Abstract 3905: Targeting RNA demethylase ALKBH5 blocks growth and improves therapy response in osteosarcoma

Abstract Background: Osteosarcoma (OS) arises primarily in children and adolescents, with a second peak in incidence in those over the age of 50. Unfortunately, outcomes of patients with metastatic or relapsed osteosarcoma have remained abysmal. Moreover, patients who survive have a highly compromised quality of life due to debilitating side effects associated with chemotherapy drugs. Virtually no new drug has been approved for treating OS in the last three decades. Recently our group showed that ALKBH5 is critical for promoting OS growth and metastasis and may thus serve as a unique driver of OS. We therefore hypothesized that epigenetic changes, specifically mRNA demethylation mediated by the RNA demethylase ALKBH5, play a pivotal role in OS growth and metastasis, and hence approaches aimed at inhibiting ALKBH5 activity could prove clinical utility for patients with OS. Methods: shRNA and CRISPR-Cas9-based knockout were used to study the effect of depleting ALKBH5 in osteosarcoma and the effect it has on growth and metastasis. Fluorescence-based high-throughput screening (HTS) assay was used to identify targets of ALKBH5. FDA-approved and LOPAC compound libraries were used. SPR analysis was used to determine the Mefloquine-ALKBH5 interaction. In vitro and In vivo intratibial orthotopic models were used to test the efficacy of Mefloquine for blocking OS growth. Experiments are currently underway to test the effect of combining Mefloquine and doxorubicin in inhibiting OS growth and metastasis. Results: We showed that upregulation of the ALKBH5 attenuates histone H2A monoubiquitination to result in the induction of key pro-tumorigenic genes and consequently unchecked cell cycle progression, incessant DNA replication, and upregulation of DSB repair. Unbiased high throughput screening using a LOPAC library identified mefloquine as a potential ALKBH5 inhibitor. SPR analysis showed the interaction of Mefloquine with ALKBH5 catalytic domain. M6A dot blot confirmed that mefloquine inhibits the catalytic activity of ALKBH5. Cell-based assays showed reduced short-term and long-term growth with the treatment of mefloquine. In vivo models showed a reduction in tumor volume upon mefloquine oral treatment. Conclusion: Our results establish RNA demethylase ALKBH5 as a novel driver of OS. This project has a direct translational relevance as an FDA-approved drug can be repurposed for treating OS patients as a monotherapy or in combination with doxorubicin and be formally tested in the clinic without much delay. Citation Format: Daisy Medina, Santosh Timilsina, Panneerdoss Subbarayalu, Prabhakar Pitta Venkata, Deepika Singh, Trong Phat Do, Shuo Zhou, Yogesh K. Gupta, Daohong Zhou, Manjeet K. Rao. Targeting RNA demethylase ALKBH5 blocks growth and improves therapy response in osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3905.