High-risk neuroblastoma (NB) is a heterogeneous and malignant childhood cancer that is frequently characterized by MYCN proto-oncogene amplification or elevated N-Myc protein (N-Myc) expression. An N-Myc downstream target gene, insulinoma associated-1 (INSM1) has emerged as a biomarker that plays a critical role in facilitating NB tumor cell growth and transformation. N-Myc activates endogenous INSM1 gene expression through binding to the E2-box of the INSM1 proximal promoter in NB. We identified a plant alkaloid, homoharringtonine (HHT), from a chemical library screening showing potent inhibition of INSM1 promoter activity. This positive-hit plant alkaloid exemplifies an effective screening approach for repurposed compound targeting INSM1 expression in NB cancer therapy. The elevated N-Myc and INSM1 expression in NB constitutes a positive-loop through INSM1 activation that promotes N-Myc stability. In the present study, the biological effects and anti-tumor properties of HHT against NB were examined. HHT either down regulates and/or interferes with the binding of N-Myc to the E2-box of the INSM1 promoter and the inhibition of PI3K/AKT-mediated N-Myc stability could lead to the NB cell apoptosis. HHT inhibition of NB cell proliferation is consistent with the INSM1 expression as higher level of INSM1 exhibits a more sensitive IC(50) value. The combination treatment of HHT and A674563 provides a better option of increasing potency and reducing cellular cytotoxicity than HHT or A674563 treatment alone. Taken together, the suppression of the INSM1-associated signaling pathway axis promotes the inhibition of NB tumor cell growth. This study developed a feasible approach for repurposing an effective anti-NB drug. CI - Copyright (c) 2023 Elsevier Inc. All rights reserved.