Differentiation Of Acute Promyelocytic Leukemia Cells Research Articles

ATPR induces acute promyelocytic leukemia cells differentiation and cycle arrest via the lncRNA CONCR/DDX11/PML-RARα signaling axis

Acute promyelocytic leukemia (APL) is a type of acute myeloid leukemia (AML) with a high mortality rate, and the production of PML-RARα fusion protein is the cause of its pathogenesis. Our group has synthesized a novel compound, 4-amino-2-trifluoromethyl-phenyl retinate (ATPR), by structural modification of All-trans retinoic acid (ATRA), which has strong cell differentiation-inducing effects and inhibits the expression of PML-RARα. In this study, acute promyelocytic leukemia NB4 cells before and after ATPR induction were analyzed by whole transcriptome microarray, and the expression of lncRNA CONCR was found to be significantly downregulated. The role of CONCR in ATPR-induced cell differentiation and cycle arrest was explored through overexpression and silencing of CONCR. And then the database was used to predict that CONCR may bind to DEAD/H-Box Helicase 11 (DDX11) protein to further explore the role of CONCR binding to DDX11. The results showed that ATPR could reduce the expression of CONCR, and overexpression of CONCR could reverse the ATPR-induced cell differentiation and cycle blocking effect, and conversely silencing of CONCR could promote this effect. RNA immunoprecipitation (RIP) experiments showed that CONCR could bind to DDX11, the protein expression levels of DDX11 and PML-RARα were elevated after overexpression of CONCR. These results suggest that ATPR can regulate the expression of DDX11 through CONCR to affect the expression of PML-RARα fusion protein, which in turn induces the differentiation and maturation of APL cells.

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.

Overexpression of CASP1 triggers acute promyelocytic leukemia cell pyroptosis and differentiation

Caspase-1 (CASP1)-mediated classical pyroptosis plays a key role in cancer development and management, however, the role of CASP1 and its regulation has not yet been documented for acute promyelocytic leukemia (APL). Here, we found that CASP1/GSDMD had lower expression in patients with APL and most other subtypes of primary de novo acute myeloid leukemia (AML) and was increased in all-trans-retinoic acid (ATRA)-treated APL cells. We showed that ATRA increases and activates CASP1 to trigger the pyroptosis and differentiation of APL cells. Mechanistically, ATRA could induce CASP1 expression via the IFNγ/STAT1 pathway in APL cells. In conclusion, ATRA-induced activation of CASP1 may serve as a suppressor in APL progression, as it triggers pyroptotic cell death and differentiation.

Roles of PADI4 in the expression of cytokines involved in inflammation and adhesion in differentiated NB4 cells treated with ATRA.

Differentiation syndrome (DS) is a common complication in patients with acute promyelocytic leukemia (APL) treated with all-trans-retinoic acid (ATRA). However, the target of ATRA during DS in patients with APL remains to be elucidated. Therefore, the current study aimed to investigate the role of peptidylarginine deiminase 4 (PADI4) in the differentiation of ATRA-induced NB4 APL cells. The results showed that PADI4 was significantly upregulated in peripheral blood samples derived from patients with APL DS compared with patients with APL only. In addition, whether ATRA could enhance the expression levels of PADI4 in NB4 cells in vitro was subsequently investigated. The results also showed that PADI4 overexpression promoted the differentiation of NB4 cells treated with ATRA, which was reversed after PADI4 silencing. To uncover the potential mechanisms underlying the above process, PADI4 overexpression induced the secretion of inflammation-related cytokines, such as TNF-α, IL-1β, IL-8, C-C motif chemokine (CCL)2, CCL4, CCR1 and intercellular adhesion molecule-1 in ATRA-treated NB4 cells. However, PADI4 knockdown in the same cells had the opposite effect. The above findings indicated that PADI4 could be involved in the differentiation of ATRA-induced NB4 cells and upregulation of cytokines.

Targeting HDAC3 to Overcome the Resistance to ATRA or Arsenic in Acute Promyelocytic Leukemia through Ubiquitination and Degradation of PML-RARα

Acute promyelocytic leukemia (APL) is driven by the oncoprotein PML-RARα, which recruits corepressor complexes, including histone deacetylases (HDACs), to suppress cell differentiation and promote APL initiation. All-trans retinoic acid (ATRA) combined with arsenic trioxide (ATO) or chemotherapy highly improves the prognosis of APL patients. However, refractoriness to ATRA and ATO may occur, which leads to relapsed disease in a group of patients. In this study, we obtained the relationship between different HDACs with APL by analyzing transcriptional dataset of leukemia and confirmed the positive association of HDAC3 with PML-RARα in the mRNA and protein levels measured in primary human APL blasts. Experiments in vitro indicated that targeting HDAC3 but not HDAC1 or HDAC2 induced more effective differentiation and apoptosis in both NB4 cells and primary human APL cells. To examine the effects of HDAC3 inhibition on APL cells in vivo, we utilized normal FVB mice transplanted with APL cells from hMRP8-PML-RARα transgenic mice and NB4 or NB4-HDAC3Cas9 xenograft mouse model. We found that pharmacological or genetically inhibition of HDAC3 extended the survival rate of mice and alleviated spleen infiltration of leukemia cells and promoted the apoptosis and differentiation of APL cells. As to the molecular mechanism, we first examined whether HDAC3 affected PML-RARα expression level and the results demonstrated that HDAC3 inhibition reduced PML-RARα protein half-life rather than transcriptional activity via the ubiquitin proteasome system. In the subsequent experiments, we constructed a series of PML-RARα mutations. By confocal imaging system, acetylation and protein-protein interaction assays, we demonstrated that HDAC3 directly bound to the LBD and DBD domains of RARA protein and modulated the acetylation of lysine 394 of PML, which reduced PIAS1-mediated PML-RARα SUMOylation and subsequent RNF4-induced ubiquitylation. HDAC3 inhibition induced an increase of K394 acetylation, and promoted PML-RARα ubiquitylation and subsequent degradation, while the effect could be rescued by proteosome inhibition. Our results further revealed that the inhibition of HDAC3 induced degradation of both wild-type and ATRA- or ATO-resistant mutant PML-RARα (A216V, L218P, ΔF286 and R276Q). Therefore, we speculated that targeting HDAC3 could induce antileukemic effects on ATO- or ATRA-resistant APL cells. In NB4-R1, NB4-R2 and primary human APL cells, inhibition of HDAC3 increased significantly the cell differentiation and apoptosis, decreased PML-RARα expression and reduced the proliferation and colony-formation units (CFUs). For experiments in vivo, ATRA-resistant APL xenograft mouse model of NB4-R2 cells and patient-derived tumor xenograft (PDX) model were utilized to evaluate the effects of targeting HDAC3. Results showed that targeting HDAC3 extended the survival of mice, decreased the leukemia cells infiltration and increased the differentiation and apoptotic cells. In conclusion, our study identifies the role of HDAC3 as a positive regulator of the PML-RARα oncoprotein by deacetylating PML-RARα and suggests that targeting HDAC3 could be a promising strategy to treat relapsed/refractory APL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A novel network pharmacology approach for leukaemia differentiation therapy using Mogrify®.

Acute myeloid leukaemia (AML) is a rapidly fatal blood cancer that is characterised by the accumulation of immature myeloid cells in the blood and bone marrow as a result of blocked differentiation. Methods which identify master transcriptional regulators of AML subtype-specific leukaemia cell states and their combinations could be critical for discovering novel differentiation-inducing therapies. In this proof-of-concept study, we demonstrate a novel utility of the Mogrify® algorithm in identifying combinations of transcription factors (TFs) and drugs, which recapitulate granulocytic differentiation of the NB4 acute promyelocytic leukaemia (APL) cell line, using two different approaches. In the first approach, Connectivity Map (CMAP) analysis of these TFs and their target networks outperformed standard approaches, retrieving ATRA as the top hit. We identify dimaprit and mebendazole as a drug combination which induces myeloid differentiation. In the second approach, we show that genetic manipulation of specific Mogrify®-identified TFs (MYC and IRF1) leads to co-operative induction of APL differentiation, as does pharmacological targeting of these TFs using currently available compounds. We also show that loss of IRF1 blunts ATRA-mediated differentiation, and that MYC represses IRF1 expression through recruitment of PML-RARα, the driver fusion oncoprotein in APL, to the IRF1 promoter. Finally, we demonstrate that these drug combinations can also induce differentiation of primary patient-derived APL cells, and highlight the potential of targeting MYC and IRF1 in high-risk APL. Thus, these results suggest that Mogrify® could be used for drug discovery or repositioning in leukaemia differentiation therapy for other subtypes of leukaemia or cancers.

PRMT5-mediated RNF4 methylation promotes therapeutic resistance of APL cells to As2O3 by stabilizing oncoprotein PML-RARα.

Acute promyelocytic leukemia (APL) is a hematological malignancy driven by the oncoprotein PML-RARα, which can be treated with arsenic trioxide (As2O3) or/and all-trans retinoic acid. The protein arginine methyltransferase 5 (PRMT5) is involved in tumorigenesis. However, little is known about the biological function and therapeutic potential of PRMT5 in APL. Here, we show that PRMT5 is highly expressed in APL patients. PRMT5 promotes APL by interacting with PML-RARα and suppressing its ubiquitination and degradation. Mechanistically, PRMT5 attenuates the interaction between PML-RARα and its ubiquitin E3 ligase RNF4 by methylating RNF4 at Arg164. Notably, As2O3 treatment triggers the dissociation of PRMT5 from PML nuclear bodies, attenuating RNF4 methylation and promoting RNF4-mediated PML-RARα ubiquitination and degradation. Moreover, knockdown of PRMT5 and pharmacological inhibition of PRMT5 with the specific inhibitor EPZ015666 significantly inhibit APL cells growth. The combination of EPZ015666 with As2O3 shows synergistic effects on As2O3-induced differentiation of bone marrow cells from APL mice, as well as on apoptosis and differentiation of primary APL cells from APL patients. These findings provide mechanistic insight into the function of PRMT5 in APL pathogenesis and demonstrate that inhibition of PRMT5, alone or in combination with As2O3, might be a promising therapeutic strategy against APL.

All-Trans-Retinoic Acid Combined With Valproic Acid Can Promote Differentiation in Myeloid Leukemia Cells by an Autophagy Dependent Mechanism.

Acute myeloid leukemia (AML) is an aggressive blood cancer with an overall survival of 30%. One form of AML, acute promyelocytic leukemia (APL) has become more than 90% curable with differentiation therapy, consisting of all-trans-retinoic acid (ATRA) and arsenic trioxide (ATO). Application of differentiation therapy to other AML subtypes would be a major treatment advance. Recent studies have indicated that autophagy plays a key role in the differentiation of ATRA-responsive APL cells. In this study, we have investigated whether differentiation could be enhanced in ATRA resistant cells by promoting autophagy induction with valproic acid (VPA). ATRA sensitive (NB4) and resistant leukemia cells (NB4R and THP-1) were co-treated with ATRA and valproic acid, followed by assessment of autophagy and differentiation. The combination of VPA and ATRA induced autophagic flux and promoted differentiation in ATRA-sensitive and -resistant cell lines. shRNA knockdown of ATG7 and TFEB autophagy regulators impaired both autophagy and differentiation, demonstrating the importance of autophagy in the combination treatment. These data suggest that ATRA combined with valproic acid can promote differentiation in myeloid leukemia cells by mechanism involving autophagy.

A practical strategy to subcutaneous administered in-situ gelling co-delivery system of arsenic and retinoic acid for the treatment of acute promyelocytic leukemia

Arsenic trioxide (ATO) combined with all trans retinoic acid (ATRA) is the first choice for the treatment of low and medium risk acute promyelocytic leukemia (APL). Clinical studies reported that the combination of ATO and ATRA could achieve a significant curative effect. However, the retinoic acid syndrome, serious drug resistance and the short half-life in vivo which lead to frequent and large dose administration limit the application of ATRA. In addition, the preparations of arsenic are conventional injections and tablets in clinic, which has poor patients' compliance caused by frequent long-term administration and serious side effects. In order to overcome the above limitations, a phospholipid phase separation gel (PPSG) loaded with ATO and ATRA was developed. ATO+ATRA-PPSG (AAP), as a biodegradable sustained-release delivery system, was the first achievement of co-delivery of hydrophilic ATO and lipophilic ATRA with high drug loading which is the main problem in the application of nano preparation. The prepared PPSG displayed high safety and biocompatibility. The drug in PPSG was released slowly and continuously in vivo and in vitro for up to 10 d, which could reduce the side effects caused by the fluctuation of blood drug concentration and solve the problem of the long treatment cycle and frequent administration. In vivo pharmacokinetics depicted that PPSG could improve the bioavailability, decrease the peak concentration, and prolong the t1/2 of ATO and ATRA. Particularly, AAP significantly inhibited the tumor volume, extended the survival period of tumor-bearing mice, and promoted the differentiation of APL cells into normal cells. Therefore, ATO+ATRA-PPSG not only could co-load hydrophilic ATO and lipophilic ATRA according to the clinical dosage, but also possessed the sustained-release and long-acting treatment effect which was expected to reduce administration time and ameliorate compliance of patients. Thus, it had great potential for clinical transformation and application.

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.

C/EBPα is indispensable for PML/RARα-mediated suppression of long non-coding RNA NEAT1 in acute promyelocytic leukemia cells.

Better understanding of the transcriptional regulatory network in acute promyelocytic leukemia (APL) cells is critical to illustrate the pathogenesis of other types of acute myeloid leukemia. Previous studies have primarily focused on the retinoic acid signaling pathway and how it is interfered with by promyelocytic leukemia/retinoic acid receptor-α (PML/RARα) fusion protein. However, this hardly explains how APL cells are blocked at the promyelocytic stage. Here, we demonstrated that C/EBPα bound and transactivated the promoter of long non-coding RNA NEAT1, an essential element for terminal differentiation of APL cells, through C/EBP binding sites. More importantly, PML/RARα repressed C/EBPα-mediated transactivation of NEAT1 through binding to NEAT1 promoter. Consistently, mutation of the C/EBP sites or deletion of retinoic acid responsive elements (RAREs) and RARE half motifs abrogated the PML/RARα-mediated repression. Moreover, silencing of C/EBPα attenuated ATRA-induced NEAT1 upregulation and APL cell differentiation. Finally, simultaneous knockdown of C/EBPα and C/EBPβ reduces ATRA-induced upregulation of C/EBPε and dramatically impaired NEAT1 activation and APL cell differentiation. In sum, C/EBPα binds and transactivates NEAT1 whereas PML/RARα represses this process. This study describes an essential role for C/EBPα in PML/RARα-mediated repression of NEAT1 and suggests that PML/RARα could contribute to the pathogenesis of APL through suppressing C/EBPα targets.

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.

Caspases Mediate Retinoic Acid–Induced Degradation of the Acute Promyelocytic Leukemia PML/RARα Fusion Protein

All-trans-retinoic acid (RA) treatment induces morphological remission in acute promyelocytic leukemia (APL) patients carrying the t(15;17) and expressing the PML/RARα product by inducing terminal differentiation of the leukemic clone. RA treatment induces downregulation of PML/RARα and reorganization of the PML-nuclear bodies. These events have been proposed to be essential for the induction of APL cell differentiation by RA. Here, we show that in the APL-derived NB4 cell line as well as in myeloid precursor U937 cells expressing the PML/RARα (U937/PR9) and in blasts from APL patients, the PML/RARα fusion protein is cleaved by a caspase 3–like activity induced by RA treatment. In fact, a caspase 3–like activity is detectable in PML/RARα expressing cells after RA treatment, and selective caspase inhibitor peptides are able to prevent the RA-induced degradation of the fusion protein in vivo and in vitro. Using recombinant caspases and PML/RARα deletion mutants we mapped a caspase 3 cleavage site (Asp 522) within the α-helix region of the PML component of the fusion protein. The extent of PML/RARα cleavage directly correlates with the ability of RA to restore the normal PML nuclear bodies (NBs) pattern. However, RA-induced differentiation is not prevented by the persistence of the fusion product and occurs in the absence of normally structured PML NBs. These results indicate that PML/RARα is directly involved in conferring RA sensitivity of APL cells and that the RA-induced reassembly of PML NBs is the consequence of the disappearance of PML/RARα.

Balance between the toxicity and anticancer activity of arsenic trioxide in treatment of acute promyelocytic leukemia

Arsenic trioxide (ATO) has a long history and it is recognized as both poison and drug for more than two thousand years. Since the establishment of ATO as a frontline therapeutic agent for acute promyelocytic leukemia (APL), the survival of APL patients have been greatly improved and APL is turned from highly fatal to highly curable disease. Mechanistically, ATO can induce PML/RARα fusion protein degradation, causing APL cell differentiation and apoptosis. On the other hand, the side effects such as differentiation syndrome, cardiac conduction abnormalities and liver toxicity are often observed during the ATO treatment of APL in clinic. It is likely that the therapeutic and adverse effects of ATO is probably associated with its distinct pattern of metabolism and direct or indirect effects on different organs. In this review, we provided a comprehensive and in-depth elaboration of the cytotoxic mechanisms of ATO and its methylated metabolites based on in vivo or in vitro studies, trying to clarify the importance of achieving balance between the toxicity and anti-leukemic activity of ATO in APL treatment.

DDX5-targeting fully human monoclonal autoantibody inhibits proliferation and promotes differentiation of acute promyelocytic leukemia cells by increasing ROS production

Acute promyelocytic leukemia (APL) therapy involves the compounds cytotoxic to both malignant tumor and normal cells. Relapsed APL is resistant to subsequent chemotherapy. Novel agents are in need to kill APL cells selectively with minimal toxicity. DDX5 has been recognized to be a novel target to suppress acute myeloid leukemia (AML). However, the role of DDX5 remains elusive in APL. Here a DDX5-targeting fully human monoclonal autoantibody named after 2F5 was prepared. It is demonstrated that 2F5 selectively inhibited APL cell proliferation without toxicity to normal neutrophil and tissues. Moreover, 2F5 was confirmed to induce G0/G1 phase arrest in APL cells, and promote APL cell differentiation combined with decreased DDX5 expression and increased reactive oxygen species (ROS) production. Knockdown of DDX5 by siRNA also inhibited proliferation, promoted cell differentiation and enhanced ROS production in APL cells. However, the ROS inhibitor reversed the effects of 2F5 on DDX5 and ROS in APL cells. Thus, we conclude that DDX5-targeting 2F5 inhibits APL cell proliferation, and promotes cell differentiation via induction of ROS. 2F5 showed the therapeutic value of fully human monoclonal autoantibody in APL, which provides a novel and valid approach for treatment of relapse/refractory APL.

ATPR induces acute promyelocytic leukemia cells differentiation and growth arrest by blockade of SHP2/Rho/ROCK1 pathway

Acute promyelocytic leukemia (APL) is a form of acute myeloid leukemia with a unique chromosome translocation t (15;17), commonly complicated by a complex coagulopathy. 4-Amino-2-trifuoromethyl-phenyl retinate (ATPR), a novel all-trans retinoic acid (ATRA) derivative, was synthesized by our group and known to possess obvious biological anti-tumor activities. It has previously been shown that ATPR could induce differentiation and inhibit proliferation of APL cells, although the mechanism responsible for this effect was not well understood. In this study, we demonstrated that ATPR remarkably inhibited the expression and activity of SHP2. Further experiments showed silencing SHP2 or using SHP2 inhibition (SHP099) enhanced the effect of ATPR on cell proliferation and maturation. In addition, we also demonstrated that Rho/ROCK1 might be regulated by SHP2. Using Y-27632, a ROCK inhibitor, further proved that ROCK1 played an important role in ATPR-induced differentiation and proliferation suppression. In conclusion, the results from this study revealed that ATPR induced APL cells terminal differentiation and growth arrest by blockade of SHP2/Rho/ ROCK1 pathway.

Inhibition of UBE2L6 attenuates ISGylation and impedes ATRA-induced differentiation of leukemic cells.

Ubiquitin/ISG15‐conjugating enzyme E2L6 (UBE2L6) is a critical enzyme in ISGylation, a post‐translational protein modification that conjugates the ubiquitin‐like modifier, interferon‐stimulated gene 15 (ISG15), to target substrates. Previous gene expression studies in acute promyelocytic leukemia (APL) cells showed that all‐trans‐retinoic acid (ATRA) altered the expression of many genes, including UBE2L6 (200‐fold) and other members of the ISGylation pathway. Through gene expression analyses in a cohort of 98 acute myeloid leukemia (AML) patient samples and in primary neutrophils from healthy donors, we found that UBE2L6 gene expression is reduced in primary AML cells compared with normal mature granulocytes. To assess whether UBE2L6 expression is important for leukemic cell differentiation—two cell line models were employed: the human APL cell line NB4 and its ATRA‐resistant NB4R counterpart, as well as the ATRA‐sensitive human AML HL60 cells along with their ATRA‐resistant subclone—HL60R. ATRA strongly induced UBE2L6 in NB4 APL cells and in ATRA‐sensitive HL60 AML cells, but not in the ATRA‐resistant NB4R and HL60R cells. Furthermore, short hairpin (sh)RNA‐mediated UBE2L6 depletion in NB4 cells impeded ATRA‐mediated differentiation, suggesting a functional role for UBE2L6 in leukemic cell differentiation. In addition, ATRA induced ISG15 gene expression in NB4 APL cells, leading to increased levels of both free ISG15 protein and ISG15 conjugates. UBE2L6 depletion attenuated ATRA‐induced ISG15 conjugation. Knockdown of ISG15 in NB4 APL cells inhibited ISGylation and also attenuated ATRA‐induced differentiation. In summary, we demonstrate the functional importance of UBE2L6 in ATRA‐induced neutrophil differentiation of APL cells and propose that this may be mediated by its catalytic role in ISGylation.

Targeting the scaffolding role of LSD1 (KDM1A) poises acute myeloid leukemia cells for retinoic acid-induced differentiation.

The histone demethylase LSD1 is deregulated in several tumors, including leukemias, providing the rationale for the clinical use of LSD1 inhibitors. In acute promyelocytic leukemia (APL), pharmacological doses of retinoic acid (RA) induce differentiation of APL cells, triggering degradation of the PML-RAR oncogene. APL cells are resistant to LSD1 inhibition or knockout, but targeting LSD1 sensitizes them to physiological doses of RA without altering of PML-RAR levels, and extends survival of leukemic mice upon RA treatment. The combination of RA with LSD1 inhibition (or knockout) is also effective in other non-APL, acute myeloid leukemia (AML) cells. Nonenzymatic activities of LSD1 are essential to block differentiation, while RA with targeting of LSD1 releases a differentiation gene expression program, not strictly dependent on changes in histone H3K4 methylation. Integration of proteomic/epigenomic/mutational studies showed that LSD1 inhibitors alter the recruitment of LSD1-containing complexes to chromatin, inhibiting the interaction between LSD1 and the transcription factor GFI1.

Nuclear import of NLS- RARα is mediated by importin α/β

The promyelocytic leukemia-retinoic acid receptor α (PML/RARα) is hypothesized to play a vital role in the pathogenesis of acute promyelocytic leukemia (APL). A previous study has demonstrated that PML/RARα is cleaved by neutrophil elastase (NE) in early myeloid cells, which leads to an increase in the nuclear localization signal (NLS) in RARα and in the incidence of APL. In this study, we explored the effects of NLS-RARα on acute myeloid leukemia (AML) cells and studied the mechanism of its localization. LV-NLS-RARα recombinant lentivirus and negative control LV-NC lentivirus were transfected into HL-60 cells and U937 cells while mutant NLS-RARα were transfected into U937 cells, and all groups were treated with 1α, 25-dihydroxyvitamin D3(1,25D3). The results showed that NLS-RARα was located mainly in the nucleus while mutant NLS-RARα was located in the cytoplasm. Overexpression of NLS-RARα downregulated the expression of CD11b, CD11c, CD14, and three forms of CEBPβ compared to the overexpression of NC and mutant NLS-RARα. It was speculated that the abnormal localization of NLS-RARα was mediated via importin-α/β in the pathogenesis of APL. By producing point mutations in the two NLSs in NLS-RARα, we showed that the nuclear import of NLS-RARα was mainly dependent on the NLS of the RARα portion. Subsequently, we found that importin-α1 (KPNA2)/importin-β1 (KPNB1) participates in the nuclear transport of NLS-RARα. Taken together, abnormal localization of NLS-RARα blocks the differentiation of APL cells, and nuclear localization of NLS-RARα depends on NLS of the RARα portion and is mediated via binding with importin-α/β.