A Novel eIF2α-Phosphorylating Agent HR-19011 Exhibits Cell Growth Inhibitory Effects in K562 Cells through Hri Activation
Authors: Kim Min Jung , Kwak Jinsook , Lee Su In , Yun Hwayoung , Jeong Jee-Yeong 
Published: December 2024

Targeted therapy exploits the unique molecular vulnerabilities of cancer cells and typically alters cellular signaling pathways to inhibit tumor growth and promote cell death. However, despite the successful application of these agents in the treatment of hematologic malignancies, intrinsic and acquired resistance remains a significant challenge.
In this study, we identified a novel squaramide derivative, HR-19011, which exhibits therapeutic effects against hematologic malignancies through the heme-regulated inhibitor (HRI) - integrated stress response (ISR) pathway. The ISR has emerged as an important regulatory target and a new strategy to enhance the efficacy of targeted therapies for hematologic malignancies.
Our findings indicate that HR-19011 demonstrates cell growth inhibition in K562 cells stably transfected with non-targeting control shRNA, with an IC50 value of 3.91 μM. In contrast, in K562 cells stably transfected with HRI-specific shRNA (K562/shHRI), the IC50 value of HR-19011 increased to 8.03 μM, suggesting that the cell growth inhibition by this compound is at least partially mediated through HRI. Additionally, the induction of ATF4 and CHOP, the dephosphorylation of S6K and 4EBP1, and the G1 cell cycle arrest were almost completely restored in K562/shHRI cells. We also demonstrated that the effectors of the ISR, ATF4 and CHOP, are important regulatory factors in the cell growth inhibition caused by HR-19011 using siRNA. The ISR-inducing effect of HR-19011 was observed in various types of leukemia cells, including K562, showing its broad therapeutic potential.
RNA sequencing analysis revealed that HR-19011 treatment strongly activates the EIF2AK1 (the gene name for HRI) response to heme deprivation and induces the ATF4-ISR in cancer cells. These findings are consistent with our experimental results. Furthermore, in vivo toxicity tests were performed in Balb/c mice, which received intraperitoneal injections of HR-19011 for one week, followed by an additional week of observation, with no significant tissue damage, weight loss, or mortality noted. In in vivo assessments using tumor-inducing mouse models, treatment with HR-19011 demonstrated remarkable anti-tumor activity.
In summary, this study provides compelling evidence that HR-19011 induces phosphorylation of eIF2α through the HRI pathway and triggers ISR, leading to effective anti-tumor activity. Targeting the HRI-eIF2α-ATF4 pathway shows significant potential as a therapeutic strategy, and further advancements in this area could greatly impact cancer treatment. We have developed a candidate lead compound with improved properties and enhanced efficacy from our active substance, HR-19011. The in vivo efficacy of this compound is expected to be completed by the first quarter of 2025, and we plan to initiate preclinical studies with it as a candidate compound in the first half of 2025.
Key words : Heme-regulated inhibitor, eIF2α phosphorylation, ATF4, Integrated stress response, HR-19011