Poster Presentation 35th Lorne Cancer Conference 2023

BRCA1 secondary splice-site mutations drive exon-skipping and PARP inhibitor resistance (#261)

Ksenija Nesic 1 2 , John J Krais 3 , Cassandra J Vandenberg 1 2 , Yifan Wang 3 , Pooja Patel 3 , Tanya Kwan 4 , Lizzie Lieshke 1 2 , Gwo-Yaw Ho 5 , Holly Barker 1 2 , Justin Bedo 1 2 , Tony Papenfuss 1 2 , Silvia Casadei 6 , Marc Radke 6 , Andrew Farrell 1 2 , Franziska Geissler 1 2 , Fan Zhang 7 , Alexander Dobrovic 7 , Inger Olesen 8 , Tom Harding 4 , Kevin Lin 4 , Elizabeth M Swisher 6 , Olga Kondrashova 9 , Clare L Scott 1 2 10 11 , Neil Johnson 3 , Matthew J Wakefield 1 2
  1. Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia
  2. University of Melbourne, Parkville, VIC, Australia
  3. Fox Chase Cancer Center, Philadelphia, Pennsylvania, United States of America
  4. Clovis Oncology, Boulder, CO, United States of America
  5. Monash University, Clayton, VIC, Australia
  6. University of Washington, Seattle, WA, United States of America
  7. University of Melbourne Department of Surgery, Austin Health, Heidelberg, Victoria, Australia
  8. The Andrew Love Cancer Centre, Barwon Health, Geelong, Victoria, Australia
  9. QIMR Berghofer Medical Research Institute, Brisbane, QLD, Australia
  10. Royal Women’s Hospital, Melbourne, VIC, Australia
  11. Peter MacCallum Cancer Center, Melbourne, VIC, Australia

BRCA1/2 secondary mutations, that restore homologous recombination DNA repair, are a well-documented mechanism of acquired PARP inhibitor (PARPi) resistance in ovarian carcinomas (OC). While certain BRCA1 splice isoforms (D11 and D11q) can contribute to PARPi resistance by splicing-out, or “skipping” deleterious exon 11 mutations in laboratory models, the true clinical impacts and underlying drivers of BRCA1 exon skipping have not yet been established. 

 

This study analysed nine OC and breast cancer Patient Derived Xenografts (PDX) with BRCA1 exon 11 mutations, where alternative isoform expression correlated with platinum and PARPi resistance in vivo, and prior clinical PARPi exposure. This included matched PDX from the same patient before and after multiple lines of chemotherapy/PARPi (#56 and #56PP respectively). We found two PDX (#56PP and #1049) had secondary BRCA1 splice site mutations (SSM’s), predicted in silico to drive alternative isoform expression. Splicing predictions were confirmed using qPCR, RNA sequencing, western blot and minigene modelling of BRCA1. These mutations appeared to be acquired following PARPi in both cases. A PARPi-resistant HGSOC cell line COV362 also harboured an exon 11 mutation with an SSM that drives high D11q expression. siRNA knockdown of D11q in this model sensitized cells to PARPi, suggesting D11q is the main driver of PARPi resistance.

 

We went on to demonstrate that these SSM’s were enriched in post-PARPi (Rucaparib) ovarian cancer patient cohorts in the ARIEL2 and ARIEL4 clinical trials. SSM’s were only found in 1% (1/115) of patients with BRCA1 exon 11 mutations prior to PARPi (but post-platinum), and this increased to 8% of patients following PARPi treatment. BRCA2 SSM’s were also found in BRCA2-mutant cases, suggesting this PARPi resistance mechanism holds relevance in the clinic beyond BRCA1.

                                                                                                            

Our findings are important because they demonstrate that splice site mutations are drivers of alternative BRCA1 splicing and resistance to the most important new treatment in ovarian cancer, PARPi, and therefore should be monitored for in the clinic, along with standard secondary mutations.