Inhibition of protein disulfide isomerase in glioblastoma causes marked downregulation of DNA repair and DNA damage response genes

Abstract

Aberrant overexpression of endoplasmic reticulum (ER)-resident oxidoreductase protein disulfide isomerase (PDI) plays an important role in cancer progression. In this study, we demonstrate that PDI promotes glioblastoma (GBM) cell growth and describe a class of allosteric PDI inhibitors that are selective for PDI over other PDI family members. <b>Methods</b>: We performed a phenotypic screening triage campaign of over 20,000 diverse compounds to identify PDI inhibitors cytotoxic to cancer cells. From this screen, <b>BAP2</b> emerged as a lead compound, and we assessed <b>BAP2</b>-PDI interactions with gel filtration, thiol-competition assays, and site-directed mutagenesis studies. To assess selectivity, we compared <b>BAP2</b> activity across several PDI family members in the PDI reductase assay. Finally, we performed <i>in vivo</i> studies with a mouse xenograft model of GBM combining <b>BAP2</b> and the standard of care (temozolomide and radiation), and identified affected gene pathways with nascent RNA sequencing (Bru-seq). <b>Results</b>: <b>BAP2</b> and related analogs are novel PDI inhibitors that selectively inhibit PDIA1 and PDIp. Though <b>BAP2</b> contains a weak Michael acceptor, interaction with PDI relies on Histidine 256 in the b' domain of PDI, suggesting allosteric binding. Furthermore, both <i>in vitro</i> and <i>in vivo</i>, <b>BAP2</b> reduces cell and tumor growth. <b>BAP2</b> alters the transcription of genes involved in the unfolded protein response, ER stress, apoptosis and DNA repair response. <b>Conclusion</b>: These results indicate that <b>BAP2</b> has anti-tumor activity and the suppressive effect on DNA repair gene expression warrants combination with DNA damaging agents to treat GBM.