General MM

DOT1L is a potential new target in MM

Epigenetic alterations involving aberrant DNA methylation and histone modification play a role in the development of multiple myeloma (MM) and potentially offer new therapeutic targets. In particular, dysregulated methylation of histone lysine residues is thought to be a key pathway in driving pathogenesis of MM. In addition, mutations in genes encoding histone modifiers have been found in patients (pts) with MM.

In a recent study, carried out by Kazuya Ishiguro and a team of researchers from the Sapporo Medical University School of Medicine in Japan, the role of histone methylation in myeloma was examined, and the findings were published in Haematologica in August 2018. Using MM cell lines and blockers of various epigenetic modifiers, the researchers found a specific link between DOT1L - an enzyme that catalyzes the mono-, di- and tri-methylation of histone H3 lysine 79 (H3K79), and the survival of MM cells. Methylation of H3K79 has been linked to active transcription and may, therefore, be a new possible target for myeloma cells.

Key Findings
DOT1L inhibition induced growth suppression, cell cycle arrest, and apoptosis
  • A number of inhibitors were tested: LSD1 inhibitors (S2101 and GSK2879552), the G9a inhibitor (UNC0638), the H3K27 methyltransferase EZH2 inhibitor (GSK126), the JMJD3 inhibitor (GSKJ1) and the DOT1L inhibitor (SGC0946)
  • Inhibitors of G9a, EZH2, and DOT1L suppressed proliferation of more than two MM cell lines at early time-points; a longer treatment duration enhanced the effect
  • DOT1L was selected for further analysis based on:
  • The strong anti-proliferative effect observed in MM cell lines when treated with DOT1L inhibitors
    • Previous experiments showing expression in normal plasma cells (PCs) that progress to monoclonal gammopathy of undetermined significance (MGUS) and smoldering MM
    • Expression at various levels in MM cell lines
  • Experiments with two DOT1L inhibitors (SGC0946 and EPZ-5676) indicated the following:
    • Significantly reduced levels of tri-methylated H3K79 [H3K79me1/me2/me3] in RPMI-8226 MM cells
    • Suppression of proliferation in MM cell lines (RPMI-8226, MM.1S, KMS-11, and KMS-12BM)
    • Increases in sub-G1 and G0-G1 phase populations and decreases in S phase populations in RPMI-8226 and MM.1S MM cells
    • Suppression of tumor formation by MM cells in SCID mice
    • Moderate suppression of cell viability of isolated CD138-positive cells taken from MM and plasma cell leukemia (PCL) pts
  • Suppression of DOTL1 using shRNA knockdown led to similar suppression of proliferation in MM cell lines as with the DOTL1 inhibitors
DOT1L inhibition of MM cells suppresses IRF4 and MYC signaling, and alters immune responses and interferon (IFN) signaling
  • ChIP-seq analysis of cells treated with DOT1L inhibitors identified a significant reduction of H3K79me2 levels in a number of genes: RPMI-8226 = 4,483 and MM.1S = 1,590
  • Gene expression levels were downregulated by > 1.5-fold in cell lines: RPMI-8226 = 912 and MM.1S = 390
  • Identification of genes in which both H3K79me2 and expression levels were significantly decreased: RPMI-8226 = 249 and MM.1S = 67; 13 of the same genes were affected in both cell lines
  • Four of these genes affected common pathways: MYC, IRF4, PRDM1, and KLF2
  • ChIP-qPCR further confirmed:
  • Decreased H3K79me2 levels in these genes in MM cells treated with DOT1L inhibitors
  • Decreased expression of these 4 genes in MM cell lines using DOT1L inhibitors
  • Enrichment of DOT1L in the IRF4-MYC signaling genes was observed in both cell lines
  • DOT1L bound to genes marked by H3K79me2 modifications
  • Up-regulation (> 1.5-fold) of probe sets (of 125 protein-coding genes) by DOT1L inhibition in RPMI-8226 cells (1255 probe sets), and MM.1S cells (492 probe sets) was shown by microarray analysis
  • Genes involved in the immune system and immune response were significantly enriched among the upregulated genes in both cells, in particular, those involved in interferon (IFN) signaling
  • DOT1L inhibitors were found to upregulate a series of IFN-stimulated genes but H3K79me2 levels were not significantly altered in these genes, suggesting a possible H3K79me2-independent mechanism
DOT1L sensitivity is potentially associated with gene mutations and a longer duration of DOT1L inhibition enhances sensitivity in MM cells
  • Differences in cell sensitivity to DOT1L inhibition between two cell lines (KMS-12PE and KMS-12BM) were investigated using targeted sequencing of a panel of 409 cancer-related genes
  • Mutations were detected in multiple histone modifier genes (EP300, KMT2C, and KMT2D) in KMS-12PE - the cell line less sensitive to DOT1L inhibition
  • MYC and IRF4 were expressed at lower levels in KMS-12PE cells, suggesting that decreased IRF4-MYC signaling is associated with the impaired antitumor effect of DOT1L inhibitors
  • KMS-12PE cells were also less sensitive to the MYC inhibitor (10058-F4) whereas the cell line sensitive to DOT1L inhibitors (KMS-12BM) was also highly sensitive to MYC inhibition
  • Treatment with DOT1L inhibitors for up to 18 days led to strong growth suppression in KMS-12PE cells
  • Gene expression microarray analysis identified 509 probe sets (401 unique genes) that were downregulated (> 1.5-fold) and 865 (739 unique genes) that were upregulated (> 1.5-fold) by MYC inhibition in KMS-12PE cells
  • A number of IRF4-MYC signaling genes were downregulated by DOT1L inhibition and genes associated with IFN signaling were upregulated
Conclusion

DOT1L appears to play a role in the survival of MM cells, and inhibition of DOT1L may target the IRF4-MYC axis. Moreover, genes associated with the immune response were also upregulated following DOT1L inhibition, and in particular IFN. Therefore, DOT1L inhibition may bolster the immune response, offering the potential for successful combination with other drugs. Notably, the time required for DOT1L inhibitors to take effect varied between different MM cell lines, suggesting a link between gene mutations and sensitivity to DOT1L inhibition. It will be interesting to see further studies assessing the mechanism of this association.

References
  1. Ishiguro K. et al. DOT1L inhibition blocks multiple myeloma cell proliferation by suppressing IRF4-MYC signaling. Haematologica. 2018 Aug 31. pii: haematol.2018.191262. DOI: 10.3324/haematol.2018.191262.
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