The transcription of the 45S ribosomal RNA (rRNA) genes by RNA Polymerase I (Pol I) is a major rate limiting step for ribosomal biogenesis, and consequently for cell growth and proliferation. We have shown that rDNA transcription can be therapeutically targeted in vivo with the small molecule CX-54611 to selectively kill B-lymphoma cells in a genetic model of spontaneous lymphoma while maintaining a viable wild type B-cell population. The therapeutic effect is a direct consequence of activation of p53-dependent apoptotic signalling2 .
The nucleolar surveillance pathway regulates p53 function in response to nucleolar stress, where p53 is stabilized and activated through the release of a number of nucleolar proteins, including nucleophosmin, ARF and a subset of ribosomal proteins (RPs), which can interact with the central acidic domain of MDM2 resulting in the suppression of p53 ubiquitination. We have recently reported that inhibition of Pol I transcription by CX-5461 increases MDM2 association with RPL5 and RP11. In addition, we have recently identified a novel p53-independent cell cycle checkpoint in response to inhibition of Pol I transcription3 . These phenotypes are associated with DNA damage – like response; activation of ATM/chk2 signaling pathways occurs rapidly following CX-5461 treatment.
This project aims to uncouple the p53 dependent and independent checkpoints identified above by utilizing MDM2 mutant mouse embryonic fibroblasts that harbor mutation in the central zinc finger of MDM2 that can specifically disrupt the interaction of MDM2 with RPL11 and L5. These MDM2 mutants retain full p53-suppressive function while escaping inhibition by ribosomal proteins L5 and L11.
We will present the results of our ongoing studies addressing whether these mutant MEFs are able to demonstrate a phenotypic response to CX-5461 treatment, and whether p53 activation can occur independently of MDM2 function.