Abstract PR15: Inhibition of RNA Polymerase I as a therapeutic strategy for cancer-specific activation of p53
Authors: Bywater Megan J. , Anderes Kenna , Huser Nanni , Proffitt Chris , Bleisath Joshua , Haddach Mustapha , Schwaebe Michael , Ryckman David , Rice William G. , Lowe Scott W. , Johnstone Ricky W. , Poortinga Gretchen , Pearson Richard B. , McArthur Grant A. , Hannan Ross D. , Sanij Elaine , Hein Nadine , Peck Abigail , Cullinane Carleen , Wall Meaghan , Cluse Leonie , Drygin Denis 
Published: June 2012

Abstract
Increased transcription of the ribosomal genes (rDNA) by RNA Polymerase I (Pol I) is a common feature of human cancer[1]. However until now no studies have directly examined the requirement for dysregulated rDNA transcription in the maintenance of the malignant phenotype. Our studies show that increased rDNA transcription is necessary for MYC oncogenic activity and can be therapeutically targeted to treat tumors. We demonstrate that restoration of hyperactivated rDNA transcription rates in Eμ-MYC lymphoma cells to the levels observed in normal B cells by knock down of Pol I transcription factors UBF and Rrn3, is rapidly selected against in vitro as determined by loss from competitive culture with parental cells. This disadvantage is due to the induction of apoptosis and can be rescued by over expression of the anti-apoptotic protein BCL2. Furthermore, treatment of Eμ-MYC lymphoma cells with a small molecule inhibitor of Pol I (CX-5461) we have recently developed[2] is able to specifically inhibit Pol I transcription (IC50=45.64nM, metabolic labeling) and rapidly induce apoptosis and subsequent cell death by 16hrs (IC50=8.4nM, PI exclusion). This apoptotic response is not an indirect consequence of ribosome insufficiency but is due to induction of the ribosome biogenesis surveillance pathway[3] characterized by rapid nucleolar disruption, as determined by immunofluorescence of Fibrillarin relocalization, and the subsequent activation of p53-dependent apoptotic signaling, as determined by increased protein levels of p53 and increased expression of p53 target genes p21, Mdm2 and Puma at the mRNA and protein level within 1hr of treatment. Using CX-5461 we show that malignant B cells have a heightened dependence on elevated rDNA transcription that can be exploited in vivo as a therapeutic target for treatment of lymphoma. Treatment of mice transplanted Eμ-MYC lymphoma with 40mg/kg CX-5461 orally every 3 days is able to delay the onset of disease (median survival of 15 days for vehicle, 31 days for drug, P&lt;0.0001), with this delay accompanied by a period of disease remission, as determined by peripheral white blood cell counts (58×10⁁3cellsμl±4.5 for vehicle, 4.2×10⁁3cellsμl±0.29 for drug, P&lt;0.001) and FACS analysis of the peripheral blood (62%tumor cells±2.3 for vehicle, 0.7%tumor cells±0.05 for drug, P&lt;0.001), and maintenance of a wild type B-cell population (20%B220+cells±1.0 for vehicle, 43%B220+cells±1.5 for drug, P&lt;0.001). Similarly in vivo, elimination of tumor cells with the inhibition of Pol I transcription results from subsequent activation of p53-dependent apoptotic signaling that specifically occurs in malignant but not normal B-lymphocytes in the spleen and bone marrow of healthy mice treated with the same dose of CX-5461. Human leukemia and lymphoma cell lines also show high sensitivity to the inhibition of Pol I transcription that is dependent on p53 mutational status. Furthermore, CX-5461 was able to delay the growth of subcutaneous MV 4;11 (human acute myeloid leukemia) xenografts in mice dosed at 125mg/kg IP weekly (tumor growth inhibition=93% compared to vehicle). Our work reveals a previously unproven paradigm that links hyperactivated rDNA transcription and nucleolar integrity to maintenance of aggressive tumors independent of ribosome levels. Critically, these results also demonstrate how activation of a ribosome biogenesis surveillance pathway by selective inhibition of rDNA transcription can be used as a novel therapeutic target for the treatment of cancer. Furthermore, patients with hematological malignancies have been identified as the appropriate first cohort for trials of CX-5461 in man commencing next year.
1. R. J. White, Nat Rev Mol Cell Biol 6, 69 (Jan, 2005).
2. D. Drygin et al., Cancer Res, (Dec 15, 2010).
3. C. Deisenroth, Y. Zhang, Oncogene 29, 4253 (Jul 29, 2010).
This abstract is also presented as Poster A35.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr PR15.