Abstract 29P
Background
Maintenance PARP inhibitors (PARPi) significantly extend overall survival in platinum sensitive, BRCA mutant high grade serous ovarian cancer (HGSOC). However, optimal duration and timing of PARPi treatment is unclear. Resistance to PARPi and subsequent lines of therapy is an emerging clinical issue. BRCA reversion mutations result in homologous recombination repair (HRR) restoration, and are the most frequently observed PARPi resistance mechanism (10-15% of patients). We explored the evolutionary dynamics between PARPi sensitive and resistant HGSOC populations, using a BRCA1 mutant murine HGSOC cell line (60577) and a daughter cell line (60577R3) generated from a 60577 murine tumour harvested after in vivo carboplatin exposure.
Methods
PARPi sensitivity was measured by cell viability assays. Population growth dynamics between 60577 and 60577R3 cells were assessed by co-culture experiments using resource deprived culture media (low serum or glucose) to expose fitness deficits. Cell populations were quantified with the Sartorius Incucyte™ Live Cell Analysis System and flow cytometry. BRCA1 status was assessed by quantitative polymerase chain reaction (qPCR) and next generation sequencing (NGS). Functional evaluation of HRR was conducted with immunofluorescence (IF) microscopy.
Results
Cell viability assays confirmed 60577 cells were PARPi sensitive and 60577R3 cells were PARPi resistant. Co-culture demonstrated competition between cell populations, with PARPi resistant cells outgrowing sensitive cells, regardless of starting ratio of sensitive:resistant cells, and resource restraint modality. qPCR indicated BRCA1 expression in resistant cells, and was confirmed by NGS. Functional restoration of HRR in PARPi resistant cells was confirmed via IF, with fewer mitotic segregation errors seen, and RAD51 foci formation observed in response to double strand breaks.
Conclusions
The mechanistic findings, along with the growth dynamics of PARPi sensitive and resistant cells in co-culture, indicate BRCA1 reversion confers fitness benefit to PARPi-resistant HGSOC subclones. This may have connotations for PARPi maintenance therapy and predicting response to subsequent lines of therapy.
Clinical trial identification
Editorial acknowledgement
Legal entity responsible for the study
The authors.
Funding
Cancer Research UK.
Disclosure
T. Graham: Financial Interests, Personal, Other, TG is named as a coinventor on patent applications that describe a method for TCR sequencing (GB2305655.9), and a method to measure evolutionary dynamics in cancers using DNA methylation (GB2317139.0). TG has received an honorarium from Genentech and consultancy fees from DAiNA therapeutics: Genentech; Financial Interests, Personal, Other: DaiNA Therapeutics. All other authors have declared no conflicts of interest.