Inducing PKM splice-switching with antisense oligonucleotides as an approach to treat hepatocellular carcinoma

项目摘要 肝癌仍然是全球最致命的癌症之一，仅次于胰腺导管。 腺癌(PDAC)，其中肝细胞癌(HCC)至少占肝癌病例的85%。 肝细胞癌最有效的治疗选择是早期到中期的肝细胞癌。不幸的是，由于 大多数肝细胞癌患者在早期没有体征和症状，直到晚期才被确诊。 因此，只能用系统疗法来治疗。晚期肝癌的一线治疗是 最近更新为阿特唑珠单抗和贝伐单抗的联合治疗，虽然这次更新是 令人鼓舞的是，无进展存活率目前保持在7个月左右。因此，更有效的战略是 治疗晚期肝癌仍然是迫切需要的。我们实验室最近发现了一种反义寡核苷酸(ASO) 它通过选择性剪接丙酮酸激酶(PKM)前mRNA来靶向葡萄糖代谢，这可以 被用作治疗肝癌的一种方法。PKM前mRNA经历了相互排斥的选择性剪接，从而导致 表达PKM1或PKM2亚型。众所周知，PKM2在肝细胞癌中优先上调。 其较低的酶活性可在糖酵解结束时造成瓶颈，从而潜在地促进肿瘤生长。 通过将上游糖酵解中间体分流到各种生物合成途径中。鉴于PKM1具有更高的 酶活性，我们基于ASO的PKM剪接交换(APSS)疗法旨在重定向替代 将PKM2剪接到PKM1，从而缓解PKM2诱导的瓶颈，减少PKM2的积累 糖酵解中间体。目前，我们的治疗已经在两个临床前模型中实现了肿瘤生长的减少。 肝细胞癌。尽管有这些有希望的结果，但我们还没有建立APSS的精确代谢解释 治疗导致肝细胞癌生长减少，并评估其与当前肝细胞癌的联合疗效。 治疗。我的假设是，我们的APSS疗法减少了肝细胞癌中丝氨酸的合成，并促进了依赖性 胞外丝氨酸，以维持嘌呤核苷酸的产生。另外，我假设这一组合 用索拉非尼治疗APSS将使对索拉非尼耐药的肝细胞癌肿瘤重新敏感。我计划在体内利用 LC-MS示踪肝癌移植瘤中的稳定同位素以获得完整的肝细胞癌轮廓 APSS治疗后的代谢反应。此外，我还将建立耐索拉非尼的肝细胞癌移植瘤 以确定我们的APSS疗法和索拉非尼之间的潜在协同作用。建议的重要意义 研究表明，它将：(I)提高我们对PKM选择性剪接在肿瘤发生中的理解；(Ii)有助于 确定增强我们的APSS治疗效果的协同疗法；以及(Iii)提供进一步的理由 用于在晚期肝癌的临床试验中对我们的APSS疗法进行最终测试。

Project Summary Liver cancer remains one of the most lethal cancers worldwide, second only to pancreatic ductal adenocarcinoma (PDAC), with hepatocellular carcinoma (HCC) making up at least 85% of liver cancer cases. The most effective treatment options for HCC are for early- to intermediate-stage HCC. Unfortunately, due to the absence of signs and symptoms in the early stages, most HCC patients are not diagnosed until advanced-stage of disease, and therefore can only be treated with systemic therapies. First-line therapy for advanced HCC was recently updated to a combination treatment of atezolizumab plus bevacizumab, and while this update is encouraging, progression-free survival currently remains around 7 months. Thus, more effective strategies to treat advanced HCC are still desperately needed. Our lab recently identified an antisense oligonucleotide (ASO) that targets glucose metabolism through alternative splicing of pyruvate kinase (PKM) pre-mRNA, which could be used as a therapy to treat HCC. PKM pre-mRNA undergoes mutually exclusive alternative splicing that results in expression of either the PKM1 or PKM2 isoform. PKM2 is well known to be preferentially upregulated in HCC. Its low enzymatic activity can create a bottleneck at the end of glycolysis that potentially promotes tumor growth by shunting upstream glycolytic intermediates into various biosynthesis pathways. Given that PKM1 has higher enzymatic activity, our ASO-based PKM splice-switching (APSS) therapy is designed to redirect alternative splicing from PKM2 to PKM1, thereby relieving the PKM2-induced bottleneck, and reducing the accumulation of glycolytic intermediates. Currently, our therapy has achieved reduced tumor growth in two pre-clinical models of HCC. Despite these promising results, we have yet to establish a precise metabolic explanation by which APSS therapy results in reduced HCC growth, as well as to evaluate its efficacy in combination with current HCC therapies. My hypothesis is that our APSS therapy reduces serine synthesis in HCC and promotes dependence on extracellular serine to sustain production of purine nucleotides. Additionally, I hypothesize that combination treatment of APSS therapy with sorafenib will re-sensitize sorafenib-resistant HCC tumors. I plan to utilize in vivo stable isotope tracing with LC-MS in HCC xenografts in order to obtain a comprehensive profile of HCC metabolism in response to APSS therapy. Additionally, I will establish sorafenib-resistant HCC xenografts in order to identify potential synergy between our APSS therapy and sorafenib. The significance of the proposed research is that it will: (i) improve our understanding of PKM alternative splicing in tumorigenesis; (ii) help to identify synergistic therapies that reinforce the effects of our APSS therapy; and (iii) provide further justification for the eventual testing of our APSS therapy in clinical trials for advanced HCC.