14. DNA methylation status is associated to lipoproteína lipase (LPL) expresión and can be modulated by FC treatment in unmutated CLL patients.

ABREU CECILIA; MORENO P; PALACIOS F; AGREGLO; LANDONI; GAMBERALE ROMINA; GIORDANO MIRTA; DIGHIERO G; OPPEZZO PABLO

houston, texas

Workshop; XIV International Workshop on CLL.; 2011

Background. Chronic lymphocytic leukemia (CLL) is a remarkably heterogeneous disease with regards to clinical course. This disease ranges from indolent to aggressive stages and remains incurable as yet. In the last years, several biologic markers such as cytogenetics, IgVH mutations, CD38 and ZAP-70 expression in CLL B-cells have provided important prognostic information. In a previous work, we reported that expression of the lipoprotein lipase (LPL) gene at the RNA level was clearly  associated to a clinical poor outcome in CLL (Oppezzo et al Blood 2005). Further  work have confirmed  these results and postulated that high expression of Zap-70, LPL and microRNA-29c is the most powerful molecular tools for CLL stratification at the time of diagnosis (Stamatopoulos et al, PlosOne, 2010). However, unlike Zap-70 and microRNA-29c, physiological role of LPL expression in CLL B-cell remains unclear. LPL is normally produced by parenchymal cells in several tissues, with the largest expression found in adipose tissue, cardiac and skeletal muscle. In addition,  LPL can augment interaction between cells where it has been shown to form a bridge between monocyte and endothelial cell surface heparan sulfate-proteoglycans. Since, modest or non expression of LPL gene has been reported in normal B-cells, its high expression in unmutated (UM) CLL B-cells, constitutes a suitable marker to the study of disease prognosis. In this work, we aimed to determine the reasons of this aberrant expression in CLL B-cells and we investigated whether this expression could be related to an irregular demethylation of this gene. Furthermore, we explore the methylation status and LPL expression after treatment with fludarabine, and cyclophosphamide (FC) and after activation with specific microenvironment signals in UM CLL patients.   Patients and Methods.  The methylation status of LPL gene was analyzed by Bisulphite DNA conversion from CLL and normal B-cells. Methylation specific primers (MSP) covering promoter and exon 1 region were designed and PCR reaction was performed. The differential methylation status was confirmed by sequencing of 10 CLL patients with or without LPL expression. As well, the same protocol was used for  in-vivo evaluation after FC treatment in 6 UM patients in order to verify LPL expression and methylation status. Finally the same methodology was used for in-vitro analysis of CLL B-cells after incubation with CD40L/IL-4 and GpG microenvironment activators.   Results. Our results show that LPL expression is associated to a differential methylation status in LPL gene. Studies in 10 different UM CLL patients, demonstrate that an important number of CpG islands placed in exon 1 of LPL gene are unmethylated and probably responsible for high expression levels of this gene. We also confirmed that UM CLL patients which respond to FC treatment show a loss of LPL gene expression and this is associated to a differential methylation status in LPL gene. Finally, we proceed to stimulate these UM CLL B-cells with specific microenvironment signals such as CD40L/IL4 and CpG. Interestingly activation with CD40L/IL-4 is able to  turn on the expression of LPL gene by DNA demethylation, in contrast to CpG activation involving TLR9 pathway.   Conclusion. Methylation status in a highly conserved region from exon 1 of LPL gene , appears to be responsible for the regulation of  LPL gene expression in normal and neoplastic CLL B-cells. The changes observed in methylation status and RNA expression of this gene following FC treatment, only in responding UM CLL patients and after specific CD40L/IL-4 activation  linked the LPL expression with the disease progression in an activated microenvironment.   Finally, measured of LPL methylation status could be simple and reproducible method for predicting prognosis in CLL as well as in treatment response. Further analyses are necessary in order to determine the exact role of LPL in CLL pathogenesis and  the importance of this methylation changes after FC treatment.