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

• 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
• 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

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），肝细胞癌（HCC）至少占肝癌病例的85％。 HCC的最有效的治疗选择是针对中级HCC的早期至中级HCC。不幸的是，由于 早期缺乏体征和症状，大多数HCC患者直到高级阶段才被诊断 疾病，因此只能通过全身疗法治疗。高级HCC的一线治疗是 最近更新到Atezolizumab Plus bevacizumab的组合处理方法，尽管此更新是 目前，令人鼓舞，无进展的生存仍保留在7个月左右。因此，更有效的策略 迫切需要治疗先进的HCC。我们的实验室最近确定了反义寡核苷酸（ASO） 通过丙酮酸激酶（PKM）pre-mRNA的替代剪接靶向葡萄糖代谢，可以 被用作治疗HCC的疗法。 PKM Pre-mRNA经历互斥的替代剪接，结果 在表达PKM1或PKM2同工型中。众所周知，PKM2在HCC中优先上调。 其低酶活性可以在糖酵解结束时产生瓶颈，从而有可能促进肿瘤生长 通过将上游糖酵解中间体转移到各种生物合成途径中。鉴于PKM1具有较高 酶活性，我们基于ASO的PKM剪接开关（APSS）疗法旨在重定向替代方案 从PKM2剪接到PKM1，从而减轻了PKM2诱导的瓶颈，并减少了 糖酵解中间体。目前，我们的疗法已在两个临床前模型中降低了肿瘤的生长 HCC。尽管这些有希望的结果，我们尚未建立一个精确的代谢解释 治疗导致HCC生长降低，并评估其与当前HCC的功效 疗法。我的假设是，我们的APSS治疗减少了HCC中的丝氨酸合成并促进依赖性 在细胞外丝氨酸上维持嘌呤核苷酸的产生。另外，我假设该组合 用索拉非尼治疗APSS治疗将重新敏感性索拉非尼的HCC肿瘤。我打算在体内使用 稳定的同位素跟踪HCC异种移植物中的LC-MS，以获得HCC的全面概况 响应APSS治疗的代谢。此外，我将在 为了确定我们的APSS治疗和索拉非尼之间的潜在协同作用。提议的意义 研究将是：（i）提高对PKM肿瘤发生中替代剪接的理解； （ii）帮助 确定增强APSS治疗作用的协同疗法； （iii）提供进一步的理由 最终测试我们的APSS治疗在晚期HCC的临床试验中。