Distinct DNA Methylation Patterns of HOX Genes in Leukemia.

The HOX subset of homeobox gene family comprises 39 genes organized in 4 clusters A-D on 4 different chromosomes and in 13 paralog groups defined across the clusters. HOX genes are important regulators of development and tissue differentiation. Dysregulated expression disturbs hematopoiesis and HOX genes are frequent partners in chromosomal translocations involved in leukemia. DNA methylation of promoter-associated CpG islands is an epigenetic modification resulting in transcriptional silencing of affected genes. Methylation pattern is faithfully copied during cell replication to subsequent cell generations and hypermethylation thus represents a permanent mark functionally equivalent to a loss-of-function mutation. Epigenetic silencing of HOX genes may disrupt normal development of blood cells and thus be involved in the development of leukemia, as was recently reported for HOXA5. We performed a comprehensive DNA methylation analysis of 22 HOX genes with CpG islands in promoter to exon 1 regions: HOXA1, A4, A5, A7, A9, A10, A11; B1, B4, B5, B7, B8, B9, B13; C4, C5, C8, C9, C10, C13; D1 and D10 in 16 leukemic cell lines, 24 samples from patients with acute myelogenous leukemia (AML), 20 samples from patients with acute lymphoblastic leukemia (ALL), and 15 control samples of normal blood cells. We used bisulfite treatment of DNA, followed by PCR and pyrosequencing to quantitatively measure levels of cytosine methylation in promoter-associated CpG islands close to transcription start sites. Nonparametric tests were used for statistical analysis. Overall, lymphoblastic leukemic cell lines (TALL, Raji, ALL1, JTAg, CEM, BJAB, Jurkat) showed the highest degree of HOX genes methylation (average 60%), followed by myeloid leukemic cell lines (HL60, KG1a, ML1, HEL, TF-1, OCI-AML3, K562, TF1i, KG1, average methylation 40%), p=0.000. Analysis of ALL and AML patient samples revealed significant differences in methylation levels of several HOX genes. Consistently with their reported overexpression in leukemia, HOX A9, B4, and also HOXB5 and HOXD10 were seldom methylated in AML patients (2/24, 0/24, 1/12, 7/24, respectively), while hypermethylation over a 10% cutoff value was observed frequently in ALL patients (10/20, 6/19, 6/17, 9/19, respectively). The differences in methylation were statistically significant (p<0.05). On the other hand, HOXA4 showed significantly higher methylation densities in AML (median 74%, range 35–95%) and ALL (median 49%, range 12–91%) than in normal controls (median 34%, range 17–59%), p<0.01. HOXC4 was more frequently methylated in normal controls (11/13) and AML (21/24) than in ALL (12/20 patients). ALL patients also showed significantly lower methylation densities of HOXC4 (median 12%, range 5–39%) than AML patients (median 32%, range 4–54%, p=0.001), underscoring the importance of HOXC4 for lymphoid cells. Unsupervised hierarchical clustering of methylation densities of all analyzed HOX genes clearly separated AML from ALL patients. We propose that epigenetic silencing of HOX genes by DNA methylation plays a role in the process of leukemic transformation of hematopoietic cells. Further analyses will help to gain a better insight into the role of HOX gene dysregulation in leukemia.