CRISPR Screening Identifies WEE1 as a Combination Target for Standard Chemotherapy in Malignant Pleural Mesothelioma.

Malignant pleural mesothelioma (MPM) is an aggressive cancer with dismal prognosis, largely due to poor response rates to and rapid relapse after first-line pemetrexed (MTA)/cisplatin chemotherapy. A better understanding of the molecular mechanisms underlying chemotherapy sensitivity and duration represents a significant but still unmet clinical need. In this study, we reported on a kinome CRISPR/Cas9 knockout screen that identified several G2-M checkpoint kinases, including WEE1, whose loss of function sensitizes MPM cells to standard chemotherapy. We further showed that deregulation of the G2-M checkpoint contributes to chemotherapy resistance, and that WEE1 inhibition synergizes with cisplatin/MTA, leading to enhanced MPM cell death in vitro and potent antitumor effects in vivo Mechanistically, WEE1 blockage overrides chemotherapy-induced G2-M cell-cycle arrest and promotes premature mitotic entry, which causes DNA damage accumulation and ultimately apoptosis. Our results suggest a new therapeutic combination for MPM, and support the application of CRISPR/Cas9-based functional genomics in identifying novel therapeutic targets to potentiate existing cancer therapies.

Tumor Initiation Capacity and Therapy Resistance Are Differential Features of EMT-Related Subpopulations in the NSCLC Cell Line A549.

Cell lines are essential tools to standardize and compare experimental findings in basic and translational cancer research. The current dogma states that cancer stem cells feature an increased tumor initiation capacity and are also chemoresistant. Here, we identified and comprehensively characterized three morphologically distinct cellular subtypes in the non-small cell lung cancer cell line A549 and challenge the current cancer stem cell dogma. Subtype-specific cellular morphology is maintained during short-term culturing, resulting in the formation of holoclonal, meroclonal, and paraclonal colonies. A549 holoclone cells were characterized by an epithelial and stem-like phenotype, paraclone cells featured a mesenchymal phenotype, whereas meroclone cells were phenotypically intermediate. Cell-surface marker expression of subpopulations changed over time, indicating an active epithelial-to-mesenchymal transition (EMT), in vitro and in vivo. EMT has been associated with the overexpression of the immunomodulators PD-L1 and PD-L2, which were 37- and 235-fold overexpressed in para- versus holoclone cells, respectively. We found that DNA methylation is involved in epigenetic regulation of marker expression. Holoclone cells were extremely sensitive to cisplatin and radiotherapy in vitro, whereas paraclone cells were highly resistant. However, inhibition of the receptor tyrosine kinase AXL, whose expression is associated with an EMT, specifically targeted the otherwise highly resistant paraclone cells. Xenograft tumor formation capacity was 24- and 269-fold higher in holo- than mero- and paraclone cells, respectively. Our results show that A549 subpopulations might serve as a unique system to explore the network of stemness, cellular plasticity, tumor initiation capacity, invasive and metastatic potential, and chemo/radiotherapy resistance.