The RNA polymerase I transcription inhibitor CX-5461 cooperates with topoisomerase 1 inhibition by enhancing the DNA damage response in homologous recombination-proficient high-grade serous ovarian cancer

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Only approximately 45% of women with ovarian cancer survive 5 years after their diagnosis1. Ovarian cancer is typically treated by surgery and chemotherapy, but the major challenge in treating women with ovarian cancer is the development of chemotherapeutic resistance, which eventually leads to relapsed disease and mortality2. This highlights the need to develop alternative treatment strategies to improve patient outcomes. High-grade serous ovarian cancer (HGSC) accounts for the majority of ovarian cancer deaths and poly-ADP ribose polymerase (PARP) inhibitors are showing great promise3 in treating the 50% of these patients that show defects in homologous recombination (HR) DNA repair. However, for the 50% of patients with intact HR, they have a dearth of treatment options.

As cancer cells rely on making more proteins than normal cells, we have devised a new strategy to inhibit ribosomal RNA gene (rDNA) transcription, an early step in the generation of ribosomes, which are the factories for making proteins. We have shown encouraging responses for the drug CX-5461 in advanced-stage blood cancer patients who were previously treated with chemotherapy4. In HR-deficient HGSC, we have demonstrated cooperation of CX-5461 with PARP inhibitors, highlighting this as an exciting combination treatment for HGSC patients5. We have also shown that CX-5461 has single-agent efficacy in PARP inhibitor-resistant models of HGSC5.

In this paper, to determine how to stop the growth of ovarian cancer cells with intact HR, we performed a genome-wide small interfering RNA (siRNA) screen in cells treated without or with CX-5461. Besides identifying a number of genes known to be important in HR, we also identified that loss of DNA topoisomerase 1 (TOP1), an enzyme that relieves the strain generated by the DNA double helix’s twisting during DNA replication and transcription, could cooperate with CX-5461 in inhibiting ovarian cancer cell proliferation. TOP1 has also been shown to play an important role at the rDNA loci6, and TOP1 inhibitors such as topotecan have been used as a salvage therapy for relapsed ovarian cancer patients though their clinical use has been limited due to haematological toxicity7.

We demonstrated the combination of CX-5461 and topotecan markedly enhanced replication stress and the DNA damage response without generating DNA double-strand breaks, leading to cell cycle arrest and decreased clonogenic survival of cancer cells (Figure 1). Importantly, when we used CX-5461 and low-dose topotecan to treat HGSC tumours in vivo, we found significant inhibition of tumour growth without observing side effects. This suggests the possibility that the toxicity often associated with using standard topotecan doses could potentially be reduced by using lower doses combined with CX-5461, thereby providing a new treatment strategy using an already approved drug for HGSC patients with HR-proficient disease, including patients who have relapsed on PARP inhibitor treatment.

We have recently identified a number of biomarkers of sensitivity to CX-5461 in HGSC including BRCA-mutated and MYC targets gene expression signatures5. Interestingly, several publications have demonstrated that defects in another DNA topoisomerase isoform TOP2 bestow resistance to CX-54618-10. We are currently very interested in exploring the mechanisms by which deficiencies in TOP1 and TOP2 confer sensitivity and resistance to CX-5461, respectively, as a greater understanding of these mechanisms will be important for the design of future clinical trials with CX-5461.

This article is published in the British Journal of Cancer at https://www.nature.com/articles/s41416-020-01158-z

Figure 1. CX-5461 inhibits RNA Pol I recruitment to upstream binding transcription factor (UBF)-bound rDNA within nucleoli, the sites of rDNA transcription. CX-5461 combined with TOP1 deficiency or inhibition induces replication stress and the DDR leading to cell cycle arrest and inhibition of tumour growth. Created with BioRender.com

References

  1. Torre, L.A., Trabert, B., DeSantis, C.E., Miller, K.D., Samimi, G., Runowicz, C.D., et al. Ovarian cancer statistics, 2018. CA: a cancer journal for clinicians 68, 284-296 (2018).
  2. Bowtell, D.D., Böhm, S., Ahmed, A.A., Aspuria, P.-J., Bast Jr, R.C., Beral, V., et al. Rethinking ovarian cancer II: reducing mortality from high-grade serous ovarian cancer. Nature reviews Cancer 15, 668-679 (2015).
  3. Audeh, M.W., Carmichael, J., Penson, R.T., Friedlander, M., Powell, B., Bell-McGuinn, K.M., et al. Oral poly (ADP-ribose) polymerase inhibitor olaparib in patients with BRCA1 or BRCA2 mutations and recurrent ovarian cancer: a proof-of-concept trial. The lancet 376, 245-251 (2010).
  4. Khot, A., Brajanovski, N., Cameron, D.P., Hein, N., Maclachlan, K.H., Sanij, E., et al. First-in-human RNA polymerase I transcription inhibitor CX-5461 in patients with advanced hematologic cancers: Results of a phase I dose-escalation study. Cancer discovery 9, 1036-1049 (2019).
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  6. Muller, M.T., Pfund, W.P., Mehta, V.B., Trask, D.K. Eukaryotic type I topoisomerase is enriched in the nucleolus and catalytically active on ribosomal DNA. EMBO J 4, 1237-1243 (1985).
  7. Armstrong, D.K. Topotecan dosing guidelines in ovarian cancer: reduction and management of hematologic toxicity. The Oncologist 9, 33-42 (2004).
  8. Bruno, P.M., Lu, M., Dennis, K.A., Inam, H., Moore, C.J., Sheehe, J., et al. The primary mechanism of cytotoxicity of the chemotherapeutic agent CX-5461 is topoisomerase II poisoning. Proc Natl Acad Sci U S A 117, 4053-4060 (2020).
  9. Olivieri, M., Cho, T., Alvarez-Quilon, A., Li, K., Schellenberg, M.J., Zimmermann, M., et al. A Genetic Map of the Response to DNA Damage in Human Cells. Cell 182, 481-496 e421 (2020).
  10. Pipier, A., Bossaert, M., Riou, J.F., Noirot, C., Nguyễn, L., Serre, R.F., et al. Transcription-associated topoisomerase activities control DNA-breaks production by G-quadruplex ligands. bioRxiv (2020).

Keefe Chan

Research Officer, Peter MacCallum Cancer Centre

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