Abstract 6450: High-throughput screening identified Brefeldin A as a compound that reduces STAT3-dependent survival in acute leukemia and neuroblastoma cells

Abstract Leukemic stem cells exhibit several dysregulated pathways, with the Janus kinase (JAK) and signal transducer and activator of transcription (STAT) pathway being among the most prominent. Additionally, phosphorylated STAT3 is observed following prolonged activation of the anaplastic lymphoma kinase (ALK) ligand-binding site, although the level of phospho-STAT3 induced by ligand-independent activation of ALK is exponentially higher. In ALK-positive tumors, ALK-STAT3 activation promotes tumor cell outgrowth and directly prevents apoptosis. The role of ALK in various ALK-expressing solid tumors, along with the therapeutic use of ALK inhibitors in treating these cancers, has been reviewed in several recent studies. Amplified or mutated ALK is found in approximately 14% of neuroblastomas (NB), one of the most common and aggressive childhood malignancies.In this study, we conducted a high-throughput chemical screen in twenty NB-derived cell lines to identify compounds that could inhibit the oncogenic activity of ALK in NB, using a curated library of around 450 compounds. Brefeldin A (BFA), an inhibitor of protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus, was identified as a promising candidate. Given the increasing evidence linking abnormal expression and activation of STAT3 with leukemia development, suggesting its potential role in leukemia pathogenesis, we next assessed the cytotoxicity and antitumor effect of BFA against 12 T-ALL and 6 AML cell lines.The antitumor effect of BFA, assessed using an MTT assay, revealed potent cytotoxicity across a broad spectrum of hematopoietic malignancies, with particularly strong efficacy observed in 10 T-ALL and 5 AML cell lines. Specifically, T-ALL and AML cell lines were significantly inhibited by BFA, with IC50 values as follows: ICH-TALL-UK, 168.1 nM; MOLT-4, 23.2 nM; Jurkat, 66.6 nM; MOLT14, 97.3 nM; and ICH-TALL-SM, 54.9 nM.In NB cell lines harboring mutated ALK, BFA exhibited notable cytotoxicity. Furthermore, BFA was found to reduce the phosphorylation of ALK in NIH3T3 cells stably expressing mutated ALK. Among the key molecules phosphorylated downstream of ALK, which are critical for the survival of anaplastic large cell lymphoma (ALCL) cells, BFA inhibited the phosphorylation of STAT3, AKT, and ERK.These results suggest that BFA can effectively target the STAT3 signaling pathway, presenting a potential therapeutic approach for leukemia treatment. Moreover, the effects of BFA on ALK-mutated NB cell lines are accompanied by a reduction in the phosphorylation of both ALK and its downstream effector, STAT3. Investigating the antitumor effects of BFA in vivo will be an important next step in the development of future therapeutic strategies. Citation Format: Mitsuteru Hiwatari, Masakazu MImaki. High-throughput screening identified Brefeldin A as a compound that reduces STAT3-dependent survival in acute leukemia and neuroblastoma 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 6450.