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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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

CHFR acts as a tumor suppressor gene, which is frequently inactivated caused by its promoter hypermethylation in various solid tumors. Although a recent study showed that CHFR hypermethylation was a frequent event in acute myeloid leukemia (AML) and correlated with adverse clinical outcome, herein, we found that CHFR methylation was a rare event in patients with myeloid malignancies (including AML, chronic myeloid leukemia, and myelodysplastic syndromes), but its expression may serve as an independent prognostic biomarker in AML. CHFR expression was assessed by real‐time quantitative PCR, whereas CHFR methylation was detected by methylation‐specific PCR and bisulfite sequencing PCR. In AML patients, lower CHFR expression was associated with lower complete remission (CR) rate, and CHFR expression was significantly increased in CR after chemotherapy. Moreover, patients with lower CHFR expression showed shorter overall survival and leukemia‐free survival, and multivariate analysis confirmed that lower CHFR expression was an independent risk factor in AML. Importantly, the prognostic value of CHFR expression was validated using the published Gene Expression Omnibus datasets. Notably, CHFR promoter was nearly unmethylated in patients with myeloid malignancies. Our findings revealed that lower CHFR expression was independently associated with unfavorable prognosis in AML. Moreover, aberrant CHFR promoter methylation was a rare event in myeloid malignances. [ABSTRACT FROM AUTHOR]

Epigenetic inactivation of GPX3 has been identified in various cancers including leukemia. Moreover, aberrant DNA methylation was also found as a dominant mechanism of disease progression in myelodysplastic syndrome ( MDS). This study intended to explore GPX3 promoter methylation and its clinical relevance in 110 patients with MDS. GPX3 methylation was examined by real-time quantitative methylation-specific PCR ( RQ- MSP) and bisulfite sequencing PCR ( BSP). GPX3 methylation was identified in 15% (17/110) MDS patients, and significantly higher than controls, and lower than acute myeloid leukemia ( AML) patients ( P = 0.024 and 0.041). GPX3 methylated patients had older age and higher frequency of DNMT3A mutation ( P = 0.015 and 0.066). Cases with GPX3 methylation showed significantly shorter overall survival ( OS) time than those with GPX3 unmethylation analyzed with Kaplan-Meier analysis ( P = 0.012). Moreover, Cox regression analysis revealed that GPX3 methylation might act as an independent prognostic indicator in MDS ( HR = 1.847, P = 0.072). GPX3 methylation density was significantly increased during the progression from MDS to secondary acute myeloid leukemia ( sAML) in three follow-up paired patients. Our study concludes that GPX3 methylation in bone marrow is associated with adverse prognosis and leukemia transformation in MDS. [ABSTRACT FROM AUTHOR]

Aberrant DNA methylation of various genes has been identified to be associated with disease progression in chronic myeloid leukemia (CML). Our study was intended to investigate DLX4 methylation pattern in different clinical stages of CML and further determine its role in regulating DLX4 expression. Real-time quantitative methylation-specific PCR and bisulfite sequencing PCR were applied to detect DLX4 methylation. 5-aza-2′-deoxycytidine (5-aza-dC) was used for demethylation studies. DLX4 was significantly hypermethylated in CML patients ( P = 0.002) especially in blastic phase (BC) stage ( P < 0.001) as compared with controls. Moreover, DLX4 methylation level in BC stage was significantly higher than in chronic phase (CP) stage ( P < 0.001). DLX4 methylation density was significantly increased during the progression of CML among the tested two patients ( P < 0.001). DLX4 hypermethylation occurred with the highest incidence in BC stage (83%), lower incidence in acute phase (AP) stage (43%), and the lowest incidence in CP stage (26%) ( P = 0.001). Moreover, t(9; 22) with additional alteration cases had significantly higher frequency of DLX4 hypermethylation compared with the other cytogenetics ( P = 0.010). Significantly negative correlation was observed between DLX4 methylation and DLX4-TV2 (the shorter DLX4 isoform) expression (R = −0.382, P = 0.001, n = 78) but not between DLX4 methylation and BP1 (the longer DLX4 isoform) expression (R = 0.134, P = 0.244, n = 78) in CML patients. Both DLX4-TV2 and BP1 mRNA were significantly increased after 5-aza-dC treatment in K562 cell line ( P < 0.001). Our study indicated that hypermethylation of DLX4 correlated with disease progression of CML. Moreover, DLX4 expression was regulated by its methylation in CML. [ABSTRACT FROM AUTHOR]

Background: Leukemia stem cell (LSC)-enriched genes have been shown to be highly prognostic in acute myeloid leukemia (AML). However, the prognostic value of tumor suppressor genes (TSGs) that are repressed early in LSC remains largely unknown.Methods: We compared the public available expression/methylation profiling data of LSCs with that of hematopoietic stem cells (HSCs), in order to identify potential tumor suppressor genes in LSC. The prognostic relevance of PCDH17 was analyzed on a cohort of 173 AML patients from The Cancer Genome Atlas (TCGA), and further validated in three independent cohorts (n = 339).Results: We identified protocadherin17 (PCDH17) and demonstrated that it was significantly down-regulated and hypermethylated in LSCs compared with HSCs. Our analyses of primary AML patient samples also confirmed these deregulations. Clinically, low PCDH17 expression was associated with female sex (P = 0.01), higher WBC (P < 0.0001), higher percentages of blasts in bone marrow (BM) and peripheral blood (PB) (P = 0.04 and < 0.001, respectively), presence of FLT3-internal tandem duplications (P = 0.002), mutated NPM1 (P = 0.02), and wild-type TP53 (P = 0.005). Moreover, low PCDH17 expression predicted worse overall survival (OS) in four independent cohorts as well as in the molecularly defined subgroups of AML patients. In multivariable analyses, low PCDH17 expression retained independent prognostic value for OS. Biologically, PCDH17 expression-associated gene signatures were characterized by deregulations of EMT- and Wnt pathway-related genes.Conclusions: PCDH17 gene was silenced by DNA methylation in AML. Low PCDH17 expression is associated with distinct clinical and biological features and improves risk stratification in patients with AML. [ABSTRACT FROM AUTHOR]