Mechanisms by which of GPCR Signaling Inhibits Acute Myeloid Leukemia

项目总结： 尽管最近癌症治疗取得了进展，但急性髓系白血病(AML)的5年总存活率 保持在30%左右。急性髓系白血病的一线护理治疗标准是化疗，但化疗对 复发的患者，往往毒性太大，不能用于老年患者。靶向治疗，如万乃馨 而地西他滨在临床上只显示出温和的效果，呼吁针对AML采取不同的方法。 我们的实验室已经证明，其中一种方法是通过激活Gs偶联的G蛋白 受体，G蛋白偶联雌激素受体(GPER)。我们实验室的研究表明，药理作用 LNS8801激活GPER诱导分化、促进生长抑制和驱动 动物模型中黑色素瘤的免疫原性，无明显毒性。我已经证明了主要的急性髓系白血病 暴露于LNS8801的细胞和AML细胞系诱导细胞死亡，这可能是通过细胞凋亡来实现的 根据Annexin V和PI染色阳性，除生长停滞外。LNS8801诱导的细胞死亡 伴随着Mcl-1的耗尽，Mcl-1是一种通常在癌症中过度表达的抗凋亡蛋白 细胞。此外，我发现对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的结构性激活来维持生存， 从而维持Mcl-1的表达。尽管Flt3抑制剂在临床上被使用，但患者不可避免地 由于抵抗力的发展而屈服于疾病。GPER激活的Flt3-ITD漏洞表明 LNS8801有望成为治疗突变的Flt3-ITD患者的一种新方法。眼见 GPER的激活可能会降低AML的凋亡阈值，我测试了LNS8801是否可以增强 阿糖胞苷的体外疗效表明，联合治疗比单独使用任何一种药物都更有效。 在这些初步数据的基础上，我将进行实验以1)确定Gs偶联的机制 GPCRs诱导AML细胞凋亡；2)检测LNS8801单独及联合应用的疗效 阿糖胞苷体内AML模型的建立。在目标1中，我将首先确定Mcl-1的枯竭是否对 在药物存在或不存在的情况下，通过基因控制Mcl-1的表达而导致的细胞死亡。接下来，我会 确定Mcl-1耗竭是否是通过AKT耗竭介导的。然后我会测试Flt3-ITD 通过测量一组Flt3-ITD的活性，突变使细胞更容易受到gpr激活的影响 和FLT3wt暴露于GPCR激动剂的初级样本。我还将在Flt3wt中过表达Flt3-ITD 和Flt3零细胞，以测试它们是否对GPCR激活变得更加敏感。接下来，在目标2中，我将测试 LNS8801在体内是否能有效抑制AML。我还将确定LNS8801是否增强了 阿糖胞苷在体内的疗效及毒性有限。综合起来，这些目标将定义Gs的机制-- 偶联GPCR诱导AML细胞凋亡并在体内检测LNS8801的疗效，这可能进一步 帮助开发治疗急性髓细胞白血病的新疗法，特别是对更脆弱和更年长的患者。

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.