The chemotherapeutic CX-5461 primarily targets TOP2B and exhibits selective activity in high-risk neuroblastoma

Abstract

Survival in high-risk pediatric neuroblastoma has remained around 50% for the last 20 years, with immunotherapies and targeted therapies having had minimal impact. Here, we identify the small molecule CX-5461 as selectively cytotoxic to high-risk neuroblastoma and synergistic with low picomolar concentrations of topoisomerase I inhibitors in improving survival in vivo in orthotopic patient-derived xenograft neuroblastoma mouse models. CX-5461 recently progressed through phase I clinical trial as a first-in-human inhibitor of RNA-POL I. However, we also use a comprehensive panel of in vitro and in vivo assays to demonstrate that CX-5461 has been mischaracterized and that its primary target at pharmacologically relevant concentrations, is in fact topoisomerase II beta (TOP2B), not RNA-POL I. This is important because existing clinically approved chemotherapeutics have well-documented off-target interactions with TOP2B, which have previously been shown to cause both therapy-induced leukemia and cardiotoxicity—often-fatal adverse events, which can emerge several years after treatment. Thus, while we show that combination therapies involving CX-5461 have promising anti-tumor activity in vivo in neuroblastoma, our identification of TOP2B as the primary target of CX-5461 indicates unexpected safety concerns that should be examined in ongoing phase II clinical trials in adult patients before pursuing clinical studies in children.

Results of the phase I CCTG IND.231 trial of CX-5461 in patients with advanced solid tumors enriched for DNA-repair deficiencies

“…Finally, our clinical results provide important context in light of recent preclinical reports 19 , 20 that CX-5461 (and other G4 ligands such as PDS) induces topoisomerase 2 (Topo2) trapping as part of the mechanism leading to double-stranded breaks and cytotoxicity 21 , 22 . In this study, CX-5461 had clinical activity without evidence of the characteristic toxicities (e.g., myelosuppression and alopecia) of topoisomerase inhibitors.…”

Labeling of heterochronic ribosomes reveals C1ORF109 and SPATA5 control a late step in human ribosome assembly

“…Ribosome biogenesis depends on ongoing rRNA transcription. Treating cells with the Pol I and TOP2B inhibitor CX-5461 ( Drygin et al, 2011 ; Pan et al, 2021 ) resulted in retention of newly labeled RPL28 within nucleoli ( Figure 1F ), further suggesting that trafficking of RPL28 from the nucleolus to the cytoplasm depends on ribosome assembly. Treatment of cells with sodium arsenite (NaAsO 2 ) results in stress granule assembly and a rapid loss of protein synthesis.…”

Glutamine deficiency in solid tumor cells confers resistance to ribosomal RNA synthesis inhibitors

“…During the preparation of this manuscript, CX-5461 was reported to possess additional inhibitory activities against Topoisomerase II 44 , 45 . This presents a limitation to our manuscript since we employed CX-5461 to inhibit pre-rRNA accumulation under ND.…”