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

基本信息

批准号：
10457285
负责人：
In Young Lee
金额：
$ 3.36万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-07-12 至 2023-07-03
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10457285
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 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 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 Western Blotting 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

项目摘要

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.

项目概要：尽管癌症治疗最近取得了进展，但急性髓系白血病 (AML) 的 5 年总体生存率仍保持在30%左右。 AML 的一线护理治疗标准是化疗，但化疗无效复发患者，且往往毒性太大而不能用于老年患者。靶向治疗，例如维奈托克和地西他滨在临床中仅显示出有限的效果，需要针对 AML 采取不同的方法。我们的实验室已经表明，这样的方法之一是通过激活 Gs 偶联的 G 蛋白偶联受体，G蛋白偶联雌激素受体（GPER）。我们实验室的工作表明，药理学 LNS8801 激活 GPER 可诱导分化、促进生长抑制并驱动在黑色素瘤中具有免疫原性，在动物模型中没有观察到毒性。我已经证明原发性 AML 暴露于 LNS8801 的细胞和 AML 细胞系会诱导细胞死亡，这可能是通过细胞凋亡基于膜联蛋白 V 和 PI 染色阳性以及生长停滞。 LNS8801诱导的细胞死亡是伴随着 Mcl-1 的消耗，Mcl-1 是一种抗凋亡蛋白，通常在癌症中过度表达细胞。此外，我发现 GPER 激活的死亡反应与突变状态高度相关 FLT3，特别是 FLT3-ITD。 FLT3-ITD 突变体通过 AKT 的组成型激活维持生存，因此维持 Mcl-1 表达。虽然FLT3抑制剂应用于临床，但患者不可避免地会出现由于抵抗力的发展而死于疾病。 GPER 激活的 FLT3-ITD 漏洞表明 LNS8801可能能够用作FLT3-ITD突变患者的新治疗方法。看见由于GPER的激活可能会降低AML中的细胞凋亡阈值，我测试了LNS8801是否可以增强阿糖胞苷的体外疗效并表明联合治疗比单独使用任何一种药物更有效。基于这些初步数据，我将进行实验以 1）定义 Gs 耦合的机制 GPCR 诱导 AML 细胞凋亡，并 2) 测试 LNS8801 单独使用以及与 LNS8801 联合使用的功效阿糖胞苷体内 AML 模型。在目标 1 中，我将首先确定 Mcl-1 的耗尽是否是导致在存在或不存在药物的情况下，通过基因操纵 Mcl-1 的表达来导致细胞死亡。接下来，我将确定 Mcl-1 耗竭是否是通过 AKT 耗竭介导的。然后我会测试是否FLT3-ITD 通过测量 FLT3-ITD 组的活力，突变使细胞更容易受到 GPCR 激活的影响和暴露于 GPCR 激动剂的 FLT3wt 初级样品。我还将在 FLT3wt 中过表达 FLT3-ITD 和 FLT3 缺失细胞来测试它们是否对 GPCR 激活变得更加敏感。接下来，在目标2中，我将测试单独使用LNS8801是否可以有效抑制体内AML。我也会确定LNS8801是否增强阿糖胞苷在体内的疗效与毒性有限。这些目标共同将定义 Gs- 的机制耦合GPCR诱导AML细胞凋亡并测试LNS8801的体内功效，这可能会进一步帮助开发针对 AML 的新疗法，特别是针对更脆弱和老年患者。