Mechanistic basis and efficacy of targeting Beta Catenin-TCF7L2-JMJD6-MYC Axis to overcome resistance to BET inhibitors [OTX015]

Promising activity of BET protein inhibitors (BETis) is compromised by adaptive or innate resistance in AML. Here, modeling of BETi- persister/resistance (BETi-P/R) in human post-MPN secondary AML (sAML) cells demonstrated accessible and active chromatin in specific super-enhancers/enhancers, which was associated with increased levels of nuclear β-catenin, TCF7L2, JMJD6, and c-Myc in BETi-P/R sAML cells. Following BETi treatment, c-Myc levels were rapidly restored in BETi-P/R sAML cells. CRISPR/Cas9-mediated knockout of TCF7L2 or JMJD6 reversed BETi-P/R, whereas ectopic overexpression conferred BETi-P/R in sAML cells; confirming the mechanistic role of the β-catenin-TCF7L2- JMJD6-MYC axis in BETi-resistance. Patient-derived, post-MPN, CD34+ sAML blasts exhibiting relative resistance to BETi, as compared to sensitive sAML blasts, displayed higher mRNA and protein expressions of TCF7L2, JMJD6 and c-Myc, and following BETi washout exhibited rapid restoration of c-Myc and JMJD6. CRISPR/Cas9 knockout of TCF7L2 and JMJD6 depleted their levels, inducing loss of viability of the sAML blasts. Disruption of co-localization of nuclear β-catenin with TBL1 and TCF7L2 by the small molecule inhibitor BC2059 combined with depletion of BRD4 by BET-PROTAC reduced c-Myc levels and exerted synergistic lethality in BETi-P/R sAML cells. This combination also reduced leukemia burden and improved survival of mice engrafted with BETi-P/R sAML cells or with patient-derived AML blasts innately resistant to BETi. Therefore, multi- targeted disruption of β-catenin-TCF7L2-JMJD6-MYC axis overcomes adaptive and innate BETi-resistance, exhibiting pre-clinical efficacy against human post-MPN sAML cells. Secondary AML HEL92.1.7 cells and their OTX persister resistant derivative (HEL OTX P/R) were treated for 8 hours with 1000 nM of OTX015 to determine the effects of BET inhibitor treatment on the global transcriptome profile in parental and persister-resistant cells. Cells were treated independently and RNA was isolated from biologic replicates (HEL92.1.7 and HEL-OTX P/R untreated [n=2 each]; HEL92.1.7 and HEL-OTX P/R OTX015-treated [n=2 each]) and RNA Sequencing was performed.

BET蛋白抑制剂（BET protein inhibitors, BETis）在急性髓系白血病（acute myeloid leukemia, AML）中展现出的颇具潜力的治疗活性，常因适应性或先天性耐药而受损。本研究通过在人骨髓增殖性肿瘤转化后继发性急性髓系白血病（post-MPN secondary AML, sAML）细胞中构建BET抑制剂耐药/持续存活（BETi-persister/resistance, BETi-P/R）模型，发现特定超级增强子/增强子区域的染色质处于可及且转录活跃的状态，该特征与BETi-P/R sAML细胞中核β-连环蛋白（β-catenin）、TCF7L2、JMJD6及c-Myc水平升高显著相关。
经BET抑制剂处理后，BETi-P/R sAML细胞内的c-Myc水平会快速恢复。通过CRISPR/Cas9介导敲除TCF7L2或JMJD6可逆转BETi-P/R表型，而过表达上述基因则可在sAML细胞中诱导BETi-P/R表型，这证实了β-连环蛋白-TCF7L2-JMJD6-MYC轴在BET抑制剂耐药中的核心机制作用。
与敏感型sAML原始细胞相比，患者来源的post-MPN CD34+ sAML原始细胞对BET抑制剂相对耐药，其TCF7L2、JMJD6及c-Myc的mRNA和蛋白表达水平更高；且在撤除BET抑制剂后，这些细胞会快速恢复c-Myc与JMJD6的表达。通过CRISPR/Cas9敲除TCF7L2和JMJD6可降低其蛋白水平，并导致sAML原始细胞的存活能力丧失。
小分子抑制剂BC2059可破坏核β-连环蛋白与TBL1、TCF7L2的共定位，联合BET-PROTAC敲低BRD4，可降低c-Myc水平，并在BETi-P/R sAML细胞中发挥协同致死效应。该联合治疗方案还可减轻植入BETi-P/R sAML细胞或先天性BET耐药患者来源AML原始细胞的小鼠的白血病负荷，并延长其存活时间。
综上，多靶点阻断β-连环蛋白-TCF7L2-JMJD6-MYC轴可克服适应性与先天性BET抑制剂耐药，在人post-MPN sAML细胞中展现出临床前治疗疗效。
为探究BET抑制剂处理对亲本细胞与耐药持续存活细胞的全局转录组谱的影响，本研究将继发性AML HEL92.1.7细胞及其OTX耐药持续存活衍生细胞（HEL OTX P/R）用1000 nM OTX015处理8小时。实验设置独立生物学重复：未处理的HEL92.1.7与HEL-OTX P/R细胞（每组n=2）、经OTX015处理的HEL92.1.7与HEL-OTX P/R细胞（每组n=2），随后分离RNA并进行RNA测序（RNA Sequencing）。