Integrated Epigenomic Profiling Reveals Aberrant DNA Hypomethylation in LGL and Demonstrates That a Combination of Genetic and Epigenetic Events Results in Leukemic Evolution in Model of Large Granular Lymphocytic Leukemia.
Authors: Opalinska J. , Daibata M. , Sohal D. , Zhou L. , Thompson R. , Pahanish P. , Affer M. , Figueroa M. , Ye K. , Greally J. , Bai F. , List A. , Melnick A. , Epling-Burnette P. , Verma A. 
Published: October 2019

Abstract
In addition to genetic alterations, epigenetic events have been recently proposed to play a role in the pathogenesis and progression of various malignancies. We wanted to study the role of both global genetic and epigenetic changes during leukemic progression by combining Whole genome methylation, Gene expression and DNA copy number alteration analyses. We used a cell line model of LGL derived from the same patient at two different stages of disease (Daibata, Int J Cancer.2004;108(6):845) for comparison. The first cell line, MOTN1, is IL-2 dependant and was derived during indolent course of LGL, while PLT-2 typifies the aggressive phenotype and is IL-2 independent in vitro. Whole genome methylation was analyzed by the HELP assay (HpaII tiny fragment Enrichment by Ligation-mediated PCR; Khulan et al, Genome Res. 2006 Aug;16(8)) that uses differential methylation specific restriction digestion by HpaII and MspI followed by amplification, 2 color labeling and cohybridization to quantitatively determine individual promoter methylation. A whole genome human promoter array (Nimblegen) was used to determine the level of methylation of 25626 gene promoters by calculating HpaII/MspI cut fragment intensity ratio. Gene expression analysis was performed using 37K oligo maskless arrays and high density array comparative genomic hybridization (aCGH) was performed at 6Kb resolution (Nimblegen). aCGH showed significantly higher sensitivity when compared to conventional cytogenetic analysis and revealed that a total of 548 genes were deleted and 635 amplified during leukemic progression. Gene amplification led to significantly increased mean expression of important genes such as CDK6, Fyn kinase, DICER, MAP3K9 and deletion led to decreased gene expression of genes implicated in many cancer pathways by Ingenuity functional analysis. Methylation analysis showed that there was a trend towards hypomethylation with disease progression. The mean change in the HpaII/MspI (HELP) Log (2) ratio was 0.67, where a negative value indicates methylation and positive value indicates hypomethylation. Several genes implicated in hematologic diseases were found to be hypomethylated and overexpressed, including CDK8, IGF binding protein 5, HDAC4 and MAPK14. Methylation and suppression of various genes belonging to the suppressive TGF-β and TNF pathways was observed during leukemic evolution. A comparison of primary LGL samples with normal NK cells and CD4 lymphocytes demonstrated a consistent trend towards global hypomethylation. Significantly, many genes hypomethylated in the cell lines were found to be hypomethylated in 2 primary patient samples of NK and T LGL as well; these included members of RAS oncogene family, and many cell cycle regulators. These results show that interplay of both genetic and epigenetic events may be responsible for transformation of malignancies. For example, in our model we observed that pro-proliferative cyclin dependant protein kinases can be overactivated by both mechanisms, CDK6 by gene amplification and CDK8 by DNA hypomethylation. These findings strongly suggest that progression of LGL from the stable to the aggressive phase is associated with significant epigenetic changes, in addition to cytogenetic alterations.