Serial transcriptome analysis and cross species integration identifies CENPE as novel neuroblastoma target. Mus musculus

Cancer genomic studies that rely on analysis of diagnostic biopsies from primary tumors may not fully identify the molecular events associated with tumor progression. We hypothesized that characterizing the transcriptome during tumor progression in the TH-MYCN transgenic neuroblastoma model would identify oncogenic drivers that would be targetable therapeutically. We quantified expression of 32,381 murine genes in 9 hyperplastic ganglia harvested at 3 time points, and 4 tumor cohorts of progressively larger size (n=6 each group) in mice homozygous for the TH-MYCN transgene. We found 93 genes that showed a linearly increasing or decreasing pattern of expression from the preneoplastic ganglia to end stage tumors. Cross-species integration identified 24 genes that were highly expressed in human MYCN amplified neuroblastomas. The genes prioritized were not exclusively driven by increasing Myc transactivation or proliferative rate. We manually curated the 24 candidates and prioritized 3 targets (Cenpe, Gpr49, Impdh2) with previously determined roles in cancer. Using siRNA knockdown in human neuroblastoma cell line models we further prioritized CENPE due to potent inhibition of cellular proliferation. Targeting of CENPE with the selective small molecular inhibitor GSK923295 showed potent inhibition of in vitro proliferation of 19 neuroblastoma derived cell lines (median IC50=41 nM; range 27-266 nM), and significantly delayed tumor growth kinetics in 3 xenograft models (p-values ranged from p<0.0001 to p=0.018). Here we provide preclinical validation that serial transcriptome analysis of a transgenic mouse model followed by cross-species integration is a useful method to identify therapeutic targets, and identify CENPE as a novel therapeutic candidate in neuroblastoma. Overall design: set 1: 24 tumor samples analyzed set 2: 9 hyperplastic ganglia harvested at 3 time points (day 0, day 7 and day 14) and 2 tumors "small" size were analysed

依赖原发性肿瘤诊断活检分析的癌症基因组学研究，或无法全面识别与肿瘤进展相关的分子事件。我们提出假说：对TH-MYCN转基因神经母细胞瘤模型的肿瘤进展过程进行转录组（transcriptome）特征分析，可识别具备治疗靶向潜力的致癌驱动因子。我们对纯合TH-MYCN转基因小鼠的3个时间点采集的9个增生性神经节，以及4组体积逐步增大的肿瘤队列（每组n=6）中的32381个小鼠基因表达量进行了定量分析。我们发现，从癌前神经节到终末期肿瘤，共有93个基因的表达呈现线性上调或下调模式。跨物种整合分析识别出24个在人源MYCN扩增型神经母细胞瘤中高表达的基因。上述优先筛选得到的基因并非仅由Myc反式激活增强或增殖速率提升所驱动。我们手动整理了这24个候选基因，并优先选出3个已被证实与癌症相关的靶点：Cenpe、Gpr49及Impdh2。在人源神经母细胞瘤细胞系模型中通过小干扰RNA（siRNA）进行基因敲低实验后，我们进一步优先筛选出CENPE，因其可强效抑制细胞增殖。使用选择性小分子抑制剂GSK923295靶向CENPE，可强效抑制19株神经母细胞瘤来源细胞系的体外增殖（半数抑制浓度IC50中位数为41 nM，范围27~266 nM），并在3个异种移植（xenograft）模型中显著延缓肿瘤生长动力学（p值范围为p<0.0001至p=0.018）。本研究提供的临床前验证结果表明：对转基因小鼠模型进行连续转录组分析，再结合跨物种整合分析，是一种有效的治疗靶点识别方法，并确认CENPE可作为神经母细胞瘤的新型治疗候选靶点。整体实验设计：组1：分析24份肿瘤样本；组2：分析3个时间点（第0天、第7天和第14天）采集的9份增生性神经节样本，以及2份小型肿瘤样本。