GENETIC AND EPIGENETIC MECHANISMS REGULATING CATALASE EXPRESSION IN CHRONIC LYMPHOCYTIC LEUKEMIA

Chronic lymphocytic leukemia (CLL) is an incurable disease charac- terized by an extremely variable clinical course. Along with the under- standing of the molecular heterogeneity of the disease, growing interest is emerging in redox metabolism in CLL. We have recently documented a differential catalase expression in CLL associated with divergent clin- ical behaviors. However, the mechanisms controlling the transcription of catalase gene are poorly understood. The main objective of this study is to investigate regulatory mechanisms underlying differential expres- sion of catalase in CLL. We investigated the role of the rs1001179 SNP and methylation levels of the catalase promoter on catalase expression in primary CLL cells, using RFLP-PCR and pyrosequencing. Catalase expression has been assessed using qPCR and flow cytometry. The rs1001179 SNP genotyping shows that CLL cells harboring the T allele exhibit a significantly higher catalase expression compared with cells bearing the CC genotype. Moreover, we show that methylation of cata- lase promoter influences catalase expression. First, CLL cells exhibit lower methylation levels compared with healthy donor (HD) B cells, in line with the higher catalase mRNA and protein levels expressed by CLL in comparison with HD B cells. Then, the methylation levels at specific CpG sites negatively correlate with the catalase gene expression level in CLL cells. Remarkably, the role of promoter methylation in regulating catalase expression was functionally validated inhibiting the activity of methyltransferase in primary CLL cells, using 2’-deoxy-5-azacitidine (DAC). Treatment of leukemic cells with DAC induces a significant in- crease in catalase gene expression, thus showing that DNA methylation controls catalase expression in CLL. Finally, we investigated the rela- tionship between the genetic and epigenetic regulatory levels, in con- trolling catalase expression using a mathematical linear model. Remarkably, the rs1001179 T allele and methylation interact in regulat- ing catalase gene expression, thus indicating that the CT/TT genotypes show a lower methylation levels and a higher catalase gene expression level. The key result of this study is to provide new insights into the knowledge of genetic and epigenetic mechanisms at the basis of differ- ential expression of catalase in CLL, which could be of crucial relevance for the development of therapies targeting redox pathways. We thank Gilead for funding support.