Therapeutics, Targets, and Chemical Biology Targeting RNA Polymerase I with an Oral Small Molecule CX-5461 Inhibits Ribosomal RNA Synthesis and Solid Tumor Growth Denis Drygin 1 , Amy Lin 1 , Josh Bliesath 1 , Caroline B. Ho 1 , Sean E. O’Brien 1 , Chris Proffitt 1 , Mayuko Omori 1 , Mustapha Haddach 1 , Michael K. Schwaebe 1 , Adam Siddiqui-Jain 1 , Nicole Streiner 1 , Jaclyn E. Quin 2,3 , Elaine Sanij 2 , Megan J. Bywater 2,3 , Ross D. Hannan 2,3,4 , David Ryckman 1 , Kenna Anderes 1 , and William G. Rice 1 Abstract Deregulated ribosomal RNA synthesis is associated with uncontrolled cancer cell proliferation. RNA polymerase (Pol) I, the multiprotein complex that synthesizes rRNA, is activated widely in cancer. Thus, selective inhibitors of Pol I may offer a general therapeutic strategy to block cancer cell proliferation. Coupling medicinal chemistry efforts to tandem cell- and molecular-based screening led to the design of CX-5461, a potent small-molecule inhibitor of rRNA synthesis in cancer cells. CX-5461 selectively inhibits Pol I–driven transcription relative to Pol II–driven transcription, DNA replication, and protein translation. Molecular studies demonstrate that CX-5461 inhibits the initiation stage of rRNA synthesis and induces both senescence and autophagy, but not apoptosis, through a p53-independent process in solid tumor cell lines. CX-5461 is orally bioavailable and demonstrates in vivo antitumor activity against human solid tumors in murine xenograft models. Our findings position CX-5461 for investigational clinical trials as a potent, selective, and orally administered agent for cancer treatment. Cancer Res; 71(4); 1418–30. Ó2010 AACR. Introduction The rate of ribosome biogenesis controls cellular growth and proliferation (reviewed in ref. 1). It, therefore, is tightly regulated in mammalian cells and is tuned to respond to extracellular stimuli such as nutrient availability and stress. During tumorigenesis, the tightly regulated relationship between extracellular signaling and ribosome biosynthesis is disrupted, and cancer cells begin the excessive produc- tion of ribosomes necessary for the protein synthesis asso- ciated with unbridled cancer growth. rRNA is a major component of the ribosome and, as such, carcinogenesis requires an increase in its synthesis (reviewed in refs. 2–5). Indeed, an increase in the synthesis of rRNA, which is transcribed in the nucleolus by RNA polymerase (Pol) I, correlates with an adverse prognosis in cancer (6). More- over, enlarged nucleoli, reflective of accelerated rRNA synthesis, have long been recognized as a marker for aggressive tumor cells (7, 8). A number of approved cancer therapeutics reportedly act through inhibition of rRNA synthesis, but none directly target the Pol I multiprotein enzyme complex (4, 9). The potential therapeutic benefit of selectively inhibiting the Pol I target so fundamental to cancer cell survival prompted the need for identification of small molecule drugs that selectively inhibit rRNA synthesis. For this purpose, we fashioned a cell-based quantitative reverse- transcription PCR (qRT-PCR) assay that differentiates between the effects of compounds on Pol I- and Pol II– driven transcription. In this article, we describe the discov- ery and characterization of CX-5461, a potent and selective inhibitor of Pol I–mediated rRNA synthesis in cancer cells that does not inhibit DNA, mRNA or protein synthesis. We reveal that CX-5461 induces autophagic cell death in cancer cells but not normal cells and exhibits potent in vivo anti- tumor activity in murine xenograft models of human solid tumors with a favorable safety profile. CX-5461 represents a fundamentally new class of small molecule–targeted anti- cancer therapeutics. Materials and Methods Materials CX-5461 and CX-5447 were synthesized by Cylene Pharma- ceuticals as off-white solid materials (99.2%–99.5% pure) and stored at room temperature as 10 mmol/L stock solutions in Authors' Affiliations: 1 Cylene Pharmaceuticals, Inc., San Diego, Califor- nia; 2 Peter MacCallum Cancer Centre; 3 Department of Biochemistry and Molecular Biology, University of Melbourne; and 4 Department of Biochem- istry and Molecular Biology, Monash University, Melbourne, Victoria, Australia Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Corresponding Author: William G. Rice, Cylene Pharmaceuticals Inc., 5820 Nancy Ridge Drive, Suite 200, San Diego CA 92121. Phone: 858- 875-5100; Fax: 858-875-5101. E-mail: wrice@cylenepharma.com doi: 10.1158/0008-5472.CAN-10-1728 Ó2010 American Association for Cancer Research. Cancer Research Cancer Res; 71(4) February 15, 2011 1418 on December 28, 2021. © 2011 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from Published OnlineFirst December 15, 2010; DOI: 10.1158/0008-5472.CAN-10-1728