Both HOX gene expression and CTCF regulation have been well demonstrated to play a critical role in regulating maintenance of leukemic stem cells (LSCs) that are known to be resistant to BET inhibitor (BETi). To investigate the regulatory role of CTCF boundary in aberrant HOX gene expression and the therapeutic sensitivity of BETi in AML, we employed CRISPR-Cas9 genome editing technology to delete 47 base pairs of the CTCF binding motif which is located between HOXA7 and HOXA9 genes (CBS7/9) in different subtypes of AML with either MLL-rearrangement or NPM1 mutation. Our results revealed that HOXA9 is significantly downregulated in response to the CBS7/9 deletion. Moreover, CBS7/9 boundary deletion sensitized the BETi treatment reaction in both MOLM-13 and OCI-AML3 cells. To further examine whether BETi therapeutic sensitivity in AML is depended on the expression level of the HOXA9 gene, we overexpressed the HOXA9 in the CBS7/9 deleted AML cell lines, which can rescue and restore the resistance to BETi treatment of the CBS7/9 KO cells by activating MAPK signaling pathway. Deletion of CBS7/9 specifically decreased the recruitment of BRD4 and RNA pol II to the posterior HOXA genes, in which, a transcription elongation factor ELL3 is the key factor in regulating HOXA gene transcription monitored by CBS7/9 chromatin boundary. Thus, disruption of CBS7/9 boundary perturbs HOXA9 transcription and regulates BETi sensitivity in AML treatment. Moreover, alteration of CTCF boundaries in the oncogene loci may provide a novel strategy to overcome the drug resistance of LSCs.

两个HOX基因表达和调节CTCF已经很好证明在调节白血病干细胞已知为是BET抑制剂（BETI）耐器（LSC）的维护的关键作用。为了研究 CTCF 边界在异常HOX基因表达中的调节作用和 BETi 在 AML 中的治疗敏感性，我们采用 CRISPR-Cas9 基因组编辑技术删除了位于HOXA7和HOXA9基因之间的 CTCF 结合基序的 47 个碱基对（CBS7 /9) 在具有MLL重排或NPM1突变的 AML 的不同亚型中。我们的结果表明HOXA9响应 CBS7/9 缺失而显着下调。此外，CBS7/9 边界缺失使 MOLM-13 和 OCI-AML3 细胞中的 BETi 处理反应敏感。为了进一步检查 BETi 在 AML 中的治疗敏感性是否取决于HOXA9基因的表达水平，我们在 CBS7/9 缺失的 AML 细胞系中过表达HOXA9，这可以挽救和恢复 CBS7/9 KO 对 BETi 治疗的抗性激活 MAPK 信号通路。CBS7/9 的缺失特异性地减少了 BRD4 和 RNA pol II 向后HOXA基因的募集，其中，转录延伸因子ELL3是调节HOXA的关键因素由 CBS7/9 染色质边界监测的基因转录。因此，CBS7/9 边界的破坏会扰乱HOXA9转录并调节 AML 治疗中的 BETi 敏感性。此外，癌基因位点中 CTCF 边界的改变可能为克服 LSC 的耐药性提供了一种新的策略。