Mechanisms by which of GPCR Signaling Inhibits Acute Myeloid Leukemia

GPCR 信号传导抑制急性髓系白血病的机制

• 批准号: 10315637
• 负责人: In Young Lee
• 金额: $ 4.6万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-12 至 2023-07-11
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10315637
• 关键词: ANXA5 gene; Acute Myelocytic Leukemia; Adult; Agonist; Animal Model; Animals; Apoptosis; Apoptotic; Biological Assay; Bioluminescence; Blood Cell Count; Caspase; Cell Death; Cell Line; Cell Proliferation; Cell Survival; Cells; Cessation of life; Cleaved cell; Clinic; Clinical; Clinical assessments; Combined Modality Therapy; Coupled; Cytarabine; Data; Decitabine; Development; Dinoprostone; Disease; Dose-Limiting; Estrogen Receptor alpha; Estrogens; Exposure to; FLT3 gene; FLT3 inhibitor; Female; Flow Cytometry; G Protein-Coupled Receptor Signaling; G-Protein-Coupled Receptors; GPER gene; GTP-Binding Protein alpha Subunits, Gs; Growth; H2 gene; Histamine; Human; Immunoblotting; In Vitro; Label; Link; MCL1 gene; Malignant Neoplasms; Measures; Mediating; Messenger RNA; Modeling; Mus; Mutation; Null Lymphocytes; Outcome; Pancreatic Ductal Carcinoma; Patients; Pharmaceutical Preparations; Pharmacology; Phase; Proteins; Proto-Oncogene Proteins c-akt; Receptor Activation; Recurrence; Regulation; Resistance development; Sampling; Signal Pathway; Signal Transduction; Stains; Subgroup; Survival Rate; TdT-Mediated dUTP Nick End Labeling Assay; Testing; Therapeutic; Toxic effect; Tumor Burden; Work; Xenograft Model; acute myeloid leukemia cell; base; cancer cell; cancer therapy; cancer type; chemotherapy; efficacy testing; experience; experimental study; genetic manipulation; immunogenicity; in vivo; leukemia; male; melanoma; mouse model; mutant; mutational status; novel therapeutic intervention; novel therapeutics; older patient; overexpression; pre-clinical; preclinical study; receptor; relapse patients; response; standard of care; targeted treatment; therapeutic evaluation; therapeutic target

Project Summary: Despite recent advances in cancer therapy the 5-year overall survival rate of Acute Myeloid Leukemia (AML) remains around 30%. The first line standard of care therapy for AML is chemotherapy, which is ineffective in relapsed patients and often too toxic to be administered to older patients. Targeted therapy such as venetoclax and decitabine have only shown modest effects in the clinic, calling for different approaches in targeting AML. Our lab has shown that one such approach is through the activation of the Gs-coupled G-protein coupled receptor, G-protein Coupled Estrogen Receptor (GPER). Work from our lab has shown that pharmacological activation of GPER with LNS8801 induces differentiation, promotes growth inhibition, and drives immunogenicity in melanoma without observable toxicity in animal models. I have shown that primary AML cells and AML cell lines that are exposed to LNS8801 induce cell death, which is likely through apoptosis based on positive Annexin V and PI staining, in addition to growth arrest. LNS8801 induced cell death is accompanied by the depletion of Mcl-1, which is an anti-apoptotic protein that is often overexpressed in cancer cells. Additionally, I found that death response to GPER activation highly correlates with the mutational status of FLT3, specifically FLT3-ITD. FLT3-ITD mutants maintain survival through constitutive activation of AKT, which consequently maintains Mcl-1 expression. Although FLT3 inhibitors are used in clinic, patients inevitably succumb to disease due to development of resistance. FLT3-ITD vulnerability to GPER activation indicates that LNS8801 may be able to be used as a novel therapeutic approach for FLT3-ITD mutant patients. Seeing that activation of GPER may lower the apoptotic threshold in AML, I tested whether LNS8801 can enhance the efficacy of cytarabine in vitro and showed that combination therapy is more effective than either drug alone. Based on these preliminary data, I will perform experiments to 1) Define the mechanism by which Gs-coupled GPCRs induce apoptosis in AML cells and to 2) test efficacy of LNS8801 alone and combination with cytarabine in vivo AML models. In aim 1, I will first determine whether the depletion of Mcl-1 is responsible for cell death by genetically manipulating the expression of Mcl-1 in the presence or absence of drug. Next, I will determine whether Mcl-1 depletion is mediated through AKT depletion. I will then test whether FLT3-ITD mutation renders cells more vulnerable to GPCR activation by measuring the viability of a panel of FLT3-ITD and FLT3wt primary samples that are exposed to GPCR agonists. I will also overexpress FLT3-ITD in FLT3wt and FLT3 null cells to test whether they become more sensitive to GPCR activation. Next, in aim 2, I will test whether LNS8801 alone can effectively inhibit AML in vivo. I will also determine whether LNS8801 enhances the efficacy of cytarabine in vivo with limited toxicity. Together, these aims will define the mechanism of Gs- coupled GPCR induced apoptosis in AML cells and test the efficacy of LNS8801 in vivo, which may further help development of novel therapeutics for AML, especially for the more vulnerable and older patients.

项目摘要： 尽管最近在癌症治疗方面取得了进步，但急性髓样白血病（AML）的5年总体生存率（AML） 仍然保持约30％。 AML护理疗法的第一行标准是化学疗法，这在 复发患者，通常过于毒性，无法对老年患者施用。靶向疗法，例如venetoclax Decitabine仅在诊所显示出适度的影响，呼吁针对AML采用不同的方法。 我们的实验室表明，一种这样的方法是通过激活GS耦合G蛋白耦合 受体，G蛋白偶联雌激素受体（GPER）。我们实验室的工作表明药理学 用LNS8801激活GPER会诱导分化，促进生长抑制和驱动 黑色素瘤的免疫原性，没有可观察到的动物模型毒性。我已经证明了主要AML 暴露于LNS8801的细胞和AML细胞系诱导细胞死亡，这可能通过凋亡 基于阳性膜联蛋白V和PI染色，除了增长停滞。 LNS8801诱导的细胞死亡是 伴随着MCL-1的耗竭，Mcl-1是一种抗凋亡蛋白，通常在癌症中过表达 细胞。此外，我发现对GPER激活的死亡反应高度与突变状态相关 flt3，特别是flt3-itd。 FLT3-ITD突变体通过AKT的构型激活保持生存 因此，它保持MCL-1表达。尽管FLT3抑制剂用于诊所，但不可避免地会患者 由于抵抗的发展而屈服于疾病。 FLT3-ITD易受GPER激活的脆弱性指示 该LNS8801可以用作FLT3-ITD突变患者的新型治疗方法。看到 GPER的激活可能会降低AML中的凋亡阈值，我测试了LNS8801是否可以增强 细胞滨在体外的功效，表明联合疗法比单独使用任何一种药物更有效。 基于这些初步数据，我将执行实验至1）定义GS耦合的机制 GPCR在AML细胞中诱导凋亡，至2）仅LNS8801的测试功效，并与 亚tarabine在体内AML模型。在AIM 1中，我将首先确定MCL-1的耗竭是否负责 细胞死亡通过在存在或不存在药物的情况下遗传操纵MCL-1的表达。接下来，我会的 确定MCL-1耗竭是否是通过AKT耗竭介导的。然后我将测试是否flt3-itd 突变通过测量FLT3-ITD面板的生存能力，使细胞更容易受到GPCR激活的影响 暴露于GPCR激动剂的FLT3WT主要样品。我还将在flt3wt中过表达flt3-itd 和FLT3无效细胞测试它们是否对GPCR激活更敏感。接下来，在AIM 2中，我将测试 仅LNS8801是否可以有效地抑制AML体内。我还将确定LNS8801是否增强 毒性有限的体内细胞押滨在体内的功效。这些目标共同定义了GS-的机制 耦合的GPCR诱导AML细胞凋亡，并测试LNS8801在体内的功效，这可能会进一步 帮助开发用于AML的新型治疗剂，特别是对于更脆弱和老年患者。