CX-5461, a Novel RNA Polymerase I Inhibitor, Is Active Against Wild-Type and Mutant p53 Myeloma Models
Authors: Lee Hans C. , Wang Hua , Li Bing-Zong , Wang Zhiqiang , Jones Richard Julian , Gu Dongmin , O'Brien Sean , Davis Richard E. , Orlowski Robert Z. 
Published: October 2019

Background
Ribosomal RNA (rRNA) forms a major component of ribosomes, which play a critical role in cellular growth and proliferation through regulation of protein synthesis. Increased rRNA transcription and ribosomal biogenesis have been associated with tumorigenesis, and recent reports have suggested that inhibition of rRNA transcription by CX-5461 (Senhwa Biosciences), a novel selective small-molecule inhibitor of RNA polymerase (pol) I, induces cell death in several human tumor types by both p53-dependent and independent mechanisms. These findings led to our hypothesis that selective RNA pol I inhibition with CX-5461 could be a rational new approach to therapy for both wild-type (wt) and mutant p53 multiple myeloma models.

Methods
Studies with CX-5461 were performed in wt p53 and mutant p53 cell lines, zinc-finger nuclease (ZFN) p53 knock-out (KO) isogenic myeloma cell lines, and bortezomib and carfilzomib-resistant myeloma cell lines.

Results
Treatment of wt p53 (MM1.S, MOLP8) and mutant p53 (U266, RPMI-8226) myeloma cell lines demonstrated a time and dose dependent decrease in cell proliferation after exposure to CX-5461 with a median inhibitory concentration (IC50) range of 50-100 nM after a 72-hour incubation. A corresponding increase in cleaved PARP, cleaved caspase-9, and cleaved caspase-3 expression was seen on Western blot, as well as increased Annexin V staining on flow cytometry analysis. Notably, the degree of Annexin V staining was less in the p53 mutant cell lines compared to the wt p53 cells at any given drug concentration, but strong apoptotic signaling could be induced in mutant p53 cell lines when using higher concentrations of CX-5461. In addition, co-culturing myeloma cells with GFP+ HS5 stromal cells to mimic the bone marrow microenvironment did decrease the therapeutic effect of CX-5461, but again could be overcome with higher drug concentrations [250-500 nM]. Similar results were seen when isogenic MM1.S ZFN p53 KO cells were used, whose sensitivity to CX-5461 was comparable to that of wt p53 cells. Finally, CX-5461 was also tested on drug-resistant myeloma cell lines that were generated by exposing cells to low concentrations of bortezomib (RMPI-8266, KAS-6/1, ANBL-6) or carfilzomib (KAS-6/1) over time. These drug-resistant cell lines showed sensitivity to CX-5461 with an IC50 in the 100-250 nM concentration range. Gene expression profiling (GEP) of isogenic MM1.S ZFN p53 KO and wt cells revealed that gene expression perturbations by CX-5461 were primarily p53-independent. Additional GEP and pathway analysis in other isogenic ZFN p53 wt and KO cell lines is currently ongoing, with a particular interest in p53-independent mechanisms that may explain the efficacy of CX-5461 in both wt and mutant p53 myeloma models.

Conclusion
RNA pol I inhibition by CX-5461 is a promising approach to myeloma therapy, with low nanomolar drug activity seen in wt p53, mutant p53, and drug-resistant myeloma cell line models, providing a rationale for translation of CX-5461 into the clinic for the treatment of multiple myeloma.

Disclosures:
O'Brien: Senhwa Biosciences, Inc: Employment. Orlowski:Bristol-Myers Squibb: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Millennium: The Takeda Oncology Company: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Onyx: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Resverlogix: Research Funding; Array Biopharma: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Genentech: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Merck: Membership on an entity’s Board of Directors or advisory committees.