A MOZ-TIF2 leukemic mouse model displays KAT6 inhibitor-sensitive H3K23 propionylation and dysregulation of a set of highly active developmental genes [RNA-seq]

Aberrant regulation of chromatin modifiers is a common occurrence across many cancer types, and a key priority is to determine how specific alteration of these proteins, often enzymes, can be targeted therapeutically. MOZ, a histone acyltransferase, is recurrently fused to coactivators CBP, p300, and TIF2 in cases of Acute Myeloid Leukemia (AML). Using either pharmacological inhibition or targeted protein degradation in a mouse model for MOZ-TIF2-driven leukemia, we show that KAT6 (MOZ/MORF) enzymatic activity and the MOZ-TIF2 protein are necessary for indefinite proliferation in cell culture. Interestingly, MOZ enzymatic activity is selectively required in MOZ-TIF2 cells, as murine MLL-AF9 leukemic cells show no sensitivity to the WM-1119 MOZ inhibitor. MOZ-TIF2 directly regulates a small subset of genes encoding developmental transcription factors, augmenting their high expression. Transcription levels of these genes positively correlate with enrichment of histone H3 propionylation at lysine 23 (H3K23pr), a recently appreciated histone acylation associated with gene activation. Unexpectedly, we also show that MOZ-TIF2 and MLL-AF9 regulate transcription of largely non-overlapping gene sets, and their cellular models exhibit distinct sensitivities to multiple small molecule inhibitors directed against AML pathways. This is despite the shared genetic pathways of wild-type MOZ and MLL. Overall, our data provide insight into how aberrant regulation of MOZ contributes to leukemogenesis. We anticipate these experiments will inform future work identifying targeted therapies in the treatment of AML and other diseases involving MOZ-induced transcriptional dysregulation. Overall design: To investigate how MOZ-TIF2 alters hematopoietic transcription, we altered MOZ/MORF and MOZ-TIF2 function through MOZ inhibition via WM-1119 treatment or MOZ-TIF2 degradation through dTAG13 treatment. We extracted nascent RNA or mRNA from mouse LSK cells containing either MOZ-TIF2 or MLL-AF9 and performed differential expression analysis comparing treated vs DMSO (control) cells mRNA was extracted from mouse LSK cells carrying either an MLL-AF9 or MOZ-TIF2 transgene. Cells were treated with either DMSO or 1 uM WM-1119 for 1 hour.

染色质调控因子（chromatin modifiers）的异常调控在多种癌症类型中普遍存在，当前的核心研究重点在于明确这些通常为酶类的蛋白质的特异性改变如何可被用于治疗靶向。组蛋白酰基转移酶（histone acyltransferase）MOZ在急性髓系白血病（Acute Myeloid Leukemia, AML）病例中，会频繁与共激活因子CBP、p300及TIF2发生融合。本研究在MOZ-TIF2驱动型白血病的小鼠模型中，分别采用药物抑制或靶向蛋白降解手段，证实KAT6（MOZ/MORF）的酶活性以及MOZ-TIF2蛋白对于细胞培养中的无限增殖是必需的。有趣的是，MOZ的酶活性仅在MOZ-TIF2细胞中具有选择性必需性：小鼠MLL-AF9白血病细胞对WM-1119这一MOZ抑制剂未表现出敏感性。MOZ-TIF2可直接调控编码发育转录因子的一小部分基因子集，增强其高表达水平。这些基因的转录水平与赖氨酸23位组蛋白H3丙酰化（H3K23pr）的富集程度呈正相关，后者是近年来被发现的与基因激活相关的组蛋白酰基化修饰。出乎意料的是，本研究还发现MOZ-TIF2与MLL-AF9调控的基因集在很大程度上不存在重叠，且它们的细胞模型对多种针对AML通路的小分子抑制剂表现出不同的敏感性。尽管野生型MOZ与MLL共享遗传通路，但上述现象依然存在。综上，本研究数据揭示了MOZ的异常调控如何促进白血病发生。我们预期本研究将为未来开发针对AML及其他涉及MOZ介导的转录失调的疾病的靶向治疗方案提供参考。

实验设计概述：为探究MOZ-TIF2如何改变造血转录过程，我们通过WM-1119处理抑制MOZ/MORF功能，或通过dTAG13处理降解MOZ-TIF2，以此调控MOZ/MORF及MOZ-TIF2的功能。我们从携带MOZ-TIF2或MLL-AF9的小鼠LSK细胞中提取新生RNA（nascent RNA）或信使RNA（mRNA），并对处理组与二甲基亚砜（DMSO，对照组）组的细胞进行差异表达分析。此外，我们从携带MLL-AF9或MOZ-TIF2转基因的小鼠LSK细胞中提取mRNA，将细胞用DMSO或1 μM WM-1119处理1小时后开展相关检测。