The EMT regulator ZEB1 couples S-phase progression to BRCA2-dependent genome maintenance, unveiling a vulnerability in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy characterized by rapid tumor progression towards metastasis and profound therapy resistance, processes frequently driven by epithelial-mesenchymal transition (EMT). The EMT-activating transcription factor ZEB1 is a key orchestrator of these malignant traits; however, its direct therapeutic targeting is challenging. Activation of EMT triggers several escape and resistance mechanisms, beneficial for tumor progression but may come at the expense of yet undiscovered molecular dependencies, as exemplified in the increased sensitivity to ferroptosis. In this study, we specifically investigated such hidden vulnerabilities that emerge upon activation of the ZEB1-driven EMT program in PDAC. To this end, we established isogenic murine PDAC cell line models (KC-M) carrying endogenous KrasG12D mutations and Cdkn2a deletion, and genetically engineered them in vitro to generate wildtype Zeb1-proficient (Z+) or Zeb1-deficient (ZΔ) clones. Genome-wide CRISPR/Cas9 screening in two independent cell lines revealed a context-specific synthetic dependency on the genome maintenance factor Brca2 in Z+ cells exclusively during TGFβ-induced EMT, but not under standard culture conditions. This dependency was absent in ZΔ counterparts undergoing an attenuated EMT. Mechanistically, Zeb1 expression enabled cells to continue cycling and enter S-phase during EMT induction. During S-phase, cells typically experience replication stress, and Brca2 mitigates such stress through fork stabilization and homologous recombination (HR) repair, suggesting a potential protective role in this context. Consequently, loss of Brca2 in TGFβ-treated Z+ cells led to G2/M accumulation, increased post-replicative cell cycle exit, and elevated genomic instability, including increased polyploidy and micronuclei formation. Further investigation of DNA damage response (DDR) factors showed that Zeb1 proficiency was associated with increased numbers of 53bp1 foci per nucleus, independent of Brca2 status, suggesting a preference for non-homologous end joining (NHEJ) related processes to handle replication stress. In contrast, ZΔ cells exhibited robust, Brca2-independent Rad51 foci formation during EMT, potentially mediated by sustained Helq expression, indicating engagement of alternative repair pathways or fork protection mechanisms. Clinically, BRCA2 dependency correlated with high ZEB1 expression and activated TGFβ signaling in human cancer cell lines. Furthermore, high BRCA2 expression was associated with worse survival in the mesenchymal (squamous) subtype of human PDAC, which is characterized by an EMT phenotype and TGFβ activity. These findings uncover a critical interplay between Zeb1-mediated plasticity and the DDR, highlighting Brca2 as a synthetic vulnerability that could be targeted to potentially prevent metastasis and chemoresistance inherent to the mesenchymal phenotype. Exploiting such acquired dependencies offers a novel therapeutic strategy for aggressive PDAC.