Epigenetic Mechanism Regulating DLX4 Expression And Its Clinical Implication In Myeloid Malignancies

Objective: The current study was aimed to investigate the pattern of DLX4（distal-less homeobox 4） expression and promoter methylation in patients with acute myeloid leukemia（AML）, chronic myeloid leukemia（CML）, and myelodysplastic syndrome（MDS）. The association between DLX4 expression and methylation, and their clinical implication were further analyzed.Methods: DLX4 transcript level was examined by real-time quantitative PCR（RQ-PCR） in the bone marrow mononuclear cells（BMMNCs） from 37 healthy donors, 102 AML patients, and 56 CML patients. Real-time quantitative methylation-specific PCR（RQ-MSP） was used to detect the level of DLX4 promoter methyaltion in 52 healthy donors, 173 AML patients, 87 CML patients, and 103 MDS patients. Bisulfite sequencing PCR（BSP） was carried out to verify the density of DLX4 methylation in representative patients. The agent 5-aza-2’-deoxycytidine（5-aza-d C） was applied for demethylation studies in leukemic cell lines（K562, THP1, and Kasumi-1）. Clinical significance of DLX4 methylation was obtained by the comparison between patients with and without DLX4 hypermethylation.Results: The transcript level of BP1（DLX4 transcript variant 1, NM₁38281.2） was significantly upregulated in AML patients compared with controls（median 0.034 vs. 0.000, P<0.001）, while DLX7（DLX4 transcript variant 2, NM₀01934.3） expression was significantly downregulated in AML patients compared to controls（median 0.001 vs. 0.265, P<0.001）. There were no significant differences in sex, age, hemoglobin（HB）, platelets（PLT）, bone marrow（BM） blasts, FAB classifications, karyotypes, karyotypic classifications, and common gene mutations between patients with low and high BP1 expression（BP1low and BP1high）（P>0.05）. However, BP1 high cases trended to have higher white blood cells（WBC） than BP1 low cases（P=0.053）. BP1 high patients had significantly lower complete remission（CR） rate than BP1 low patients after induction therapy among both all and non-M3 AML（P=0.039 and P=0.009, respectively）. Kaplan-Meier analyses revealed that BP1 high patients had significantly shorter overall survival（OS） time than BP1 low patients in both non-M3-AML（P=0.001） and cytogenetically normal AML（CN-AML）（P=0.040） but not in all AML（P=0.067）. Cox regression multivariate analyses demonstrated that BP1 overexpression was an independent prognostic factor in both whole AML（P=0.020） and non-M3-AML（P=0.014） but not in CN-AML patients（P=0.054）. No significant differences were observed in all clinical and laboratory parameters between patients with low and high DLX7 expression（P>0.05）. Moreover, there were also no significant differences in CR rate and OS time between patients with low and high DLX7 expression（P>0.05）. DLX4 promoter region 1（the Cp G islands located at the promoter of BP1） presented fully unmethylated in both controls and AML patients. However, DLX4 promoter region 2（the Cp G islands located at the promoter of DLX7） was significantly hypermethylated（reported as DLX4 methylation） in AML patients compared with controls（P<0.001）. There were no significant differences in sex, age, peripheral blood cells, BM blasts, FAB classifications, WHO classifications, karyotypes, karyotypic classifications, and common gene mutations between patients with and without DLX4 hypermethylation（P>0.05）. However, DLX4 hypermethylated patients showed significantly higher frequency of U2AF1 mutation compared with DLX4 non-hypermethylated patients（P=0.043）. Both all AML and non-M3-AML patients with DLX4 hypermethylation presented significantly lower CR rate than those with DLX4 non-hypermethylation（P=0.001 and <0.001, respectively）. DLX4 hypermethylated cases had significantly shorter OS time than DLX4 non-hypermethylated cases among both all AML（P=0.003）, non-M3-AML（P=0.001）, and CN-AML（P=0.032）. Cox regression multivariate analyses confirmed that DLX4 methylation was an independent risk factor in both all AML（P=0.001） and non-M3-AML patients（P<0.001）. DLX4 methylation was negatively associated with DLX7（R=-0.202, P=0.021） but not BP1（R=-0.049, P=0.582） expression in AML patients. DLX7 and BP1 expression were significantly increased after 5-aza-d C treatment in leukemic cell lines THP1 and Kasumi-1.The level of DLX4 methylation was significantly increased in CML patients as compared with controls（P=0.002）. Moreover, DLX4 methylation level in CML in blastic phase stage（BC-CML） was significantly higher than in CML in chronic phase stage（CP-CML） and CML in accelerated phase stage（AP-CML）（P<0.001）. No significant differences were found between the two groups with respect to age, sex, peripheral blood cells, and BCR-ABL transcript（P>0.05）. DLX4 hypermethylation occurred with the highest incidence in BC-CML（83%）, lower incidence in AP-CML（43%）, and the lowest incidence in CP-CML（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）. DLX4 methylation density was significantly increased during the progression of CML among the tested two patients（P<0.001）. Significant negative correlation was observed between DLX4 methylation and DLX7 expression（R=-0.382, P=0.001） but not between DLX4 methylation and BP1 expression（R=0.134, P=0.244） in CML patients. Both DLX7 and BP1 expression were significantly increased after 5-aza-d C treatment in leukemic cell line K562.DLX4 was significantly hypermethylated in MDS patients than controls（P<0.001）. No significant differences were observed between the hypermethylated and non-hypermethylated MDS patients in age, WBC, PLT, WHO classifications, FAB classifications, IPSS risks, and common gene mutations（P>0.05）. However, DLX4 hypermethylated patients tended to have higher HB than DLX4 non-hypermethylated patients（P=0.079）. Moreover, there was a trend that male patients, poor karyotype patients, and IPSS Int-2/High patients had a higher frequency of DLX4 hypermethylation（P=0.067, 0.065, and 0.068, respectively）. DLX4 hypermethylated patients had significantly shorter OS time than DLX4 non-hypermethylated patients（P=0.004）. Cox regression analyses confirmed the prognostic value of DLX4 methylation in MDS patients（P<0.001）.Conclusions:（1）BP1 overexpression was a frequent event and was associated with poor prognosis in AML, whereas low DLX7 expression was a common event in AML but not correlated with adverse prognosis.（2）Abnormal methylation of DLX4 promoter region 1 was not observed in AML patients, whereas hypermethylation of DLX4 promoter region 2 was a frequent event and was negatively associated with DLX7 expression in AML as well as CML.（3）Hypermethylation of DLX4 pomoter region 2 was associated with unfavorable clinical outcome in AML as well as MDS patients and correlated with disease progression in CML patients.