DNA methylation of micro- & long non-coding RNA in chronic lymphocytic leukemia

Abstract

B-cell chronic lymphocytic leukemia (CLL) is characterized accumulation in bone marrow, peripheral blood and secondary lymphoid tissues of neoplastic, mature small B lymphocytes. It is the most common adult leukemia in the western countries but much rarer in Asian countries with an incidence of 3.9/100,000/year in the United States and about 0.45/100,000/year in Hong Kong. Despite being an indolent illness in the majority, it remains an incurable disease with some patients pursuing an aggressive clinical course. Therefore, to optimize treatment outcome, identification of new prognostic markers, and hence better risk-stratification is needed.

In the human genome, apart from protein-coding genes (PCGs), with the development of high-throughput RNA sequencing, non-coding RNAs (ncRNAs) with little or no protein-coding capacity were discovered. Based on transcript size, ncRNAs are generally grouped into two major classes: small ncRNAs and lncRNAs. miRNAs, being single-stranded non-coding RNAs measuring 19-25 nucleotides (nt), is an important type of small ncRNAs, which serve as post-transcriptional regulators of gene expression via mRNA cleavage or translational repression of their corresponding target PCGs. lncRNAs, mRNA-like transcripts ranging from 200 nt to ~100 kilobases, have been shown to regulate the expression of their target genes through epigenetic, transcriptional or post-transcriptional mechanisms. Indeed, dysregulation of miRNAs and lncRNAs have been implicated in human cancers, suggesting a potential oncogenic or tumor suppressive role for ncRNAs. Apart from histone modification, DNA methylation is another important epigenetic modification leading to gene silencing without altering DNA sequence, and hence impacting cellular signaling and biological processes. In this thesis, the hypothesis that aberrant promoter methylation of tumor suppressor miRNAs or lncRNAs might contribute to CLL leukemogenesis is tested.

By the candidate gene approach, the role of DNA methylation of tumor suppressor miRNAs, such as miR-34 family, miR-9 family and miR-3151, together with the lncRNA BM742401 was investigated in CLL. The tumor suppressor role of miR-34b/c, miR-9-3, miR-3151 and BM742401 were demonstrated in CLL cell lines. Remarkably, restoration of miR-9 in CLL cells led to downregulation of NFκB1, which is a known target of miR-9, thereby testifying a role of miR-9-3 methylation in the constitutive activation of NFκB signaling pathway in CLL. Moreover, miR-3151 methylation protected CLL cells from apoptosis through over-expression of both of its direct PCG targets MADD and PIK3R2, leading to constitutive activation of MEK/ERK and PI3K/AKT signaling respectively, and consequently upregulation of MCL1. Furthermore, despite infrequent inactivating TP53 mutation in CLL at diagnosis, methylation of miR-34b/c, which are direct transcriptional targets of TP53, is implicated in the perturbation of the TP53-regulated tumor suppressor network. Finally, in primary CLL samples at diagnosis, frequent methylation of miR-3151 and BM742401 and modest methylation of miR-34b/c and miR-9-3 were demonstrated. Of note, miR-9-3 methylation was shown associated with advanced Rai stage (≥ stage 2) in CLL patients, suggesting miR-9-3 methylation as a potential poor risk factor for survival.

In conclusion, hypermethylation of tumor suppressor ncRNAs including miR-34b/c, miR-9-3, miR-3151 and lncRNA BM742401 is associated with constitutive activation of NFκB, MEK/ERK and PI3K/AKT signaling, and hence implicated in CLL pathogenesis.