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

Cytoplasmic Restriction of Mutated SOD1 Impairs the DNA Repair Process in Spinal Cord Neurons

“…Unfortunately, primary cultured SOD1G93A genotype neurons are not the most suitable samples to study the protective function of PDI on DNA damage, because it is impossible to artificially induce DNA damage and overexpress PDI evenly in all neurons. In the glioblastoma multiform tumor model, PDI is directly associated with DNA repair process by maintaining a specific level of DNA repair enzymes [66,67]. Thus, increasing PDI that activates the SOD1G93A protein transport into the nucleus could enhance the nucleic translocation of DNA repair enzymes by reducing ER stress.…”

Endoplasmic Reticulum stress-dependent expression of ERO1L promotes aerobic glycolysis in Pancreatic Cancer

“…Endoplasmic reticulum oxidoreductase 1 alpha (ERO1L) is a flavin adenine nucleotide-containing enzyme that exists in the ER and favors disulfide bond formation of secreted proteins and cell surface proteins by accepting electrons from reduced protein disulfide isomerase (PDI) and passing them on to molecular oxygen 8 - 11 . Emerging evidence has demonstrated that ERO1L is highly expressed in various types of human cancers, and that high expression is associated with worse clinical outcome 12 - 15 .…”

Activation of the Unfolded Protein Response via Inhibition of Protein Disulfide Isomerase Decreases the Capacity for DNA Repair to Sensitize Glioblastoma to Radiotherapy

“…We hypothesized that the dependence of tumor cells on PDI activity provides a rationale for its therapeutic targeting in GBM, irrespective of oncogenic mutation status. We recently described propynoic acid carbamoyl methyl amides (PACMA) as irreversible inhibitors of PDI, a first-in-class, safe, and efficacious targeted anticancer agents (24), as well as second-generation PDI inhibitors BAP2 (25). Because the prolyl 4-hydroxylase subunit beta (P4HB) gene family that encodes PDI proteins, comprises 21 genes, varying in size, expression, localization, and enzymatic function (26), of which PDIA1 is believed to be the most highly expressed in GBM (19) and the primary target of our current small molecule agents, it is essential that we demonstrate the selective dependence of GBM cells on PDI to validate it as a therapeutic target.…”