SLC22A3 methylation-mediated gene silencing predicts adverse prognosis in acute myeloid leukemia.

BACKGROUND: We screened out several hypermethylated solute carrier (SLC) family genes in acute myeloid leukemia by reduced representation bisulfite sequencing. SLC22A3 encodes an organic cation transport protein, which is critical for drug transportation and cellular detoxification. SLC22A3 is significantly downregulated and associated with tumor progression and worse prognosis in a variety of solid tumors. However, there are no data available regarding the role of SLC22 in AML. This study aimed to explore the regulatory mechanism of DNA methylation on SLC22A3 expression, as well as its clinical significance in AML prognosis. RESULTS: SLC22A3 was identified as the sole prognosis-associated gene among SLCs based on TCGA and Beat AML databases. Bone marrow mononuclear cells (BMMNCs) from AML, MDS patients, and healthy donors were enrolled in this study. SLC22A3 methylation was significantly increased in AML compared with controls and MDS patients; meanwhile, the expression level of SLC22A3 was decreased. SLC22A3 hypermethylation presented an obvious association with some specific clinical characteristics and affected the survival time of AML patients as an independent risk indicator. SLC22A3 expression changed regularly as the disease complete remissions and relapses. Demethylation drug 5-aza-2'-deoxycytidine (DAC) activated transcription and increased mRNA expression of SLC22A3 in leukemia cell lines and AML fresh BMMNCs. Knockdown of SLC22A3 in leukemia cells enhanced cell proliferation and suppressed cell apoptosis. Data from public programs were used for auxiliary screening of probable molecular mechanisms of SLC22A3 in the antileukemia effect. CONCLUSIONS: Our results showed that increased methylation and decreased expression of SLC22A3 may be indicators of poor prognosis in AML. Methylation-silenced SLC22A3 expression may have potential guiding significance on antileukemia effect of DAC.

SLIT2 promoter hypermethylation predicts disease progression in chronic myeloid leukemia.

BACKGROUND: Aberrant DNA methylation plays a crucial role in the progression of myeloid neoplasms. Previously, our literature reported that slit guidance ligand 2 (SLIT2) promoter methylation was associated with disease progression and indicated a poor prognosis in patients with myelodysplastic syndrome. Herein, we further investigated the clinical implications and role of SLIT2 promoter methylation in patients with chronic myeloid leukemia (CML). METHODS: The level of SLIT2 promoter methylation was determined in 104 CML patients, and its clinical significance was analyzed. Moreover, demethylation studies were performed in K562 cells to determine the epigenetic mechanism by which SLIT2 promoter methylation is regulated in CML. RESULTS: The level of SLIT2 promoter methylation was similar between CML patients and controls. However, deeper analysis revealed that the SLIT2 promoter methylation level in the accelerated phase (AP) and blast crisis (BC) was markedly higher than that in the chronic phase (CP) and controls. Additionally, a marked difference was identified between the SLIT2 promoter hypermethylated and non-hypermethylated groups among CML patients grouped by clinical stage. The frequency of SLIT2 hypermethylation was markedly increased with the progression of clinical stage, that is, it was the lowest in CP samples (12/80, 15%), higher in AP samples (4/8, 50%) and the highest in BC samples (11/16, 69%). Importantly, the level/density of SLIT2 promoter methylation was significantly higher in the advanced stage than in the early stage among the 6 tested paired CML patients. Epigenetically, the expression of the SLIT2-embedded non-coding genes SLIT2-IT1 and miR-218 expression was decreased in patients with CML. SLIT2 promoter hypermethylated cases had a markedly lower SLIT2-IT1 expression level than SLIT2 promoter non-hypermethylated cases. Moreover, SLIT2-IT1 and miR-218 expression was remarkably upregulated in a dose-dependent manner after demethylation treatment of K562 cells. CONCLUSIONS: Hypermethylation of the SLIT2 promoter is correlated with disease progression in CML. Furthermore, SLIT2 promoter methylation may function by regulating the expression of the SLIT2-embedded non-coding genes SLIT2-IT1 and miR-218 during CML progression.

Whole-Genome DNA Methylation Sequencing Reveals Epigenetic Changes in Myelodysplastic Syndromes.

Epigenetic dysregulation of cancer-associated genes has been identified to contribute to the pathogenesis of myelodysplastic syndromes (MDS). However, few studies have elucidated the whole-genome DNA methylation in the initiation pathogenesis of MDS. Reduced representation bisulfite sequencing was performed in five de novo MDS patients and four controls to investigate epigenetic alterations in MDS pathogenesis. The mean global methylation in five MDS patients showed no significant difference compared with the four controls. In depth, a total of 1,459 differentially methylated fragments, including 759 hypermethylated and 700 hypomethylated fragments, were identified between MDS patients and controls. Targeted bisulfite sequencing further identified that hypermethylation of DLEU7, FOXR1, LEP, and PANX2 were frequent events in an additional cohort of MDS patients. Subsequently, LEP hypermethylation was confirmed by real-time quantitative methylation-specific PCR in an expanded cohort of larger MDS patients. In clinics, LEP hypermethylation tended to be associated with lower bone marrow blasts and was significantly correlated with U2AF1 mutation. Survival analysis indicated that LEP hypermethylation was associated with a markedly longer survival time but was not an independent prognostic biomarker in MDS patients. Functional studies revealed pro-proliferative and anti-apoptotic effects of leptin in the MDS cell line SKM-1, and it was significantly associated with cell growth and death as well as the Toll-like receptor and NF-kappa B signaling pathways. Collectively, our findings demonstrated that whole-genome DNA methylation analysis identified novel epigenetic alterations such as DLEU7, FOXR1, LEP, and PANX2 methylations as frequent events in MDS. Moreover, LEP might play a role in MDS pathogenesis, and LEP hypermethylation was associated with longer survival but not as an independent prognostic biomarker in MDS.

DNA methylation-mediated differential expression of DLX4 isoforms has opposing roles in leukemogenesis.

BACKGROUND: Previously, we reported the expression of DLX4 isoforms (BP1 and DLX7) in myeloid leukemia, but the functional role of DLX4 isoforms remains poorly understood. In the work described herein, we further determined the underlying role of DLX4 isoforms in chronic myeloid leukemia (CML) leukemogenesis. METHODS: The expression and methylation of DLX4 isoforms were detected by real-time quantitative PCR (RT-qPCR) and real-time quantitative methylation-specific PCR (RT-qMSP) in patients with CML. The functional role of DLX4 isoforms was determined in vitro and in vivo. The molecular mechanism of DLX4 isoforms in leukemogenesis was identified based on chromatin immunoprecipitation with high-throughput sequencing (ChIP-Seq)/assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-Seq) and RNA sequencing (RNA-Seq). RESULTS: BP1 expression was increased in patients with CML with unmethylated promoter, but DLX7 expression was decreased with hypermethylated promoter. Functionally, overexpression of BP1 increased the proliferation rate of K562 cells with S/G2 promotion, whereas DLX7 overexpression reduced the proliferation rate of K562 cells with G1 arrest. Moreover, K562 cells with BP1 overexpression increased the tumorigenicity in NCG mice, whereas K562 cells with DLX7 overexpression decreased the tumorigenicity. Mechanistically, a total of 91 genes including 79 messenger RNAs (mRNAs) and 12 long noncoding RNAs (lncRNAs) were discovered by ChIP-Seq and RNA-Seq as direct downstream targets of BP1. Among the downstream genes, knockdown of RREB1 and SGMS1-AS1 partially revived the proliferation caused by BP1 overexpression in K562 cells. Similarly, using ATAC-Seq and RNA-Seq, a total of 282 genes including 151 mRNA and 131 lncRNAs were identified as direct downstream targets of DLX7. Knockdown of downstream genes PTPRB and NEAT1 partially revived the proliferation caused by DLX7 overexpression in K562 cells. Finally, we also identified and validated a SGMS1-AS1/miR-181d-5p/SRPK2 competing endogenous RNA (ceRNA) network caused by BP1 overexpression in K562 cells. CONCLUSIONS: The current findings reveal that DNA methylation-mediated differential expression of DLX4 isoforms BP1 and DLX7 plays opposite functions in leukemogenesis. BP1 plays an oncogenic role in leukemia development, whereas DLX7 acts as a tumor suppressor gene. These results suggest DLX4 as a therapeutic target for antileukemia therapy.

SLIT2 promoter hypermethylation-mediated SLIT2-IT1/miR-218 repression drives leukemogenesis and predicts adverse prognosis in myelodysplastic neoplasm.

Epigenetic modifications have been found to play crucial roles in myelodysplastic neoplasm (MDS) progression. Previously, we investigated genome-wide DNA methylation alterations during MDS evolution to acute myeloid leukemia (AML) by next-generation sequencing (NGS). Herein, we further determined the role and clinical implications of an evident methylation change in CpG islands at the SLIT2 promoter identified by NGS. First, increased SLIT2 promoter methylation was validated in 11 paired MDS/AML patients during disease evolution. Additionally, SLIT2 promoter methylation was markedly increased in MDS/AML patients compared with controls and was correlated with poor clinical phenotype and outcome. Interestingly, SLIT2 expression was particularly upregulated in AML patients and was not correlated with SLIT2 promoter methylation. However, the SLIT2-embedded genes SLIT2-IT1 and miR-218 were downregulated in AML patients, which was negatively associated with SLIT2 promoter methylation and further validated by demethylation studies. Functionally, SLIT2-IT1/miR-218 overexpression exhibited antileukemic effects by affecting cell proliferation, apoptosis and colony formation in vitro and in vivo. Mechanistically, SLIT2-IT1 may function as a competing endogenous RNA by sponging miR-3156-3p to regulate BMF expression, whereas miR-218 may directly target HOXA1 in MDS progression. In summary, our findings demonstrate that SLIT2 promoter hypermethylation is associated with disease evolution in MDS and predicts poor prognoses in both MDS and AML. Epigenetic inactivation of SLIT2-IT1/miR-218 by SLIT2 promoter hypermethylation could be a promising therapeutic target in MDS.

Reduced expression of lncRNA DLEU7-AS1 is a novel favorable prognostic factor in acute myeloid leukemia.

The objective of our study was to measure DLEU7-AS1 expression in de novo acute myeloid leukemia (AML) whilst also analyzing its clinical relevance. We used gene expression data from The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), Cancer Cell Line Encyclopedia (CCLE) and Genotype-Tissue Expression project (GTEx) to assess the expression profile of DLEU7-AS1 in pan-cancers, cancer cell lines and normal tissues. Reverse transcription-quantitative PCR was used to measure DLEU7-AS1 expression in bone marrow from 30 normal individuals and 110 patients with de novo AML. DLEU7-AS1 expression was found to be markedly reduced in the AML samples of the TCGA pan-cancer datasets. In our PCR validation, DLEU7-AS1 expression was significantly decreased in the AML samples compared with that in controls (P<0.001). Low DLEU7-AS1 expression (DLEU7-AS1low) correlated positively with lower blood platelet counts (P=0.029). In addition, low DLEU7-AS1 expression was more frequently observed in the intermediate (58%; 44/76) and favorable karyotypes (65%; 15/23) compared with that in the poor karyotype (10%; 1/10; P=0.005). In particular, patients with high expression levels of DLEU7-AS1 (DLEU7-AS1high) showed lower complete remission rates (P=0.002) than patients with DLEU7-AS1low. Survival analysis revealed that patients with DLEU7-AS1low had longer overall survival (OS) than patients with DLEU7-AS1high (P<0.05). Multivariate Cox analysis demonstrated that in patients with non-acute promyelocytic leukemia (non-M3) who were ≤60 years old, DLEU7-AS1 expression was an independent prognostic factor for OS. Furthermore, we found distinct correlations among the expression of DLEU7-AS1, infiltration by immune cells and immune checkpoint genes in AML.

Comprehensive analysis of SPAG1 expression as a prognostic and predictive biomarker in acute myeloid leukemia by integrative bioinformatics and clinical validation.

BACKGROUND: Recently, an increasing number of studies have reported that sperm-associated antigen (SPAG) proteins play crucial roles in solid tumorigenesis, and may serve as potentially helpful biomarkers for cancer diagnosis and prognosis. However, very few studies systematically investigated the expression of SPAG family members and their clinical significance in acute myeloid leukemia (AML). METHODS: The expression of SPAGs and their prognostic significance in AML were determined by a systematic analysis on data gathered from public databases, and the results were validated in clinical samples. RESULTS: Using public data, we identified only increased SPAG1 expression negatively associated with survival in AML by Cox regression (P < 0.001) and Kaplan-Meier analysis (P < 0.001). The prognostic value of SPAG1 expression was further confirmed in other independent cohorts. Clinically, higher SPAG1 expression was significantly correlated with white blood cell counts (P = 0.014) and French-American-British (FAB) subtypes (P = 0.024). Moreover, higher SPAG1 expression was more common in + 8 patients (P = 0.034), rarely found with t(8;21) (P = 0.014), and correlated with FLT3 (P < 0.001) and DNMT3A mutations (P = 0.001). Despite these associations, multivariate analysis confirmed the independent prognostic value of SPAG1 expression in AML (P < 0.001). Notably, AML patients with higher SPAG1 expression may benefit from hematopoietic stem cell transplantation (HSCT), whereas patients with lower SPAG1 expression appeared less likely to benefit. Finally, we further validated that SPAG1 expression was significantly increased in newly diagnosed AML patients compared with normal controls (P < 0.001) and with AML patients who achieved complete remission (P < 0.001). Additionally, SPAG1 expression could act as a potentially helpful biomarker for the diagnosis and prognosis of AML (P < 0.001 and = 0.034, respectively). CONCLUSIONS: Our findings demonstrated that SPAG1 overexpression may serve as an independent prognostic biomarker and may guide the choice between HSCT and chemotherapy in patients with AML.

Distinct associations of NEDD4L expression with genetic abnormalities and prognosis in acute myeloid leukemia.

BACKGROUND: There is mounting evidence that demonstrated the association of aberrant NEDD4L expression with diverse human cancers. However, the expression pattern and clinical implication of NEDD4L in acute myeloid leukemia (AML) remains poorly defined. METHODS: We systemically determined NEDD4L expression with its clinical significance in AML by both public data and our research cohort. Moreover, biological functions of NEDD4L in leukemogenesis were further tested by in vitro experiments. RESULTS: By the public data, we identified that low NEDD4L expression was correlated with AML among diverse human cancers. Expression of NEDD4L was remarkably decreased in AML compared with controls, and was confirmed by our research cohort. Clinically, low expression of NEDD4L was correlated with greatly lower age, higher white blood cells, and higher bone marrow/peripheral blood blasts. Moreover, NEDD4L underexpression was positively correlated with normal karyotype, FLT3 and NPM1 mutations, but negatively associated with complex karyotype and TP53 mutations. Importantly, the association between NEDD4L expression and survival was also discovered in cytogenetically normal AML patients. Finally, a number of 1024 RNAs and 91 microRNAs were identified to be linked to NEDD4L expression in AML. Among the negatively correlated microRNAs, miR-10a was also discovered as a microRNA that may directly target NEDD4L. Further functional studies revealed that NEDD4L exhibited anti-proliferative and pro-apoptotic effects in leukemic cell line K562. CONCLUSIONS: Our findings indicated that NEDD4L underexpression, as a frequent event in AML, was associated with genetic abnormalities and prognosis in AML. Moreover, NEDD4L expression may be involved in leukemogenesis with potential therapeutic target value.

[Overexpression of LncRNA ITGB2-AS1 Predicts Adverse Prognosis in Acute Myeloid Leukemia].

OBJECTIVE: LncRNA ITGB2-AS1 has been found to play important roles in the occurrence and development of human solid tumors. However, its role in hematological diseases, especially acute myeloid leukemia (AML), remains unclear. The aim of this study was to identify the expression pattern of ITGB2-AS1 in AML patients and to further explore its clinical significance. METHODS: ITGB2-AS1 expression was analyzed in public datasets (including TCGA and GSE63270) and further validated in a cohort of 109 AML patients by real-time quantitative PCR (RT-qPCR). RESULTS: The level of ITGB2-AS1 was up-regulated among two independent cohorts (TCGA, P<0.05; GSE63270, P<0.05), which was confirmed by the data from 109 AML patients enrolled in this study (P<0.05). Clinically, high ITGB2-AS1 expression was associated with older age (P=0.023) and lower complete remission (CR) rate (P=0.005). Multivariate analysis identified that high ITGB2-AS1 expression was an independent prognostic factor not only for CR rate (P=0.027) but also for overall survival (OS) time (P=0.011), and ITGB2-AS1 was positively correlated with ITGB2 expression in both TCGA (r=0.74, P<0.001) and clinical data detected in this study (r=0.881, P<0.001). High ITGB2 expression was also associated with older age (P=0.02) and lower CR rate (P=0.020). Moreover, high ITGB2 expression predicted worse OS (P=0.028). CONCLUSION: ITGB2-AS1 is overexpressed in AML and predicts poor prognosis in AML patients.

Abnormal expression and methylation of PRR34-AS1 are associated with adverse outcomes in acute myeloid leukemia.

It was previously reported that PRR34-AS1 was overexpressed in some solid tumors. PRR34-AS1 promoter was shown to have a differential methylation region (DMR), and was hypomethylated in acute myeloid leukemia (AML). Therefore, the present study used real-time quantitative PCR (RQ-PCR) to explore the expression characteristics of PRR34-AS1 in AML. In addition, the correlation between the expression of PRR34-AS1 and clinical prognosis of AML was determined. The findings of this study indicated that high PRR34-AS1 expression was bound up with shorter overall survival (OS) in AML patients (p = 0.002). Moreover, patients with high expression of PRR34-AS1 had significantly lower complete remission (CR) rate compared with those with low expression of PRR34-AS1 after induction chemotherapy. Furthermore, multivariate analysis confirmed that PRR34-AS1 expression was an independent factor affecting CR in whole-AML, non-APL-AML, and CN-AML patients (p = 0.032, 0.039, and 0.036, respectively). Methylation-specific PCR (MSP) and bisulfite sequencing PCR (BSP) were used to explore the methylation status of PRR34-AS1. PRR34-AS1 promoter showed a pattern of hypomethylation in AML patients compared with normal controls (p = 0.122). Notably, of whole-AML and non-APL-AML patients, PRR34-AS1 hypomethylated patients presented a significantly shorter OS than those with a hypermethylated PRR34-AS1 (p = 0.010 and 0.037, respectively). Multivariate analysis confirmed that the hypomethylation of PRR34-AS1 served as an independent prognostic indicator in both whole-cohort AML and non-APL-AML categories (p = 0.057 and 0.018, respectively). In summary, the findings of this study showed that abnormalities in PRR34-AS1 are associated with poor prognosis in AML. Therefore, monitoring this index may be important in the prognosis of AML and can provide information on effective chemotherapy against the disease.

Identification and validation of obesity-related gene LEP methylation as a prognostic indicator in patients with acute myeloid leukemia.

BACKGROUND: Obesity confers enhanced risk for multiple diseases including cancer. The DNA methylation alterations in obesity-related genes have been implicated in several human solid tumors. However, the underlying role and clinical implication of DNA methylation of obesity-related genes in acute myeloid leukemia (AML) has yet to be elucidated. RESULTS: In the discovery stage, we identified that DNA methylation-associated LEP expression was correlated with prognosis among obesity-related genes from the databases of The Cancer Genome Atlas. In the validation stage, we verified that LEP hypermethylation was a frequent event in AML by both targeted bisulfite sequencing and real-time quantitative methylation-specific PCR. Moreover, LEP hypermethylation, correlated with reduced LEP expression, was found to be associated with higher bone marrow blasts, lower platelets, and lower complete remission (CR) rate in AML. Importantly, survival analysis showed that LEP hypermethylation was significantly associated with shorter overall survival (OS) in AML. Moreover, multivariate analysis disclosed that LEP hypermethylation was an independent risk factor affecting CR and OS among non-M3 AML. By clinical and bioinformatics analysis, LEP may be also regulated by miR-517a/b expression in AML. CONCLUSIONS: Our findings indicated that the obesity-related gene LEP methylation is associated with LEP inactivation, and acts as an independent prognostic predictor in AML.

Genome-wide methylation sequencing identifies progression-related epigenetic drivers in myelodysplastic syndromes.

The potential mechanism of myelodysplastic syndromes (MDS) progressing to acute myeloid leukemia (AML) remains poorly elucidated. It has been proved that epigenetic alterations play crucial roles in the pathogenesis of cancer progression including MDS. However, fewer studies explored the whole-genome methylation alterations during MDS progression. Reduced representation bisulfite sequencing was conducted in four paired MDS/secondary AML (MDS/sAML) patients and intended to explore the underlying methylation-associated epigenetic drivers in MDS progression. In four paired MDS/sAML patients, cases at sAML stage exhibited significantly increased methylation level as compared with the matched MDS stage. A total of 1090 differentially methylated fragments (DMFs) (441 hypermethylated and 649 hypomethylated) were identified involving in MDS pathogenesis, whereas 103 DMFs (96 hypermethylated and 7 hypomethylated) were involved in MDS progression. Targeted bisulfite sequencing further identified that aberrant GFRA1, IRX1, NPY, and ZNF300 methylation were frequent events in an additional group of de novo MDS and AML patients, of which only ZNF300 methylation was associated with ZNF300 expression. Subsequently, ZNF300 hypermethylation in larger cohorts of de novo MDS and AML patients was confirmed by real-time quantitative methylation-specific PCR. It was illustrated that ZNF300 methylation could act as a potential biomarker for the diagnosis and prognosis in MDS and AML patients. Functional experiments demonstrated the anti-proliferative and pro-apoptotic role of ZNF300 overexpression in MDS-derived AML cell-line SKM-1. Collectively, genome-wide DNA hypermethylation were frequent events during MDS progression. Among these changes, ZNF300 methylation, a regulator of ZNF300 expression, acted as an epigenetic driver in MDS progression. These findings provided a theoretical basis for the usage of demethylation drugs in MDS patients against disease progression.

Methylation-independent CRIP1 expression is a potential biomarker affecting prognosis in cytogenetically normal acute myeloid leukemia.

Abnormal expression of CRIP1 has been identified in numerous solid tumors. However, CRIP1 expression and its regulation are little known in acute myeloid leukemia (AML). The purpose of this study was to evaluate the expression and regulation of CRIP1 and the clinical implications of CRIP1 aberration in AML. Real-time quantitative PCR was carried out to detect the level of CRIP1 expression in 138 AML patients and 38 controls. CRIP1 methylation was detected by methylation-specific PCR and bisulfite sequencing PCR. Five public available AML datasets from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) were further analyzed. The level of CRIP1 expression was up-regulated in AML patients compared with controls (P = 0.045). CRIP1 high patients had a significantly lower complete remission (CR) rate than CRIP1 low patients (P = 0.020). CRIP1 high group had a shorter overall survival (OS) and leukemia-free survival (LFS) than CRIP1 low group in cytogenetically normal AML (CN-AML) patients (P = 0.007 and 0.012, respectively). Multivariate analysis further confirmed that high CRIP1 expression was an independent risk factor for LFS in CN-AML patients (P = 0.005). However, we found that CRIP1 expression was not associated with the status of its promoter, which was nearly fully unmethylated both in controls and AML patients. Furthermore, our results were validated using the published GEO datasets and TCGA datasets. Our findings suggest that high CRIP1 expression is independently related with unfavorable prognosis in CN-AML.

Identification and validation of prognosis-related DLX5 methylation as an epigenetic driver in myeloid neoplasms.

The deregulated DLX gene family members DLX1/2/3/4/5/6 (DLXs) caused by DNA methylation has been demonstrated in various cancers with therapeutic target value. However, the potential role of DLXs methylation in myeloid neoplasms such as acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) remains to be elucidated. Clinical significance of DLXs methylation/expression was analyzed in patient with AML and MDS. The functional roles of DLXs were determined in vitro. In the identification stage, we found that lower DLX5 expression was correlated with prognosis in AML among all DLXs analyzed by The Cancer Genome Atlas datasets. In the validation stage, we revealed that reduced DLX5 expression was frequently occurred, and was also correlated with promoter hypermethylation in AML evaluated by targeted bisulfite sequencing. Epigenetic studies also showed that DLX5 promoter DNA methylation was associated with its expression. By quantitative polymerase chain reaction, we also validated that DLX5 hypermethylation was frequent event in both AML and MDS, and also correlated with MDS transformation to leukemia. Moreover, DLX5 hypermethylation was associated with lower rate of complete remission and shorter time of leukemia-free/overall survival, and was also confirmed by Logistic/Cox regression analysis. Functional studies revealed the antiproliferative and pro-apoptotic effects of DLX5 in MDS-derived AML cell-line SKM-1. Finally, bioinformatics analysis demonstrated that DLX5 functioned in leukemogenesis may be through the association with PI3K/Akt signaling pathway. Collectively, our findings demonstrated that DLX5 methylation, negatively correlated DLX5 expression, was a potential prognostic and predictive indicator in patients with AML and MDS, which could also act as an epigenetic driver in myeloid neoplasms.

Expression and prognosis analysis of DNMT family in acute myeloid leukemia.

DNA methyltransferases (DNMTs) by regulating DNA methylation play crucial roles in the progression of hematologic malignancies, especially for acute myeloid leukemia (AML). Accumulating investigations have identified the high incidence of DNMT3A mutation in AML, and it is correlated with poor prognosis. Although a few studies have shown the expression of DNMTs and their clinical significance in AML, the results remain to be discussed. Herein, we systemically analyzed the DNMTs expression and their relationship with clinic-pathological features and prognosis in AML patients. DNMTs expression especially for DNMT3A/3B was closely associated with AML among various human cancers. DNMT3A expression was increased in AML patients, whereas DNMT3B expression was decreased. Significant associations between DNMT3A/B expression and clinic-pathological features/gene mutations were observed. Kaplan-Meier analysis showed that DNMT3A expression was associated with better overall survival (OS) and leukemia-free survival (LFS) among whole-cohort AML, and independently affected OS determined by Cox repression multivariate analysis. Notably, patients that received hematopoietic stem cell transplantation (HSCT) showed significantly better OS and LFS in DNMT3A lower-expressed groups, whereas patients in DNMT3A higher-expressed groups did not. By bioinformatics analysis, DNMT3A expression was found to be positively correlated with several leukemia-associated genes/microRNAs, and DNMT3A was identified as direct targets of miR-429 and miR-29b in AML. Collectively, our study demonstrated that DNMT3A/3B showed significant expression differences in AML. DNMT3A expression acted as a potential prognostic biomarker and may guide treatment choice between chemotherapy and HSCT in AML.

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.

Hypomethylation of MIR-378 5'-flanking region predicts poor survival in young patients with myelodysplastic syndrome.

BACKGROUND: Previous studies have disclosed up-regulation of MIR-378 in acute myeloid leukemia (AML), and might consequently affect the outcome of the patients. Correspondingly, hypomethylation of MIR-378 was also identified in AML, particularly for FAB-M2 subtype with t(8;21) chromosomal translocation. Nevertheless, the methylation status of MIR-378 has not been illustrated in myelodysplastic syndrome (MDS). Herein we designed to understand the methylation pattern of MIR-378 involved in MDS and clinical interrelation thereof. METHODS: Real-time quantitative methylation-specific PCR (RQ-MSP) was performed to evaluate the methylation degree of MIR-378 5'-flanking region on bone marrow mononuclear cells collected from 95 de novo MDS patients. Five gene mutations (IDH1, IDH2, DNMT3A, U2AF1, and SF3B1) were detected by high-resolution melting analysis to further evaluate the clinical relevance of hypomethylation of MIR-378. RESULTS: Unmethylated level of MIR-378 5'-flanking region was significantly higher in MDS patients than that in controls (p = .034). Hypomethylated MIR-378 was identified in 20 of 95 (21%) cases with MDS. Male patients appeared to be more frequent to harbor MIR-378 hypomethylation compared to female patients (15/55, 27.3% vs. 4/40, 10.0%, p = .04). There was no significant difference in age, white blood cell counts, platelet counts, hemoglobin concentration, and karyotypes between the patients with and without MIR-378-hypomethylation. Distinct distribution of five gene mutations was not observed in the two groups as well. However, MIR-378-hypomethylated patients had significantly shorter overall survival than those without MIR-378 hypomethylation (p = .036). Moreover, among patients <60 years, hypomethylation of MIR-378 was confirmed to be an independent adverse prognostic factor by both Kaplan-Meier and Multivariate Cox analyses. CONCLUSION: Hypomethylation of MIR-378 5'-flanking region is an adverse prognosticator in MDS, particularly in patients <60 years.

DOK6 promoter methylation serves as a potential biomarker affecting prognosis in de novo acute myeloid leukemia.

BACKGROUND: Downstream of tyrosine kinase 6 (DOK6), which is specifically expressed in the nervous system, was previously recognized as an adapter only in neurite outgrowth. Recent studies also demonstrated the potential role of DOK6 in solid tumors such as gastric cancer and breast cancer. However, previous studies of DOK6 have not dealt with its roles in myeloid malignancies. Herein, we verified the promoter methylation status of DOK6 and further explored its clinical implication in de novo acute myeloid leukemia (AML). METHODS: A total of 100 newly diagnosed adult AML patients were involved in the current study. DOK6 expression and methylation were detected by real-time qPCR and methylation-specific PCR (MSP), respectively. Bisulfite sequencing PCR (BSP) was performed to assess the methylation density of the DOK6 promoter. RESULTS: Downstream of tyrosine kinase 6 promoter methylation was significantly increased in AML patients compared to controls (P = .037), whereas DOK6 expression significantly decreased in AML patients (P < .001). The expression of DOK6 was markedly up-regulated after treated by 5-aza-2'-deoxycytidine (5-aza-dC) in THP-1 cell lines. The methylation status of the DOK6 promoter was associated with French-American-British classifications (P = .037). There was no significant correlation existed between DOK6 expression and its promoter methylation (R = .077, P = .635). Interestingly, of whole-AML and non-APL AML patients, both have a tendency pertaining to the DOK6 methylation group and a significantly longer overall survival (OS) than the DOK6 unmethylation group (P = .042 and .036, respectively). CONCLUSION: Our study suggested that DOK6 promoter hypermethylation was a common molecular event in de novo AML patients. Remarkably, DOK6 promoter methylation could serve as an independent and integrated prognostic biomarker not only in non-APL AML patients but also in AML patients who are less than 60 years old.

SOX30 methylation correlates with disease progression in patients with chronic myeloid leukemia.

BACKGROUND: Our previous study has reported that aberrant SOX30 methylation was associated with poor prognosis in AML, and it correlated with disease progression in MDS. Herein, we further determined SOX30 methylation and its clinical significance in the other myeloid malignance - chronic myeloid leukemia (CML). METHODS: SOX30 methylation was examined by real-time quantitative methylation-specific PCR and bisulfite sequencing PCR, whereas SOX30 expression was detected by real-time quantitative PCR. RESULTS: SOX30 methylation was identified in 11% (10/95) CML patients. SOX30 methylation was associated with lower hemoglobin and platelets (P=0.006 and 0.032, respectively). Importantly, significant differences were observed in the distributions of clinical stages and cytogenetics (P=0.006 and 0.002, respectively). The frequency of SOX30 methylation in chronic phase (CP) stage occurred with lowest frequency (4/74, 5%), higher in accelerated phase (AP) stage (1/7, 14%), and the highest in blast crisis (BC) stage (12/31, 39%). In addition, SOX30 methylated patients tended to have a higher level of BCR-ABL transcript than SOX30 non-methylated patients (P=0.063). In two paired CML patients, SOX30 methylation showed lower density in CP stage (19% and 17%, respectively), and was significantly increased in BC stage (89% and 69%, respectively) during disease progression. Additionally, SOX30 methylated CML patients presented a lower SOX30 transcript level than SOX30 non-methylated CML patients (P=0.046). CONCLUSION: Our study revealed that SOX30 methylation correlated with disease progression in chronic myeloid leukemia.

Promoter methylation of the candidate tumor suppressor gene TCF21 in myelodysplastic syndrome and acute myeloid leukemia.

Transcription factor 21 (TCF21) has been identified as a candidate tumor suppressor gene which was epigenetically inactivated in a variety of human cancers. However, TCF21 methylation pattern remains unknown in hematologic malignancies. The aim of this study was to investigate TCF21 methylation and its clinical relevance in myelodysplastic syndrome (MDS) and non-M3 acute myeloid leukemia (AML). A total cohort of 33 MDS patients, 100 non-M3 AML patients and 25 healthy donors were enrolled in the study. Targeted bisulfite sequencing assay was performed to identify the methylation pattern of CpG islands within the promoter of TCF21 gene. The bioinformatics analyses were based on The Cancer Genome Atlas (TCGA) database and Gene Expression Omnibus (GEO). The results showed that there were significant differences in the methylation levels of TCF21 between MDS, non-M3 AML and controls (P = 0.003 and < 0.001, respectively). TCF21 hypermethylation might be served as a promising biomarker which could distinguish MDS/AML from normal controls (P < 0.001 and = 0.003, respectively). There was a significant difference in cytogenetic risk categories between TCF21 hypermethylation and non-hypermethylation AML patients (P = 0.032). Notably, TCF21 hypermethylation occurred frequently in AML patients with adverse risk category, compared with those with favorable and intermediate categories, respectively (67% vs 44% and 29%). TCF21 non-hypermethylation AML patients showed a higher probability of normal karyotype than abnormal karyotype (P = 0.003). The rate of DNMT3A gene mutation was significantly higher in the non-hypermethylation AML patients than that in the hypermethylation (8/44 vs 0/34, P = 0.020). These results suggested that aberrant DNA promoter methylation of TCF21 was frequent event in MDS and non-M3 AML, and TCF21 hypermathylation was associated with adverse risk karyotype in AML.