Hypermethylation of HOX Genes Is Associated with Longer Survival in Acute Myeloid Leukemia
Authors: Jelinek Jaroslav , Kroeger Heike , He Rong , Saraf Amanda , Lau Samantha F , Nguyen Tuong-Van , Thomas Michael W , Shpall Elizabeth J , Kornblau Steven , Bueso-Ramos Carlos , Kantarjian Hagop M , Issa Jean-Pierre J 
Published: April 2020

Abstract
HOX genes regulate developmental processes and tissue differentiation. The HOX family consists of 39 genes forming paralogs assembled in 4 separate clusters (A, B, C & D) on 4 chromosomes. Translocations or dysregulation of HOX genes are frequently found in leukemia. Since DNA methylation of CpG islands at transcriptional start sites of genes leads to permanent epigenetic silencing equivalent to loss-of-function mutations, we sought to investigate methylation of HOX genes in acute myeloid leukemia (AML). We performed a comprehensive DNA methylation analysis by bisulfite pyrosequencing of 31 HOX genes in 24 AML patients and characterized a subset of 10 HOX genes hypermethylated in AML (HOXA4, HOXA5, HOXA6, HOXA7, HOXA9, HOXB13, HOXC4, HOXC10, HOXD1 & HOXD9). We next measured DNA methylation of these HOX genes in the bone marrow obtained at the time of diagnosis in another set of 67 AML patients. Hypermethylation exceeding the upper 95% confidence limit established in 23 normal control blood samples was observed in AML patients in proportions shown after gene names: HOXA4, 84%; HOXA5, 35%; HOXA6, 53%; HOXA7, 19%; HOXA9, 26%; HOXB13, 62%; HOXC4, 51%; HOXC10, 39%; HOXD1, 82%; and HOXD9, 75%. Hypermethylation of HOXA5 and HOXA6 genes was associated with longer overall survival (P=0.05 and 0.007, respectively). Additionally, methylation of HOXA5, HOXA6, HOXA9, HOXC4, and HOXD9 genes significantly correlated with methylation of genes that we previously showed to be associated with good prognosis in AML, namely NOR1 and SLC26A4 (Kroeger et al., Blood, 2007, 110: ASH Annual Meeting Abstract 595). In order to better understand the impact of DNA methylation on HOX gene silencing, we analyzed mRNA expression of the above 10 HOX genes in 24 patients with available RNA by TaqMan real time PCR and compared it to normal adult and cord blood cells. HOXA5, HOXA6, HOXA7, HOXA9, and HOXC10 mRNAs were overexpressed in 5, 7, 8, 12, and 4 patients (21%, 29%, 33%, 50%, and 17%, respectively). Overexpression of HOXA5 and HOXA7 mRNAs was associated with significantly shortened survival (P&lt;0.001 and P=0.005, respectively), and HOXA6 mRNA overexpression showed a trend to worse survival (P=0.09). Associations of increased expression of HOXA5, HOXA6, and HOXA7 with a poor outcome suggest functional significance and are in a good agreement with our observation of a good prognosis in patients with hypermethylated HOXA5 and HOXA6 genes. As expected, expression levels of HOX genes were inversely correlated to their methylation status and no significant expression was observed in genes methylated over 50%. HOXA5 gene was an exception, since high expression of HOXA5 mRNA was observed in AML patients with average methylation densities of 66-87% detected by bisulfite pyrosequencing. Bisulfite genomic sequencing of cloned PCR products revealed the presence of alleles completely free from methylation and thus available for expression beside alleles fully methylated and presumably silenced. In conclusion, our data on HOX gene methylation in AML provide additional support to our concept of a distinct category of ‘epigenetic AML’ with pronounced epigenetic gene silencing that is associated with longer survival and greater potential for cure by chemotherapy.