A new mouse model for testing splice-switching therapies in IGF-driven cancers

摘要 肝细胞癌（HCC）被预测为第六大最常见的诊断癌症， 全球第四大癌症死亡原因。发病率和死亡率都高出2至3倍 因此，肝癌在男性死亡人数中居第二位。仅在美国， 估计到2020年将有42，810名成年人（31，762名男性和11，048名女性）被诊断患有原发性肝癌。 这些统计数据，加上肝癌的死亡率在过去的一年中增加了43%， 十年，需要非常规的治疗方法。基因组研究已经确立了 然而，只有约25%的肿瘤具有已知的靶向驱动因子。另 另一方面，高通量测序技术的最新进展已经揭示了惊人数量的 与肿瘤发生相关的可变剪接变体，涉及肿瘤中的剪接失调 表型。因此，我们将注意力转向肝癌中的选择性剪接或异常剪接转录本 “剪接体”来鉴定新的治疗靶点。 胰岛素受体已经独特地进化为经历选择性剪接以产生两种亚型： 长度INSR-B和外显子11跳过INSR-A同种型。来自TCGA肝癌队列的数据以及我们自己的数据 多个内部患者组显示正常肝组织主要表达胰岛素受体B同种型， 而人HCC患者样品表达更多的INSR-A。INSR-A除了与胰岛素结合外， 对IGF 2的亲和力异常高，并加速肿瘤细胞标志如增殖和 血管生成我们的数据进一步表明，INSR-B向INSR-A的这种转化是在以下条件下发生的： 应激条件如缺氧。这些观察结果与HCC特别相关，因为1）Hif 1a具有 显示出显著升高，并与HCC的恶化进展相关; 2）IGF 2已被 称为HCC的表观遗传致癌驱动因子。因此，我们假设，改变剪接模式， 肝癌中的INSR将消除下游的增殖信号传导并阻碍肿瘤的发生过程。 为了实现治疗干预，我们建议使用剪接转换寡核苷酸（SSO）技术， 恢复肝细胞中正常的INSR剪接模式。在这项提案中，我们的目标是产生一个临床相关的 HCC的小鼠模型，忠实地再现了在人类条件下观察到的INSR剪接变化。的 目前的HCC小鼠模型不表达INSR选择性剪接亚型，因此不能预测 对靶向IGF途径的治疗的反应性。因此，迫切需要新的小鼠模型 这将允许准确测试治疗方式。

ABSTRACT Hepatocellular Carcinoma (HCC) is predicted to be the sixth most commonly diagnosed cancer and the fourth leading cause of cancer death worldwide. Rates of both incidence and mortality are 2 to 3 times higher among men and thus liver cancer ranks second in terms of deaths for males. In the United States alone, an estimated 42,810 adults (31,762 men and 11,048 women) will be diagnosed with primary liver cancer in 2020. These statistics, combined with the fact that the death rate of liver cancer has increased by 43% in the last decade, necessitates unconventional treatment approaches. Genomic studies have established the landscape of molecular changes in HCC, however, only ~25% of tumors harbor known targetable drivers. On the other hand, recent advances in high throughput sequencing technologies have uncovered a surprising number of alternatively spliced variants associated with tumorigenesis, implicating de-regulated splicing in the tumor phenotype. Hence, we have turned our attention to the alternatively or aberrantly spliced transcripts in the HCC “spliceome” to identify new therapeutic targets. Insulin receptor has uniquely evolved to undergo alternative splicing to produce two isoforms: the full- length INSR-B and exon 11 skipped INSR-A isoform. Data from TCGA liver cancer cohorts as well as our own multiple in-house patient cohorts show that normal liver tissue primarily expresses the insulin receptor B isoform, whereas human HCC patient samples express more INSR-A. INSR-A, in addition to binding to insulin, has abnormally high affinity for IGF2 and accelerates the onset of tumor-cell hallmarks like proliferation and angiogenesis. Our data further show that this conversion of INSR-B to INSR-A takes place in the presence of stress conditions such as hypoxia. These observations are particularly relevant to HCC because 1) Hif1a has been shown to be significantly elevated and associated with worse progression in HCC and 2) IGF2 has been referred to as an epigenetic onco-driver of HCC. We therefore hypothesize that altering the splice pattern of INSR in liver cancer will abrogate the proliferative signaling downstream and impede the tumorigenic process. To achieve therapeutic intervention, we propose to use splice-switching oligonucleotide (SSO) technology to restore the normal INSR splicing pattern in liver cells. In this proposal, we aim to generate a clinically relevant mouse model of HCC that faithfully recapitulates the INSR splicing changes seen in the human condition. The current HCC mouse models do not express INSR alternatively spliced isoforms and thus do not predict responsiveness to therapies targeting the IGF pathway. There is therefore a critical need for new mouse models of HCC that will allow accurate testing of therapeutic modalities.