A novel Menin-MLL inhibitor induces specific chromatin changes and eradicates disease in models of MLL-rearranged leukemia [ChIP-seq]

Treatment of cells carrying MLL-rearrangements with VTP-50469 (specific Menin-MLL1 inhibitor) displaces Menin from high molecular weight complexes and chromatin genome-wide. Since VTP-50469 block Menin interaction with MLL1 we tested using chip-seq if treatment with VTP-50469 also displaces MLL1 or MLL-fusions from chromatin. We found that the VTP-50469 treatment displaced MLL-fusions from only a subset of MLL-fusion binding sites. Since DOT1L is associated with MLL-AF9 we then tested if displacement of MLL1 also leads to loss of DOT1L association with chromatin on MLL-AF9 binding sites. We found that DOT1L binds to thousands of genes, treatment with VTP-50469 leads to genome wide loss of DOT1L binding including the same subset of MLL-fusion binding sites. Using ChIP-seq we examined Menin, MLL1, and DOT1L genome binding sites as well as methylation of lysine 79 on Histone H3 (H3K79me2) in cell lines carrying MLL-rearrangements MOLM13 (MLL-AF9), RS4;11 (MLL-AF4) and ML-2 (MLL-AF6) treated with VTP-50469 (100-330nM) or DMSO for 2-4 days.

针对携带MLL重排（MLL-rearrangements）的细胞，使用VTP-50469（特异性Menin-MLL1抑制剂）进行处理，可在全基因组范围内将Menin从高分子量复合物及染色质中解离。由于VTP-50469能够阻断Menin与MLL1的相互作用，我们通过染色质免疫共沉淀测序（ChIP-seq，Chromatin Immunoprecipitation Sequencing）检测了VTP-50469处理是否同样会将MLL1或MLL融合蛋白从染色质上解离。实验结果显示，VTP-50469仅能使MLL融合蛋白从部分MLL融合蛋白结合位点处发生解离。鉴于DOT1L与MLL-AF9存在关联，我们进一步探究了MLL1的解离是否会导致DOT1L在MLL-AF9结合位点处与染色质的结合出现丢失。研究发现，DOT1L可结合数千个基因，经VTP-50469处理后，全基因组范围内DOT1L的结合均出现显著丢失，涵盖了前述MLL融合蛋白结合的全部位点。我们采用ChIP-seq技术，对经VTP-50469（100~330nM）或二甲基亚砜（DMSO，Dimethyl Sulfoxide）处理2~4天的携带MLL重排的细胞系——包括MOLM13（MLL-AF9）、RS4;11（MLL-AF4）及ML-2（MLL-AF6）——中Menin、MLL1及DOT1L的全基因组结合位点，以及组蛋白H3第79位赖氨酸二甲基化修饰（H3K79me2）进行了检测。