Abstract
CX-5461 is a G-quadruplex (G4) ligand currently in trials with initial indications of clinical activity in cancers with defects in homologous recombination repair. To identify more genetic defects that could sensitize tumors to CX-5461, we tested synthetic lethality for 480 DNA repair and genome maintenance genes to CX-5461, pyridostatin (PDS), a structurally unrelated G4-specific stabilizer, and BMH-21, which binds GC-rich DNA but not G4 structures. We identified multiple members of HRD, Fanconi Anemia pathways, and POLQ, a polymerase with a helicase domain important for G4 structure resolution. Significant synthetic lethality was observed with UBE2N and RNF168, key members of the DNA damage response associated ubiquitin signaling pathway. Loss-of-function of RNF168 and UBE2N resulted in significantly lower cell survival in the presence of CX-5461 and PDS but not BMH-21. RNF168 recruitment and histone ubiquitination increased with CX-5461 treatment, and nuclear ubiquitination response frequently co-localized with G4 structures. Pharmacological inhibition of UBE2N acted synergistically with CX-5461. In conclusion, we have uncovered novel genetic vulnerabilities to CX-5461 with potential significance for patient selection in future clinical trials.
Highlights
Folded G-quadruplex (G4) structures arise transiently at guanine-rich sequences in the DNA and RNA of multiple organisms, including microbes, yeast, plants, C. elegans, mouse and h umans[1,2,3,4,5,6,7,8,9]
We discovered that the loss of factors UBE2N and RNF168, involved in DNA damage response (DDR)-related ubiquitin signaling, sensitizes cells to G4 stabilizers
The quality of the pooled library was determined by next-generation sequencing which revealed that more than 99% of single guide RNA (sgRNA) were present in the library with less than an eightfold difference in the representation of 80% of them (Supplementary information Fig. 1a) and by functional assessment of known essential genes
Summary
Folded G-quadruplex (G4) structures arise transiently at guanine-rich sequences in the DNA and RNA of multiple organisms, including microbes, yeast, plants, C. elegans, mouse and h umans[1,2,3,4,5,6,7,8,9]. The G4 stabilizing activity of CX-5461 inducing DNA damage, in part by replication stalling, was shown to be synthetically lethal with loss of homologous recombination repair. Since double stranded break induction is a key mechanism for some of the G4 stabilization drugs, a more complete understanding of DNA repair synthetic lethality beyond the known homologous recombination repair pathways could lead to expanded clinical indications. We address this for CX5461 and associated control compounds by screening at depth for synthetic lethal interactions with 480 genes, including core DNA repair genes. We further demonstrated that the ubiquitination pathway was activated on cellular treatment with G4 stabilizers and that CX-5461, in combination with a UBE2N inhibitor, increased cancer cell toxicity
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