Poster Presentation 45th Lorne Genome Conference 2024

New method development to identify and characterise novel breast and ovarian cancer treatment     (#108)

Shiella Amelia Soetomo 1 2 , Michael Sharp 1 2 , Wayne Crismani 1 2
  1. St Vincent's Institute, Melbourne, VIC, Australia
  2. The University of Melbourne, Melbourne, VIC, Australia

BRCA1/2-mutated breast and ovarian cancer can be treated with effective poly-ADP ribose polymerase (PARP) inhibitors because of synthetic lethality between PARP and BRCA1/2, leading to precision treatment for BRCA1/2-mutated cancer1. We are going to find new drug targets using synthetic lethality as alternative therapies for PARP inhibitors. Therefore, we are developing a suitable screening method to identify and characterise the new drug targets for the specific killing of BRCA1/2-mutated cancer. 

One method that is used to observe synthetic lethality is sulforhodamine (SRB) assay. This study started by using this traditional cytotoxicity assay to determine the respective sensitivities of SUM149.PT.BRCA1-/- and SUM149.A22.BRCA1+/- cells to the PARP inhibitor. The two genotypes were grown separately, treated with PARP inhibitor, coloured with SRB, and observed using a microplate reader2. The results showed that SUM149.PT.BRCA1-/- is more sensitive to the PARP inhibitor than SUM149.A22.BRCA1+/-, demonstrating synthetic lethality between BRCA1 and PARP. 

On the other hand, we have successfully developed a two-colour competitive growth assay that can assess synthetic lethality with higher sensitivity. Both SUM149.PT.BRCA1-/- and SUM149.A22.BRCA1+/- cells that express different coloured transgenes were mixed, exposed to PARP inhibitor, and quantified using flow cytometry. This assay generated great results, with the outgrowth of the SUM149.A22.BRCA1+/- cells due to the drug targeting the SUM149.PT.BRCA1-/- cells. Additional to cell survival, fold-change of area under the curve (AUC) SUM149.PT.BRCA1-/- to the AUC SUM149.A22.BRCA1+/- was calculated to determine method sensitivity. The fold-change of the competitive growth assay is 2.4 times higher than that in SRB assay, indicating that the new assay has higher sensitivity than SRB assay. 

In conclusion, this competitive growth assay is more sensitive and more representative of what happens in human body. More importantly, this assay can be used to help discover and characterise new targeted breast and ovarian cancer treatments.

  1. Neiger, H. E., Siegler, E. L., Shi, Y. Breast cancer predisposition genes and synthetic lethality. (2021). International Journal of Molecular Sciences, 22(11), 5614. https://doi.org/10.3390/ijms22115614
  2. Vichai, V., Kirtikara, K. (2006). Sulforhodamine B colorimetric assay for cytotoxicity screening. Nature Protocols, 1, 1112–1116. https://doi.org/10.1038/nprot.2006.179