Abstract 6648: Novel integrated stress response (ISR) kinase inhibitors have activity in multiple cancer cells
Authors: Aurelien Laguerre, Martin Smrcina, Ivy Nai-Jung Hung, Devin K. Porter, Nathan G. Hedrick, Dmitri Rozanov, Mark R. Hansen, Vikas K. Goel
Journal: Cancer Research (2025)

Abstract The integrated stress response (ISR) is an adaptive cellular program that facilitates survival and proliferation under conditions of cellular stress and can be appropriated to contribute to the tumorigenesis of multiple cancers. ISR is activated in response to various cues in the tumor microenvironment and cell-intrinsic factors but is ultimately regulated by four core serine/threonine kinases: GCN2 (activated by amino acid deprivation), HRI (in response to heme deficiency), PERK (unfolded protein response stress), and PKR (dsRNA stress response of infection and oxidative stress). These four ISR kinases play a central role in cancer cell equilibrium, allowing them to proliferate by adapting to adverse conditions, immune evasion, and metastasis. All ISR kinases phosphorylate a common downstream effector, eIF2a, at Ser51. peIF2aS51 shuts down cap-dependent translation and results in the up-regulation of ATF4, a transcription factor that controls the transcription of genes essential for cancer cells to adapt to stress and nutrient deprivation. Due to the observation that adaptive compensation and rewiring in cancer cell pathways can occur in response to inhibition of single signaling nodes, it has been suggested that simultaneous inhibition of multiple ISR kinases may be required to improve anti-cancer response or counteract drug resistance. We developed an initial high-throughput ATF4 immunofluorescence (IF) assay to screen chemical matters in low- to no-glutamine culture conditions. We identified three potential subseries of compounds of the type II kinase inhibitor class. Structure modeling and docking suggested that these subseries differ in inhibitory profiles against four ISR kinases, and we decided to focus on two of these subseries to develop potent ISR inhibitors further. Multiple hits (ZF125, ZF163, ZF284, ZF302) demonstrated low nM potency in the cellular target engagement assay (ATF4 IF) and biochemical kinase assay against three ISR kinases. RNA transcriptomic analysis confirmed that ZF-ISR inhibitors downregulate several ISR kinase target genes, such as DDIT3 and SLC1A5, ATF4, CEBPb, and TRIB3. Our lead molecule has also demonstrated high selectivity in a select kinome screen (40 kinases) at 1uM, except for ISR kinases. Zafren’s lead ISR (ZF-ISR) inhibitors have shown a growth inhibitory effect in multiple cancer cells with 10mU/ml of ASNase of ASNase. Interestingly, most of these cancer cell lines harbor RAS/RAF/MPAK pathway mutations. DepMAP analysis revealed other potential gene signatures as combination strategies with ZF-ISR inhibitors. As determined by multiple ADME assays, ZF-ISR inhibitors have also shown favorable drug-like property profiles. In-vivo tumor growth inhibition studies are in progress. Citation Format: Aurelien Laguerre, Martin Smrcina, Ivy Nai-Jung Hung, Devin K. Porter, Nathan G. Hedrick, Dmitri Rozanov, Mark R. Hansen, Vikas K. Goel. Novel integrated stress response (ISR) kinase inhibitors have activity in multiple cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6648.