Evaluating the Synergistic Therapeutic Effects of Retinoic Acid and the Proteasome Inhibitors in Neuroblastoma

Neuroblastoma (NB) is the most common extracranial solid tumor in children, with high-risk patients experiencing 5-year survival rates of only 40-50% despite intensive multimodal therapy including retinoic acid (RA) maintenance treatment. While RA therapy promotes differentiation and prevents relapses, its efficacy is limited by resistance development, necessitating novel combination strategies. Proteasome inhibitors (bortezomib, carfilzomib, ixazomib) have shown promise in various malignancies by disrupting protein degradation pathways and promoting apoptosis, with our preliminary data demonstrating potent synergistic anti-tumor effects when combined with isotretinoin (13-cis-retinoic acid) across multiple NB cell lines and patient-derived xenografts. Notably, carfilzomib exhibits a synergistic anti-tumor effect with isotretinoin in NB preclinical mouse model. We hypothesize that proteasome inhibitors will potentiate RA therapy in NB by enhancing RA-induced differentiation and apoptosis. This proposal will systematically evaluate proteasome inhibitor-RA combination therapy through three specific aims using genetically diverse NB models that recapitulate key clinical biomarkers (MYCN amplification, TP53 mutations, ALK status). Because the complex tumor microenvironment, systemic toxicities, pharmacokinetics, and metastatic cascades of neuroblastoma cannot be adequately recapitulated by in vitro or computational alternative systems, the use of live vertebrate animal models is required to validate these therapeutic approaches prior to clinical translation. All proposed animal experiments have been fully approved by the Institutional Animal Care and Use Committee (IACUC) at the Children's National Research Institute (07/31/2025-07/31/2028). Aim 1 will assess tumor growth inhibition by evaluating all three proteasome inhibitors combined with isotretinoin in subcutaneous xenograft models, measuring anti-tumor efficacy, synergistic interactions, and mechanistic endpoints including apoptosis markers and pathway analysis. Aim 2 will determine survival benefit and anti-metastatic activity by advancing the most effective combination to intravenous injection models using luciferase-expressing cells, with primary endpoints of overall survival and metastatic burden assessed by bioluminescence imaging. Aim 3 will comprehensively assess toxicity and tolerability in healthy mice over 4 weeks, evaluating treatment-related mortality, organ toxicity, and safety parameters to establish therapeutic windows for clinical translation. This innovative approach represents the first systematic evaluation of proteasome inhibitor-RA combination therapy in NB, employing advanced methodologies including real-time metastatic assessment, comprehensive combination index analysis, and toxicological evaluation. Expected outcomes include identification of the optimal proteasome inhibitor-RA combination with >50% tumor growth inhibition, ≥30% survival improvement, ≥50% reduction in metastatic burden, and acceptable toxicity profiles supporting clinical translation. Success will generate definitive preclinical data for IND application and Phase I clinical trial initiation, offering new hope for children with high-risk NB and potentially benefiting patients with treatment-resistant malignancies. Project Number: 1R21CA302806-01A1 | Fiscal Year: 2026 | NIH Institute/Center: National Cancer Institute (NCI) | Principal Investigator: Jianhua Yang | Institution: CHILDREN'S RESEARCH INSTITUTE, WASHINGTON, DC | Award Amount: $467,968 | Activity Code: R21 | Study Section: Special Emphasis Panel[ZRG1 CTH-V (81)] View on NIH RePORTER: https://reporter.nih.gov/project-details/11371019