Pinometostat (EPZ-5676) is a potent DOT1L histone methyltransferase inhibitor (Ki=80 pM). Pinometostat has antitumor activity and can be used in experiments to study a variety of leukemia treatments.

DOT1L:80 pM(Ki)

方法: MLL-r 和 non-MLL-r AML 细胞用 Pinometostat (0.1-10 µM) 处理 4-16 天，通过 flow cytometry 检测细胞增殖。
结果: 携带 MLL-AF4 或 MLL-AF9 融合物的 MV4-11、MOLM-13 和 NOMO-1 细胞系在治疗 8 天后细胞数量急剧减少，IC50 值低于 1 µM。相比之下，均缺乏 MLL-r 的 U-937 或 HL-60 细胞的增殖不受 Pinometostat 的影响。令人惊讶的是，DOT1L 抑制对MLL-AF9 阳性 THP-1 细胞没有影响，而它显著降低了非 MLL-r OCI-AML3 细胞系的增殖。[1]
方法: 人白血病细胞 MV4-11 用 Pinometostat (0.06-1000 nM) 处理 4 天，通过 Western Blot 检测靶点蛋白表达水平。
结果: 在 Pinometostat 浓度增加的情况下，表达 MLL-AF4 的 MV4-11 的整体细胞甲基化 H3K79 水平的浓度依赖性降低。[2]

方法: 为检测体内抗肿瘤活性，将 JSH-23 (50 mg/kg，2% DMSO+30%PEG 300+5% Tween80+63% PBS) 腹腔注射给携带 MDA-MB-468 肿瘤的 NSG 小鼠，每两天一次，给药 6 次。
结果: Pinometostat 在 10 周内显著降低了肿瘤体积和原发性肿瘤的转移。[3]

EPZ-5676 is dissolved in DMSO. To analyse inhibition of histone methylation in MV4-11 cells following EPZ-5676 treatment, extracted histones (400 ng) are fractionated on a 10-20% Tris HCl gels with Tris-Glycine SDS running buffer under denaturing conditions and transferred to nitrocellulose filters. Filters are cut into strips and incubated for 1 hour in blocking buffer at room temperature (RT) and then incubated overnight at 4°C in blocking buffer. Filters are washed 3 times for 5 minutes with wash buffer (Phosphate buffered saline (PBS) including 0.01% Tween 20 (PBST)) and incubated with infrared tagged secondary antibody at RT for 1 hour. Filters are washed in PBST and reprobed for 1 hour at RT with the appropriate total histone antibody control (mouse anti-histone H3 (1:20,000), CST 3638, or mouse anti-histone H4 (1:10,000), CST 2935). Filters are washed again in PBST and incubated with infrared tagged secondary antibody (IRDye 800Cw donkey-anti-mouse IgG (1:20,000), Li-Cor 926-32212) at RT for 1 hour. After a final ish in PBST, filters are scanned using the Odyssey infared imager (Li-cor). To analyse inhibition of H3K79 methylation in peripheral blood mononuclear cells (PBMCs) from rats dosed with EPZ-5676, 20 μL of PBMC whole cell lysate is fractionated on denaturing gels and analysed by immunoblotting with antibodies to H3K79me2 or total H3. Signal intensities specific for the H3K79me2 antibody and total histone H3 control antibody are quantified using Odyssey software. The H3K79me2 signal intensity is normalized by dividing it by the total histone H3 control signal intensity in the same lane.