Fig. 1

Splice-site mutations (SSMs) drive alternative splicing and PARPi resistance in PDX and cell line models of ovarian and breast cancer. A Patient #56 timeline, showing generation of the matched HGSOC PDX #56 (chemo-naïve) and #56PP (post-chemotherapy/PARPi patient). Created with BioRender.com. CR = Complete response; PD = Progressive disease; SD = Stable disease; C6 = cycle 6. B In vivo treatment data for HGSOC PDX #56 (previously published [3]), classified as PARPi responsive (P = 0.005). C HGSOC PDX #56PP was derived from patient #56 following multiple lines of therapy, including PARPi inhibitor, and was refractory to rucaparib (P = 0.375). Mean PDX tumor volume (mm³) ± 95% CI (hashed lines are individual mice) and corresponding Kaplan–Meier survival analysis. Censored events are represented by crosses on Kaplan–Meier plot; n = individual mice. Detailed patient clinical data can be found in Supplementary Table 1. Details of time to harvest, time to progression and Log-Rank test P values for each PDX can be found in Supplementary Table 2. D Relative △11 and △11q expression for each PDX (mean ± SD). PDX #56PP had the highest △11 expression relative to all other PDX (P = 0.0079 compared to matched PDX #56), while PDX #049 and #264 had the highest △11q levels relative to other PDX (classifications in Supplementary Tables 3 and 4). E △11q levels in PARPi responsive PDX models (< 2 prior lines of platinum in patient) were lower than levels in non-responsive PDX models (≥ 2 prior lines of platinum + PARPi) (P = 0.0007). Statistical comparisons of gene expression were made using an unpaired t-test with Welch’s correction. F Lysates from nuclear extracts from 3 independent tumors were probed for BRCA1 expression by immunoblotting. Bands at the anticipated sizes for full length (FL) BRCA1 and the △11/△11q isoforms are marked. Tubulin immunoblotting is included as a loading control. Gels were run simultaneously with cell line lysates included as controls for each gel. MDA-MB-231 [231] cells are a BRCA1 wild-type control with full-length (FL) and △11q expression, UWB1.289 (U) and COV362 (C) cells with exon 11 variants and △11/△11q expression. G Schematic of BRCA1 mini-gene design, and splicing outcomes predicted for each secondary splice site mutation found in PDX and cell line models. H Splicing predictions for each secondary splice-site mutation modelled by the mini-gene were confirmed by immunoblotting for the HA tag. The PDX #56PP deletion was confirmed to drive high ∆11 and potentially also isoform ∆(9,10,11q) expression. COV362 and PDX #049 secondary splice site mutations were confirmed to drive high ∆11q relative to the wild-type (WT) BRCA1 control and the primary deleterious BRCA1 mutation found in PDX #206. I Cells exposed to sgSS (SS) were found to have elevated BRCA1 D11q protein compared to untreated (-) and sgRosa (Ro) cells. J Example image of colony forming assays of UWB1.289 or SUM149 cells exposed to sgSS or sgRosa treated with DMSO control or 1µM Rucaparib. K Quantification of n = 3 colony forming experiments described in part (J). Mean ± SEM plotted; Ns = Not statistically significant; **p < 0.01; using unpaired, two-tailed t-test. L Representative image of RAD51 foci in UWB1.289 or SUM149 cells exposed to sgSS or sgRosa treated with 10 Gy dose of irradiation. M Quantification of nuclei with > 5 RAD51 foci in UWB1.289 (P = 0.11 compared to control) or SUM149 (P = 0.02 compared to control) cells exposed to sgSS or sgRosa and treated with 10 Gy dose of irradiation. Mean ± SEM plotted; Ns = Not statistically significant; *p < 0.05; using unpaired, two-tailed t-test