IGF1R gene 3'UTR variants in high-risk pediatric neuroblastoma

描述(申请人提供)：对于高危神经母细胞瘤(NB)患者，存活率保持在40%以下，幸存者患有终身治疗相关残疾。这就需要开发新的靶向疗法。胰岛素样生长因子1受体(IGF1R)小分子抑制剂在体外和体外对NB细胞的抗肿瘤作用 活着。然而，考虑到IGF1R基因明显缺乏体细胞突变，还没有确定哪一类NB患者将从这种方法中受益。我们假设IGF1R的表达是由3‘UTR区的变化控制的，这会影响microRNAs的结合。在我们的初步实验中，我们首先关注了遗传变异rs3833015，这是在约50%的NB患者中发现的一个小的2-nT缺失。通过荧光素酶传感器论文、常用的NB细胞系的免疫印迹和28例原发神经母细胞瘤的基因集浓缩分析，证实了这种3‘UTR变异体对IGF1R表达的积极影响。此外，rs3833015过表达的基因对IGF1R/MAPK通路的成员也有很强的富集性，该通路不仅在细胞增殖中发挥着重要作用，而且在化疗后的细胞生存中也发挥着重要作用。同时，在无偏向表达-数量性状基因座(EQTL)研究过程中，我们发现了另一个突变体rs3743251，它创建了一个新的miR结合位点，并与IGF1R mRNA水平下降相关。我们目前的总体假设是rs3833015和rs374251改变IGF1信号(分别向上和向下)，导致对传统化疗药物和小分子IGF1R抑制剂的耐药性改变。我们将在以下两个目标中追求这一假说。1.研究rs3833015和rs3743251对IGF1R表达的调节作用机制，寻找与IGF1R表达上调/缺失相关的新的SNP。2.探讨IGF1R突变体对神经母细胞瘤对小分子IGF1R抑制剂和化疗药物敏感性的影响。为此，我们将使用Kaplan-Meier分析将IGF1R3‘UTR型与NB患者的生存期相关联，并比较和对比rs3833015和rs374251肿瘤对传统疗法(环磷酰胺和拓扑替康)和IGF1R抑制剂的反应。这项研究的主要影响是，它将允许对参加IGF1R抑制剂临床试验的患者进行前瞻性分层，并对这些药物产生更强劲的反应，从而开启miR药物基因组学时代。

DESCRIPTION (provided by applicant): For patients with high-risk neuroblastoma (NB) survival remains below 40%, and survivors suffer from life-long treatment-related disabilities. This necessitates the development of new targeted therapies. Small-molecule inhibitors of insulin-like growth factor 1 receptor (IGF1R) exhibit antitumor effects on NB cells in vitro and in vivo. Yet given the apparent lack of somatic mutations in the IGF1R gene, it remains to be determined what subset of NB patients would benefit from this approach. We hypothesized that IGF1R expression is controlled by alterations in the 3'UTR which would affect binding by microRNAs. In our Preliminary Experiments, we first focused on the genetic variant rs3833015, which is a small 2-nt deletion found in ~50% of NB patients. The positive effects of this 3'UTR variant on IGF1R expression were established using luciferase sensor essays, immunoblotting of commonly available NB cell lines, and transcriptome profiling of 28 primary neuroblastomas using Gene Set Enrichment Analysis. Furthermore, rs3833015-overexpressed genes were also strongly enrichment for members of the IGF1R/MAPK pathway, which plays a well-established role not only in cell proliferation but also in cell survival in the face of chemotherapy. In paralel, during the course of unbiased expression-quantitative trait loci (eQTL) studies, we identified another variant, rs3743251, which creates a new miR-binding site and correlates with decreased IGF1R mRNA levels. Our current overall hypothesis is that rs3833015 and rs374251 change IGF1 signaling (up and down, respectively), resulting in altered resistance to conventional chemotherapeutic agents and small-molecule IGF1R inhibitors. We will pursue this hypothesis in the following two Aims. 1. To determine the mechanisms of IGF1R deregulation by rs3833015 and rs3743251 and to identify new SNPs linked to IGF1R over/under-expression. 2. To investigate the contribution of IGF1R variants to neuroblastoma sensitivity to small-molecule IGF1R inhibitors and chemotherapeutic agents. To this end, we will correlate IGF1R 3'UTR genotypes with survival of NB patients using Kaplan-Meier analysis and compare and contrast responses of rs3833015 and rs374251 tumors to conventional therapeutics (cyclophosphamide and topotecan) and IGF1R inhibitors. The main impact of this study is that it would allow prospective stratification of patients enrolled in clinical trials of IGF1R inhibitors nd more robust responses to these drugs, ushering in the era of miR pharmacogenomics.