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[Preprint]. 2023 Aug 28:2023.03.20.23287465. Originally published 2023 Mar 21. [Version 2] doi: 10.1101/2023.03.20.23287465

Figure 2. High BRCA1 Δ11q/Δ11 isoform expression observed in PARPi-resistant PDX models.

Figure 2.

(A) Schematic for design of quantitative two-step PCR (qRT-PCR) assays. (B) Relative total BRCA1 expression for each PDX (mean ± SD). Δ11q-high cell line MDA-MB-231 (11) was included as a technical control on each qRT-PCR plate, while UWB1.289 and COV362 were included as additional cell line controls. PDX #36 was included as a Δ11q-high PDX control. (C) Relative Δ11 and Δ11q expression for each PDX (mean ± SD). PDX #56PP had the highest Δ11 expression relative to other PDX (P=0.0079 compared to matched PDX #56), while PDX #049 and #264 had the highest Δ11q levels relative to other PDX (classifications in Supp. Tables 3 and 4). (D) Δ11q 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). (E) RNA sequencing results for a subset of models with either high Δ11q (cell line COV362, PDX #049), high Δ11 (PDX #56PP) or low Δ11/Δ11q (#206) and presented as sashimi plots. BRCA1 exon regions are displayed at bottom of plot, and sequencing read coverage across each exon is represented as a histogram above. The lines connecting exons represent splicing events detected, and the numbers indicate the number of reads assigned to a given event. The blue arrow indicates the exon 11p region, which is retained in the Δ11q isoform. (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 Δ11/Δ11q 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 Δ11q expression, UWB1.289 (U) and COV362 (C) cells with exon 11 variants and Δ11/Δ11q expression. Statistical comparisons were made using an unpaired t-test with Welch’s correction.