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

尽管我们对急性髓细胞白血病的遗传起源的了解取得了进展，但治疗方案仍然存在 30年来基本没有变化，临床结果仍然很差。白血病干细胞代表着 化疗耐药并在治疗后重新启动AML的原始细胞群体；因此，这一子集 必须根除爆炸才能治愈疾病。不幸的是，专门针对LSC开发的疗法有 尚待临床验证。我们和其他人最近发现了一种新的AML抗原CD97，它可以表达 在绝大多数人类AML中。我们最近发表的研究揭示了CD97的几个特征 提示它可能是AML的一个很好的治疗靶点：1)CD97是最常见的表达之一 AML抗原；2)CD97调节原始细胞的生长、存活和分化；3)CD97调节LSC功能，AS 在原发AML的系列移植实验中得到证实；以及，4)HSC不需要CD97 功能，提示CD97靶向治疗药物毒性低。突显其临床重要性的CD97 mRNA 在急性髓系白血病中，表达是无病和总存活率的独立预测因子。 CD97是一种黏附型G蛋白偶联受体，其特征是具有一个长的细胞外配体- 结合结构域和GPCR-自蛋白分解诱导(增益)结构域，可以诱导可能或可能 不需要胞外结构域的脱落。选择性剪接产生的CD97亚型不同于 配体结合区的组成，但目前尚不清楚各种CD97亚型是否起中介作用 AML中的唯一或重叠角色。我们的总体假设是不同的CD97亚型扮演着不同的角色 通过其独特的配体结合和/或信号特性，在白血病发生和LSC自我更新中发挥重要作用。我们的 具体目标是确定CD97亚型在白血病转化和LSC功能中的作用 确定CD97活性的分子和结构要求，并利用新的人类合成 不同表位特异性的抗CD97抗体评价CD97功能的研究 测试它们的抗白血病活性。我们将确定CD97所需的各种结构亚域的作用 对于利用我们新的CD97抗体的LSC功能，CD97构建表达多种自然产生的和 CD97的工程化结构变体以及小鼠和人的体内外互补模型 AML。 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，我们 在研究CD97信号转导机制和LSCs功能方面具有独特的优势。总而言之， 这些研究将极大地提高我们对LSC自我调节的分子机制的理解。 更新并帮助加快CD97抗体疗法向临床的转化。最后，这些研究可能会 更广泛的后果，因为CD97在其他人类癌症中发挥着疾病修正的作用。

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.