ALK异常型神经母细胞瘤原发性耐药机制的磷酸化蛋白组学研究

Neuroblastoma is the most common solid tumor in children with high fatality. Genomic sequencing revealed 7~10% of the sporadic neuroblastoma patients bearing mutated anaplastic lymphoma kinase (ALK). Mutated ALK results in disregulated downstream signaling pathway which plays an vital role in the tumorgenesis of neuroblastoma. On the other hand, mutated ALK also provides an alternative druggable target for personal therapy. Whereas, recent clinical trial revealed that some but not all the ALK positive neuroblastoma patients show complete response suggesting the existence of de novo resistance to ALK specific inhibitor (Crizotinib). The limited knowledge of the diverse ALK downstream signaling pathway in neuroblastoma largely hinder our understanding of the drug resistance. Our preliminary data showed the downstream signaling pathway of ALK in NB were largely different with that in adult ALK positive tumor. Based on the preliminary funding and the established quantitative phosphoproteomics platform, we will further study the phosphoproteome of the Crizotinib sensitive and resistant cell lines to investigate the mechanism of de novo resistance to Crizotinib in NB. We will further integrate biochemistry, molecular biology and approaches to validate and explore the potential mechanism. This project will provide the a global picture of downstream signaling pathway of diverse disregulated ALK in NB providing a step stone for overcoming the de novo drug resistance to Crizotinib in NB.

神经母细胞瘤（NB）是婴幼儿中最常见的高致死率实体肿瘤。测序结果表明突变型间变性淋巴瘤激酶（ALK）约占到7-10%NB样本。异常活性ALK引起的磷酸化信号通路异常对NB致病起到了关键的作用，同时也提供特异的治疗靶点。然而，近期临床研究显示部分ALK阳性NB存在原发性耐药。目前，NB中ALK下游的信号通路不明确且呈现多样性，极大地阻碍了对这种原发性耐药分子机制的认识。申请人前期的研究显示NB中ALK下游信号通路与成人肿瘤存在显著差异，在此基础上本课题提出利用已建立的定量磷酸化蛋白质组学技术进一步对敏感型及耐药型ALK的NB细胞株的磷酸化修饰谱进行研究，探讨ALK不同的下游信号通路及多样性的调控网络，深入探讨ALK原发性耐药的分子机制。在此基础上，利用生物化学，分子生物学及细胞生物学的方法对潜在的耐药机制进行进一步的验证及探索，为克服NB中原发性ALK耐药实现靶向治疗提供理论基础。

研究表明神经母细胞瘤中的酪氨酸激酶受体ALK可作为治疗靶标之一。然而，临床试验表明仅有少数ALK阳性神经母细胞瘤患者对ALK特异性抑制剂克唑替尼显示良好的临床效果。目前ALK异常的神经母细胞瘤精确分子机制仍然不清楚，进一步限制了靶向ALK的临床应用。本项研究中，我们利用深度定量磷酸化蛋白质组学方法，探究克唑替尼处理后的神经母细胞瘤细胞株的下游磷酸化蛋白调控机制。我们通过蛋白质组学方法鉴定到超过19,500种蛋白质，在每种细胞系中定量分析了大约10,000个磷酸化位点，最终确定了450-790个具有显著差异的磷酸化蛋白调节节点。生物信息学分析提示RAS/JNK信号通路处于ALK蛋白下游，进一步的实验证明ALK/JNK信号通路可以被ALK或JNK特异性抑制剂抑制，并可通过诱导细胞周期停滞和细胞凋亡抑制神经母细胞瘤的生长。同时我们也发现了PDCD4位于ALK调控的下游，参与肿瘤增殖过程。本项研究通过蛋白质组学的方法研究了ALK特异性抑制剂克唑替尼处理后的ALK异常型神经母细胞瘤的磷酸化调控网络，为靶向治疗ALK异常的神经母细胞瘤患者提供了特异性靶点。