TrkB with its ligand, brain-derived neurotrophic factor (BDNF), are overexpressed in the majority of high-risk neuroblastomas (NB). Entrectinib is a novel pan-TRK, ALK, and ROS1 inhibitor that has shown excellent preclinical efficacy in NB xenograft models, and recently it has entered phase 1 trials in pediatric relapsed/refractory solid tumors. We examined entrectinib-resistant NB cell lines to identify mechanisms of resistance. Entrectinib-resistant cell lines were established from five NB xenografts initially sensitive to entrectinib therapy. Clonal cell lines were established in increasing concentrations of entrectinib and had >10X increase in IC50. Cell lines underwent genomic and proteomic analysis using whole-exome sequencing, RNA-Seq, and proteomic expression profiling with confirmatory RT-PCR and Western blot analysis. There was no evidence of NTRK2 (TrkB) gene mutation in any resistant cell lines. Inhibition of TrkB was maintained in all cell lines at increasing concentrations of entrectinib (target independent). PTEN pathway downregulation and ERK/MAPK pathway upregulation were demonstrated in all resistant cell lines. One of these clones also had increased IGF1R signaling, and two additional clones had increased P75 expression, which likely increased TrkB sensitivity to ligand. In conclusion, NB lines overexpressing TrkB developed resistance to entrectinib by multiple mechanisms, including activation of ERK/MAPK and downregulation of PTEN signaling. Individual cell lines also had IGF1R activation and increased P75 expression, allowing preservation of downstream TrkB signaling in the presence of entrectinib. An understanding of changes in patterns of expression can be used to inform multimodal therapy planning in using entrectinib in phase II/III trial planning.

中文翻译：

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神经母细胞瘤异种移植模型中恩曲替尼耐药的机制

TrkB 及其配体脑源性神经营养因子 (BDNF) 在大多数高危神经母细胞瘤 (NB) 中过度表达。Entrectinib 是一种新型泛 TRK、ALK 和 ROS1 抑制剂，在 NB 异种移植模型中显示出优异的临床前疗效，最近它已进入儿科复发/难治性实体瘤的 1 期试验。我们检查了耐恩曲替尼的 NB 细胞系，以确定耐药机制。从最初对 entrectinib 治疗敏感的五个 NB 异种移植物建立了 entrectinib 抗性细胞系。克隆细胞系在增加浓度的 entrectinib 中建立，IC50 增加 > 10 倍。使用全外显子组测序、RNA-Seq 和蛋白质组表达谱与验证性 RT-PCR 和蛋白质印迹分析对细胞系​​进行基因组和蛋白质组学分析。在任何耐药细胞系中都没有 NTRK2 (TrkB) 基因突变的证据。TrkB 的抑制在所有细胞系中在 entrectinib 浓度增加时保持（不依赖于靶标）。PTEN 通路下调和 ERK/MAPK 通路上调在所有抗性细胞系中均得到证实。其中一个克隆也增加了 IGF1R 信号，另外两个克隆增加了 P75 表达，这可能增加了 TrkB 对配体的敏感性。总之，过表达 TrkB 的 NB 系通过多种机制产生了对 entrectinib 的抗性，包括 ERK/MAPK 的激活和 PTEN 信号传导的下调。单个细胞系也有 IGF1R 激活和 P75 表达增加，允许在 entrectinib 存在下保留下游 TrkB 信号。