Development of image-based high-content screening (HCS) assays for novel cancer therapy

Abstract

Xeroderma pigmentosum group B (XPB), one of the core subunits of the transcription factor TFIIH complex, is required to remove DNA damage via nucleotide excision repair (NER). Inhibiting XPB and blocking NER can sensitize cancer cells to chemotherapy. However, understanding the molecular mechanisms of XPB inhibitors and XPB is limited owing to the lack of research tools. Here, we report a clickable probe, TL-alkyne (TL-alk), that specifically labels XPB with fast kinetics in live cells. Using this probe, we established an image-based high-content screening (HCS) assay to quantify the drug occupancy of XPB at single-cell resolution and automated data analysis. Potential XPB inhibitors that bind to the same active site compete with the probe and reduce the reporter signals. In a screening library containing 1953 Food and Drug Administration (FDA) drugs, pelitinib was identified as a new XPB binder. Pelitinib effectively suppressed XPB ATPase activity and NER, and synergized with platinum-based alkylating compounds to kill cancer cells. Our study not only provides a novel clickable chemical probe for easy and specific labeling of XPB but also underscores the power of bioorthogonal probes in HCS assays. The novel XPB inhibitor discovered will add a new tool compound to study XPB biology and show therapeutic potential in enhancing chemotherapy. Major Histocompatibility Complex Class I (MHC-I) molecules expressed on the surface of nucleated cells play an essential role in boosting T cell-mediated cytotoxicity against cancer by presenting antigens to CD8+ T cells. However, many human cancers, such as melanoma, NSCLC, colorectal, and cervical cancers, have been observed to exhibit MHC-I loss or downregulation. This is considered the primary mechanism that allows cancer cells to evade immune surveillance. Restoring cell surface MHC-I can potentially increase tumor sensitivity to immunotherapy and enhance the effectiveness of combination therapies with immune checkpoint inhibitors (ICIs). To identify MHC-I up-regulators, we developed an Image-based HCS platform. Using this platform, we identified Resminostat, an HDAC inhibitor, as a potential regulator of cell surface MHC-I in both human and mouse melanoma cell lines by stimulating the nuclear factor-kappa B (NF-κB) signaling pathway. Importantly, our methodology is not limited to this specific application, and can be extended to identify other drugs with immunomodulatory effects. This broad applicability significantly enhances the potential impact of our research on the field of immunotherapy.