Abstract 6447: Preclinical efficacy of GCN2 inhibition by KAS-1155 in targeting the integrated stress response in rhabdomyosarcoma

The integrated stress response (ISR) is an crucial adaptive mechanism that enables the cells to survive microenvironment stress, such as nutrient deficiency. General control non-derepressible 2 (GCN2) and its relevant pathway is essential and pivotal for ISR activation, in which the cells undergo amino acid deficiency. GCN2 phosphorylates Eukaryotic Translation Initiation Factor α (eIF2α) and enhances Activating Transcription Factor 4 (ATF4) expression to regulate the genes involved in physiological responses including cellular adaptation and survival. The roles of GCN2 in cancer survival have been studied and suggested as a target of cancer treatment. Rhabdomyosarcoma is one of the most common cancer types in childhood and adolescence, however, there was little progress in the treatment development over the past two decades. This study evaluated the efficacy KAS-1155, a novel GCN2 inhibitor in two representative rhabdomyosarcoma cell lines. Two rhabdomyosarcoma cell lines (RD, RH-30) were treated with KAS-1155 which is a GCN2 inhibitor produced and provided by Korea Research Institute of Chemical Technology (KRICT). RD was cultured in DMEM, transferred to glutamine-free media for 8 hours, and treated with KAS-1155. The RH30 was initially grown in RPMI media, and the cell line was sub-cultured twice in the glutamine-free media to establish a resistant cell population. This glutamine-starvation-resistant RH30 cell line was treated with KAS-1155 in the same conditions as RD. Cellular viability was assessed using Cell Counting Kit-8(CCK-8) assays. GCN2 pathway activation, including phosphorylated GCN2 (p-GCN2), eIF2α, and ATF4 expression, was analyzed by western blotting. In the glutamin-free media, p-GCN2 was detected after 8 hours in RD cells and, persisted up to 40 hours. In RH30, the p-GCN2 was detectable at 8 hours, and peaked at 40 hours ,and then maintained elevated until 72 hours. Following the 14 hours and 49 hours of glutamine-free media exposure for RD and RH30 respectively, p-GCN2, p-eIF2α and ATF were suppressed successfully with the treatment of KAS-1155 at the concentration of 0.1μM and 1μM for each cell line. Viability assays demonstrated IC50 values of 1.06 μM for RD cells and 1.87 μM for RH30 cells, indicating moderate growth suppression by KAS-1155. GCN2 pathway activation represented the adaptive response in rhabdomyosarcoma cell lines under glutamine-free, amino acid starvation condition. The KAS-1155 inhibited RD and RH30 proliferation suppression at moderate concentrations. This suggests the GCN2 inhibitor may have a role in the treatment of rhabdomyosarcoma, especially in the resistant cell types. Further preclinical investigation is needed to clarify its therapeutic potential and proper use of the GCN2 inhibitor in the rhabdomyosarcoma cell lines. Citation Format: Da Yeon Kang, Hae Won Lee, Jong Woo Yoon, Jung-Nyoung Heo, Youngki Choi, Seung Min Hahn, Won Ki Ahn, Jung Woo Han. Preclinical efficacy of GCN2 inhibition by KAS-1155 in targeting the integrated stress response in rhabdomyosarcoma [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 6447.