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

用反义寡核苷酸诱导 PKM 剪接转换作为治疗肝细胞癌的方法

基本信息

批准号：
10464020
负责人：
Dillon Matthew Voss
金额：
$ 3.91万
依托单位：
STATE UNIVERSITY NEW YORK STONY BROOK
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-06-01 至 2027-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10464020
关键词：
Alternative Splicing Amino Acid Transporter Anabolism Antisense Oligonucleotide Therapy Antisense Oligonucleotides Apoptosis BAY 54-9085 CASP3 gene Carbon Cell Line Clinical Trials Combined Modality Therapy Data Dependence Disease Enzyme Inhibition Enzymes Future Genes Glucose Glycolysis Goals Growth In Vitro Incidence Knowledge Label Malignant Epithelial Cell Malignant Neoplasms Malignant neoplasm of liver Measures Metabolic Metabolism Pancreatic Ductal Adenocarcinoma Pathway interactions Patients Persons Pharmacology Pre-Clinical Model Primary carcinoma of the liver cells Production Progression-Free Survivals Protein Isoforms Proteins Purine Nucleotides Pyruvate Kinase RNA Splicing Research Resistance Serine Signs and Symptoms Stains Systemic Therapy Testing Therapeutic Tissues United States Update Western Blotting Work Xenograft Model Xenograft procedure base bevacizumab design effective therapy extracellular glucose metabolism improved in vivo liver cancer model mRNA Precursor metabolic phenotype nucleotide metabolism response stable isotope synergism tumor tumor growth tumor metabolism tumor microenvironment tumorigenesis

项目摘要

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.

项目概要肝癌仍然是全球最致命的癌症之一，仅次于胰腺导管癌腺癌 (PDAC)，其中肝细胞癌 (HCC) 至少占肝癌病例的 85%。 HCC 最有效的治疗方案是针对早期至中期 HCC。不幸的是，由于早期没有体征和症状，大多数 HCC 患者直到晚期才被诊断出来因此只能通过全身治疗来治疗。晚期 HCC 的一线治疗是最近更新为阿特珠单抗加贝伐珠单抗的联合治疗，虽然此更新是令人鼓舞的是，目前无进展生存期仍约为 7 个月。因此，更有效的策略仍然迫切需要治疗晚期 HCC。我们的实验室最近发现了一种反义寡核苷酸（ASO）通过丙酮酸激酶 (PKM) 前体 mRNA 的选择性剪接来靶向葡萄糖代谢，这可以用作治疗 HCC 的疗法。 PKM pre-mRNA 经历相互排斥的选择性剪接，导致 PKM1 或 PKM2 同工型的表达。众所周知，PKM2 在 HCC 中优先上调。其酶活性低，可能会在糖酵解结束时形成瓶颈，从而可能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 剪接转换 (APSS) 疗法旨在重定向替代疗法 PKM2 剪接至 PKM1，从而缓解 PKM2 引起的瓶颈，并减少糖酵解中间体。目前，我们的疗法已在两种临床前模型中实现了肿瘤生长的减少肝癌。尽管取得了这些有希望的结果，但我们尚未建立 APSS 的精确代谢解释治疗可减少 HCC 生长，并评估其与当前 HCC 结合的疗效疗法。我的假设是我们的 APSS 疗法减少了 HCC 中的丝氨酸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。此外，我假设该组合使用索拉非尼进行 APSS 治疗将使索拉非尼耐药的 HCC 肿瘤重新变得敏感。我打算在体内使用在 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 APSS 治疗的代谢反应。此外，我将在以下国家建立索拉非尼耐药的 HCC 异种移植物为了确定我们的 APSS 疗法和索拉非尼之间的潜在协同作用。拟议的意义研究表明它将：（i）提高我们对肿瘤发生中 PKM 选择性剪接的理解； (二) 帮助确定增强 APSS 疗法效果的协同疗法； (iii) 提供进一步的理由在晚期 HCC 的临床试验中最终测试我们的 APSS 疗法。