Role of rDNA chromatin in sensitizing OVCA to RNA polymerase I transcription inhibitors

Abstract

The ribosomal RNA (rRNA) genes (rDNA) are arrayed in multiple tandem repeats on the 5 acrocentric chromosomes, and are transcribed by RNA Polymerase I (Pol I) which gives rise to the 47S pre-rRNA, the precursor of 18S, 5.8S and 28S rRNAs. rDNA transcription accounts for around 35-60% of all cellular transcription and is a highly energy consuming process as well as a key determinant of cellular growth and proliferation rates. The highly repetitive and actively transcribed nature of rDNA gives rise to a high recombinogenic potential. Further, structural dynamics and variation in the rDNA loci have been reported in over 50% of solid human cancers (Stults et al. 2009).

Although the rate of rDNA transcription is likely to be rate limiting in ribosome biogenesis, only a subset of rRNA genes are transcribed from “active” rDNA at any given time. rDNA chromatin exists in active or silent forms. Active rDNA chromatin is “open” and bound by the upstream binding transcription factor (UBF), which is essential for establishing and maintaining active rDNA states (Sanij et al. 2008).

We have reported that rDNA silencing increases during terminal differentiation of granulocytes due to decreased UBF levels (Sanij et al. 2015). Conversely, our studies utilizing MYC-driven B-lymphoma mouse model, demonstrated reactivation of silent rDNA as MYC-driven B-cells progress towards malignancy. We have also demonstrated that inhibition of rDNA transcription by the novel Pol I transcription inhibitor CX-5461 can selectively kill MYC-driven B-lymphoma cells in vivo, while sparing wild-type B-cells (Bywater et al. 2012). Although, CX-5461 activates p53-dependent and p53-independent cellular stress response pathways leading to apoptosis, senescence and cell cycle arrest, the mechanisms underlying tumour cell sensitivity to CX-5461 remain unclear.

We have undertaken a systematic approach across a panel of ovarian cancer (OVCA) cell lines to examine their sensitivity to CX-5461. We have demonstrated that OVCA cell lines display similar sensitivities to Pol I transcription inhibition, however, they exhibit differential cellular proliferative growth responses, associated with an immediate or a delayed cell cycle delay and arrest. Furthermore, our studies demonstrate that increased ratio of active to inactive active rDNA chromatin and not rDNA transcription rate per se determines the growth inhibitory sensitivity to CX-5461 treatment of OVCA cells.

We utilized RNAi based knock-down of UBF to modulate rDNA chromatin as well as ZFN and CRISPR-Cas9 genome editing knock-out (KO) of rDNA repeats approaches to investigate whether rDNA chromatin states and/or rDNA copy number play a role in determining sensitivity to CX-5461. We demonstrated the important role of rDNA in ribosome biogenesis and genome wide instability. Specifically, reducing rDNA copy number can modulate rDNA chromatin states which possibly mediate global genome instability. We propose that rDNA chromatin states could potentially serve as a biomarker to identify OVCA patients that may benefit from CX-5461 treatment.