Characterization of chromatin changes induced by Menin-MLL inhibition in NPM1c mutant acute myeloid leukemia

NPM 1c是最常见的细胞遗传学正常的AML类型。尽管发病率很高，但白血病发生的分子机制仍然知之甚少，缺乏靶向治疗方案。我们小组最近的数据表明，NPM 1c可以诱导髓系祖细胞的自我更新，并在小鼠模型中产生发展为迟发性AML的白血病前克隆。我们使用该小鼠模型来证明Menin-MLL抑制在PDX模型中根除NPM 1c突变体白血病前植入以及完全发展的AML方面是非常有效的。我们的数据表明，Menin及其与MLL的相互作用对于维持NPM 1c突变细胞的白血病自我更新至关重要。基因表达分析显示，Menin抑制导致HOX辅因子MEIS 1和PBX 3的表达迅速丧失。出乎意料的是，我们没有观察到HOXA/B表达的任何损失，这是在ML重排白血病的情况下使用较旧的Menin抑制剂分子的其他组所报告的。为了理解表观遗传景观的差异，我们想要彻底分析由Menin-MLL抑制诱导的染色质变化。首先，我们将研究破坏Menin和MLL相互作用后染色质占据率的变化。NPM 1c突变细胞也显示对组蛋白甲基转移酶DOT 1 L抑制剂有反应。因此，我们将研究DOT 11的行为以及沉积在MLL和DOT 1 L（H3 K4和H3 K79）上的活化组蛋白标记，以研究这些因子是否在Menin抑制后改变它们的分布。将分析染色质景观中的这些变化与通过RNAseq获得的基因表达变化的相关性。 通过所提出的实验，我们将更深入地了解Menin-MLL破坏后NPM 1c AML中的染色质动力学。

NPM1c is the most common type of cytogenetically normal AML. Despite the high prevalence, the molecular mechanisms of leukemogenesis remain poorly understood and targeted therapy options are lacking. Recent data from our group has shown that NPM1c can induce self-renewal in myeloid progenitor cells and give rise to preleukemic clones that develop late onset AML in mouse models. We used this mouse model to demonstrate that Menin-MLL inhibition is highly effective in eradicating NPM1c mutant pre-leukemic engraftment as well as fully developed AML in PDX models. Our data suggest that Menin and its interaction with MLL are essential for maintaining leukemic self-renewal of NPM1c mutant cells. Gene expression analysis revealed that Menin inhibition leads to a rapid loss of expression of HOX Co-factors MEIS1 and PBX3. Unexpectedly, we did not observe any loss of HOXA/B expression which was reported by other groups using older Menin inhibitor molecules in the context of MLL-rearranged leukemias. To understand the difference in the epigenetic landscape we want to thoroughly analysis chromatin changes induced by Menin-MLL inhibition. First and foremost we will study the chromatin occupancy changes in Menin and MLL after disrupting their interaction. NPM1c mutant cells were also shown to be responsive to histone methyltransferase DOT1L inhibitors. Therefore, we will investigate the behavior of DOT1l and the activating histone marks deposited MLL and DOT1L (H3K4 and H3K79) in order to investigate whether these factors are changing their distribution upon Menin inhibition. These changes in the chromatin landscape will be analyzed for their correlation with changes in gene expression obtained by RNAseq. With the proposed experiments, we will gain deeper insight into the chromatin dynamics in NPM1c AML after disruption of the Menin-MLL.