Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, Alabama, 35294, USA.
Department of Radiation Oncology and Molecular Radiation Sciences and Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
J Biol Chem. 2022 Jan;298(1):101450. doi: 10.1016/j.jbc.2021.101450. Epub 2021 Nov 25.
Cancer cells are dependent upon an abundance of ribosomes to maintain rapid cell growth and proliferation. The rate-limiting step of ribosome biogenesis is ribosomal RNA (rRNA) synthesis by RNA polymerase I (Pol I). Therefore, a goal of the cancer therapeutic field is to develop and characterize Pol I inhibitors. Here, we elucidate the mechanism of Pol I inhibition by a first-in-class small-molecule BMH-21. To characterize the effects of BMH-21 on Pol I transcription, we leveraged high-resolution in vitro transcription assays and in vivo native elongating transcript sequencing (NET-seq). We find that Pol I transcription initiation, promoter escape, and elongation are all inhibited by BMH-21 in vitro. In particular, the transcription elongation phase is highly sensitive to BMH-21 treatment, as it causes a decrease in transcription elongation rate and an increase in paused Pols on the ribosomal DNA (rDNA) template. In vivo NET-seq experiments complement these findings by revealing a reduction in Pol I occupancy on the template and an increase in sequence-specific pausing upstream of G-rich rDNA sequences after BMH-21 treatment. Collectively, these data reveal the mechanism of action of BMH-21, which is a critical step forward in the development of this compound and its derivatives for clinical use.
癌细胞依赖大量核糖体来维持快速的细胞生长和增殖。核糖体生物发生的限速步骤是 RNA 聚合酶 I（Pol I）合成核糖体 RNA（rRNA）。因此，癌症治疗领域的一个目标是开发和表征 Pol I 抑制剂。在这里，我们阐明了第一类小分子 BMH-21 抑制 Pol I 的机制。为了研究 BMH-21 对 Pol I 转录的影响，我们利用了高分辨率体外转录测定和体内天然延伸转录测序（NET-seq）。我们发现 BMH-21 在体外抑制 Pol I 转录起始、启动子逃逸和延伸。特别是，转录延伸阶段对 BMH-21 处理高度敏感，因为它会降低转录延伸速率并增加核糖体 DNA（rDNA）模板上暂停的 Pol。体内 NET-seq 实验通过揭示 BMH-21 处理后模板上 Pol I 占有率降低以及富含 G 的 rDNA 序列上游的序列特异性暂停增加，补充了这些发现。总之，这些数据揭示了 BMH-21 的作用机制，这是该化合物及其衍生物临床应用开发的重要一步。
