Genome-Wide RNAi Screen Identifies The Mechanistic Role For DOT1L In MLL-Rearranged Leukemia

Rearrangement of Mixed-Lineage Leukemia (MLL) gene defines a genetically distinguishable subset of aggressive leukemias with poor prognosis. Recent studies exhibit promising activity of small-molecule inhibitors of the H3K79 methyltransferase DOT1L (disruptor of telomeric silencing-1 like) against leukemias bearing MLL-translocations (Daigle et. al. 2011 Cancer Cell). However, the mechanisms underlying the epigenetic addiction of MLL-fusion oncogenic program to H3K79-methylation remain unclear.

A number of labs have recently shown the expression of MLL-fusion target genes including HOXA cluster and MEIS1 is strongly dependent on DOT1L activity, whereas most of the normal genes do not require H3K79 methylation for their expression. To further investigate this unique subordination of MLL-fusion leukemic program to DOT1L/H3K79-methylation, we sought to identify genes, whose suppression would rescue the Dot1l loss-of-function phenotype in mouse MLL-AF9 cells. We conducted a suppressor screen by introducing a mouse genome-wide shRNA library (92,425 shRNA targeting 16,469 mouse genes) into Dot1l^f/f-MLL-AF9 leukemic cells harboring tamoxifen-inducible Cre recombinase. Relative frequencies of integrated shRNA sequences were assessed by high throughput sequencing. Our results revealed that knockdown of sirtuin 1 (Sirt1), a histone deacetylase, significantly antagonizes the lethality cause by loss of Dot1l/H3K79me2 in MLL-AF9 leukemia.

To understand the function of Sirt1 in MLL-rearranged leukemia, we performed genome-wide expression profiling and revealed that Sirt1 is essential for complete silencing of the MLL-AF9 driven oncogenic program including Hoxa7 and Meis1 upon inhibition of Dot1l. ChIP-seq analyses indicate that Sirt1 localizes to MLL-AF9 targets and mediates loss of H3K9ac upon suppression of Dot1l. Because Sirt1 is known to interact with epigenetic repressive enzymes such as polycomb repressive complex (PRC) members and Suv39h1, we them examine whether Sirt1 participates in suppression of MLL-AF9 targets by recruiting these silencing mechanisms to the loci. ChIP-seq analyses showed that suppression of Dot1l activity resulted in increased H3K9me2 and H3K27me3 levels near the transcriptional start site of MLL-AF9 target genes. Remarkably, knockdown of Sirt1 blocks the accumulation of H3K9me2 and maintains the chromatin accessibility at MLL-AF9 targets after Dot1l suppression, whereas the increase of H3K27me3 at those loci was minimally affected. Additionally, we identified that histone methyltransferase Suv39h1, a well characterized Sirt1 binding partner, is responsible for H3K9 di-methylation and gene silencing of Hoxa7 and Meis1 in response to DOT1L inhibitor. Finally, we showed that pharmacological activation of Sirt1 and inhibition of Dot1l synergistically suppresses the leukemogenetic potential of MLL-AF9 cells in a secondary leukemia drug exposure model.

Our results suggest that aberrant methylation of H3K79 may render leukemic transformation by inhibition of repressive mechanisms (including SIRT1/SUV39H1-mediated heterochromatin formation and others) at MLL-fusion targets. Combination treatment model further implies that patients with leukemia bearing MLL-translocations may benefit from synergistic therapies that simultaneously target both DOT1L and SIRT1 or other epigenetic regulators.