Phosphoproteomics reveals ALK promote cell progress via RAS/JNK pathway in neuroblastoma

// Kai Chen 1, 2, * , Fan Lv 1, 2, * , Guofeng Xu 1 , Min Zhang 1, 2 , Yeming Wu 1, 2 , Zhixiang Wu 1, 2 1 Department of Pediatric Surgery, Xinhua Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, China 2 Division of Pediatric Oncology, Shanghai Institute of Pediatric Research, Shanghai, China * These authors have contributed equally to this work Correspondence to: Yeming Wu, email: wuymsh@163.com Zhixiang Wu, email: zhixiangwu@yahoo.com Keywords: neuroblastoma, ALK, phosphoproteomics, target therapy, pediatric oncology Received: March 25, 2016 Accepted: September 26, 2016 Published: October 07, 2016 ABSTRACT Emerging evidence suggests receptor tyrosine kinase ALK as a promising therapeutic target in neuroblastoma. However, clinical trials reveal that a limited proportion of ALK-positive neuroblastoma patients experience clinical benefits from Crizotinib, a clinically approved specific inhibitor of ALK. The precise molecular mechanisms of aberrant ALK activity in neuroblastoma remain elusive, limiting the clinical application of ALK as a therapeutic target in neuroblastoma. Here, we describe a deep quantitative phosphoproteomic approach in which Crizotinib-treated neuroblastoma cell lines bearing aberrant ALK are used to investigate downstream regulated phosphoproteins. We identified more than 19,500—and quantitatively analyzed approximately 10,000—phosphorylation sites from each cell line, ultimately detecting 450–790 significantly-regulated phosphorylation sites. Multiple layers of bioinformatic analysis of the significantly-regulated phosphoproteins identified RAS/JNK as a downstream signaling pathway of ALK, independent of the ALK variant present. Further experiments demonstrated that ALK/JNK signaling could be inactivated by either ALK- or JNK-specific inhibitors, resulting in cell growth inhibition by induction of cell cycle arrest and cell apoptosis. Our study broadly defines the phosphoproteome in response to ALK inhibition and provides a resource for further clinical investigation of ALK as therapeutic target for the treatment of neuroblastoma.