Abstract 1613: Dimerization-induced activation of the integrated stress response kinase PERK by an investigational small molecule modulator, DP-9024
Authors: Al-Ani Gada , Rudeen Aaron J. , Groer Qi , Elliott Kristin M. , Kearney Patrick C. , Zwicker Jeffery D. , Ahn Yu Mi , Bulfer Stacie L. , Heiniger Cale L. , Hood Molly M. , Javid Salim , Large Joshua W. , Petty Max D. , Stoltz Kristen L. , Bourdonnec Bertrand Le , Smith Bryan D. , Flynn Daniel L. 
Published: April 2023

Background: The Integrated Stress Response (ISR) is a major adaptive stress response pathway in cancers. The ISR kinase family member PERK controls one of the three arms of the Unfolded Protein Response (UPR). The UPR is considered an Achilles’ heel in B-cell cancers. Myelomas and B-cell lymphomas are dependent on a well-balanced UPR pathway to cope with the high demand for protein folding and their secretory nature. Given the double-edge sword nature of the UPR, the activation of PERK and downstream pathway can have cytoprotective or cytotoxic effects. In B-cell cancers the UPR is at close to maximum cytoprotective capacity, such that further pharmacological stimulation of PERK drives a cytotoxic outcome leveraged to induce antitumoral effects. Methods: Recombinant WT and mutant PERK constructs were assayed in the presence of DP-9024. Structures of compound-bound PERK were determined by X-ray crystallography. Kinome profiling was determined using enzymatic and cellular assays. Cellular modulation of the ISR/UPR pathway (phospho-GCN2, PERK, ATF4, CHOP) or the apoptosis pathway (cleaved-PARP, cleaved-Caspase 3/7) was measured by Western blot or ELISA. The level of DP-9024-induced PERK activation was determined using a cellular nanoBRET dimerization assay utilizing WT and mutant PERK constructs. Results: DP-9024 was designed as a selective and potent modulator of PERK and GCN2. DP-9024 was found to upregulate the ISR/UPR pathway (ATF4, CHOP). The mechanism by which DP-9024 induced the UPR pathway was found to be through dimerization-dependent activation of PERK. Utilizing recombinant biophysical and cellular assays of WT and mutant PERK constructs, we found that DP-9024 directly binds to a switch control site in the kinase domain of PERK that governs dimerization and that the binding of the compound to one monomer was sufficient to induce dimerization-mediated activation of the unoccupied monomer. This paradoxical stimulation of the unbound PERK monomer is reminiscent of the phenomenon observed with some BRAF inhibitors.1 X-ray crystallography studies revealed that PERK crystalizes as a dimer with both monomers bound to compound, due to the high concentration of compound used during crystallization. DP-9024-mediated PERK dimerization and transactivation led to the activation of downstream pathways (ATF4, CHOP), apoptotic pathway (Caspase 3/7, PARP1), and growth arrest in cell lines with high levels of endoplasmic reticulum (ER) stress such as multiple myeloma and B-cell lymphoma. Conclusions: Paradoxical stimulation of the ISR family member kinase PERK, through direct binding and dimerization by DP-9024, led to unresolved ER stress that can potentially be leveraged as a novel mechanism to induce growth arrest in UPR vulnerable cancers, including myelomas and B-cell lymphomas. References: 1. Poulikakos et al. 2010. Nature 464:427-30

Citation Format: Gada Al-Ani, Aaron J. Rudeen, Qi Groer, Kristin M. Elliott, Patrick C. Kearney, Jeffery D. Zwicker, Yu Mi Ahn, Stacie L. Bulfer, Cale L. Heiniger, Molly M. Hood, Salim Javid, Joshua W. Large, Max D. Petty, Kristen L. Stoltz, Bertrand Le Bourdonnec, Bryan D. Smith, Daniel L. Flynn. Dimerization-induced activation of the integrated stress response kinase PERK by an investigational small molecule modulator, DP-9024 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1613.