Abstract
DOT1L, the only known histone H3-lysine 79 (H3K79) methyltransferase, has been shown to be essential for the survival and proliferation of mixed-linkage leukemia (MLL) gene rearranged leukemia cells, which are often resistant to conventional chemotherapeutic agents. To study the functions of DOT1L in MLL-rearranged leukemia, SYC-522, a potent inhibitor of DOT1L developed in our laboratory, was used to treat MLL-rearranged leukemia cell lines and patient samples. SYC-522 significantly inhibited methylation at H3K79, but not H3K4 or H3K27, and decreased the expression of two important leukemia-relevant genes, HOXA9 and MEIS1, by more than 50%. It also significantly reduced the expression of CCND1 and BCL2L1, which are important regulators of cell cycle and anti-apoptotic signaling pathways. Exposure of MLL-rearranged leukemia cells to this compound caused cell cycle arrest and promoted differentiation of those cells, both morphologically and by increased CD14 expression. SYC-522 did not induce apoptosis, even at 10 µM for as long as 6 days. However, treatment with this DOT1L inhibitor decreased the colony formation ability of primary MLL-rearranged AML cells by up to 50%, and promoted monocytic differentiation. Notably, SYC-522 treatment significantly increased the sensitivity of MLL-rearranged leukemia cells to chemotherapeutics, such as mitoxantrone, etoposide and cytarabine. A similar sensitization was seen with primary MLL-rearranged AML cells. SYC-522 did not affect chemotherapy-induced apoptosis in leukemia cells without MLL-rearrangement. Suppression of DOT1L activity inhibited the mitoxantrone-induced increase in the DNA damage response marker, γH2AX, and increased the level of cPARP, an intracellular marker of apoptosis. These results demonstrated that SYC-522 selectively inhibited DOT1L, and thereby altered gene expression, promoted differentiation, and increased chemosensitivity by preventing DNA damage response. Therefore, inhibition of DOT1L, in combination with DNA damaging chemotherapy, represents a promising approach to improving outcomes for MLL-rearranged leukemia.
Highlights
Acute leukemia with somatic rearrangements of the gene Mixedlineage leukemia (MLL) involves a chromosomal translocation that fuses the MLL gene at 11q23 with one of .70 reported fusion partners [1]
We have evaluated the compound in leukemia cell-based functional assays
Cell cycle analysis confirmed that DOT1L inhibition by SYC-522 caused MV4-11 cells to be arrested at the G0/G1 phase after a 3-day treatment (Fig. S1), with the percentage of MV4-11 cells in the G0/G1 phase significantly increased, while the percentage in the S phase was decreased. These results suggested that SYC-522 treatment could affect the cell cycle and apoptosis signaling in both types of leukemia cells, indicating that DOT1L inhibitors may have some degree of activity against leukemia cells without MLL rearrangement
Summary
Acute leukemia with somatic rearrangements of the gene Mixedlineage leukemia (MLL) involves a chromosomal translocation that fuses the MLL gene at 11q23 with one of .70 reported fusion partners [1]. For MLL fusion proteins, the 59 end of the MLL gene is fused to the 39 portion of its partners, such as AF4, AF9, AF10 or ENL [3]. MLLrearranged leukemia accounts for ,75% of infant and ,10% child/adult acute leukemias [4]. This type of leukemia has a poor prognosis and high risk of relapse. The 5-year event-free survival rates of infants with MLL-rearranged acute lymphoid leukemia (ALL) are only 30–40% [5], and the 5-yearevent-free survival rates for patients with MLL-rearranged acute myeloid leukemia (AML) are 34–61% [6]. There is a pressing need to find new drugs to treat patients with MLL-rearranged leukemia
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