| Objective DOK1/2/5 are closely related members of downstream of tyrosine kinase(DOK)family genes,which are found to be frequently rearranged in several hematopoietic cancers and plays important role in tumor development.This study was aimed to explore expression,promoter methylation,and clinical significance of DOK1/2/5 in acute myeloid leukemia(AML),chronic myeloid leukemia(CML),and myelodysplastic syndrome(MDS).Methods The expression of DOK1/2/5was detected by real-time quantitative PCR(RQ-PCR).Methylation level of DOK1/2/5was detected by real-time quantitative methylation-specific PCR(RQ-MSP),methylation density was verified by bisulfite sequencing PCR(BSP).Leukemic cell lines(THP-1 and K562)were treated with demethylated agent decitabine to further validate whether DOK1/2/5 methylation regulating expression.Results DOK1/2/5 expression levels in AML patients were significantly down regulated than those in normal controls(P=0.041,P=0.016,and P<0.001).DOK1 and DOK2 high and low expression levels were not significantly different in clinical parameters of AML patients(P>0.05).DOK5 expression levels were higher in male than in female AML patients(P=0.031).Survival analyses showed that the overall survival time of all AML and non-M3 AML patients with DOK1 low expression were significantly shorter than DOK1 high expression patients(P=0.006 and P=0.015).Meanwhile,leukemia-free survival time was also significantly shorter in DOK1 low expressed all AML and non-M3 AML patients than those with high expressed patients(P=0.018 and P=0.009).Follow-up data after chemotherapy showed that the complete remission rate was significantly lower in the DOK2 low expression patients than in the high expression patients(P=0.046).Furthermore,overall survival and leukemia-free survival were significantly shorter in all AML(P<0.001 and P=0.022)and non-M3 AML(P<0.001 and P=0.006)patients with low DOK2 expression.Multivariate analyses further revealed that low expression of DOK1 and DOK2 were independent prognostic factors in AML patients.Although the expression of DOK5 was lower in AML patients than in controls,DOK5 expression level was not related tooverall survival and leukemia-free survival of AML patients(P>0.05).RQ-MSP showed that DOK1 and DOK2 methylation were significantly higher in AML patients(P=0.047 and P=0.048).While,the difference of DOK5 methylation level in AML patients and controls was not statistically significant(P=0.301).There were no significant difference in laboratory parameters,karyotype classification,karyotype,and gene mutations were found between DOK1 and DOK2 hypermethylationin and hypomethylation in AML patients(P>0.05).Survival analyses found that overall survival and leukemia-free survival of DOK1 hypermethylated patients were significantly shorter than those hypomethylated patients in all AML cohort(P=0.026 and P=0.043),whereas DOK1 methylation levels were not associated with overall survival and leukemia-free survival time in non-M3 AML patients(P>0.05).DOK2 hypermethylated patients in all AML and non-M3 AML were associated with significantly shorter overall survival time(P=0.005 and P=0.016),but not related with leukemia-free survival time in those patients(P>0.05).The complete remission rate of DOK2 hypermethylated AML patients was significantly lower than those hypomethylated patients(P=0.011).Multivariate analyses showed that hypermethylation of DOK1 and DOK2 were independent prognositic factors in AML patients.Correlation analyses found that DOK1 and DOK2 gene expression and methylation were negatively correlated in AML patients(R=-0.341,P=0.001 and R=-0.252,P=0.011).However,DOK5 expression was not associated with methylation in AML patients(R=0.049,P=0.791).THP-1 leukemia cell line treated with demethylation agent revealed that DOK1 and DOK2 expression levels were up-regulated with decitabine treatment.Meanwhile,the methylation levels and methylation densities were significantly decreased after treatment.DOK1/2/5 expression among CML patients and controls showed no significant difference(P=0.704,P=0.210,and P=0.736).Methylation levels in controls and CML patients detected by RQ-MSP showed that DOK2 methylation in CML patients was significantly higher(P<0.001).Whereas,DOK1 and DOK5 methylations were not significantly different between CML patients and controls(P=0.659 and P=0.624).In addition,no significant differences in clinical characteristics such as sex,age,whiteblood count,hemoglobin,platelet count,and karyotype were found between DOK2 hypermethylated and hypomethylated patients(P>0.05).Furthermore,there was no significant difference between DOK2 hypermethylated and hypomethylated patients in CML disease stages: chronic phase,accelerated phase,and blast crisis phase were found(P>0.05).After decitabine demethylation treatment of leukemia cell line K562,the expression level of DOK2 was up-regulated,and both methylation level and density were significantly decreased.Methylation of DOK1/2/5 in MDS patients showed that DOK1 and DOK2 methylation levels were significantly higher in MDS patients than in controls(P=0.017 and P<0.001),whereas DOK5 methylation in MDS patients and controls showed no significant difference(P=0.073).No significant difference in gender,age,white blood count,hemoglobin,platelet count,and bone marrow blast between DOK1 and DOK2 hypermethylated and hypomethylated MDS patients were identified(P>0.05).In addition,clinical parameters such as karyotype classification,IPSS score,and gene mutations were also not associated with DOK1 and DOK2 methylation levels(P>0.05).Survival analyses showed that DOK1 and DOK2 hypermethylated MDS patients had relatively shorter overall survival time,but the differences were not statistically significant(P=0.974 and P=0.305).Conclusion DOK1 and DOK2 were down-regulated in AML patients and were independent prognositic factors.Lower expression of DOK5 was not related with the prognosis of AML patients.Hypermethylation of DOK1 and DOK2 were common molecular events in AML and were associated with poor prognosis.Moreover,DOK1 and DOK2 expression and methylation were negatively correlated in AML.Hypermethylation of the DOK1 and DOK2 were also detected in MDS patients,but not associated with patients’ prognosis.DOK1/2/5 expression in CML patients found no difference between controls.DOK2 was found hypermethylated in CML,but was not associated with disease progression. |
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