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

摘要 据预测，肝细胞癌是第六种最常见的癌症， 全球第四大癌症死亡原因。发病率和死亡率都高出2到3倍。 因此，在男性死亡人数中，肝癌位居第二。仅在美国，一个 据估计，2020年将有42,810名成年人(31,762名男性和11,048名女性)被诊断为原发性肝癌。 这些统计数字，再加上过去一年中肝癌死亡率增加了43% 十年，有必要采用非常规治疗方法。基因组研究已经确定了这种景观 然而，在肝细胞癌的分子变化中，只有~25%的肿瘤具有已知的靶向驱动因素。另一方面 另一方面，高通量测序技术的最新进展揭示了数量惊人的 与肿瘤发生相关的选择性剪接变异体，与肿瘤中的剪接缺失有关 表型。因此，我们将注意力转向肝细胞癌中交替或异常剪接的转录本。 “拼接体”以确定新的治疗靶点。 胰岛素受体经过独特的进化，经历了选择性剪接，产生了两种异构体：完整的 长度为INSR-B和外显子11跳过了INSR-A亚型。来自TCGA肝癌队列以及我们自己的数据 多个内部患者队列显示，正常肝组织主要表达胰岛素受体B亚型， 而人肝细胞癌患者样本表达更多的INSR-A。INSR-A除了与胰岛素结合外，还具有 对IGF2的异常高亲和力，并加速肿瘤细胞特征的开始，如增殖和 血管生成。我们的数据进一步表明，INSR-B到INSR-A的这种转换发生在 应激条件，如缺氧。这些观察结果与肝癌特别相关，因为1)HIF1a 已被证明显著升高，并与肝细胞癌的恶化有关，以及2)IGF2已被证明是 被认为是肝细胞癌的表观遗传致癌因素。因此，我们假设改变剪接模式 在肝癌中，INSR将取消下游的增殖信号，阻碍肿瘤的发生过程。 为了实现治疗干预，我们建议使用剪接开关寡核苷酸(SSO)技术来 使肝细胞恢复正常的INSR剪接模式。在这项提案中，我们的目标是产生一种临床相关的 小鼠的肝细胞癌模型，忠实地概括了在人类条件下看到的INSR剪接变化。这个 目前的肝癌小鼠模型不表达INSR选择性剪接异构体，因此不能预测 对针对IGF途径的治疗的反应性。因此，迫切需要新的鼠标模型 这将使治疗方式的准确测试成为可能。

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.