Targeting miR‐193a‐AML1‐ETO‐β‐catenin axis by melatonin suppresses the self‐renewal of leukaemia stem cells in leukaemia with t (8;21) translocation

AML1‐ETO, the most common fusion oncoprotein by t (8;21) in acute myeloid leukaemia (AML), enhances hematopoietic self‐renewal and leukemogenesis. However, currently no specific therapies have been reported for t (8;21) AML patients as AML1‐ETO is still intractable as a pharmacological target. For this purpose, leukaemia cells and AML1‐ETO‐induced murine leukaemia model were used to investigate the degradation of AML1‐ETO by melatonin (MLT), synthesized and secreted by the pineal gland. MLT remarkedly decreased AML1‐ETO protein in leukemic cells. Meanwhile, MLT induced apoptosis, decreased proliferation and reduced colony formation. Furthermore, MLT reduced the expansion of human leukemic cells and extended the overall survival in U937T‐AML1‐ETO‐xenografted NSG mice. Most importantly, MLT reduced the infiltration of leukaemia blasts, decreased the frequency of leukaemia stem cells (LSCs) and prolonged the overall survival in AML1‐ETO‐induced murine leukaemia. Mechanistically, MLT increased the expression of miR‐193a, which inhibited AML1‐ETO expression via targeting its putative binding sites. Furthermore, MLT decreased the expression of β‐catenin, which is required for the self‐renewal of LSC and is the downstream of AML1‐ETO. Thus, MLT presents anti‐self‐renewal of LSC through miR‐193a‐AML1‐ETO‐β‐catenin axis. In conclusion, MLT might be a potential treatment for t (8;21) leukaemia by targeting AML1‐ETO oncoprotein.     

Epigenetic inactivation of the hsa-miR-203 in haematological malignancies

miR-203 is a tumour suppressor microRNA (miRNA). We studied the methylation of hsa-miR-203 in 150 samples including acute myeloid leukaemia (AML), acute lymphoblastic leukaemia (ALL), chronic myeloid leukaemia (CML), chronic lymphocytic leukaemia (CLL) and non-Hodgkin's lymphoma (NHL) by methylation-specific PCR, and miRNA expression by stem-loop RT-qPCR. hsa-miR-203 promoter was unmethylated in normal controls but homozygously methylated in two AML and four lymphoma cell lines, in which 5-Aza-2'-deoxycytidine treatment led to promoter demethylation and miR-203 re-expression. Restoration of miR-203 expression in lymphoma cells inhibited cellular proliferation and increased cell death, suggesting an inherent tumour suppressor activity. In primary samples, hsa-miR-203 methylation was absent in CML but detected in 5.0% ALL, 10.0% AML, 42.0% CLL and 38.8% of NHL (including six [60.0%] natural killer-cell, nine [40.9%] B-cell and four [23.5%] T cell NHL). Moreover, hsa-miR-203 methylation was associated with hypermethylation of hsa-miR-34a, -124a and -196b in NHL but not CLL. In CLL, hsa-miR-203 methylation was associated with a higher presenting Hb level (P = 0.033). The projected 10 year overall survival of the CLL patients was 58.2%, which was impacted by Rai stage and high-risk karyotypes but not hsa-miR-203 methylation. hsa-miR-203 was more frequently methylated in lymphoid than myeloid malignancies (P = 0.002). In conclusion, miR-203, a tumour suppressor gene, was hypermethylated in a tumour-specific manner with gene silencing. hsa-miR-203 was more frequently hypermethylated in lymphoid than myeloid malignancies. In NHL, hsa-miR-203 methylation was associated with concomitant methylation of other tumour suppressor miRNAs. The frequent hsa-miR-203 methylation in lymphoid malignancies suggested a pathogenetic role of hsa-miR-203 methylation.     

Identification of predictive genetic signatures of Cytarabine responsiveness using a 3D acute myeloid leukaemia model

This study reports the establishment of a bone marrow mononuclear cell (BMMC) 3D culture model and the application of this model to define sensitivity and resistance biomarkers of acute myeloid leukaemia (AML) patient bone marrow samples in response to Cytarabine (Ara‐C) treatment. By mimicking physiological bone marrow microenvironment, the growth conditions were optimized by using frozen BMMCs derived from healthy donors. Healthy BMMCs are capable of differentiating into major hematopoietic lineages and various types of stromal cells in this platform. Cryopreserved BMMC samples from 49 AML patients were characterized for ex vivo growth and sensitivity to Ara‐C. RNA sequencing was performed for 3D and 2D cultures to determine differential gene expression patterns. Specific genetic mutations and/or gene expression signatures associated with the ability of the ex vivo expansion and response to Ara‐C were elucidated by whole‐exome and RNA sequencing. Data analysis identified unique gene expression signatures and novel genetic mutations associated with sensitivity to Ara‐C treatment of proliferating AML specimens and can be used as predictive therapeutic biomarkers to determine the optimal treatment regimens. Furthermore, these data demonstrate the translational value of this ex vivo platform which should be widely applicable to evaluate other therapies in AML.     

Epigenetic dysregulation of ID4 predicts disease progression and treatment outcome in myeloid malignancies

Promoter hypermethylation‐mediated inactivation of ID4 plays a crucial role in the development of solid tumours. This study aimed to investigate ID4 methylation and its clinical relevance in myeloid malignancies. ID4 hypermethylation was associated with higher IPSS scores, but was not an independent prognostic biomarker affecting overall survival (OS) in myelodysplastic syndrome (MDS). However, ID4 hypermethylation correlated with shorter OS and leukaemia‐free survival (LFS) time and acted as an independent risk factor affecting OS in acute myeloid leukaemia (AML). Moreover, ID4 methylation was significantly decreased in the follow‐up paired AML patients who achieved complete remission (CR) after induction therapy. Importantly, ID4 methylation was increased during MDS progression to AML and chronic phase (CP) progression to blast crisis (BC) in chronic myeloid leukaemia (CML). Epigenetic studies showed that ID4 methylation might be one of the mechanisms silencing ID4 expression in myeloid leukaemia. Functional studies in vitro showed that restoration of ID4 expression could inhibit cell proliferation and promote apoptosis in both K562 and HL60 cells. These findings indicate that ID4 acts as a tumour suppressor in myeloid malignancies, and ID4 methylation is a potential biomarker in predicting disease progression and treatment outcome.     

Development and validation of a 10‐gene prognostic signature for acute myeloid leukaemia

Acute myeloid leukaemia (AML) is the most common type of adult acute leukaemia and has a poor prognosis. Thus, optimal risk stratification is of greatest importance for reasonable choice of treatment and prognostic evaluation. For our study, a total of 1707 samples of AML patients from three public databases were divided into meta‐training, meta‐testing and validation sets. The meta‐training set was used to build risk prediction model, and the other four data sets were employed for validation. By log‐rank test and univariate COX regression analysis as well as LASSO‐COX, AML patients were divided into high‐risk and low‐risk groups based on AML risk score (AMLRS) which was constituted by 10 survival‐related genes. In meta‐training, meta‐testing and validation sets, the patient in the low‐risk group all had a significantly longer OS (overall survival) than those in the high‐risk group (P < .001), and the area under ROC curve (AUC) by time‐dependent ROC was 0.5854‐0.7905 for 1 year, 0.6652‐0.8066 for 3 years and 0.6622‐0.8034 for 5 years. Multivariate COX regression analysis indicated that AMLRS was an independent prognostic factor in four data sets. Nomogram combining the AMLRS and two clinical parameters performed well in predicting 1‐year, 3‐year and 5‐year OS. Finally, we created a web‐based prognostic model to predict the prognosis of AML patients ( https://tcgi.shinyapps.io/amlrs_nomogram/ ).     

Acquired TET2 mutation in one patient with familial platelet disorder with predisposition to AML led to the development of pre‐leukaemic clone resulting in T2‐ALL and AML‐M0

Familial platelet disorder with predisposition to acute myeloid leukaemia (FPD/AML) is characterized by germline RUNX1 mutations, thrombocytopaenia, platelet dysfunction and a risk of developing acute myeloid and in rare cases lymphoid T leukaemia. Here, we focus on a case of a man with a familial history of RUNX1^R174Q mutation who developed at the age of 42 years a T2‐ALL and, 2 years after remission, an AML‐M0. Both AML‐M0 and T2‐ALL blast populations demonstrated a loss of 1p36.32‐23 and 17q11.2 regions as well as other small deletions, clonal rearrangements of both TCRγ and TCRδ and a presence of 18 variants at a frequency of more than 40%. Additional variants were identified only in T2‐ALL or in AML‐M0 evoking the existence of a common original clone, which gave rise to subclonal populations. Next generation sequencing (NGS) performed on peripheral blood‐derived CD34⁺ cells 5 years prior to T2‐ALL development revealed only the missense TET2^P1962T mutation at a frequency of 1%, which increases to more than 40% in fully transformed leukaemic T2‐ALL and AML‐M0 clones. This result suggests that TET2^P1962T mutation in association with germline RUNX1^R174Q mutation leads to amplification of a haematopoietic clone susceptible to acquire other transforming alterations.     

miR‐203 inhibits proliferation of HCC cells by targeting survivin

To validate whether down‐regulation of microRNA‐203 (miR‐203) in hepatocellular carcinoma (HCC) is involved in HCC progression by targeting survivin. MiR‐203 mimics was transfected into HepG2 cells to enhance miR‐203 expression, and miR‐203 inhibitor was transfected into HepG2 cells to inhibit miR‐203 expression. The effect of up‐regulation and down‐regulation of miR‐203 on survivin expression of HepG2 cells was evaluated using Western blot assay. The effect of miR‐203 or survivin expression on the proliferation of HepG2 cells was detected using the CKK‐8 assay. Over‐expression of miR‐203 significantly inhibited the expression of survivin in HepG2 cells (p < 0·05), and down‐expression of miR‐203 significantly promoted the expression of survivin in HepG2 cells (p < 0·05). Both over‐expression of miR‐203 and down‐regulation of survivin suppressed proliferation of HepG2 cells significantly compared with negative control. Low expression of miR‐203 contributes to the progression of HCC via targeting survivin. Copyright © 2012 John Wiley & Sons, Ltd.     

Matrine induces Akt/mTOR signalling inhibition‐mediated autophagy and apoptosis in acute myeloid leukaemia cells

Pharmacological modulation of autophagy has been referred to as a promising therapeutic strategy for cancer. Matrine, a main alkaloid extracted from Sophora flavescens Ait, has antitumour activity against acute myelocytic leukaemia (AML). Whether autophagy is involved in antileukaemia activity of matrine remains unobvious. In this study, we demonstrated that matrine inhibited cell viability and colony formation via inducing apoptosis and autophagy in AML cell lines HL‐60, THP‐1 and C1498 as well as primary AML cells. Matrine promoted caspase‐3 and PARP cleavage dose‐dependently. Matrine up‐regulated the level of LC3‐II and down‐regulated the level of SQSTM1/p62 in a dose‐dependent way, indicating that autophagy should be implicated in anti‐AML effect of matrine. Furthermore, the autophagy inhibitor bafilomycin A1 relieved the cytotoxicity of matrine by blocking the autophagic flux, while the autophagy promoter rapamycin enhanced the cytotoxicity of matrine. Additionally, matrine inhibited the phosphorylation of Akt, mTOR and their downstream substrates p70S6K and 4EBP1, which led to the occurrence of autophagy. In vivo study demonstrated that autophagy was involved in antileukaemia effect of matrine in C57BL/6 mice bearing murine AML cell line C1498, and the survival curves showed that mice did benefit from treatment with matrine. Collectively, our findings indicate that matrine exerts antitumour effect through apoptosis and autophagy, and the latter one might be a potential therapeutic strategy for AML.     

Up‐regulation of SPINT2/HAI‐2 by Azacytidine in bone marrow mesenchymal stromal cells affects leukemic stem cell survival and adhesion

The role of tumour microenvironment in neoplasm initiation and malignant evolution has been increasingly recognized. However, the bone marrow mesenchymal stromal cell (BMMSC) contribution to disease progression remains poorly explored. We previously reported that the expression of serine protease inhibitor kunitz‐type2 (SPINT2/HAI‐2), an inhibitor of hepatocyte growth factor (HGF) activation, is significantly lower in BMMSC from myelodysplastic syndromes (MDS) patients compared to healthy donors (HD). Thus, to investigate whether this loss of expression was due to SPINT2/HAI‐2 methylation, BMMSC from MDS and de novo acute myeloid leukaemia (de novo AML) patients were treated with 5‐Azacitidine (Aza), a DNA methyltransferase inhibitor. In MDS‐ and de novo AML‐BMMSC, Aza treatment resulted in a pronounced SPINT2/HAI‐2 levels up‐regulation. Moreover, Aza treatment of HD‐BMMSC did not improve SPINT2/HAI‐2 levels. To understand the role of SPINT2/HAI‐2 down‐regulation in BMMSC physiology, SPINT2/HAI‐2 expression was inhibited by lentivirus. SPINT2 underexpression resulted in an increased production of HGF by HS‐5 stromal cells and improved survival of CD34⁺ de novo AML cells. We also observed an increased adhesion of de novo AML hematopoietic cells to SPINT2/HAI‐2 silenced cells. Interestingly, BMMSC isolated from MDS and de novo AML patients had increased expression of the integrins CD49b, CD49d, and CD49e. Thus, SPINT2/HAI‐2 may contribute to functional and morphological abnormalities of the microenvironment niche and to stem/progenitor cancer cell progression. Hence, down‐regulation in SPINT2/HAI‐2 gene expression, due to methylation in MDS‐BMMSC and de novo AML‐BMMSC, provides novel insights into the pathogenic role of the leukemic bone marrow microenvironment.     

EZH2 dysregulation: Potential biomarkers predicting prognosis and guiding treatment choice in acute myeloid leukaemia

Accumulating studies have proved EZH2 dysregulation mediated by mutation and expression in diverse human cancers including AML. However, the expression pattern of EZH2 remains controversial in acute myeloid leukaemia (AML). EZH1/2 expression and mutation were analysed in 200 patients with AML. EZH2 expression was significantly decreased in AML patients compared with normal controls but not for EZH1 expression. EZH2 mutation was identified three of the 200 AML patients (1.5%, 3/200), whereas none of the patients harboured EZH1 mutation (0%, 0/200). EZH2 expression and mutation were significantly associated with ?7/del(7) karyotypes. Moreover, lower EZH2 expression was associated with older age, higher white blood cells, NPM1 mutation, CEBPA wild‐type and WT1 wild‐type. Patients with EZH2 mutation showed shorter overall survival (OS) and leukaemia‐free survival (LFS) than patients without EHZ2 mutation after receiving autologous or allogeneic haematopoietic stem cell transplantation (HSCT). However, EZH2 expression has no effect on OS and LFS of AML patients. Notably, in EZH2 low group, patients undergone HSCT had significantly better OS and LFS compared with patients only received chemotherapy, whereas no significant difference was found in OS and LFS between chemotherapy and HSCT patients in EZH2 high group. Collectively, EZH2 dysregulation caused by mutation and under‐expression identifies specific subtypes of AML EZH2 dysregulation may be acted as potential biomarkers predicting prognosis and guiding the treatment choice between transplantation and chemotherapy.     

Signalling mechanisms that regulate metabolic profile and autophagy of acute myeloid leukaemia cells

Acute myeloid leukaemia (AML) comprises a heterogeneous group of hematologic neoplasms characterized by diverse combinations of genetic, phenotypic and clinical features representing a major challenge for the development of targeted therapies. Metabolic reprogramming, mainly driven by deregulation of the nutrient‐sensing pathways as AMPK, mTOR and PI3K/AKT, has been associated with cancer cells, including AML cells, survival and proliferation. Nevertheless, the role of these metabolic adaptations on the AML pathogenesis is still controversial. In this work, the metabolic status and the respective metabolic networks operating in different AML cells (NB‐4, HL‐60 and KG‐1) and their impact on autophagy and survival was characterized. Data show that whereas KG‐1 cells exhibited preferential mitochondrial oxidative phosphorylation metabolism with constitutive co‐activation of AMPK and mTORC1 associated with increased autophagy, NB‐4 and HL‐60 cells displayed a dependent glycolytic profile mainly associated with AKT/mTORC1 activation and low autophagy flux. Inhibition of AKT is disclosed as a promising therapeutical target in some scenarios while inhibition of AMPK and mTORC1 has no major impact on KG‐1 cells’ survival. The results highlight an exclusive metabolic profile for each tested AML cells and its impact on determination of the anti‐leukaemia efficacy and on personalized combinatory therapy with conventional and targeted agents.     

Germ cell survival in C. elegans and C. remanei is affected when the dead box rna helicases Vbh‐1 or Cre‐VBH‐1 are silenced

The Vasa family of proteins comprises several conserved DEAD box RNA helicases important for mRNA regulation whose exact function in the germline is still unknown. In Caenorhabditis elegans, there are six known members of the Vasa family, and all of them are associated with P granules. One of these proteins, VBH‐1, is important for oogenesis, spermatogenesis, embryo development, and the oocyte/sperm switch in this nematode. We decided to extend our previous work in C. elegans to sibling species Caenorhabditis remanei to understand what is the function of the VBH‐1 homolog in this gonochoristic species. We found that Cre‐VBH‐1 is present in the cytoplasm of germ cells and it remains associated with P granules throughout the life cycle of C. remanei. Several aspects between VBH‐1 and Cre‐VBH‐1 function are conserved like their role during oogenesis, spermatogenesis, and embryonic development. However, Cre‐vbh‐1 silencing in C. remanei had a stronger effect on spermatogenesis and spermatid activation than in C. elegans. An unexpected finding was that silencing of vbh‐1 in the C. elegans caused a decrease in germ cell apoptosis in the hermaphrodite gonad, while silencing of Cre‐vbh‐1 in C. remanei elicited germ cell apoptosis in the male gonad. These data suggest that VBH‐1 might play a role in germ cell survival in both species albeit it appears to have an opposite role in each one. genesis 1–18 2012. © 2012 Wiley Periodicals, Inc.     

Silencing of lncRNA UCA1 curbs proliferation and accelerates apoptosis by repressing SIRT1 signals by targeting miR‐204 in pediatric AML

The long noncoding RNA urothelial carcinoma‐associated 1 (UCA1) has been reported to sustain the proliferation of acute myeloid leukemia (AML) cells through downregulating cell cycle regulators p27^kip1. Yet, the foundational mechanism of UCA1 in AML pathologies remains unclear. Herein, we found an escalation of UCA1 expression and suppression of miR‐204 expression in pediatric AML patients and cells. UCA1 silencing suppressed cell proliferative abilities, promoted apoptotic rates, decreased Ki67, and increased cleaved caspase‐3 in AML cells. Moreover, UCA1 sponged miR‐204 and suppressed its expression. UCA1 overexpression inversed the miR‐204 suppressed proliferation and promoted apoptosis. UCA1 also boosted the expression of SIRT1, a miR‐204 target, via the sponging interaction. Furthermore, miR‐204 inhibited inducible nitric oxide synthase and cyclooxygenase‐2 expression, while UCA1 overexpression inversed the inhibitory effects in AML cells. Our findings concluded that UCA1 downregulation repressed cell proliferation and promoted apoptosis through inactivating SIRT1 signals by upregulating miR‐204 in pediatric AML.     

血清miR-203、miR-217表达与急性髓系白血病患者预后的关系

摘要 目的：探究血清miR-203、miR-217表达与急性髓系白血病(AML)患者预后的关系。方法：选择2010年4月至2014年4月我院诊治的101例AML患者作为AML组,AML组根据治疗效果进一步分为完全缓解组和复发组,选择同期在我院体检的101例健康者作为健康组。采用荧光定量PCR检测各组的血清miR-203、miR-217表达水平,分析血清miR-203、miR-217表达水平与患者临床病理特征的关系,采用Kaplan-Meier法分析不同血清miR-203、miR-217表达水平AML患者的预后。结果：与健康组相比,AML组的血清miR-203、miR-217表达水平明显更低（P<0.05）。与完全缓解组相比,复发组的血清miR-203、miR-217表达水平明显更低（P<0.05）。血清miR-203表达水平与AML患者白细胞计数相关（P<0.05）,而血清miR-217表达水平与AML患者血小板计数相关（P<0.05）。血清miR-203相对高表达和miR-217相对高表达的AML患者5年生存率分别高于血清miR-203相对低表达和miR-217相对低表达患者（Log Rank ^miR-203 =17.870,Log Rank ^miR-217 =28.926,均P=0.000）。结论：血清miR-203、miR-217的表达水平与AML密切相关,检测血清miR-203、miR-217表达水平可能有助于评估AML患者的预后。     

Leukaemic transformation by CALM-AF10 involves upregulation of Hoxa5 by hDOT1L

Chromosomal translocation is a common cause of leukaemia and the most common chromosome translocations found in leukaemia patients involve the mixed lineage leukaemia (MLL) gene. AF10 is one of more than 30 MLL fusion partners in leukaemia. We have recently demonstrated that the H3K79 methyltransferase hDOT1L contributes to MLL-AF10-mediated leukaemogenesis through its interaction with AF10 (ref. 5). In addition to MLL, AF10 has also been reported to fuse to CALM (clathrin-assembly protein-like lymphoid-myeloid) in patients with T-cell acute lymphoblastic leukaemia (T-ALL) and acute myeloid leukaemia (AML). Here, we analysed the molecular mechanism of leukaemogenesis by CALM-AF10. We demonstrate that CALM-AF10 fusion is both necessary and sufficient for leukaemic transformation. Additionally, we provide evidence that hDOT1L has an important role in the transformation process. hDOT1L contributes to CALM-AF10-mediated leukaemic transformation by preventing nuclear export of CALM-AF10 and by upregulating the Hoxa5 gene through H3K79 methylation. Thus, our study establishes CALM-AF10 fusion as a cause of leukaemia and reveals that mistargeting of hDOT1L and upregulation of Hoxa5 through H3K79 methylation is the underlying mechanism behind leukaemia caused by CALM-AF10 fusion.     

Wogonoside induces depalmitoylation and translocation of PLSCR1 and N‐RAS in primary acute myeloid leukaemia cells

Acute myeloid leukaemia (AML) comprises a range of disparate genetic subtypes, involving complex gene mutations and specific molecular alterations. Post‐translational modifications of specific proteins influence their translocation, stability, aggregation and even leading disease progression. Therapies that target to post‐translational modification of specific proteins in cancer cells represent a novel treatment strategy. Non‐homogenous subcellular distribution of PLSCR1 is involved in the primary AML cell differentiation. However, the nuclear translocation mechanism of PLSCR1 remains poorly understood. Here, we leveraged the observation that nuclear translocation of PLSCR1 could be induced during wogonoside treatment in some primary AML cells, despite their genetic heterogeneity that contributed to the depalmitoylation of PLSCR1 via acyl protein thioesterase 1 (APT‐1), an enzyme catalysing protein depalmitoylation. Besides, we found a similar phenomenon on another AML‐related protein, N‐RAS. Wogonoside inhibited the palmitoylation of small GTPase N‐RAS and enhanced its trafficking into Golgi complex, leading to the inactivation of N‐RAS/RAF1 pathway in some primary AML cells. Taken together, our findings provide new insight into the mechanism of wogonoside‐induced nuclear translocation of PLSCR1 and illuminate the influence of N‐RAS depalmitoylation on its Golgi trafficking and RAF1 signalling inactivation in AML.