Global Methylation Analysis Reveals Novel Candidate Tumor Suppressor Genes In Natural Killer Cell Lymphomas

Natural killer cell lymphomas (NKCLs) are aggressive neoplasms with poor prognosis. A few candidate tumor suppressor genes were identified with locus specific methylation analysis. However, no genome-wide study has been performed on NKCL cases to identify the spectrum of epigenetically silenced candidate genes contributing to the neoplastic transformation of NK cells.

Here, we analyzed genome-wide methylation profile in NKCL cases (n=12) and NK cell lines (n=2) by performing methyl-sensitive cut counting (MSCC). The methylation profile of malignant NK samples (n=14) were compared with IL2 activated normal NK cells (n=3) to identify genes with promoter hypermethylation. HG U133+ II DNA Microarray was performed on 6 NKCL cases, 2 NK cell lines and resting or activated normal NK cells (n=4).q-MSP was performed on NK cell lines (n=7) or NKCL cases (n=8) for selected genes to validate the promoter methylation identified by MSSC. q-RT-PCR was performed on NK cell lines to validate epigenetic silencing. Decitabine treatment and q-RT-PCR was performed on NK cell lines to assess reactivation of genes with promoter methylation. Apoptosis assay was performed by Annexin V-PE staining. Cell survival assay was performed on Asparaginase treated NK cell lines to show the correlation of Asparagine synthetase (ASNS) mRNA expression and sensitivity to the asparaginase treatment.

We observed significant global promoter hypermethylation in malignant NK samples compared with activated NK cells. Using stringent criteria which included significant correlation between promoter methylation and gene expression, we identified 152 genes showing recurrent promoter hypermethylation and transcriptional repression in NKCL cases compared with normal NK cells. 34.4% (51 of 152) of the silenced genes have been previously shown to be tumor suppressor gene in at least one cancer type. Gene Ontology and pathway analysis of the methylated/downregulated genes showed enrichment of the cell cycle inhibitors, pro-apoptotic genes such as BIM and DAPK1, DNA methylation modifiers such as TET2 and DNMT3A, metabolic pathway genes, transcription factors, negative regulators of JAK/STAT signaling such as SOCS6 and IL12Rb2, WNT pathway and NK-cell activation pathways. Decitabine treatment of NK cell lines reactivated expression of all 10 selected genes. Ectopic expression of BIM in two BIM-null NK cell lines led to increased apoptosis and eventual elimination of BIM-transduced cells. Reintroduction of BIM also sensitized NK cell lines to chemotherapy-induced apoptosis. NK cell lines with ASNS methylation and repression showed increased sensitivity to treatment with L-asparaginase, an agent highly effective in the treatment of NKCLs.

We identified novel candidate tumor suppressor genes epigenetically silenced in NKCLs which may contribute to the pathogenesis of NKCLs. Reactivation of these silenced genes may be therapeutically effective in NKCL patients. In addition, we showed that epigenetic inactivation of ASNS has an association with response to Asparaginase treatment. Promoter methylation status of ASNS in NKCL patients may be used in the decision of which patients are more likely to respond to asparaginase containing chemotherapeutic regimens.

No relevant conflicts of interest to declare.