Development of Microbial-Based Therapies to Suppress Macropinocytosis in Kras-Driven Cancers

项目摘要 胰腺导管腺癌 (PDAC) 是一种高度侵袭性疾病，预后不佳 预后。 PDAC 近乎普遍的致癌驱动因素是小 GTP 酶蛋白的组成型激活 Kras 可诱导多个下游信号级联反应，共同促进细胞快速增殖， 转移和治疗耐药。为了克服这些活动的高能量需求，克拉斯还 触发代谢适应，促进细胞外来源的营养物清除，例如通过 巨胞饮作用。巨胞饮作用是细胞外物质被非特异性吞噬并被吞噬的过程。 然后在溶酶体中降解产生肿瘤细胞用于生物合成的最终产物。这个过程 本质上赋予了对多种合成代谢抑制剂的抵抗力。 Syndecan-1 是一种硫酸乙酰肝素蛋白多糖 (HSPG) 在细胞表面上调，是 PDAC 中巨胞饮作用的关键介质， 其他由 Kras 驱动的癌症，包括膀胱癌、肺癌、前列腺癌、结肠癌和乳腺癌。除了调解之外 大分子运输，HSPGs可以在肿瘤细胞外基质（ECM）中结合并调节 许多信号分子（例如生长因子和细胞因子）与其同源受体的相互作用。 HSPG 的促肿瘤活性可通过对其乙酰肝素进行酶促修饰来精确调节 硫酸盐（HS）部分。哺乳动物乙酰肝素酶利用 β-(1,4)-糖苷键的水解裂解 葡萄糖醛酸和葡萄糖胺之间的作用，促进生长因子和酶的释放 ECM 重塑、侵袭和转移。与哺乳动物肝素酶相比，细菌肝素酶 III (HepIII) 通过独特的 β 消除机制解聚 HSPG，该机制在 α-(1,4)- 处裂解 糖苷键。各种研究已证实 HSPG 的 HepIII 修饰可抑制新血管形成， 巨胞饮作用、肿瘤生长和转移。然而，无法限制肿瘤组织的 HepIII 活性 长期以来禁止将其用作治疗剂。使用减毒、肿瘤靶向鼠伤寒沙门氏菌 (ST) 载体，我们通过 严格调控的诱导型启动子。我们已经证实 ST-HepIII 具有抑制高亲和力 HS 的能力 Kras 突变肿瘤的相互作用、巨胞饮作用和生长。在此应用中，我们将： 1) 确定 ST-HepIII 治疗对体内代谢物利用率和代谢相关基因途径的影响； 2） 确定合成代谢抑制剂与 ST-HepIII 组合的抗肿瘤功效； 3) 开发和 表征在肿瘤诱导启动子下表达 HepIII 的重组 ST，以获得更大的临床可行性。 完成这些目标将使我们能够开发出一类新型的肿瘤靶向药物，能够抑制 对于 PDAC 和其他 Kras 驱动的癌症的生存至关重要的代谢过程。我们的代理可能会被使用 抵消对针对合作合成代谢过程的标准护理疗法的获得性耐药性和 对难治性癌症患者的生存产生积极影响。

PROJECT SUMMARY Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive disease with dismal prognosis. A near-universal oncogenic driver of PDAC is the constitutive activation of the small GTPase protein Kras, which induces multiple downstream signaling cascades that together facilitate rapid cell proliferation, metastasis and therapeutic resistance. To surmount the high energy demands of these activities, Kras also triggers metabolic adaptations to promote nutrient scavenging from extracellular sources, such as through macropinocytosis. Macropinocytosis is a process by which extracellular material is non-specifically engulfed and then degraded in lysosomes to produce end-products utilized by tumor cells for biosynthesis. This process essentially confers resistance to a myriad of anabolic inhibitors. Syndecan-1 is a heparan sulfate proteoglycan (HSPG) upregulated on the surface of cells that serves as the key mediator of macropinocytosis in PDAC and other Kras-driven cancers that includes bladder, lung, prostate, colon and breast. In addition to mediating macromolecular transport, HSPGs can be found in the tumor extracellular matrix (ECM) binding to and regulating the interaction of numerous signaling molecules (e.g. growth factors and cytokines) with their cognate receptors. The pro-tumorigenic activities of HSPGs are exquisitely regulated by enzymatic modification of their heparan sulfate (HS) moieties. Mammalian heparanases employ hydrolytic cleavage of the beta-(1,4)-glycosidic bond between glucuronic acid and glucosamine to promote the release of growth factors and enzymes involved in ECM remodeling, invasion and metastasis. In contrast to mammalian heparanases, bacterial heparinase III (HepIII) depolymerizes HSPGs through a unique beta-elimination mechanism that cleaves at the alpha-(1,4)- glycosidic bond. Various studies have confirmed HepIII modification of HSPGs suppresses neovascularization, macropinocytosis, tumor growth and metastasis. However, the inability to restrict HepIII activity to tumor tissue has long prohibited its use as a therapeutic agent. Using attenuated, tumor-targeting Salmonella typhimurium (ST) vectors, we have developed the first recombinant ST expressing functional HepIII (ST-HepIII) through a tightly regulated, inducible promoter. We have confirmed the ability of ST-HepIII to suppress high-affinity HS interactions, macropinocytosis, and growth of Kras-mutant tumors. In this application, we will: 1) Determine the impact of ST-HepIII treatment on metabolite availability and metabolic-associated gene pathways in vivo; 2) Determine anti-tumor efficacy of anabolic inhibitors in combination with ST-HepIII; and 3) Develop and characterize recombinant STs expressing HepIII under tumor-inducible promoters for greater clinical feasibility. Completing these aims will allow us to develop a novel class of tumor-targeting agents capable of suppressing a metabolic process essential to the survival of PDAC and other Kras-driven cancers. Our agents may be used to counteract acquired resistance to standard-of-care therapies that target cooperative anabolic processes and positively impact survival for patients with difficult-to-treat cancers.