Hyperactivation of RNA polymerase I (Pol I) transcription of ribosomal RNA (rRNA) genes (rDNA) is a key determinant of growth and proliferation and a consistent feature of cancer cells. We have demonstrated that inhibition of rDNA transcription by the Pol I transcription inhibitor CX-5461 selectively kills tumor cells in vivo. Moreover, the first-in human trial of CX-5461 has demonstrated CX-5461 is well-tolerated in patients and has single-agent anti-tumor activity in hematologic malignancies. However, the mechanisms underlying tumor cell sensitivity to CX-5461 remain unclear. Understanding these mechanisms is crucial for the development of predictive biomarkers of response that can be utilized for stratifying patients who may benefit from CX-5461. The rDNA repeats exist in four different and dynamic chromatin states: inactive rDNA can be either methylated silent or unmethylated pseudo-silent; while active rDNA repeats are described as either transcriptionally competent but non-transcribed or actively transcribed, depending on the level of rDNA promoter methylation, loading of the essential rDNA chromatin remodeler UBF and histone marks status. In addition, the number of rDNA repeats per human cell can reach hundreds of copies. Here, we tested the hypothesis that the number and/or chromatin status of the rDNA repeats, is a critical determinant of tumor cell sensitivity to Pol I therapy. We systematically examined a panel of ovarian cancer (OVCA) cell lines to identify rDNA chromatin associated biomarkers that might predict sensitivity to CX-5461. We demonstrated that an increased proportion of active to inactive rDNA repeats, independent of rDNA copy number, determines OVCA cell line sensitivity to CX-5461. Further, using zinc finger nuclease genome editing we identified that reducing rDNA copy number leads to an increase in the proportion of active rDNA repeats and confers sensitivity to CX-5461 but also induces genome-wide instability and sensitivity to DNA damage. We propose that the proportion of active to inactive rDNA repeats may serve as a biomarker to identify cancer patients who will benefit from CX-5461 therapy in future clinical trials. The data also reinforces the notion that rDNA instability is a threat to genomic integrity and cellular homeostasis.

核糖体 RNA (rRNA) 基因 (rDNA) 的 RNA 聚合酶 I (Pol I) 转录过度激活是生长和增殖的关键决定因素，也是癌细胞的一致特征。我们已经证明，Pol I 转录抑制剂 CX-5461 抑制 rDNA 转录可选择性杀死肿瘤细胞体内。此外，CX-5461的首次人体试验表明CX-5461在患者中具有良好的耐受性，并且在血液恶性肿瘤中具有单药抗肿瘤活性。然而，肿瘤细胞对 CX-5461 敏感的机制仍不清楚。了解这些机制对于开发反应预测生物标志物至关重要，这些生物标志物可用于对可能受益于 CX-5461 的患者进行分层。 rDNA 重复序列以四种不同的动态染色质状态存在：非活性 rDNA 可以是甲基化沉默或非甲基化伪沉默；而活性 rDNA 重复序列被描述为有转录能力但非转录或活性转录，具体取决于 rDNA 启动子甲基化水平、必需的 rDNA 染色质重塑子 UBF 的负载和组蛋白标记状态。此外，每个人体细胞的rDNA重复次数可以达到数百个拷贝。在这里，我们测试了以下假设：rDNA 重复的数量和/或染色质状态是肿瘤细胞对 Pol I 治疗敏感性的关键决定因素。我们系统地检查了一组卵巢癌 (OVCA) 细胞系，以确定可能预测对 CX-5461 敏感性的 rDNA 染色质相关生物标志物。我们证明，活性与非活性 rDNA 重复序列比例的增加（与 rDNA 拷贝数无关）决定了 OVCA 细胞系对 CX-5461 的敏感性。 此外，使用锌指核酸酶基因组编辑，我们发现减少 rDNA 拷贝数会导致活性 rDNA 重复序列比例增加，并赋予对 CX-5461 的敏感性，但也会诱导全基因组不稳定性和对 DNA 损伤的敏感性。我们建议，活性 rDNA 重复序列与非活性 rDNA 重复序列的比例可以作为生物标志物，以识别在未来的临床试验中将从 CX-5461 治疗中受益的癌症患者。这些数据还强化了 rDNA 不稳定性对基因组完整性和细胞稳态构成威胁的观点。