Poster Presentation 36th Lorne Cancer Conference 2024

The nucleolar stress response leads to replication stress that can be harnessed to treat PAPRi-resistant ovarian cancer (#211)

Jiachen Xuan 1 , Ruofei Liu 1 , Shalini Sundramurthi Chelliah 1 , Diannita Kwang 1 , Kezia Gitareja 1 , Junqi Pan 1 , Kaylene Simpson 2 , Elizabeth Christie 2 , Clare Scott 3 , Keefe Chan 2 , Rick Pearson 2 , Jian Kang 1 , Elaine Sanij 1 2
  1. St Vincent's Institute Medical Research, Fitzroy, VIC, Australia
  2. Research Division, Peter MacCallum Cancer Institute, Melbourne, VIC, Australia
  3. The Walter and Eliza Hall Institute, Parkville, VIC, Australia

The nucleoli, the site of RNA polymerase I (Pol I) transcription of ribosomal RNA genes (rRNA) and ribosome subunit assembly, coordinate cellular response to various stresses. Damage at rRNA genes and perturbations in ribosome biogenesis trigger a nucleolar stress response, leading to inhibition of key processes in the nucleus, including DNA replication. Indeed, we have shown that the inhibitor of Pol I transcription CX-5461 induces nucleolar DNA damage response (nDDR) and DNA replication stress at distinct genomic region1.

 

CX-5461 has demonstrated therapeutic benefits in various preclinical cancer models, including ovarian cancer, and in Phase I clinical trials2-8. We have shown that CX-5461-mediated replication stress is linked to replication fork degradation and that it overcomes the protection of stalled replication fork, a mechanism of acquired resistance to chemotherapy and poly-ADP ribose polymerase inhibitors (PARPi) in ovarian cancer. Therefore, further investigation of the mechanism by which nDDR lead to replication stress can uncover a new class of cancer therapeutics by leveraging the nucleolar stress response to maximize replication stress in cancer cells.

 

Here, we identify that CX-5461 treatment leads to recruitment of topoisomerase 2A (TOP2A) to stalled replication forks in high grade serous ovarian cancer (HGSOC) cells. In an alternative approach of activating nDDR, the induction of DNA damage at the nucleoli using inducible-Cas9 and sgRNAs targeting rRNA gene sequences, also induces global replication stress associated with TOP2A trapping. We further perform a boutique CRISPR-Cas9 synthetic lethal screen of CX-5461 and DNA repair genes and identify a unique synthetic lethal interaction with RAD54L. We demonstrate that RAD54L binds to stalled replication forks in response to CX-5461 and that depletion of RAD54L further destabilizes replication forks and enhances nucleolar and global DNA damage, supporting a protective role of RAD54L in resolving CX-5461-mediated replication stress. Lastly, we demonstrate that CX-5461 synergizes with ATR inhibitors in inhibiting PARPi-resistant patient-derived HGSOC cells in vitro and ID8 ovarian tumour growth in vivo via enhancing replication stress. Taken together, our study uncovers novel mechanisms by which nucleolar stress causes replication stress that can be harnessed for cancer therapy with promising potential for treating PARPi- and chemo-resistant ovarian cancer.

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