Abstract Background: Osteosarcoma (OS) is the most common form of bone cancer in youth, with a second incidence peak observed in adults over the age of 50. Current OS treatments rely on aggressive chemotherapy and surgery, which remain largely ineffective for patients with metastatic or relapsed disease. Approximately 20% of patients present with metastases at the time of diagnosis, with a five-year survival rate below 30%. This highlights an urgent need for a novel drug to treat OS. Recently, our group identified the RNA demethylase ALKBH5 as a key promoter of OS growth and metastasis. Using high throughput FDA approved drug library, we identified mefloquine as a small-molecule inhibitor of ALKBH5. Importantly, we discovered that mefloquine improves the efficacy of immunotherapy in OS. These findings indicate that mefloquine may serve as an effective therapeutic for treating osteosarcoma patients. Methods: shRNA and CRISPR-Cas9 based knockout were used to assess the effects of ALKBH5 depletion on osteosarcoma growth and metastasis. Fluorescence-based high-throughput screening (HTS) of FDA-approved and LOPAC compound libraries identified mefloquine as a potential ALKBH5 inhibitor. RNA sequencing was performed to identify genes altered in mefloquine-treated OS cells. In vitro human and murine OS models were used to identify the inhibitory potency of mefloquine. Surface plasmon resonance (SPR) and m6A dot blot assays were used to validate mefloquine’s interaction with ALKBH5. In vivo orthotopic intratibial and tail vein mouse models were used to test the efficacy of mefloquine for reducing OS growth, metastasis, and improving immunotherapy response. Ongoing experiments aim to further elucidate the mechanisms of mefloquine-mediated OS suppression. Results: Here, we demonstrate that mefloquine is a promising inhibitor of ALKBH5, suppressing OS growth and metastasis, and improving immunotherapy response. Loss of ALKBH5 in vitro and in vivo significantly reduced osteosarcoma cell growth and tumor burden. RNA sequencing revealed that mefloquine treatment led to upregulation of immune-related genes. In vivo mouse models showed reduction of tumor volume and metastatic burden in mice treated with mefloquine. Conclusion: Our results establish mefloquine as a promising inhibitor of ALKBH5 in osteosarcoma, effectively suppressing tumor growth and metastasis while enhancing antitumor immune responses. As an FDA approved drug for malaria, mefloquine could be repurposed as a safe and effective therapy for OS. Citation Format: Victoria Chen Mai, Daisy Medina, Manjeet K. Rao. Targeting RNA demethylase ALKBH5 with mefloquine enhances antitumor immunity and reduces osteosarcoma progression and metastasis abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 437.
Chen et al. (Fri,) studied this question.