In the ongoing battle against BCR-ABL+ leukemia, despite significant advances with tyrosine kinase inhibitors (TKIs), the persistent challenges of drug resistance and the enduring presence of leukemic stem cells (LSCs) remain formidable barriers to achieving a cure. In this study, we demonstrated that Disulfiram (DSF) induces ferroptosis to synergize with TKIs in inhibiting BCR-ABL+ cells, particularly targeting resistant cells and LSCs, using cell models, mouse models, and primary cells from patients. We elucidated the mechanism by which DSF promotes GPX4 degradation to induce ferroptosis through immunofluorescence, co-immunoprecipitation (CO-IP), RNA sequencing, lipid peroxidation assays, and rescue experiments. Here, we present compelling evidence elucidating the sensitivity of DSF, an USA FDA-approved drug for alcohol dependence, towards BCR-ABL+ cells. Our findings underscore DSF’s ability to selectively induce a potent cytotoxic effect on BCR-ABL+ cell lines and effectively inhibit primary BCR-ABL+ leukemia cells. Crucially, the combined treatment of DSF with TKIs selectively eradicates TKI-insensitive stem cells and resistant cells. Of particular note is DSF’s capacity to disrupt GPX4 stability, elevate the labile iron pool, and intensify lipid peroxidation, ultimately leading to ferroptotic cell death. Our investigation shows that BCR-ABL expression induces alterations in cellular iron metabolism and increases GPX4 expression. Additionally, we demonstrate the indispensability of GPX4 for LSC development and the initiation/maintenance of BCR-ABL+ leukemia. Mechanical analysis further elucidates DSF’s capacity to overcome resistance by reducing GPX4 levels through the disruption of its binding with HSPA8, thereby promoting STUB1-mediated GPX4 ubiquitination and subsequent proteasomal degradation. Furthermore, the combined treatment of DSF with TKIs effectively targets both BCR-ABL+ blast cells and drug-insensitive LSCs, conferring a significant survival advantage in mouse models. In summary, the dual inhibition of GPX4 and BCR-ABL presents a promising therapeutic strategy to synergistically target blast cells and drug-insensitive LSCs in patients, offering potential avenues for advancing leukemia treatment.

中文翻译：

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GPX4 和 BCR-ABL 酪氨酸激酶的联合靶向选择性损害 BCR-ABL+ 白血病干细胞


在与 BCR-ABL+ 白血病的持续斗争中，尽管酪氨酸激酶抑制剂 （TKI） 取得了重大进展，但耐药性的持续挑战和白血病干细胞 （LSC） 的持续存在仍然是实现治愈的巨大障碍。在这项研究中，我们使用细胞模型、小鼠模型和来自患者的原代细胞证明，双硫仑 （DSF） 诱导铁死亡与 TKI 协同抑制 BCR-ABL+ 细胞，特别是靶向耐药细胞和 LSC。我们通过免疫荧光、免疫共沉淀 （CO-IP）、RNA 测序、脂质过氧化测定和拯救实验阐明了 DSF 促进 GPX4 降解以诱导铁死亡的机制。在这里，我们提供了令人信服的证据，阐明了 DSF（一种美国 FDA 批准的酒精依赖药物）对 BCR-ABL+ 细胞的敏感性。我们的研究结果强调了 DSF 能够选择性地诱导对 BCR-ABL+ 细胞系产生有效的细胞毒作用，并有效抑制原代 BCR-ABL+ 白血病细胞。至关重要的是，DSF 与 TKI 的联合治疗选择性地根除对 TKI 不敏感的干细胞和耐药细胞。特别值得注意的是 DSF 破坏 GPX4 稳定性、提升不稳定铁池和加剧脂质过氧化的能力，最终导致铁死亡细胞。我们的研究表明，BCR-ABL 表达诱导细胞铁代谢的改变并增加 GPX4 表达。此外，我们证明了 GPX4 对 LSC 发展和 BCR-ABL+ 白血病的启动/维持是必不可少的。 机械分析进一步阐明了 DSF 通过破坏 GPX4 与 HSPA8 的结合来降低 GPX4 水平来克服耐药性的能力，从而促进 STUB1 介导的 GPX4 泛素化和随后的蛋白酶体降解。此外，DSF 与 TKIs 的联合治疗有效地靶向 BCR-ABL+ 原始细胞和药物不敏感的 LSC，在小鼠模型中具有显着的生存优势。总之，GPX4 和 BCR-ABL 的双重抑制提供了一种有前途的治疗策略，可以协同靶向患者的原始细胞和对药物不敏感的 LSC，为推进白血病治疗提供潜在途径。