ATF3 drives synergy between protein disulfide isomerase (PDI) inhibitors and epigenetic cancer therapy

The clinical potential of epigenetic cancer therapy has not been fully realized. Histone deacetylase inhibitors (HDACi), for example, only benefit a minor fraction of all cancer patients despite thousands of preclinical studies demonstrating their antitumor effects. In this study we show that a new structural class of protein disulfide isomerase (PDI) that was discovered by our lab dramatically enhances the antitumor effects of HDACi and select other epigenetic modifiers. We set out to identify synergistic interactions between our lead PDI inhibitor, E64FC26, and FDA-approved drugs in the NCI Approved Oncology Drug Set VIII of ~130 marketed drugs. We identified robust and previously uncharacterized synergy with all four HDACi in the set (i.e., panobinostat, romidepsin, vorinostat, and belinostat) in a broad range of solid and heme-malignancies. For example, E64FC26 potentiated panobinostat-induced cytotoxicity by 240-fold in PANC-1 pancreatic cancer cells and by 20-fold in T98G glioblastoma cells. Synergy with HDACi involved an apoptotic response measured by the activation of caspase 3, 8, 9 and the cleavage of the caspase 3 substrate PARP. PDI mediates proper protein folding, and western blotting and confocal microscopy confirmed the accumulation of misfolded poly-ubiquitinated protein aggregates in treated cells, which we showed was a critical component underlying the synergistic effects of the combination. To further understand the mechanism, we profiled the transcriptomes of combination treated cells using comparative RNA-Seq. These experiments revealed a convergence on ATF3 and DDIT3, two candidates in the ER stress pathway. RNA-Seq results were confirmed by qPCR and western blotting analysis in a range of tumor types. ATF3 played an apparent dominant role in driving the synergy between PDI and HDAC inhibitors, as knockdown of ATF3 completely ameliorated synergy between the two drug classes. We further clarified this molecular mechanism to show that PDI inhibition induces an ER stress response that leads to the induction of ATF3, which is further potentiated in the presence of HDACi, leading to the synergistic effects of the drug combination. These effects were not limited to HDACi, as similar results were observed in combinations with other epigenetic targeted therapies. We went on to characterize the efficacy and tolerability of PDI and HDAC inhibitor combinations in multiple mouse models, including pancreatic and glioblastoma xenograft models as well as multiple myeloma xenotransplant models. In summary, this study presents the potent antitumor combination of PDI and HDAC inhibitors and demonstrates a key mechanistic role of the ATF3 transcription factor. The combination of our developmental PDI inhibitor and HDACi offers a new dual therapeutic strategy and the opportunity to amplify and rescue previously unrealized activity of epigenetic therapy in oncology. Citation Format: Ravyn M. Duncan, Leticia Reyes, Katelyn Moats, Reeder M. Robinson, Holly A. Stessman, Nathan G. Dolloff. ATF3 drives synergy between protein disulfide isomerase (PDI) inhibitors and epigenetic cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3840.