NPM-ALK and ALK in Lymphoma and Normal Development

间变性淋巴瘤激酶（ALK）受体酪氨酸激酶（RTK）通过非霍奇金淋巴瘤（NHL）和称为炎性肌纤维母细胞瘤（IMT）的肉瘤中的染色体重排而改变，以产生组成型活性ALK融合蛋白。 ALK融合蛋白的表达与NHL或IMT的发生之间存在直接的因果关系。相反，抑制ALK活性可通过实验逆转恶性表型。 ALK的正常功能尚不完全清楚，并且ALK敲除小鼠未表现出大体或组织病理学异常。 ALK信号转导的研究已经确定了几种先前已知的激酶底物在介导ALK信号传导中的重要性，但激酶正常或致癌信号传导所需的完整底物谱尚不清楚。 鉴于ALK作为定向治疗靶点的良好验证，本提案的目标1a旨在开发ALK的ATP竞争性小分子抑制剂，并确定其在临床前肿瘤模型中的特异性、毒性和疗效。 为了为ALK抑制剂的合理、基于结构的设计提供指导，目的1b中的研究将确定apo、无活性和三磷酸化、完全活性ALK KD的X射线晶体学结构，以及与选定抑制剂共结晶的ALK KD的结构。 被称为白细胞酪氨酸激酶（LTK）的RTK和ALK具有显著的同源性和部分重叠的表达模式，表明可能存在功能冗余。 为了更好地了解这些激酶在生长和发育中的功能，并评估其同时抑制的后果（即使使用高度特异性的ATP竞争性ALK抑制剂也可能发生），将在目标1c中生成缺乏Alk和Ltk的小鼠并进行表征。 最近的ALK信号转导研究已经确定了一种新的底物，ALK相互作用核蛋白3（ANP-3），具有改变细胞凋亡和细胞周期转运的能力，特别是有丝分裂的进入和退出。 目标2a中的研究将寻求确定ANP-3在凋亡性死亡和细胞周期的正常调节中的确切作用，特别关注M期转换，并了解致癌ALK融合导致ANP-3失调的机制和后果。 最后，在目标2b中，将评估Anp-3体内失活对胚胎发育、细胞周期完整性和凋亡控制的表型影响。 这些研究将提高对正常ALK功能和ALK融合蛋白在恶性肿瘤中所采用的机制的基本理解，同时还确定了可用作基础研究工具的小分子ALK抑制剂，并最终可能用于治疗应用。

The anaplastic lymphoma kinase (ALK) receptor tyrosine kinase (RTK) is altered by chromosomal rearrangements in non- Hodgkin's lymphoma (NHL) and the sarcoma known as inflammatory myofibroblastic tumor (IMT) to produce constitutively active ALK fusion proteins. A direct causal relationship exists between the expression of ALK fusions and the genesis of NHL or IMT. Conversely, inhibition of ALK activity reverses the malignant phenotype experimentally. The normal functions of ALK are incompletely known, and Alk knockout mice exhibit no gross or histopathologic abnormalities. Studies of ALK signal transduction have identified several previously known kinase substrates as important in mediating ALK signaling, but the complete spectrum of substrates required for normal or oncogenic signaling by the kinase is not yet clear. Given the excellent validation of ALK as a target for directed therapies, Aim 1a of this proposal seeks to develop ATP-competitive small molecule inhibitors of ALK and to determine their specificity, toxicity and efficacy in preclinical tumor models. To provide guidance for the rational, structure-based design of ALK inhibitors, the studies in Aim 1b will determine the X-ray crystallographic structure of the apo, inactive and tris-phosphorylated, fully active ALK KD, as well as the structure of the ALK KD co- crystallized with selected inhibitors. The RTK known as leukocyte tyrosine kinase (LTK) and ALK share remarkable homology and a partially overlapping expression pattern, suggesting possible functional redundancy. To better understand the function of these kinases in growth and development and assess the consequences of their simultaneous inhibition (as may occur pharmacologically even with highly specific ATP-competitive ALK inhibitors), mice that lack both Alk and Ltk will be generated and characterized in Aim 1c. Recent ALK signaling studies have identified a novel substrate, ALK-interacting nuclear protein 3 (ANP-3), that possesses the ability to alter both apoptosis and cell cycle transit, especially mitotic entry and exit. The studies in Aim 2a will seek to determine the exact role of ANP-3 in the normal regulation of apoptotic death and the cell cycle, focusing especially on M-phase transit, and to understand the mechanism and consequences of ANP-3 deregulation by oncogenic ALK fusions. Finally, in Aim 2b, the phenotypic effects of Anp-3 inactivation in vivo on embryonic development, cell cycle integrity and apoptosis control will be assessed. These studies will improve basic understanding of normal ALK functions and the mechanisms employed by ALK fusion proteins in malignancy, while also identifying small molecule ALK inhibitors for use as basic research tools and ultimately perhaps for therapeutic applications.