Extended Data Fig. 9. Brca2-deficient tumors acquire PARPi resistance without restoration of RAD51 foci formation.
(a) Schematic depicting the conditional BRCA2 allele of the KB2P (K14CRE;Brca2f/f;p53f/f) spontaneous tumor model. (b) Outline of the PARPi intervention study. A spontaneous BRCA2-/p53-deficient tumor was generated and re-transplated into syngeneic wild-type mice. When the tumors reached 200 mm3, they were treated either with vehicle or PARPi AZD2461. (c) PARPi response curve of the KB2P tumor (relative tumor volume (rtv) vs. days). The treatment for 28 consecutive days was started when the tumor reached 200 mm3 (rtv=100%). In response to the treatment the tumor shrank but eventually grew back. When it reached 100% rtv the treatment was repeated (as indicated with red arrows) for another 28 days. This regime was continued until the tumor became resistant to PARPi (black arrow). (d) The stability of acquired resistance of the KB2P tumor was confirmed by re-transplanting matched naive and resistant tumors and treating animals either with vehicle or AZD2461 (only one 28-day cycle). Kaplan-Meier survival curve indicates that resistant tumors did not respond to the AZD2461 treatment, while naive tumors exhibited high sensitivity, indicative of a stable genetic mechanism of resistance. (e, f) IR-induced RAD51 foci were detected by immunofluorescence in the KB2P donor: RAD51 foci formation was undetectable in naive and resistant tumors, suggesting that HR restoration is not an underlying mechanism of PARPi resistance. Spontaneous tumors from K14 Cre; p53f/f mice treated with IR were used as positive control for RAD51 foci. Unirradiated K14 Cre; p53f/f cells were used as a negative control. (g) Replication fork progression rates measured by tract lengths in µm of CldU (red) and IdU (green) in PARPi-naive or PARPi-resistant tumors. Numbers in red indicate the mean and standard deviation for each sample. 125 replication forks were analyzed for each condition. (h) Western blot analysis for PTIP and MRE11 levels in PARPi-naive or PARPi-resistant tumors. Tubulin is used as loading control.