Poly-(ADP-ribose) polymerase (PARP) inhibitors (PARPis) selectively kill BRCA1/2-deficient cells, but their efficacy in BRCA-deficient patients is limited by drug resistance. Here, we used derived cell lines and cells from patients to investigate how to overcome PARPi resistance. We found that the functions of BRCA1 in homologous recombination (HR) and replication fork protection are sequentially bypassed during the acquisition of PARPi resistance. Despite the lack of BRCA1, PARPi-resistant cells regain RAD51 loading to DNA double-stranded breaks (DSBs) and stalled replication forks, enabling two distinct mechanisms of PARPi resistance. Compared with BRCA1-proficient cells, PARPi-resistant BRCA1-deficient cells are increasingly dependent on ATR for survival. ATR inhibitors (ATRis) disrupt BRCA1-independent RAD51 loading to DSBs and stalled forks in PARPi-resistant BRCA1-deficient cells, overcoming both resistance mechanisms. In tumor cells derived from patients, ATRis also overcome the bypass of BRCA1/2 in fork protection. Thus, ATR inhibition is a unique strategy to overcome the PARPi resistance of BRCA-deficient cancers.

TGF-β secreted by tumor stroma induces epithelial-to-mesenchymal transition (EMT) in cancer cells, a reversible phenotype linked to cancer progression and drug resistance. However, exposure to stromal signals may also lead to heritable changes in cancer cells, which are poorly understood. We show that epithelial cells failing to undergo proliferation arrest during TGF-β-induced EMT sustain mitotic abnormalities due to failed cytokinesis, resulting in aneuploidy. This genomic instability is associated with the suppression of multiple nuclear envelope proteins implicated in mitotic regulation and is phenocopied by modulating the expression of LaminB1. While TGF-β-induced mitotic defects in proliferating cells are reversible upon its withdrawal, the acquired genomic abnormalities persist, leading to increased tumorigenic phenotypes. In metastatic breast cancer patients, increased mesenchymal marker expression within single circulating tumor cells is correlated with genomic instability. These observations identify a mechanism whereby microenvironment-derived signals trigger heritable genetic changes within cancer cells, contributing to tumor evolution.

Single-cell technologies have described heterogeneity across tissues, but the spatial distribution and forces that drive single-cell phenotypes have not been well defined. Combining single-cell RNA and protein analytics in studying the role of stromal cancer-associated fibroblasts (CAFs) in modulating heterogeneity in pancreatic cancer (pancreatic ductal adenocarcinoma [PDAC]) model systems, we have identified significant single-cell population shifts toward invasive epithelial-to-mesenchymal transition (EMT) and proliferative (PRO) phenotypes linked with mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 3 (STAT3) signaling. Using high-content digital imaging of RNA in situ hybridization in 195 PDAC tumors, we quantified these EMT and PRO subpopulations in 319,626 individual cancer cells that can be classified within the context of distinct tumor gland "units." Tumor gland typing provided an additional layer of intratumoral heterogeneity that was associated with differences in stromal abundance and clinical outcomes. This demonstrates the impact of the stroma in shaping tumor architecture by altering inherent patterns of tumor glands in human PDAC.