The clinically aggressive alveolar rhabdomyosarcoma (RMS) subtype is characterized by expression of the oncogenic fusion protein PAX3-FOXO1, which is critical for tumorigenesis and cell survival. Here, we studied the mechanism of cell death induced by loss of PAX3-FOXO1 expression and identified a novel pharmacologic combination therapy that interferes with PAX3-FOXO1 biology at different levels. Depletion of PAX3-FOXO1 in fusion-positive (FP)-RMS cells induced intrinsic apoptosis in a NOXA-dependent manner. This was pharmacologically mimicked by the BH3 mimetic navitoclax, identified as top compound in a screen from 208 targeted compounds. In a parallel approach, and to identify drugs that alter the stability of PAX3-FOXO1 protein, the same drug library was screened and fusion protein levels were directly measured as a read-out. This revealed that inhibition of Aurora kinase A most efficiently negatively affected PAX3-FOXO1 protein levels. Interestingly, this occurred through a novel specific phosphorylation event in and binding to the fusion protein. Aurora kinase A inhibition also destabilized MYCN, which is both a functionally important oncogene and transcriptional target of PAX3-FOXO1. Combined treatment with an Aurora kinase A inhibitor and navitoclax in FP-RMS cell lines and patient-derived xenografts synergistically induced cell death and significantly slowed tumor growth. These studies identify a novel functional interaction of Aurora kinase A with both PAX3-FOXO1 and its effector MYCN, and reveal new opportunities for targeted combination treatment of FP-RMS. SIGNIFICANCE: These findings show that Aurora kinase A and Bcl-2 family proteins are potential targets for FP-RMS.

中文翻译：

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Aurora A激酶抑制作用会破坏PAX3-FOXO1和MYCN的稳定性，并与Navitoclax协同作用，从而导致横纹肌肉瘤细胞死亡。

临床上具有侵略性的肺泡横纹肌肉瘤（RMS）亚型的特征是致癌融合蛋白PAX3-FOXO1的表达，这对于肿瘤发生和细胞存活至关重要。在这里，我们研究了PAX3-FOXO1表达缺失引起的细胞死亡机制，并确定了一种新的药物联合疗法，可在不同水平上干扰PAX3-FOXO1生物学。融合阳性（FP）-RMS细胞中PAX3-FOXO1的耗尽以NOXA依赖性方式诱导内在凋亡。这在药理学上被BH3仿制药navitoclax模仿，在208种目标化合物的筛选中被鉴定为顶级化合物。采用并行方法，并鉴定出改变PAX3-FOXO1蛋白稳定性的药物，筛选了相同的药物库，并直接测量了融合蛋白的水平作为读数。这表明抑制Aurora激酶A最有效地负面影响PAX3-FOXO1蛋白水平。有趣的是，这是通过融合蛋白中新的特异性磷酸化事件并与融合蛋白结合而发生的。极光激酶A的抑制也会使MYCN不稳定，MYCN既是功能上重要的癌基因，又是PAX3-FOXO1的转录靶标。在FP-RMS细胞系中与Aurora激酶A抑制剂和navitoclax联合治疗与患者来源的异种移植物协同诱导细胞死亡，并显着减慢肿瘤的生长。这些研究确定了Aurora激酶A与PAX3-FOXO1及其效应物MYCN的新型功能相互作用，并揭示了针对性联合治疗FP-RMS的新机会。意义：