The chemotherapeutic agent CX-5461 irreversibly blocks RNA polymerase I initiation and promoter release to cause nucleolar disruption, DNA damage and cell inviability

Abstract

In the search for drugs to effectively treat cancer, the last 10 years have seen a resurgence of interest in targeting ribosome biogenesis. CX-5461 is a potential inhibitor of ribosomal RNA synthesis that is now showing promise in phase I trials as a chemotherapeutic agent for a range of malignancies. Here, we show that CX-5461 irreversibly inhibits ribosomal RNA transcription by arresting RNA polymerase I (RPI/Pol1/PolR1) in a transcription initiation complex. CX-5461 does not achieve this by preventing formation of the pre-initiation complex nor does it affect the promoter recruitment of the SL1 TBP complex or the HMGB-box upstream binding factor (UBF/UBTF). CX-5461 also does not prevent the subsequent recruitment of the initiation-competent RPI–Rrn3 complex. Rather, CX-5461 blocks promoter release of RPI–Rrn3, which remains irreversibly locked in the pre-initiation complex even after extensive drug removal. Unexpectedly, this results in an unproductive mode of RPI recruitment that correlates with the onset of nucleolar stress, inhibition of DNA replication, genome-wide DNA damage and cellular senescence. Our data demonstrate that the cytotoxicity of CX-5461 is at least in part the result of an irreversible inhibition of RPI transcription initiation and hence are of direct relevance to the design of improved strategies of chemotherapy.

Bidirectional cooperation between Ubtf1 and SL1 determines RNA Polymerase I promoter recognition in cell and is negatively affected in the UBTF-E210K neuroregression syndrome

“…At later times, Ubtf became highly condensed and partially segregated from RPI while FBL dispersed throughout the nucleus (120 h and 144 h post 4-HT in Figures 1D and S3). These changes were consistent with the nucleolar changes previously observed on inactivation of RPI transcription either by Rrn3 gene deletion or CX-5461 drug inhibition (24,28).…”

“…Hence, as observed for the other RPI basal factors Ubtf and Rrn3, Taf1b is an essential factor in mouse. Conditional deletion of taf1b in MEF and mES cell culture was also found to arrest rDNA transcription and to cause severe disruption of nucleolar structure characteristic of nucleolar stress (24,28). Depletion of Taf1b also prevented promoter recruitment of Taf1c and TBP subunits of SL1 and hence PIC formation at both the 47S pre-rRNA and the Enhancer-associated Spacer rDNA promoters.…”

Ribosomal RNA Transcription Regulation in Breast Cancer

“…Here, it was found that the mechanism of cell death is through Topoisomerase II poisoning [ 129 ]. Mars et al also found that CX-5461 blocks the release of RNAPI-RRN3 and blocks transcription initiation [ 130 ]. Perhaps CX-5461 has multiple mechanisms of action that could depend on cell context—an interesting avenue to explore.…”

Commuting to Work: Nucleolar Long Non-Coding RNA Control Ribosome Biogenesis from Near and Far

“…At the exit from mitosis, pre-rRNA synthesis resumes and the nucleolus reassembles its tripartite structure, when CDK1 activity is suppressed and RNAPI transcription is reactivated [ 36 , 37 , 38 ]. Along the same lines, the nucleolus disintegrates upon specific induction of nucleolar DNA damage or selective impairment of RNAPI transcription [ 39 , 40 , 41 , 42 ]. The inhibition of early steps in pre-rRNA processing also leads to nucleolar stress, along with the nucleoplasmic translocation of nucleolar proteins and the formation of ‘nucleolar caps’ [ 16 , 43 ].…”