Targeting ALKBH5 with mefloquine as a therapeutic strategy to enhance antitumor immunity in osteosarcoma.

Abstract

e14596 Background: Osteosarcoma (OS) is the most common primary bone malignancy in children and adolescents, with a second incidence peak in older adults. Most treatments require intensive chemotherapy and surgery, with outcomes for patients with metastatic or relapsed disease remain poor, with five-year survival rates below 30%. Approximately 20% of patients present with metastatic disease at diagnosis, underscoring the urgent need for novel, clinically actionable therapeutic strategies. We recently identified the RNA demethylase ALKBH5 as a key driver of OS growth, metastasis, and immune evasion. Through high-throughput screening of FDA-approved compounds, we identified mefloquine as a small-molecule inhibitor of ALKBH5, with potential to enhance antitumor immune responses and improve immunotherapy efficacy in OS. Methods: Depletion of ALKBH5 was achieved using shRNA and CRISPR-Cas9 approaches to evaluate its role in OS growth and metastasis. Fluorescence-based high-throughput screening of FDA-approved and LOPAC libraries identified mefloquine as a candidate ALKBH5 inhibitor. Transcriptomic changes following mefloquine treatment were assessed by RNA sequencing. In vitro studies were conducted using human and murine OS cell models. Direct interaction between mefloquine and ALKBH5 was validated using surface plasmon resonance and m6A dot blot assays. Therapeutic efficacy was evaluated in orthotopic intratibial and experimental metastasis mouse models, including assessment of tumor growth, metastatic burden, and response to immunotherapy. Results: Genetic loss of ALKBH5 significantly reduced OS cell proliferation, primary tumor growth, and metastatic burden in vitro and in vivo. Mefloquine treatment phenocopied ALKBH5 depletion, suppressing tumor growth and metastasis across multiple OS models. RNA sequencing revealed upregulation of immune-related pathways following mefloquine treatment. In vivo, mefloquine significantly reduced primary tumor volume and metastatic disease and enhanced response to immunotherapy. Conclusions: These findings identify ALKBH5 as a clinically relevant regulator of osteosarcoma progression and antitumor immunity. Mefloquine, an FDA-approved antimalarial drug, demonstrates potent antitumor and immunomodulatory activity through ALKBH5 inhibition and represents a promising repurposing strategy for osteosarcoma treatment. This work provides a strong preclinical rationale for further clinical investigation of ALKBH5-targeted therapies, including combination strategies with immunotherapy, in patients with osteosarcoma.