Effect of combination treatment of RNA polymerase I transcription inhibitor CX-5461 and histone deacetylase inhibitor panobinostat on gene expression in human multiple myeloma cell lines MM.1S and JJN3. Effect of combination treatment of RNA polymerase I transcription inhibitor CX-5461 and histone deacetylase inhibitor panobinostat on gene expression in human multiple myeloma cell lines MM.1S and JJN3

The high rates of protein synthesis and processing render multiple myeloma (MM) cells vulnerable to perturbations in protein homeostasis. The induction of proteotoxic stress by targeting protein degradation with proteasome inhibitors (PI) has revolutionized the treatment of MM. However, resistance to PI is inevitable and represents an ongoing clinical challenge. Our first-in-human study of the selective inhibitor of RNA polymerase I transcription of ribosomal RNA genes, CX-5461 has demonstrated a potential signal for anti-tumor activity in three of six heavily pre-treated MM patients. Here we show that CX-5461 has potent antimyeloma activity in PI-resistant MM preclinical models in vitro and in vivo. In addition to inhibiting ribosome biogenesis, CX-5461 causes topoisomerase II trapping and replication-dependent DNA damage, leading to G2/M cell cycle arrest and apoptotic cell death. Surprisingly, the addition of PI does not enhance the therapeutic benefit of CX-5461. In contrast, CX-5461 shows synergistic interaction with the histone deacetylase inhibitor panobinostat in both the Vk*MYC and the 5T33-KaLwRij mouse models of MM by targeting ribosome biogenesis and protein synthesis through distinct mechanisms. Our findings thus provide strong evidence to facilitate the clinical development of targeting the ribosome to treat relapsed and refractory MM. Overall design: To investigate the molecular mechanisms underlying the synergistic response to CX-5461 and panobinostat, we performed RNAseq analysis in both p53-WT MM1.S and p53-null JJN-3 cells. MM.1S cells were treated with vehicle, 500 nM CX-5461, 50 nM panobinostat or the combination for 24 hr and cellular RNA was extracted for 3’ RNA sequencing analysis (n=3).

蛋白质合成与加工的高负荷状态使得多发性骨髓瘤（multiple myeloma, MM）细胞极易受到蛋白质稳态扰动的影响。通过蛋白酶体抑制剂（proteasome inhibitor, PI）靶向蛋白质降解以诱导蛋白毒性应激，这一策略已彻底革新了MM的治疗方案。然而，对PI的耐药性不可避免，仍是当前临床面临的棘手难题。

我们针对核糖体RNA基因RNA聚合酶I转录的选择性抑制剂CX-5461开展的首个人体研究，在6例经多线治疗的MM患者中有3例显示出潜在的抗肿瘤活性信号。本研究证实，CX-5461在体外及体内的PI耐药MM临床前模型中均具有强效的抗骨髓瘤活性。

除抑制核糖体生物发生外，CX-5461还可引发拓扑异构酶II捕获及复制依赖性DNA损伤，进而导致G2/M期细胞周期阻滞与细胞凋亡。令人意外的是，联用PI并未增强CX-5461的治疗获益。

与之相反，在MM的Vk*MYC与5T33-KaLwRij小鼠模型中，CX-5461与组蛋白去乙酰化酶抑制剂帕比司他（panobinostat）展现出协同作用，二者通过不同机制靶向核糖体生物发生与蛋白质合成。

因此，本研究结果为靶向核糖体治疗复发难治性MM的临床开发提供了强有力的证据支撑。

整体实验设计：为探究CX-5461与帕比司他协同应答的分子机制，我们分别在p53野生型MM1.S细胞与p53缺失型JJN-3细胞中开展了RNA测序（RNA-seq）分析。将MM.1S细胞分别以溶剂对照、500 nM CX-5461、50 nM 帕比司他或二者联合处理24小时，随后提取细胞总RNA进行3'端RNA测序分析（n=3）。