Genome-wide DNA methylation of a proliferative CLL subset reveals upregulation of antiapoptosis/proliferation and drug resistance related genes

ABREU CECILIA; PALACIOS FLORENCIA; PRIETO DANIEL; MORANDE PABLO; GREIF GONZALO; FERNÁNDEZ-CALERO TAMARA; GABÚS RAÚL; DIGHIERO GUILLERMO; OPPEZZO PABLO

Sydney

Workshop; XVI International Workshop on Chronic Lymphocytic Leukaemia; 2015

Chronic Lymphocytic Leukemia (CLL) has a strikingly variable clinical course and althoughavailable drugs are able of induce disease remission, most patients inevitably relapse.Evidences suggest that the microenvironment has a critical role in CLL cell survival,accumulation and drug resistance through the maintenance of a proliferating B-cellsubpopulation. In previous work, our group described a proliferative small tumoral subsetpresent in the peripheral blood of unmutated and progressive CLL patients expressing highlevels of the enzyme AID (activation-induced cytidine deaminase). Concomitantly with themutagenic capacity of AID and their association with the development of many human B-cellmalignancies, this enzyme has been also related with DNA demethylation. To gain insightinto the origins of this proliferative subset and the putative role of AID in epigenetic generegulation in these cells, we isolated by cell sorter analysis both subsets and performedgenome wide DNA-methylation through CpG island microarrays (244K-arrays Agilent). Ourresults display 322 genes that are differentially demethylated in the proliferative subsetcompared with their quiescent counterpart. Data crossing-over between genome wide DNAmethylationand mRNA arrays expression interrogating these subsets, allowed us to select 15demethylated/overexpressed genes related to proliferation or antiapoptotic function. Fromthese 15 selected genes, 5 of them were furthermore validated by bisulphite sequencing andqRT-PCR (GIMAP1, GIMAP5, PRKCH, MYO1G and STARD10). To explore the biologicrelevance of this observation in CLL we focus on two key molecules selected by theirimportance in tumor survival: a) GIMAP5 (GTPase IMAP family member 5) an intracellularprotein associated with tumor progression by its interaction with antiapoptotic Bcl-2 familyproteins and b) PRKCH (protein kinase C eta), which also has a role in apoptosis regulationand drug resistance in several cancers. Our studies concerning the expression of these twomolecules at protein level show that both are overexpressed in the proliferative fractionconfirming the data obtained in the methyloma and transcriptoma arrays analysis. At this timewe are focused in the biological role of GIMAP5 and PRKCH expression in the proliferativeCLL pool, as well as their clinical impact in the CLL evolution.