Epigenetic dysregulation of transcriptional program in MLL-EEN leukemia

Abstract

Aberrant DNA methylation is a typical feature of cancers, which is usually associated with deregulation of tumor suppressor genes and oncogenes. Mixed lineage leukemia (MLL)-rearranged leukemia is characterized by the presence of MLL fusion proteins resulting from chromosomal translocation between MLL and its partner genes. The MLL fusion proteins have lost the MLL histone methyltransferase SET domain, leading to alteration of epigenetic functions. Many genome-wide DNA methylation profiling studies showed significant global alteration in MLL-rearranged leukemia patients, suggesting that MLL fusion proteins mediate leukemia through dysregulation of DNA methylation status. Our previous work showed that MLL-EEN fusion protein can enhance the self-renewal capacity of murine hematopoietic progenitor cells through mediating DNA hypomethylation at HoxA family gene promoters. Nevertheless, how the aberrant DNA methylation alters the transcriptional program in MLL-EEN leukemia cells remains unclear. We performed RNA-seq to study the transcriptional program associated with MLL-EEN in murine hematopoietic stem and progenitor cells (HSPCs). We observed 1023 upregulated and 317 downregulated protein coding genes (PCGs) upon MLL-EEN overexpression. Interestingly, key regulators of DNA methylation, including Dnmt3a, Dnmt3b and Tet family genes, were downregulated, suggesting their involvement in the dysregulation of DNA methylation in MLL-EEN-driven leukemia. Gene ontology analysis showed that signaling pathways crucial to hematopoietic stem cell functions, such as Wnt and NFκb, were significantly enriched. Motif enrichment analysis also showed that the promoters of dysregulated genes were enriched with the binding sites of hematopoietic specific transcriptional factors, such as PU.1 and C/EBP family. In addition, we have identified 3016 upregulated and 1357 downregulated long non-coding RNAs (lncRNAs) in MLL-EEN HSPCs. lncRNAs, Hotairm1 and linc-p21, which was previously reported playing important roles in hematopoiesis and leukemogenesis, were also found deregulated. Notably, the deregulated lncRNAs showed positive correlation with the expression of nearby PCGs (±5 kb to lncRNAs), further demonstrating the transcriptional regulatory role of lncRNAs. MBD-seq analysis showed global DNA hypermethylation in MLL-EEN HSPC. Promoters of genes involved in myeloid differentiation and cell cycle regulation were hypermethylated, presumably associated with their downregulation mediated by MLL-EEN. We are currently investigating how the aberrant DNA methylation mediated by MLL-EEN can affect lncRNA transcription, which subsequently alters the expression of PCGs and leads to leukemic transformation. Taken together, our study provides new insights into the epigenetic regulation of transcriptional program in MLL-rearranged leukemia, which could facilitate the development of novel therapeutic strategies.