Simple SummaryRibosome biosynthesis has emerged as an excellent target for inhibition of cancer cell growth and proliferation. We used a high throughput screen to identify potent inhibitors of ribosome synthesis in malignant melanoma cells. Here, we show that the newly identified ribosome biogenesis inhibitor, RBI2, inhibits multiple cancer cell types by a mechanism of action that is distinct from previously described compounds. Using chromatin IP, isotopic labelling, and RNA sequencing, we conclude that treatment with RBI2 does not inhibit transcription initiation by RNA polymerase I, but appears to induce rapid polyadenylation and degradation of ribosomal RNA. This mechanism of action would be unique from previously described inhibitors of RNA polymerase I.AbstractCancer cells are especially sensitive to perturbations in ribosome biogenesis as they rely on finely tuned protein homeostasis to facilitate their rapid growth and proliferation. While ribosome synthesis and cancer have a well-established relationship, ribosome biogenesis has only recently drawn interest as a cancer therapeutic target. In this study, we exploited the relationship between ribosome biogenesis and cancer cell proliferation by using a potent ribosome biogenesis inhibitor, RBI2 (Ribosome Biogenesis Inhibitor 2), to perturb cancer cell growth and viability. We demonstrate herein that RBI2 significantly decreases cell viability in malignant melanoma cells and breast cancer cell lines. Treatment with RBI2 dramatically and rapidly decreased ribosomal RNA (rRNA) synthesis, without affecting the occupancy of RNA polymerase I (Pol I) on the ribosomal DNA template. Next-generation RNA sequencing (RNA-seq) revealed that RBI2 and previously described ribosome biogenesis inhibitor CX-5461 induce distinct changes in the transcriptome. An investigation of the content of the pre-rRNAs through RT-qPCR revealed an increase in the polyadenylation of cellular rRNA after treatment with RBI2, constituting a known pathway by which rRNA degradation occurs. Northern blotting revealed that RBI2 does not appear to impair or alter rRNA processing. Collectively, these data suggest that RBI2 inhibits rRNA synthesis differently from other previously described ribosome biogenesis inhibitors, potentially acting through a novel pathway that upregulates the turnover of premature rRNAs.