Department of Molecular Medicine and Medical Biotechnologies, University of Naples Federico II, Naples, Italy.
CEINGE, Biotecnologie Avanzate, Naples, Italy.
J Cell Mol Med. 2020 Jun;24(11):6459-6471. doi: 10.1111/jcmm.15297. Epub 2020 Apr 26.
Neuroblastoma (NB) is the most common extracranial neoplasm in children. The overall outcome for high-risk NB patients is still unacceptable, therefore, it is critical to deeply understand molecular mechanisms associated with NB, which in turn can be utilized for developing drugs towards the treatment of NB. Protein kinases (TKs) play an essential role in the regulation of cell survival and proliferation. Different kinases, such as anaplastic lymphoma kinase (ALK), Aurora kinase, RET receptor tyrosine kinase, are potential therapeutic targets in various cancers, including NB. We analysed a cohort of 45 high-risk NB patients and 9 NB cell lines by a targeted-(t)NGS custom gene panel (genes codifying for the kinase domains of 90 TKs). We identified somatic variants in four TK genes (ALK, EPHB4, LMTK3 and EPHB6) in NB patients and we functionally characterized an interesting somatic variant, V871I, in EPHB4 gene. EPHB4 plays a crucial role in cardiovascular development and regulates vascularization in cancer-promoting angiogenesis, tumour growth and metastasis. Several EPHB4 mutations have previously been identified in solid and haematological tumour specimens but EPHB4 mutations were not described until now in NB. Interestingly, a re-analysis of public CGH-array showed that the EPHB4 gain is associated with advanced diseases in NB. We further demonstrated that higher EPHB4 expression is correlated to stage 4 of NB and with poor overall survival. Additionally, we also revealed that the EPHB4-V871I accounts for increased proliferation, migration and invasion properties in two NB cell lines by acting on VEGF, c-RAF and CDK4 target genes and by increasing the phosphorylation of ERK1-2 pathway. The use of two EPHB4 inhibitors, JI-101 and NVP-BHG712, was able to rescue the phenotype driven by the variant. Our study suggested that EPHB4 is a promising therapeutic target in high-risk NB.
神经母细胞瘤（NB）是儿童最常见的颅外肿瘤。高危 NB 患者的总体预后仍然不能令人接受，因此，深入了解与 NB 相关的分子机制至关重要，这反过来又可以用于开发治疗 NB 的药物。蛋白激酶（TKs）在细胞存活和增殖的调节中起着重要作用。不同的激酶，如间变性淋巴瘤激酶（ALK）、Aurora 激酶、RET 受体酪氨酸激酶，是包括 NB 在内的各种癌症的潜在治疗靶点。我们通过靶向（t）NGS 定制基因panel（编码 90 种 TK 激酶结构域的基因）分析了 45 例高危 NB 患者和 9 株 NB 细胞系。我们在 NB 患者中发现了四个 TK 基因（ALK、EPHB4、LMTK3 和 EPHB6）中的体细胞变异，并对 EPHB4 基因中的一个有趣的体细胞变异 V871I 进行了功能表征。EPHB4 在心血管发育中起着至关重要的作用，并调节肿瘤促进血管生成、肿瘤生长和转移中的血管生成。以前在实体瘤和血液肿瘤标本中已经发现了几个 EPHB4 突变，但直到现在在 NB 中才描述了 EPHB4 突变。有趣的是，对公共 CGH-array 的重新分析表明，EPHB4 增益与 NB 中的晚期疾病有关。我们进一步证明，较高的 EPHB4 表达与 NB 的第 4 期有关，并与总生存率差有关。此外，我们还揭示了 EPHB4-V871I 通过作用于 VEGF、c-RAF 和 CDK4 靶基因并增加 ERK1-2 通路的磷酸化，导致两种 NB 细胞系增殖、迁移和侵袭特性增加。两种 EPHB4 抑制剂 JI-101 和 NVP-BHG712 的使用能够挽救由变体驱动的表型。我们的研究表明，EPHB4 是高危 NB 的一个有前途的治疗靶点。
