Clinical Study Of NKD2 Expression And Methylation In Myeloid Malignancies

ObjectiveIn recent years,a large number of studies have reported the role and mechanism of NKD2 in several solid tumors.However,the pattern of NKD2 expression and clinical relevance in myeloid malignancies has not been explored yet.Therefore,we focused on the NKD2 expression in bone marrow mononuclear cells as well as methylation and their clinical significance in de novo patients with acute myeloid leukemia(AML),chronic myeloid leukemia(CML)and myelodysplastic syndrome(MDS)in the current study to explore its impact on patient’s prognosis and survival.It can also provide guidance for clinical treatment and disease monitoring,and provide important clues for exploring new biological markers and potential therapeutic targets.Methods1.Real-time quantitative PCR(RQ-PCR)was used to detect NKD2 transcript level in24 normal controls,113 AML,71 CML and 37 MDS patients.2.NKD2methylationlevelwasdetectedbyreal-timequantitative methylation-specific PCR(RQ-MSP)in 35 normal controls,146 AML,82 CML and90 MDS patients.3.bisulfite sequencing PCR(BSP)were performed to detect the level of NKD2methylation in myeloid malignances.4.Demethylation agent 5-aza-dC was used to treat leukemia cell lines(THP-1 and K562)and construct a dosing cell model to evaluate whether the hypermethylation of NKD2 is a regulatory mechanism for its abnormal expression.Results1 NKD2 expression and methylation in AML patientsThe transcript level of NKD2 in AML patients was significantly lower than controls(P=0.039).There were no significant differences in sex,age,white blood cell(WBC),hemoglobin(HB),platelets and BM blasts between the NKD2~lowow and NKD2~highigh groups(P>0.05).No significant differences were found between the two groups in karyotypic classifications and gene mutation(P>0.05).Moreover,there was no significant difference in complete remission(CR)rate between NKD2~lowow patients and NKD2~highigh patients in whole AML,non-APL AML(AML without acute promyelocytic leukemia)and CN-AML(Cytogenetically normal AML)patients(P>0.05).However,Kaplan-Meier survival analysis found that patients with low NKD2 expression tend to have a shorter overall survival(OS)time than those with high NKD2 expression in whole AML(P=0.081).In addition,among CN-AML patients,cases in NKD2~lowow expression group had significantly shorter OS time than those in NKD2~highigh expression group(P=0.029).We further performed Cox regression analysis to determine the prognostic value of NKD2 expression in CN-AML patients.Univariate analysis disclosed that low NKD2 expression was an independent risk factor in OS among CN-AML patients(P=0.022).Gene expression profiling(GEP)data from two independent cohorts of adult CN-AML patients(162 and 78)showed that patients with low NKD2 expression had shorter OS time as compared to NKD2~highigh group(P=0.033 and 0.053,respectively).In follow-up AML patients,the expression of NKD2 was up-regulated in patients who achieved CR after induction therapy(P=0.024).Furthermore,NKD2 methylation level in AML patients was significantly higher than controls(P=0.033).However,there were no significant differences in clinical manifestations and laboratory features between the two groups(P>0.05).We found that patients in NKD2 hypermethylation group had a significantly lower complete remission(CR)rate than those in NKD2non-hypermethylation group(P=0.024).In addition,cases in NKD2 hypermethylation group had significantly shorter OS time than those in NKD2 non-hypermethylation group among whole AML and non-APL AML(P=0.005,P=0.034).Univariate analysis disclosed that NKD2 hypermethylation was an independent risk factor in OS among whole AML patients(P=0.006).Moreover,there was a significant negative correlation between NKD2 expression and its promoter methylation in AML patients(R=-0.218,P=0.029).We further selected THP1 leukemia cell line treated with different concentrations of 5-aza-dC to verify the epigenetic mechanism.NKD2 promoter methylation level was decreased,meanwhile,NKD2 expression was significantly up-regulated after 5-aza-dC treatment,suggesting that NKD2 expression may be regulated by its methylation in AML.At the same time,the correlation between NKD2 and canonical Wnt/β-catenin(β-catenin,a protein encoded by CTNNB1 gene)signaling pathway was also detected in our study,but the results suggested that there was no significant relevance between CTNNB1 and NKD2expression.2 NKD2 expression and methylation in CML patientsThe transcript level of NKD2 in CML patients was significantly lower than controls(P=0.002).There was no significant difference in BCR-ABL1 transcript between two groups(P>0.05).No significant difference was found between CML and controls in NKD2 methylation level(P>0.05).There was no significant relevance between NKD2 expression and its methylation.We further selected K562 leukemia cell line treated with different concentrations of 5-aza-dC to verify the epigenetic mechanism.NKD2 transcript level increased while its methylation level decreased with the increase of drug concentration,indicating that NKD2 expression may be partially regulated by its methylation in CML.3 NKD2 expression and methylation in MDS patientsThe transcript level of NKD2 in MDS patients was significantly lower than controls(P=0.001).There were no significant differences between NKD2 expression and laboratory features as well as common gene mutations(P>0.05).Kaplan-Meier survival analysis found no significant association in OS time between two groups.No significant difference was found between MDS and controls in NKD2 methylation level(P>0.05).However,there was a significant negative correlation between NKD2expression and its promoter methylation in MDS patients(R=-0.705,P=0.034).Conclusions1.NKD2 low expression is a common event in myeloid malignancies.2.Decreased NKD2 expression is associated with adverse outcome in CN-AML patients.It can also be used for condition monitoring of disease treatment.3.NKD2 hypermethylation was an independent risk factor in OS among whole AML patients.4.Decreased NKD2 expression inactivated by promotor hypermethylation is a common event in AML and MDS patients.