Mechanisms by which of GPCR Signaling Inhibits Acute Myeloid Leukemia

项目摘要： 尽管癌症治疗取得了最新进展，但急性髓系白血病（AML）的5年总生存率 仍保持在30%左右。AML的一线标准治疗是化疗，化疗对急性粒细胞白血病无效。 复发的患者，并且通常毒性太大而不能施用于老年患者。靶向治疗，如venetoclax 和地西他滨在临床上仅显示出适度的效果，需要不同的方法来靶向AML。 我们的实验室已经表明，其中一种方法是通过激活G-偶联的G-蛋白偶联的 G蛋白偶联雌激素受体（GPER）。我们实验室的研究表明， 用LNS 8801激活GPER诱导分化，促进生长抑制，并驱动 在动物模型中没有可观察到的毒性。我已经证明原发性AML 细胞和AML细胞系暴露于LNS 8801诱导细胞死亡，这可能是通过细胞凋亡 基于阳性膜联蛋白V和PI染色，除了生长停滞。LNS 8801诱导的细胞死亡 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GPER激活的死亡反应与突变状态高度相关， 尤其是FLT 3-ITD。FLT 3-ITD突变体通过AKT的组成型激活维持存活， 其因此维持Mcl-1表达。尽管FLT 3抑制剂在临床上得到应用，但患者不可避免地 由于抵抗力的发展而死于疾病。FLT 3-ITD对GPER激活的脆弱性表明 LNS 8801可以作为FLT 3-ITD突变患者的一种新的治疗方法。看到 GPER的激活可能降低AML的凋亡阈值，我测试了LNS 8801是否能增强AML的凋亡阈值。 阿糖胞苷的体外疗效，并显示联合治疗比单独使用任何一种药物更有效。 基于这些初步的数据，我将进行实验，以1）定义的机制，GS耦合 GPCR在AML细胞中诱导细胞凋亡，并且2）测试单独的LNS 8801和与GPCR组合的LNS 8801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 在药物存在或不存在的情况下，通过遗传操纵Mcl-1的表达来导致细胞死亡。接下来我会 确定Mcl-1消耗是否通过AKT消耗介导。然后，我将测试FLT 3-ITD是否 通过测量一组FLT 3-ITD的活力，突变使细胞更容易受到GPCR活化的影响。 和暴露于GPCR激动剂的FLT 3 wt初级样品。我还将在FLT 3 wt中过表达FLT 3-ITD 和FLT 3缺失细胞，以测试它们是否对GPCR活化变得更敏感。接下来，在目标2中，我将测试 LNS 8801单独是否能有效抑制体内AML。我还将确定LNS 8801是否能增强 阿糖胞苷在体内的疗效和有限的毒性。总之，这些目标将定义G的机制- 偶联GPCR诱导AML细胞凋亡并测试LNS 8801的体内功效，这可能进一步 帮助开发AML的新疗法，特别是针对更脆弱和老年患者。

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.