Therapeutic Targeting of RNA Polymerase I With the Small-Molecule CX-5461 for Prevention of Arterial Injury–Induced Neointimal Hyperplasia

Abstract

Objective-RNA polymerase I (Pol I)-dependent rRNA synthesis is a determinant factor in ribosome biogenesis and thus cell proliferation. The importance of dysregulated Pol I activity in cardiovascular disease, however, has not been recognized. Here, we tested the hypothesis that specific inhibition of Pol I might prevent arterial injury-induced neointimal hyperplasia. Approach and Results-CX-5461 is a novel selective Pol I inhibitor. Using this tool, we demonstrated that local inhibition of Pol I blocked balloon injury-induced neointima formation in rat carotid arteries in vivo. Neointimal development was associated with augmented rDNA transcriptional activity as evidenced by the increased phosphorylation of upstream binding factor-1. The beneficial effect of CX-5461 was mainly mediated by inducing G2/M cell cycle arrest of proliferating smooth muscle cells without obvious apoptosis. CX-5461 did not induce p53 stabilization but increased p53 phosphorylation and acetylation and activated the ataxia telangiectasia mutated/ataxia telangiectasia and Rad3-related (ATR) pathway. Inhibition of ATR, but not of ataxia telangiectasia mutated, abolished the cytostatic effect of CX-5461 and p53 phosphorylation. In addition, inhibition of p53 or knockdown of the p53 target GADD45 mimicked the effect of ATR inhibition. In vivo experiments showed that the levels of phospho-p53 and acetyl-p53, and activity of the ataxia telangiectasia mutated/ATR pathway were all augmented in CX-5461-treated vessels. Conclusions-Pol

Abnormal Ribosome Biogenesis Partly Induced p53-Dependent Aortic Medial Smooth Muscle Cell Apoptosis and Oxidative Stress

“…cx-5461 accelerated the occurrence of AD, inhibited the proliferation of ASMCs, and induced apoptosis. In a recent study, Ye et al reported that cx-5461 prevented aortic intima hyperplasia, indicating its clinical potential against atherosclerosis and stenosis [44]. In contrast to our study, however, they showed that cx-5461 only inhibited ASMC proliferation and did not induce apoptosis [43].…”

Therapeutic efficacy of the novel selective RNA polymerase I inhibitor CX‐5461 on pulmonary arterial hypertension and associated vascular remodelling

“…protein (Boulon et al, 2010). Similar to our findings in aortic smooth muscles (Ye et al, 2017), however, CX-5461 did not increase the total amount of p53 protein; instead it induced a robust increase in p53 phosphorylation (Figure 5d,e). Moreover, pretreatment with the selective p53 inhibitor pifithrin-α attenuated the inhibitory effect of CX-5461 on the cell cycle (see Figure 5b).…”

Inhibition of nucleolar stress response by Sirt1: A potential mechanism of acetylation‐independent regulation of p53 accumulation

“…In addition to the activation of the p53 pathway, NSR may also signal to other pathways implicated in regulating cell cycle and apoptosis (James et al, ). Interestingly, we and other groups have observed in different cells that induction of NSR by selective inhibition of RNA polymerase I triggers activation of the ATR pathway, possibly independent of DNA strand breaks (Ma & Pederson, ; Quin et al, ; Ye et al, ). Hence, it is plausible that the nucleolus serves as a signaling hub, while Sirt1 may intervene with multiple pathways by regulating the occurrence of NSR.…”

“…Treatment with resveratrol attenuated NSR induced by H 2 O 2 , while this effect of resveratrol was blunted by EX‐527 (Figure e). We and others have shown that NSR induction by inhibiting RNA polymerase I triggers cell cycle arrest (Negi & Brown, ; Ye et al, ). To test whether Sirt1 could also inhibit NSR‐associated cellular functional changes, we performed flow cytometry analysis; we showed that ActD treatment caused G2/M blockade, which was attenuated by Sirt1 overexpression (Figure f).…”