MLL-fusion-mediated activation of FLT3-ITD lesions promotes leukemogenesis

MLL-rearranged (MLL-r) leukemia, a particularly intractable disease, depends on DOT1L-mediated H3K79 methylation. Depletion of this transcriptionally activating mark by DOT1L loss-of-function or high concentrations of highly-specific inhibitor pinometostat (=1 mM EPZ5676) leads to the downregulation of HOXA9 and MEIS1, and consequent reduction leukemia survival. . Yet, some MLL-r cell lines are inexplicably susceptible to low-dose pinometostat, below the threshold for downregulating these canonical oncogenic drivers. Here we define alternative pathways disrupted by low-dose pinometostat (10 nM), a concentration that reduces proliferation of the MLL-r MV4;11 cell line without affecting HOXA9 and MEIS1 expression and downregulates a subset of MLL-fusion targets including FLT3, one of the most commonly mutated genes in leukemia. Using quantitative ICeChIP-seq, we observe profound H3K79me2 depletion at downregulated MLL-r targets, with resulting increases in transcriptionally activating H3K4me3 at promoters and reductions in repressive H3K27me3. The presence of co-occurring activating FLT3 mutations, portend greater cytotoxicity to inhibitor treatment. Although downregulation of polycomb components modestly contributes to reductions in proliferation, overexpression of constitutively active STAT5A, a target of FLT3-ITD-signalling, nearly completely rescues proliferation, accounting for the bulk of cytotoxicity from H3K79me2 depletion. We also observe a dependence of FLT3-STAT5A signaling on MLL function, suggesting that the FLT3 locus is exquisitely sensitivity to both H3K79me2 and H3K4me3 depletion and arguing that combinations of DOT1L, MLL1 and FLT3 inhibitors should be explored for treating the ~30% of all leukemias that carry FLT3 mutations. Overall design: Examination of histone modifications by calibrated ChIP-seq in MV4;11 cells with and without methyltransferase inhibitor

MLL重排型（MLL-rearranged, MLL-r）白血病是一类极具难治性的疾病，其存活依赖于DOT1L介导的H3K79甲基化修饰。通过DOT1L功能丧失或高浓度高特异性抑制剂皮诺莫司他（pinometostat，即1 mM浓度的EPZ5676）去除这一转录激活型修饰，可导致HOXA9与MEIS1基因的表达下调，进而削弱白血病细胞的存活能力。然而，部分MLL-r细胞系却未明原因地对低剂量皮诺莫司他表现出易感特性，该剂量尚未达到下调上述经典致癌驱动基因的阈值。本研究明确了10 nM低剂量皮诺莫司他所干扰的替代通路：该浓度可在不影响HOXA9与MEIS1表达的前提下，抑制MLL-r细胞系MV4;11的增殖，并下调一组MLL融合靶基因，其中包括FLT3（FMS样酪氨酸激酶3，FLT3）——白血病中最常见的突变基因之一。借助定量ICeChIP-seq（quantitative ICeChIP-seq），我们在下调的MLL-r靶基因位点处检测到显著的H3K79me2缺失，同时伴随启动子区域转录激活标记H3K4me3水平升高，以及抑制性组蛋白修饰H3K27me3水平降低。共存的激活型FLT3突变预示着肿瘤细胞对抑制剂治疗的细胞毒性反应更强。尽管多梳复合体（polycomb）组分的下调会轻度促进增殖抑制，但过表达FLT3-ITD信号通路的靶标——组成型激活的STAT5A（信号转导与转录激活因子5A，STAT5A），几乎完全逆转了增殖抑制效应，这表明H3K79me2缺失所介导的细胞毒性大部分源于该通路。我们还发现FLT3-STAT5A信号通路依赖于MLL的功能，这提示FLT3基因座对H3K79me2与H3K4me3的缺失均极为敏感，同时表明应当探索DOT1L、MLL1与FLT3抑制剂的联合疗法，用于治疗约占所有白血病30%的FLT3突变型白血病。整体实验设计：在添加与未添加甲基转移酶抑制剂的MV4;11细胞中，采用校准型染色质免疫沉淀测序（calibrated ChIP-seq）检测组蛋白修饰水平。