Abstract
Background: The role of dysregulation of the proto-oncogene MYC in both early and late myeloma progression events is well established. Among key MYC -downstream targets is upregulation of ribosomal biogenesis, resulting in increased protein translational capacity and biomass accumulation that is characteristic of neoplastic cells. Thus, given the relationship between myeloma pathobiology, MYC dysregulation, and ribosomal biogenesis, we hypothesized that selective targeting of ribosomal RNA (rRNA) transcription with the small molecule RNA polymerase (pol) I inhibitor CX-5461 (Senhwa Biosciences) may represent a novel therapeutic strategy in myeloma.
Methods: Studies with CX-5461 were performed in human myeloma cell lines, isogenic p53 wild-type (wt) and knock-out (KO) p53 cells generated using sequence-specific zinc-finger nucleases, drug-resistant cell lines, primary patient samples, and myeloma murine xenograft models using NOD-SCID IL2Rgnull mice.
Results: CX-5461 treatment of p53 wt (MM1.S, MOLP-8) and p53 mutant (U266, RPMI-8226) myeloma cell lines demonstrated a time- and dose-dependent decrease in cell proliferation with a median inhibitory concentration (IC50) at nM levels after 72 hours. 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, although this was more pronounced in p53 wt versus mutant cell lines. CX-5461 also retained activity in a panel of cell lines resistant to standard myeloma therapeutic agents (bortezomib, carfilzomib, lenalidomide, and doxorubicin) and in primary patient samples, including a heavily pretreated relapsed/refractory patient and a de novo plasma cell leukemia patient with del 17p. In vivo studies using a systemic isogenic MM1.S p53 wt and KO myeloma murine xenograft model demonstrated significant improvement in median overall survival in the CX-5461-treated p53 wt cohort (41 days vs. not reached, P .05), although outcomes were more modest in the p53 KO cohort with only a trend towards improved survival (P.1) in the drug-treated mice.
To probe the p53-independent effects of CX-5461, gene expression profiling and gene set enrichment analysis was performed on isogenic MM1.S and MOLP-8 p53 wt and KO myeloma cell lines treated with CX-5461 or vehicle. These results suggested downregulation of MYC downstream targets as one p53-independent effect of RNA pol I inhibition. qPCR and Western blot studies revealed rapid downregulation of MYC at the transcript level within 1-hour of CX-5461 treatment followed by decreases in MYC protein levels.
Previous studies have suggested ribosomal biogenesis is tightly controlled by an auto-regulatory feedback mechanism in which ribosomal proteins such as RPL5 and RPL11 can bind to the 3'UTR of MYC mRNA and facilitate its degradation through the RNA-induced silencing complex (RISC). Because RNA pol I inhibition is known to induce a nucleolar stress response and increase the availability of free ribosomal proteins, RISC-mediated degradation of MYC mRNA was explored as one possible mechanism of CX-5461-mediated MYC downregulation. Indeed, treatment with CX-5461 led to increased pull-down of RPL5 when immunoprecipitated with the RISC subunit TAR (HIV-1) RNA Binding Protein 2 (TARBP2) compared to vehicle-treated controls, and RNA immunoprecipitation assays with the catalytic RISC subunit, Argonaute 2 (AGO2), demonstrated enrichment of MYC mRNA with CX-5461 treatment. These results suggest that CX-5461 may induce degradation of MYC through the cooperative binding of ribosomal proteins, RISC subunits, and MYC mRNA.
Finally, to evaluate the role of MYC expression and ribosomal biogenesis in relation to CX-5461 sensitivity, MYC was overexpressed in the H1112 myeloma cell line, which at baseline does not harbor a MYC translocation. MYC overexpression in H1112pCDH-myc cells led to increased basal pre-rRNA transcript levels compared to H1112pCDH cells, and furthermore, led to enhanced sensitivity to CX-5461.
Conclusion: RNA pol I inhibition by CX-5461 is a promising target in myeloma therapy, with downregulation of MYC representing one mechanism of action. Moreover, increased MYC expression enhances sensitivity to CX-5461, providing rationale for the clinical translation of CX-5461 for the treatment of myeloma and other MYC-driven cancers.
Disclosures
O'Brien: Senhwa Biosciences, Inc.: Employment. Keats:Translational Genomic Research Institute: Employment. Orlowski:Bristol-Myers Squibb: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Spectrum Pharmaceuticals: Research Funding; Janssen Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Acetylon: Membership on an entity's Board of Directors or advisory committees; Onyx Pharmaceuticals: Consultancy, Research Funding; Millennium Pharmaceuticals: Consultancy, Research Funding; Forma Therapeutics: Consultancy; Genentech: Consultancy; BioTheryX, Inc.: Membership on an entity's Board of Directors or advisory committees; Array BioPharma: Consultancy, Research Funding.
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