Zhou Cheng, Zhao Liang, Zhou Ming, Wu Chao, Liu Guanghua, Long Jiangwen, Shi Yuanxiang, Liu Can
Department of Hematology, Hunan Provincial People's Hospital, The First Affiliated Hospital of Hunan Normal University, Changsha, Hunan, 410005, China.
Department of Hematology, Xiangya Hospital, Central South University, Changsha, Hunan, 410008, China.
Cancer Cell Int. 2024 Dec 26;24(1):430. doi: 10.1186/s12935-024-03626-5.
Drug resistance remains a significant obstacle to Acute myeloid leukemia (AML) successful treatment, often leading to therapeutic failure. Our previous studies demonstrated that Glioma-associated oncogene-1 (GLI1) reduces chemotherapy sensitivity and promotes cell proliferation in AML cells. GANT61, an inhibitor of GLI1, emerges as a promising candidate in AML treatment. This study aims to explore the effects of the combination of GANT61 and Adriamycin (ADR) on AML cells resistance and elucidate the mechanisms through which GANT61 may potentiate the sensitivity of AML cells to ADR.
AML cell lines and AML primary cells were studied to evaluate effects and mechanisms of GANT61. Flow cytometry assays were used to verify apoptosis. Cell Counting Kit-8 (CCK-8) and EDU staining were used to observe changes in cell viability and the cytotoxic effect to different drugs. The transcriptomic profiles of HL-60/ADR cells with or without GANT61 treatment were compared via RNA-Seq analysis. Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses and Gene Set Enrichment Analysis (GSEA) were performed for differentially expressed genes (DEGs) to reveal the underlying mechanisms of GANT61 in AML cells. GLI1, BCL2, Bax protein and mRNA expression levels were assessed by Western blot and Real-time polymerase chain reaction (RT-PCR).
Our studies found that the combination of GANT61 and ADR synergistically inhibits proliferation while enhancing apoptosis in HL-60/ADR cells, and does not significantly exacerbate myelosuppression. Mechanistically, GSEA revealed enrichment of the gene set associated with the KEGG term "Apoptosis" and "Lysosome" in GANT61 treated cells. Meanwhile, "Apoptosis" was identified as the third most relevant pathway enriched by lysosomal DEGs, and BCL2 expression showed a negative correlation with these lysosomal DEGs in AML patients. RT-PCR and Western blot analysis disclosed that GANT61 significantly restrained BCL2 expression in AML cells. Lastly, we proved that venetoclax, a BCL2 inhibitor, co-treatment with GANT61 improved ADR sensitivity in HL-60/ADR cells.
GANT61 effectively reversed ADR resistance in HL-60/ADR cells by upregulating lysosome activities and downgrading BCL2 expression, providing a new treatment strategy with acceptable toxicity for AML-resistant patients.
耐药性仍然是急性髓系白血病（AML）成功治疗的重大障碍，常常导致治疗失败。我们之前的研究表明，胶质瘤相关致癌基因1（GLI1）降低了AML细胞的化疗敏感性并促进其细胞增殖。GLI1抑制剂GANT61成为AML治疗中一个有前景的候选药物。本研究旨在探讨GANT61与阿霉素（ADR）联合应用对AML细胞耐药性的影响，并阐明GANT61增强AML细胞对ADR敏感性的机制。
研究AML细胞系和AML原代细胞以评估GANT61的作用和机制。采用流式细胞术检测来验证细胞凋亡。使用细胞计数试剂盒-8（CCK-8）和EdU染色来观察细胞活力变化以及对不同药物的细胞毒性作用。通过RNA测序分析比较有或没有GANT61处理的HL-60/ADR细胞的转录组图谱。对差异表达基因（DEG）进行京都基因与基因组百科全书（KEGG）分析和基因集富集分析（GSEA），以揭示GANT61在AML细胞中的潜在机制。通过蛋白质免疫印迹法和实时聚合酶链反应（RT-PCR）评估GLI1、BCL2、Bax蛋白和mRNA表达水平。
我们的研究发现，GANT61与ADR联合应用可协同抑制HL-60/ADR细胞的增殖，同时增强其凋亡，且不会显著加重骨髓抑制。从机制上讲，GSEA显示在GANT61处理的细胞中与KEGG术语“凋亡”和“溶酶体”相关的基因集富集。同时，“凋亡”被确定为溶酶体DEG富集的第三大相关通路，并且在AML患者中BCL2表达与这些溶酶体DEG呈负相关。RT-PCR和蛋白质免疫印迹分析表明，GANT61显著抑制AML细胞中BCL2的表达。最后，我们证明BCL2抑制剂维奈克拉与GANT61联合处理可提高HL-60/ADR细胞对ADR的敏感性。
GANT61通过上调溶酶体活性和下调BCL2表达有效逆转了HL-60/ADR细胞对ADR的耐药性，为AML耐药患者提供了一种毒性可接受的新治疗策略。
