ABSTRACT: In recent years, several small molecule cytotoxic drugs have been identified as potential inhibitors of ribosome biogenesis (Drygin et al., 2011; Peltonen et al., 2014a; Peltonen et al., 2014b). CX-5461 is one such drug that has also demonstrated anticancer potential for a wide range of malignancies (Bywater et al., 2012; Cornelison et al., 2017; Devlin et al., 2015; Drygin et al., 2011; Hald et al., 2019; Hein et al., 2017; Ismael et al., 2019; Lawrence et al., 2018; Lee et al., 2017; Negi and Brown, 2015; Taylor et al., 2019; Xu et al., 2017; Yan et al., 2017) (Haddach et al., 2012), and is presently under phase I trials for the treatment of both hematological cancers and solid tumours (Group, 2016; Khot et al., 2019). CX-5461 was initially characterized as an inhibitor of RNA Polymerase I (RPI/PolR1/PolI) that is responsible for the synthesis of the major ribosomal RNAs and the initial step in ribosome biogenesis (Drygin et al., 2011). Since RPI and its corresponding core transcription factors are dedicated to this task alone, they present ideal molecular targets by which to modulate ribosome biogenesis. However, the specificity of CX-5461 has been questioned and it has been suggested that this drug may also act by stabilizing DNA G-quadruplexes or by “poisoning” topoisomerase II (Topo II). Thus, the primary target of this drug and its mode of action are still in doubt. Here we used Deconvolution-ChIP-Seq in NIH3T3 and HEK293T cells treated for different times with CX-5461. The data show that the primary target of CX5461 is the initiation of ribosomal RNA gene (rDNA) transcription. CX-5461 blocks transcription initiation in vitro and in vivo by arresting RNA polymerase I (RPI/Pol1) within the preinitiation complex. In contrast to previous suggestions, CX-5461 does not effect recruitment of the TBP-TAF complex SL1 to the rDNA promoter, the recruitment of the initiation competent RPI-Rrn3 complex or ongoing transcription elongation, arguing against a role for G-quadruplex stabilization or topoisomerase II poisoning. Inhibition of transcription by CX-5461 is not reversible, the RPI-Rrn3 complex remains arrested in the preinitiation complex even after drug removal. This leads to nucleolar stress, extensive DNA damage and cell senescence. Our data show that the cytotoxicity of CX-5461 is the downstream result of the highly specific inhibition of rDNA transcription. The observation that this inhibition is irreversible will be important for the future design of chemotherapeutic strategies and the avoidance of drug resistance.