PO.ET02.11 · 实验与分子治疗
用甲氟喹（mefloquine）靶向 RNA 去甲基化酶 ALKBH5 可增强抗肿瘤免疫并抑制骨肉瘤进展与转移
Targeting RNA demethylase ALKBH5 with mefloquine enhances antitumor immunity and reduces osteosarcoma progression and metastasis
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
背景：骨肉瘤（OS）是青少年中最常见的骨癌类型，在 50 岁以上成人中可见第二个发病高峰。目前 OS 的治疗依赖于强化化疗和手术，但对转移性或复发患者仍基本无效。约 20% 的患者在确诊时已发生转移，五年生存率低于 30%。这凸显了亟需一种治疗 OS 的新药。近期，我们团队发现 RNA 去甲基化酶 ALKBH5 是 OS 生长和转移的关键促进因子。通过高通量 FDA 批准药物库筛选，我们鉴定出甲氟喹为 ALKBH5 的小分子抑制剂。重要的是，我们发现甲氟喹可改善 OS 免疫治疗的疗效。这些发现表明，甲氟喹可能成为治疗骨肉瘤患者的有效疗法。
方法：采用基于 shRNA 及 CRISPR-Cas9 的敲除方法评估 ALKBH5 缺失对骨肉瘤生长和转移的影响。通过对 FDA 批准药物库及 LOPAC 化合物库进行基于荧光的高通量筛选（HTS），鉴定出甲氟喹为潜在的 ALKBH5 抑制剂。开展 RNA 测序以鉴定甲氟喹处理的 OS 细胞中发生改变的基因。采用体外人源及鼠源 OS 模型确定甲氟喹的抑制效力。采用表面等离子共振（SPR）及 m6A 斑点印迹实验验证甲氟喹与 ALKBH5 的相互作用。采用体内原位胫骨内注射及尾静脉小鼠模型检验甲氟喹在减少 OS 生长、转移及改善免疫治疗应答方面的疗效。正在进行的实验旨在进一步阐明甲氟喹介导 OS 抑制的机制。
结果：本研究表明，甲氟喹是一种有前景的 ALKBH5 抑制剂，可抑制 OS 的生长和转移，并改善免疫治疗应答。体外及体内 ALKBH5 的缺失均显著减少了骨肉瘤细胞生长和肿瘤负荷。RNA 测序显示甲氟喹处理导致免疫相关基因上调。体内小鼠模型显示，接受甲氟喹治疗的小鼠肿瘤体积和转移负荷减少。
结论：我们的结果确立了甲氟喹作为骨肉瘤中一种有前景的 ALKBH5 抑制剂，可有效抑制肿瘤生长和转移，同时增强抗肿瘤免疫应答。作为一种已获 FDA 批准用于疟疾的药物，甲氟喹可被重新定位为 OS 的一种安全有效的疗法。
查看英文原文 English 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.
利益披露 Disclosure
V. C. Mai, None.