DOT1L Inhibition Sensitizes MLL-Rearranged AML to Chemotherapy

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.

DOT1L（组蛋白H3-赖氨酸79，即histone H3-lysine 79, H3K79，已知唯一的甲基转移酶）已被证实对混合谱系白血病（mixed-linkage leukemia, MLL）基因重排白血病细胞的存活与增殖至关重要，此类细胞通常对常规化疗药物产生耐药性。为研究DOT1L在MLL重排白血病中的功能，本实验室开发的强效DOT1L抑制剂SYC-522被用于处理MLL重排白血病细胞系及患者样本。SYC-522可显著抑制H3K79位点的甲基化，但对H3K4或H3K27位点的甲基化无显著影响；同时可使两个关键白血病相关基因HOXA9与MEIS1的表达水平下调超过50%。此外，该化合物还可显著降低细胞周期调控与抗凋亡信号通路的重要调节因子CCND1及BCL2L1的表达水平。将MLL重排白血病细胞暴露于SYC-522后，可引发细胞周期阻滞，并从形态学与CD14表达上调两方面促进细胞分化。即便以10微摩尔浓度处理长达6天，SYC-522也未诱导细胞凋亡。不过，该DOT1L抑制剂可使原发性MLL重排急性髓系白血病（acute myeloid leukemia, AML）细胞的集落形成能力最高降低50%，并促进单核细胞分化。值得注意的是，SYC-522处理可显著增强MLL重排白血病细胞对米托蒽醌（mitoxantrone）、依托泊苷（etoposide）及阿糖胞苷（cytarabine）等化疗药物的敏感性，该增敏效应在原发性MLL重排AML细胞中同样得以验证。对于无MLL基因重排的白血病细胞，SYC-522不会影响化疗诱导的细胞凋亡过程。抑制DOT1L活性可阻断米托蒽醌诱导的DNA损伤反应标志物γH2AX的上调，并提升细胞内凋亡标志物剪切型多聚ADP核糖聚合酶（cPARP）的表达水平。上述实验结果证实，SYC-522可选择性抑制DOT1L活性，进而改变基因表达谱、促进细胞分化，并通过阻断DNA损伤反应提升肿瘤细胞的化疗敏感性。因此，将DOT1L抑制与DNA损伤性化疗联合应用，有望为MLL重排白血病患者带来更优的治疗结局。