Investigating the p53-independent responses to inhibition of RNA Polymerase I transcription by CX-5461
AffiliationBiochemistry and Molecular Biology
MetadataShow full item record
Document TypePhD thesis
Access StatusOpen Access
© 2017 Dr. Jaclyn Quin
Increased rates of DNA-dependent RNA Polymerase I (Pol I) transcription of the 47S pre-ribosomal RNA (rRNA) genes are observed in almost all cancer types. Cancer cells may require high rates of Pol I transcription and ribosome biogenesis to achieve their unrestrained growth and proliferative capacity, thus presenting a therapeutic window for selectively targeting cancer cells with inhibitors of Pol I transcription. Our laboratory helped develop and validate a first-in-class small molecule selective inhibitor of Pol I transcription, CX-5461 (Senhwa Biosciences). Here, we have investigated the response of cells at defined stages of malignant transformation to inhibition of Pol I transcription, utilising a panel of isogenically matched BJ fibroblast cell lines. We compared the response of non-transformed and transformed cells of the same genetic background, and demonstrated that CX-5461 can selectively induce cell death in cancer cell lines in vitro. We investigated the phenotypic response of a nontransformed BJ fibroblast cell line minimally immortalized with hTERT (BJ-T) to CX- 5461, and demonstrated that they display a proliferation defect. The proliferation defect is associated with the activation of p53 and a p53-dependent G1 cell cycle checkpoint, as well as p53-independent S-phase and G2 cell cycle checkpoints and senescence. Escape from cell cycle arrest in transformed BJ fibroblast cell lines is associated with increased rates of cell death in response to CX-5461. To identify pathways mediating the p53-independent responses to inhibition of Pol I transcription, we have performed RNA-sequencing analysis in CX-5461 treated BJ-T cells in which p53 was silenced (BJ-T p53shRNA). The analysis identified ATM (Ataxia-telangiectasia mutated) / ATR (ATM and RAD3-related) signaling and transcriptional programs associated with senescence to be modulated following treatment with CX-5461. Further, we have demonstrated that inhibition of Pol I transcription by CX-5461 rapidly and potently activates the ATM/ATR kinase signaling pathways in the absence of global DNA damage. Combined ATM/ATR inhibition and CX-5461 treatment results in bypass of CX-5461 mediated S-phase and G2 arrest, and induced cell death in the BJ-T p53shRNA cell line. We investigated the mechanisms by which inhibition of Pol I transcription by CX-5461 activates the ATM/ATR signaling pathways. We demonstrated that inhibition of Pol I transcription initiation by CX-5461 results in ‘exposed’ rRNA genes (rDNA) that are in an open chromatin conformation but devoid of Pol I. Inhibition of Pol I transcription by CX-5461 also results in reorganization of nucleolar structure and translocation of proteins to and from the nucleoli. We observed increased levels of NBS1 (Nijmegen Breakage Syndrome 1) activation by ATM specifically within the nucleoli during S/G2. We propose CX-5461 treatment induces an unusual chromatin structure at the rDNA that is sufficient to activate ATM/ATR in the nucleoli. Finally, we have shown that DNA damage repair is attenuated following treatment with CX-5461. Together, our studies identify activation of ATM/ATR signaling as a key p53-independent pathway of response to inhibition of Pol I transcription, that can be targeted to improve the efficacy of CX-5461 in cancer therapy.
Keywordsribosome; ribosomal RNA; rRNA; ribosomal RNA genes; rDNA; RNA Polymerase I; Pol I; CX-5461; cancer; nucleoli; nucleolar stress signaling; p53; DNA damage response; DDR; ATM; ATR
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