PAX translocations remodel mitochondrial metabolism through altered leucine usage in rhabdomyosarcoma [Cut&Run]

Alveolar rhabdomyosarcoma (ARMS) patients harboring PAX3-FOXO1 and PAX7-FOXO1 fusion proteins exhibit a greater incidence of tumor relapse, metastasis, and poor survival outcome, thereby underscoring the urgent need to develop effective therapies to treat this subtype of childhood cancer. To uncover mechanisms that contribute to tumor initiation, we develop a muscle progenitor model and use epigenomic approaches to unravel genome rewiring events mediated by PAX3/7 fusion proteins. Among the key targets of PAX3/7 fusion proteins, we identify a cohort of oncogenes, FGF receptors, tRNA-modifying enzymes, and genes essential for mitochondrial metabolism and protein translation, which we successfully targeted in preclinical trials. We identify leucine usage as a key factor driving the growth of aggressive PAX-fusion tumors, as limiting its bioavailability impaired oxidative phosphorylation and mitochondrial metabolism, delaying tumor progression and improving survival in vivo. Our data provide a compelling list of actionable targets and suggest promising new strategies to treat this tumor. Overall design: Human induced pluripotent stem cells (iPS) were differentiated to muscle progenitors through the inducible expression of Pax7 with doxycycline. Next, we introduce the expression of the oncogenic fusion proteins Pax3-FOXO1 or Pax7-FOXO1 (Flag tagged) using lentiviruses and stopped Pax7 expression for 3 days to study the roles of the fusion proteins in gene regulation. We extracted RNA for gene expression analysis by RNA-seq. We performed Cut&Run followed by sequencing using antibodies against Falg, H3K4me1, H3K4me3 and H3K27ac. Finally, we extracted DNA to perform promoter-capture HiC (pcHiC) and HiC to explore the changes in chromatin topology exerted by the expression of the fusion proteins.

携带PAX3-FOXO1与PAX7-FOXO1融合蛋白的腺泡状横纹肌肉瘤（ARMS）患者，肿瘤复发、转移发生率更高，生存预后更差，这凸显了开发针对该儿童癌症亚型的有效治疗手段的迫切需求。为揭示驱动肿瘤发生的潜在机制，我们构建了肌肉祖细胞模型，并利用表观基因组学方法解析PAX3/7融合蛋白介导的基因组重编程事件。在PAX3/7融合蛋白的关键靶标中，我们鉴定出一组致癌基因、成纤维细胞生长因子（FGF）受体、tRNA修饰酶，以及线粒体代谢和蛋白质翻译所必需的基因，并在临床前试验中成功靶向这些靶点。我们发现亮氨酸利用是驱动侵袭性PAX融合肿瘤生长的关键因素，限制其生物利用度会损伤氧化磷酸化与线粒体代谢，延缓体内肿瘤进展并改善生存期。本研究数据提供了一系列具有临床转化价值的可靶向靶点，并为该肿瘤的治疗提出了颇具前景的新策略。
实验设计概述：通过多西环素诱导Pax7表达，将人诱导多能干细胞（iPS）定向分化为肌肉祖细胞。随后，我们利用慢病毒载体引入带有Flag标签的致癌融合蛋白Pax3-FOXO1或Pax7-FOXO1，并暂停Pax7表达3天，以研究融合蛋白在基因调控中的作用。我们提取RNA进行RNA测序（RNA-seq）以分析基因表达谱；采用针对Flag、H3K4me1、H3K4me3及H3K27ac的抗体进行Cut&Run测序；最终提取DNA开展启动子捕获Hi-C（pcHiC）与Hi-C实验，以探究融合蛋白表达对染色质拓扑结构的改变。