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

项目摘要 肝癌仍然是世界上最致命的癌症之一，仅次于胰腺导管癌。 腺癌（PDAC），其中肝细胞癌（HCC）占肝癌病例的至少85%。 HCC最有效的治疗选择是早期至中期HCC。不幸的是，由于 由于在早期阶段没有体征和症状，大多数HCC患者直到晚期才被诊断 因此，只能通过全身治疗来治疗。晚期肝癌的一线治疗是 最近更新为atezolizumab+贝伐珠单抗的联合治疗，虽然该更新是 令人鼓舞的是，无进展生存期目前保持在7个月左右。因此，更有效的战略， 仍然迫切需要治疗晚期HCC。我们实验室最近发现了一种反义寡核苷酸（阿索） 通过丙酮酸激酶（PKM）前体mRNA的选择性剪接靶向葡萄糖代谢， 用作治疗HCC的疗法。PKM前体mRNA经历相互排斥的选择性剪接， 在PKM 1或PKM 2亚型的表达中。众所周知，PKM 2在HCC中优先上调。 它的低酶活性可以在糖酵解结束时产生瓶颈，从而可能促进肿瘤生长 通过将上游糖酵解中间产物分流到各种生物合成途径中。由于PKM 1具有更高的 酶的活性，我们的基于ASO的PKM剪接转换（APSS）疗法旨在重新定向替代 从PKM 2剪接到PKM 1，从而缓解PKM 2诱导的瓶颈，并减少 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， HCC。尽管有这些有希望的结果，我们还没有建立一个精确的代谢解释， 治疗导致HCC生长减少，以及评估其与当前HCC联合治疗的疗效 治疗我的假设是，我们的APSS疗法减少了肝癌中丝氨酸的合成， 以维持嘌呤核苷酸的产生。另外，我假设 用索拉非尼进行APSS治疗将使索拉非尼抗性HCC肿瘤再敏感。我计划在体内使用 在HCC异种移植物中使用LC-MS进行稳定同位素示踪，以获得HCC的全面概况 代谢对APSS治疗的反应。此外，我将建立索拉非尼耐药的HCC异种移植物， 以确定我们的APSS治疗和索拉非尼之间的潜在协同作用。建议的意义 这项研究将：（i）提高我们对肿瘤发生中PKM选择性剪接的理解;（ii）有助于 确定加强我们的APSS治疗效果的协同疗法;（iii）提供进一步的理由 用于在晚期肝癌临床试验中最终测试我们的APSS疗法。

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.