Expression of the deleted in liver cancer-1 gene is regulated by DNA methylation and is a target for therapy in Waldenström’s Macroglobulinemia

Abstract

19505 Background: The deleted in liver cancer-1 (DLC-1) gene encodes a Rho GTPase activating protein (RhoGAP) with tumor-suppressor activity. Hypermethylation of the DLC-1 promoter leads to transcriptional silencing of DLC-1, and its re-expression blocks proliferation and/or induces apoptosis. We therefore sought to determine the methylation status of the DLC-1 promoter and its impact on DLC-1 transcription and tumor growth in Waldenström’s macroglobulinemia (WM). Materials and Methods: The methylation status of the DLC-1 promoter was analyzed in WM (BCWM.1 and WM-WSU), multiple myeloma (INA6, MM1S, MM1R, RPMI 8226 and U266), and bone marrow CD19+ cells from 19 WM patients and 4 healthy individuals using methylation-specific PCR. The methylation pattern was subsequently determined by cloning and bisulfite DNA sequencing. DLC-1 mRNA expression was measured by quantitative RT-PCR. Results: The DLC- 1 promoter was completely methylated in WM-WSU and partially methylated in BCWM.1 WM cells. In myeloma cells, complete methylation was observed in RPMI 8226 and U266 cells; partial methylation in INA6 cells; and was undetectable in MM.1S and MM.1R cells. In WM and myeloma cells with complete methylation, DLC-1 transcripts were not expressed, whereas transcript expression was decreased in WM and myeloma cells with partial DLC-1 methylation. Treatment with the DNA methyltransferase inhibitor decitabine resulted in up-regulation of DLC-1 in BCWM.1 (7.4 fold) and re-expression of DLC-1 in WM-WSU WM cells. Importantly, decitabine abrogated cell growth in BCWM.1 and WM-WSU cells, and induced apoptosis of WM-WSU WM cells. Of the 19 WM patients examined, 13 (68%) had methylation in the DLC-1 promoter region. No methylation was seen in healthy individuals, and DLC-1 transcript expression was significantly lower in WM patients relative to the normal controls (p = 0.014). Conclusions: Methylation of CpG islands in the DLC-1 promoter region is a frequent epigenetic modification and contributes to down-regulation of DLC-1 in WM. Treatment with decitabine leads to up-regulation of DLC-1 expression and inhibits growth and/or induces apoptosis in WM cells setting the framework for the study of demethylating agents in WM. No significant financial relationships to disclose.