Adhesion GPCR regulation of acute myeloid leukemia stem cells - Resubmission - 1

尽管我们对AML遗传起源的理解有所进步，但治疗选择仍然存在。 30年来基本上没有变化，临床结果仍然很差。白血病干细胞（LSC）代表 对化疗耐药并在治疗后重新启动AML的原始细胞群体;因此， 必须根除爆炸才能治愈疾病。不幸的是，专门针对LSC开发的疗法 尚待临床验证。我们和其他人最近发现了一种新的AML抗原CD 97， 在绝大多数人类AML中。我们最近发表的研究揭示了CD 97的几个特征， 提示它可能是AML的一个极好的治疗靶点：1）CD 97是AML中最常见的表达之一， AML抗原; 2）CD 97调节原始细胞生长、存活和分化; 3）CD 97调节LSC功能，如 在原发性AML的系列移植实验中证实;和，4）HSC不需要CD 97 功能，表明CD 97靶向治疗剂的低毒性。强调其临床重要性，CD 97 mRNA 表达是AML患者无病生存期和总生存期的独立预测因子。 CD 97是一种粘附类G蛋白偶联受体（aGPCR），其特征是一个长的细胞外配体- 结合结构域和GPCR-自身蛋白水解诱导（GAIN）结构域，其可以诱导信号， 不需要细胞外结构域脱落。通过选择性剪接产生的CD 97亚型在以下方面不同： 虽然CD 97与配体结合结构域的组成密切相关，但目前尚不清楚各种CD 97亚型是否介导了CD 97与配体结合结构域的结合， 在AML中扮演独特或重叠的角色。我们的总体假设是，不同的CD 97亚型发挥不同的作用， 由于其独特的配体结合和/或信号传导特性，在白血病发生和LSC自我更新中起作用。我们 具体目标是确定CD 97亚型在白血病转化和LSC功能中的作用， 鉴定CD 97活性的分子和结构要求，并利用新的人合成 具有不同表位特异性的抗CD 97的抗体（sAb），以评估CD 97的功能以及 测试其抗白血病活性。我们将确定所需的CD 97的各种结构子域的作用， 对于利用我们的新型CD 97 Ab的LSC功能，表达多种天然存在的和 CD 97的工程化结构变体，以及小鼠和人的互补的体外和体内模型 急性髓细胞白血病 鉴于我们团队在LSC生物学、抗体工程和aGPCR生物学方面的互补专业知识， 在研究LSC中CD 97信号传导和功能的机制方面具有独特的地位。总的来说， 这些研究将极大地增加我们对调节LSC自身的分子机制的理解， 更新和帮助加快翻译的CD 97抗体疗法的临床。最后，这些研究可能 更广泛的后果，因为CD 97在其他人类癌症中发挥疾病修饰作用。

Despite advances in our understanding of the genetic origins of AML, treatment options have remained essentially unchanged for 30 years, and clinical outcomes remain poor. Leukemia stem cells (LSCs) represent the population of blasts that are resistant to chemotherapy and re-initiate AML after therapy; thus, this subset of blasts must be eradicated to cure disease. Unfortunately, therapies developed specifically to target LSCs have yet to be validated in the clinic. We and others recently identified a novel AML antigen, CD97, that is expressed in the vast majority of human AMLs. Our recently published studies have revealed several features of CD97 that suggest that it may be an excellent therapeutic target in AML: 1) CD97 is one of the most commonly expressed AML antigens; 2) CD97 regulates blast growth, survival, and differentiation; 3) CD97 regulates LSC function, as demonstrated in serial transplantation experiments of primary AML; and, 4) CD97 is not required for HSC function, suggesting low toxicity of CD97-targeting therapeutics. Highlighting its clinical importance, CD97 mRNA expression is an independent predictor of disease-free and overall survival in AML. CD97 is an adhesion class G-protein coupled receptor (aGPCR) characterized by a long, extracellular ligand- binding domain and a GPCR-Autoproteolysis-INducing (GAIN) domain that can induce signals that may or may not require extracellular domain shedding. Isoforms of CD97 produced by alternative splicing differ in the composition of the ligand-binding domain, but at present, it is unclear if the various CD97 isoforms mediate unique or overlapping roles in AML. Our overall hypothesis is that the various CD97 isoforms play distinct roles in leukemogenesis and LSC self-renewal by virtue of their unique ligand binding and/or signaling properties. Our specific goals are to determine the role of CD97 isoforms in leukemic transformation and LSC function, to identify the molecular and structural requirements for CD97 activity, and to utilize novel human synthetic antibodies (sAbs) against CD97 with different epitope specificities to evaluate the function of CD97 as well as test their anti-leukemic activity. We will determine the roles of the various structural subdomains of CD97 required for LSC function utilizing our novel CD97 Abs, CD97 constructs expressing multiple naturally occurring and engineered structural variants of CD97, and complementary in vitro and in vivo models of mouse and human AML. Given our team's complementary expertise in LSC biology, antibody engineering, and aGPCR biology, we are uniquely positioned to investigate the mechanisms of CD97 signaling and function in LSCs. Collectively, these studies will dramatically increase our understanding of the molecular mechanisms that regulate LSC self- renewal and help expedite translation of CD97 antibody therapies to the clinic. Finally, these studies may have broader consequences since CD97 plays disease-modifying roles in other human cancers.