Abstract A159: Epigenetic silencing of the M-type phospholipase A2 receptor gene in leukemic cells.

Abstract

Abstract Background: The M-type phospholipase A2 receptor (PLA2R) plays a crucial role in a number of different signaling pathways and may act as tumor-suppressor. In the current study the expression and methylation of the PLA2R gene in the myeloid U937 cell line and blood leukocytes of patients with leukemia were analyzed. Methods. Expression of PLA2R in U937 cells was studied using RT-quantitative PCR. To determine methylation levels of the PLA2R locus, bisulfite-modified DNA fragments were sequenced. Identification of different 5′-CpG methylation sites in normal and leukemic patients was followed by methylation specific-high resolution melting (MS-HRM) analysis to quantify PLA2R methylations. Results: Our data establish that expression of PLA2R is completely down-regulated in U937 cells. Simultaneously, a 100% methylation of PLA2R promoter and down-stream regions was found in this cell line; after exposure of the cells to 5-aza-2 -deoxycytidine, PLA2R was re-expressed. MS-HRM analyzes in 34 patients with different types of leukemia indicated an average PLA2R methylation of 30%±18%, whereas in 52 normal subjects methylation levels were below 10%. In a preliminary study of high-risk myelodysplastic syndrome patients (MDS), three patients had increased PLA2R methylations. In the course of treatment with azacitidine (Vidaza) as methyltransferase inhibitor, two of these patients were responders in contrast to one patient who was a non-responder with increased methylation levels in blood leukocytes during treatment. Another patient with minimal residual disease after treatment of AML underwent preemptive treatment with azacitidine to avoid hematological relapse (HR). After two azacitidine cycles a minor cell fraction with 100% methylated PLA2R was completely diminished. However, three weeks later methylation of the PLA2R promoter increased to 50%, with 9% blasts detectable in blood samples. After further azacitidine treatment cycle, blasts diminished in blood samples but the methylation of PLA2R in blood leukocytes remained at 35%. During the following seven weeks with no further azacitidine treatment blasts increased up to 44% suggesting that the treatment with azacitidine was effective but not efficient enough to prevent a HR in this patient. Conclusions: The study shows for the first time that the PLA2R promoter and down-stream regions in leukemic cells are hypermethylated and that the determination of PLA2R methylation by MS-HRM may be useful as a biomarker in the diagnosis and therapeutic treatment of high-risk MDS and AML patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A159.