated to achieve complete remission (CR) or cure. Nonetheless, increasing evidence emphasized the plasticity of differentiated blasts undergoing interconversion into LICs. We exploited murine models of acute promyelocytic leukemia (APL), a subtype of acute myeloid leukemia driven by the promyelocytic leukemia/retinoic acid receptor (PML-RARα) oncofusion protein, which recruits histone deacetylase (HDAC)-containing complexes. We studied APLs with different LIC frequencies and investigated the effect of two HDAC inhibitors: valproic acid (VPA), with relative selectivity towards class I HDAC enzymes and vorinostat/suberoylanilide hydroxamic acid (SAHA) (pan-HDAC inhibitor) in combination with all-trans retinoic acid (ATRA), on the bulk APL cells and APL LICs. Indeed, we found that while VPA differentiates the bulk APL cells, SAHA selectively targets LICs. ATRA + VPA + SAHA combination efficiently induced CR in an APL model with lower LIC frequency. Substituting ATRA with synthetic retinoids as etretinate which promotes APL differentiation without downregulating PML/RARα compromised the therapeutic benefit of ATRA + VPA + SAHA regimen. Altogether, our study emphasizes the therapeutic power of co-targeting the plasticity and heterogeneity of cancer -herein demonstrated by tackling LICs and bulk leukemic blasts - to achieve and maintain CR.

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

AbstractEpigenetic 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.

Overexpression of KMT2E Is Associated with Higher Granulocytic Differentiation Rate in Response to ATRA-Based Treatment in Acute Promyelocytic Leukemia Cell Lines

Introduction: In the clinical setting, low KMT2E expression has been associated with poor prognosis in both acute myeloid leukemia (Damm et al. J Clin Oncol. 2011 Feb 20;29(6):682-9) and acute promyelocytic leukemia (APL) (Lucena-Araujo et al. Br J Haematol. 2014 Aug;166(4):540-9). Member of the Trithorax-group, the KMT2E is implicated in the positive transcriptional control of a specific set of genes related to hematopoiesis and differentiation processes in many cell types. Functional analyses showed that the encoded protein is involved in terminal myeloid differentiation and facilitate retinoic-acid induced granulopoiesis in human promyelocytes. Using lentiviral gene transfer, we hypothesized that the forced expression of KMT2E is implicated in the ATRA-induced granulocytic differentiation in APL. Aims: To investigate the effects of overexpression of KMT2E gene in cell viability and signaling in response to ATRA plus anthracycline or arsenic trioxide (ATO) treatment in APL cell lines. Methods: NB4 and NB4-R2 (resistant to ATRA treatment) cell lines were transduced with pMEG or pMEG- KMT2E lentiviral vectors and cultured with stable selection using puromycin. Before all functional experiments, cells were tested for KMT2E gene and protein expression by qPCR and confocal microscopy, respectively. The cells transduced with KMT2E and control cells were evaluated for cell viability (MTT assay), proliferation (Ki-67 staining), cell cycle (flow cytometry), mitochondrial reactive oxygen species (ROS) production (flow cytometry) in basal conditions and under treatment with ATRA (1 μM), ATO (0.187, 0.375, 0.75, 1.5 and 3 μM), and the association of ATO (1 μM) plus ATRA (1 μM) or cytarabine (150 nM). The granulocytic differentiation in response to ATRA treatment was evaluated based on the CD11b expression (flow cytometry) plus morphological analysis (Leishman staining). The expression of the main target genes (p21, MDM2, and BMI1) encoding for proteins known to be involved in KMT2E function were evaluated by qPCR. Results: Overexpression of KMT2E was significantly associated with higher cell proliferation rates in basal or ATO and cytarabine treatment compared to vehicle treated cells (all P<.001). KMT2E overexpression significantly increase cell proliferation in NB4, but not in NB4-R2 cells (Ki-67 mean intensity of fluorescence (MFI) value - pMEG: 489±10 vs KMT2E : 1432±25; P<.001). The distribution of cells in the phases of the cell cycle was similar between groups. Using a nonlinear regression analysis, IC50 for ATO was of 2.96 and 6 μM for NB4 pMEG and KMT2E transduced cells, respectively. NB4- KMT2E cells showed lower levels of ROS generation upon ATO treatment (1.5 μM) compared to controls (ROS - MFI value - pMEG: 270.6±6 vs KMT2E : 84±11; P <.001). In the attempt to assess the role of KMT2E in granulocytic differentiation, NB4- KMT2E cells were treated with ATRA 1 μM. The cells containing an extra copy of KMT2E gene showed more differentiation rate in 72, 96 and 120 hours compared to empty vector transduced cells (all p<.05). Considering the expression of target genes of KMT2E, only p21 showed an increase in KMT2E cells treated with ATRA. Conclusion: In summary, the overexpression of KMT2E is associated with higher proliferation capacity and increased resistance to ATO treatment, associated with less ROS generation. DisclosuresNo relevant conflicts of interest to declare.

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.

Induced lineage promiscuity undermines the efficiency of all-trans-retinoid-acid-induced differentiation of acute myeloid leukemia

SummaryAll-trans retinoid acid (ATRA) can induce terminal differentiation of acute promyelocytic leukemia (APL), also known as the M3 subtype of acute myeloid leukemia (AML). However, non-APL types of AML respond poorly to ATRA-induced differentiation, and the mechanism underlying cell-type-specific resistance against ATRA remains unclear. Here, we use single-cell transcriptome analysis to compare the differentiation trajectories of two AML cell types during ATRA treatment. We show that in NB4 (APL/AML-M3) cells, ATRA activates canonical myeloid lineage factors—including SPI1, CEBPE, and STAT1—to direct near-normal differentiation toward mature granulocytes. By contrast, in HL60 (AML-M2) cells, ATRA-induced differentiation is incomplete and promiscuous, which is characterized by coinduction of both myelopoiesis and lymphopoiesis gene expression programs, as well as transient activation of cis-regulatory elements associated with myeloid differentiation. Our study suggests that the differentiation inducing capacity of ATRA in certain subtypes of AML may be compromised by therapy-induced lineage promiscuity.

Reducing FASN expression sensitizes acute myeloid leukemia cells to differentiation therapy

Fatty acid synthase (FASN) is the only human lipogenic enzyme available for de novo fatty acid synthesis and is often highly expressed in cancer cells. We found that FASN mRNA levels were significantly higher in acute myeloid leukemia (AML) patients than in healthy granulocytes or CD34+ hematopoietic progenitors. Accordingly, FASN levels decreased during all-trans retinoic acid (ATRA)-mediated granulocytic differentiation of acute promyelocytic leukemia (APL) cells, partially via autophagic degradation. Furthermore, our data suggest that inhibition of FASN expression levels using RNAi or (-)-epigallocatechin-3-gallate (EGCG) accelerated the differentiation of APL cell lines and significantly re-sensitized ATRA refractory non-APL AML cells. FASN reduction promoted translocation of transcription factor EB (TFEB) to the nucleus, paralleled by activation of CLEAR network genes and lysosomal biogenesis. Together, our data demonstrate that inhibition of FASN expression in combination with ATRA treatment facilitates granulocytic differentiation of APL cells and may extend differentiation therapy to non-APL AML cells.

Dual DNA and protein tagging of open chromatin unveils dynamics of epigenomic landscapes in leukemia.

The architecture of chromatin regulates eukaryotic cell states by controlling transcription factor access to sites of gene regulation. Here we describe a dual transposase/peroxidase approach, integrative DNA And Protein Tagging (iDAPT), which detects both DNA (iDAPT-seq) and protein (iDAPT-MS) associated with accessible regions of chromatin. In addition to direct identification of bound transcription factors, iDAPT enables the inference of their gene regulatory networks, protein interactors, and regulation of chromatin accessibility. We applied iDAPT to profile the epigenomic consequences of granulocytic differentiation of acute promyelocytic leukemia, yielding previously undescribed mechanistic insights. Our findings demonstrate the power of iDAPT as a platform for studying the dynamic epigenomic landscapes and their transcription factor components associated with biological phenomena and disease.

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.

Transcriptional and Metabolic Dissection of ATRA-Induced Granulocytic Differentiation in NB4 Acute Promyelocytic Leukemia Cells.

Acute promyelocytic leukemia (APL) is a hematological disease characterized by a balanced reciprocal translocation that leads to the synthesis of the oncogenic fusion protein PML-RARα. APL is mainly managed by a differentiation therapy based on the administration of all-trans retinoic acid (ATRA) and arsenic trioxide (ATO). However, therapy resistance, differentiation syndrome, and relapses require the development of new low-toxicity therapies based on the induction of blasts differentiation. In keeping with this, we reasoned that a better understanding of the molecular mechanisms pivotal for ATRA-driven differentiation could definitely bolster the identification of new therapeutic strategies in APL patients. We thus performed an in-depth high-throughput transcriptional profile analysis and metabolic characterization of a well-established APL experimental model based on NB4 cells that represent an unevaluable tool to dissect the complex mechanism associated with ATRA-induced granulocytic differentiation. Pathway-reconstruction analysis using genome-wide transcriptional data has allowed us to identify the activation/inhibition of several cancer signaling pathways (e.g., inflammation, immune cell response, DNA repair, and cell proliferation) and master regulators (e.g., transcription factors, epigenetic regulators, and ligand-dependent nuclear receptors). Furthermore, we provide evidence of the regulation of a considerable set of metabolic genes involved in cancer metabolic reprogramming. Consistently, we found that ATRA treatment of NB4 cells drives the activation of aerobic glycolysis pathway and the reduction of OXPHOS-dependent ATP production. Overall, this study represents an important resource in understanding the molecular “portfolio” pivotal for APL differentiation, which can be explored for developing new therapeutic strategies.

NTAL is associated with treatment outcome, cell proliferation and differentiation in acute promyelocytic leukemia

Non-T cell activation linker (NTAL) is a lipid raft-membrane protein expressed by normal and leukemic cells and involved in cell signaling. In acute promyelocytic leukemia (APL), NTAL depletion from lipid rafts decreases cell viability through regulation of the Akt/PI3K pathway. The role of NTAL in APL cell processes, and its association with clinical outcome, has not, however, been established. Here, we show that reduced levels of NTAL were associated with increased all-trans retinoic acid (ATRA)-induced differentiation, generation of reactive oxygen species, and mitochondrial dysfunction. Additionally, NTAL-knockdown (NTAL-KD) in APL cell lines led to activation of Ras, inhibition of Akt/mTOR pathways, and increased expression of autophagy markers, leading to an increased apoptosis rate following arsenic trioxide treatment. Furthermore, NTAL-KD in NB4 cells decreased the tumor burden in (NOD scid gamma) NSG mice, suggesting its implication in tumor growth. A retrospective analysis of NTAL expression in a cohort of patients treated with ATRA and anthracyclines, revealed that NTAL overexpression was associated with a high leukocyte count (P = 0.007) and was independently associated with shorter overall survival (Hazard Ratio: 3.6; 95% Confidence Interval: 1.17–11.28; P = 0.026). Taken together, our data highlights the importance of NTAL in APL cell survival and response to treatment.

RIG-I regulates myeloid differentiation by promoting TRIM25-mediated ISGylation

Retinoic acid-inducible gene I (RIG-I) is up-regulated during granulocytic differentiation of acute promyelocytic leukemia (APL) cells induced by all-trans retinoic acid (ATRA). It has been reported that RIG-I recognizes virus-specific 5'-ppp-double-stranded RNA (dsRNA) and activates the type I interferons signaling pathways in innate immunity. However, the functions of RIG-I in hematopoiesis remain unclear, especially regarding its possible interaction with endogenous RNAs and the associated pathways that could contribute to the cellular differentiation and maturation. Herein, we identified a number of RIG-I-binding endogenous RNAs in APL cells following ATRA treatment, including the tripartite motif-containing protein 25 (TRIM25) messenger RNA (mRNA). TRIM25 encodes the protein known as an E3 ligase for ubiquitin/interferon (IFN)-induced 15-kDa protein (ISG15) that is involved in RIG-I-mediated antiviral signaling. We show that RIG-I could bind TRIM25 mRNA via its helicase domain and C-terminal regulatory domain, enhancing the stability of TRIM25 transcripts. RIG-I could increase the transcriptional expression of TRIM25 by caspase recruitment domain (CARD) domain through an IFN-stimulated response element. In addition, RIG-I activated other key genes in the ISGylation pathway by activating signal transducer and activator of transcription 1 (STAT1), including the modifier ISG15 and several enzymes responsible for the conjugation of ISG15 to protein substrates. RIG-I cooperated with STAT1/2 and interferon regulatory factor 1 (IRF1) to promote the activation of the ISGylation pathway. The integrity of ISGylation in ATRA or RIG-I-induced cell differentiation was essential given that knockdown of TRIM25 or ISG15 resulted in significant inhibition of this process. Our results provide insight into the role of the RIG-I-TRIM25-ISGylation axis in myeloid differentiation.

Tetrandrine Induces HL-60 Cell Differentiation

To investigate the differentiation of acute promyelocytic leukemia (APL) cells induced by Tetrandrine targeting MUC1. HL-60 cells at logarithmic growth phase were choosen as object of reaserch and were divided into four groups: control, ATRA, Tetrandrine and ATRA+Tetrandrine group. Cell morphologic changes in each group were observed by microscopy. The change of cell differentiation ability was analyzed by nitro-blue tetrazolium (NBT) reduction test. The expression of cell surface differentiation antigen CD11b was measured by flow cytometry. The expression of MUC1 protein was detected by Western blot. The differentiation of HL-60 cell could be induced by Tetrandrine. The NBT reduction-positive rate of ATRA+Tetrandrine group was significantly higher than that in ATRA group and Tetrandrine group (P<0.05). The percentage of CD11b positive cells in ATRA+Tetrandrine group(43.62％±1.38％) was significantly higher than that in Tetrandrine group(15.25％±2.11％), ATRA group (28.84％±7.53％) and control group(8.16％±1.01％) (P<0.05). The content of MUC1 protein in Tetrandrine group was significantly lower than that in control group and ATRA group (P<0.05). Tetrandrine and ATRA can synergize to promote the differentiation and maturation of HL-60 cells, and the mechanism may be related with MUC1 expression.