Abstract 610: ClpP-dependent and -independent activation of HRI kinase by small molecules

Abstract HRI was initially identified as a kinase essential for maintaining heme-globin balance within red blood cells as well as for controlling the ISR in response to oxidative stress. HRI also responds to a broad range of stresses such as osmotic stress, heat shock, proteasome inhibition. The recently discovered unexpected functions of HRI include innate immunity, translational control of immune evasion in cancer by upregulating PD-L1, proteostasis, mitochondrial stress, inhibition of histone H3 lysine 27 (H3K27) demethylase (KDM6A) and iron deficiency. Importantly, recent evaluation of patient data uncovered high expression of HRI mRNA in a subset of epithelial tumors versus normal tissues. Elevated expression of HRI protein in these tumor cells lead to cell death when BIRC6 ubiquitin complex is inhibited, which mediates degradation of HRI and is required for the survival of the tumors. These further broaden the importance of this member of the eIF2α kinase family as a cancer therapeutic strategy. Dordaviprone (ONC201), an imipridone small molecule, binds to and activates mitochondrial protease ClpP leading to integrated stress response (ISR) and ATF4 transcription factor activation. PG3, a prodigiosin analog, induces ATF4 and pro-apoptotic PUMA. Our data indicate that PG3 activates ATF4 through ISR via eIF2α kinase HRI. ALAS1 (5'-aminolevulinate synthase 1) catalyzes the first rate-limiting step in heme (Iron-protoporphyrin) biosynthesis. ONC201 treatment leads to potent downregulation and inhibition of ALAS1, indicating that ONC201 inhibits heme biosynthesis. It is well known that reduced heme results in activation of the HRI kinase. We show that an inhibitor of heme biosynthesis or knockdown of ALAS1 results in HRI activation, while silencing of HRI or knockout of HRI gene potently inhibit the eIF2α phosphorylation and upregulation of ATF4, CHOP and PUMA by PG3 and imipridones. Knockdown of ClpP rescues ONC201-induced downregulation of ALAS1 which blocks ONC201-induced upregulation of CHOP. Also, silencing of ClpP significantly reduced PARP cleavage in HCT116 p53−/- and MDA-MB-468 cancer cells. Our studies identify a novel link between ClpP activation induced by ONC201 treatment and ATF4 upregulation, via the ClpP/ALAS1/HRI/ATF4 pathway. However, PG3 treatment did not lead to degradation of ALAS1, indicating that PG3 does not activate ClpP. PG3 potently induced cell apoptosis through ISR via HRI/ATF4/PUMA pathway independent of ClpP. We are further investigating the targets of PG3 and the signaling pathway that leads to PG3-induced activation of HRI. Our results suggest that different small molecule inducers of the ISR such as ONC201 and PG3 can achieve an anti-tumor effect through different pathways converging on kinase HRI ultimately leading to ATF4 activation and tumor cell death. Citation Format: Xiaobing Tian, Praveen Srinivasan, Wafik S. El-Deiry. ClpP-dependent and -independent activation of HRI kinase by small molecules [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 610.