Validation of the Pre-BCR Signaling Complex in pre-B ALL Cell Model by Two-Color Single Particle Tracking and Peptidomimetic Inhibition

通过双色单粒子追踪和拟肽抑制验证 pre-B ALL 细胞模型中的 Pre-BCR 信号复合物

• 批准号: 9233061
• 负责人: Michael Frank Erasmus
• 金额: $ 0.9万
• 依托单位: UNIVERSITY OF NEW MEXICO HEALTH SCIS CTR
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-03-10 至 2017-05-12
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9233061
• 关键词: Acute; Acute Lymphocytic Leukemia; Affinity; Antibodies; Antigens; Apoptosis; B-Cell Leukemia; B-Cell Receptor Binding; B-Lymphocytes; Bacteriophages; Binding; Biological Assay; Biophysics; Blast Cell; Bone Marrow; Cancerous; Cell Maturation; Cell Survival; Cell model; Cell surface; Cells; Cellular biology; Childhood; Childhood Leukemia; Co-Immunoprecipitations; Color; Complex; Computational Biology; Cytoplasmic Tail; Cytoprotection; Development; Developmental Process; Diffusion; Dimerization; Disease; Effector Cell; Event; Galactose Binding Lectin; Galectin 1; Goals; Growth; Homo; ITAM; Image; Imaging technology; Immune system; Immunoglobulin A; Libraries; Ligands; Ligation; Light; Link; Mature B-Lymphocyte; Measures; Mediating; Membrane; Molecular; Natural Killer Cells; Outcome; Patients; Peptide antibodies; Peptides; Phagocytes; Phagocytosis; Phase; Phosphorylation; Receptor Signaling; Receptors, Antigen, B-Cell; Recruitment Activity; Resistance; SYK gene; Signal Transduction; Site; Stromal Cells; Surface; Testing; Therapeutic; Therapeutic Agents; Therapeutic antibodies; Training; Up-Regulation; Validation; Western Blotting; Yeasts; antigen binding; assay development; base; chemotherapeutic agent; crosslink; cytotoxicity; design; dimer; expectation; experimental study; glycosylation; imaging modality; immunoglobulin B; inhibitor/antagonist; innovation; killings; leukemia; macrophage; novel; oncology; particle; peptidomimetics; pre-B cell receptor; prevent; protein protein interaction; public health relevance; receptor; surrogate light chain; therapeutic target; uptake; young adult; Acute; Acute Lymphocytic Leukemia; Affinity; Antibodies; Antigens; Apoptosis; B-Cell Leukemia; B-Cell Receptor Binding; B-Lymphocytes; Bacteriophages; Binding; Biological Assay; Biophysics; Blast Cell; Bone Marrow; Cancerous; Cell Maturation; Cell Survival; Cell model; Cell surface; Cells; Cellular biology; Childhood; Childhood Leukemia; Co-Immunoprecipitations; Color; Complex; Computational Biology; Cytoplasmic Tail; Cytoprotection; Development; Developmental Process; Diffusion; Dimerization; Disease; Effector Cell; Event; Galactose Binding Lectin; Galectin 1; Goals; Growth; Homo; ITAM; Image; Imaging technology; Immune system; Immunoglobulin A; Libraries; Ligands; Ligation; Light; Link; Mature B-Lymphocyte; Measures; Mediating; Membrane; Molecular; Natural Killer Cells; Outcome; Patients; Peptide antibodies; Peptides; Phagocytes; Phagocytosis; Phase; Phosphorylation; Receptor Signaling; Receptors, Antigen, B-Cell; Recruitment Activity; Resistance; SYK gene; Signal Transduction; Site; Stromal Cells; Surface; Testing; Therapeutic; Therapeutic Agents; Therapeutic antibodies; Training; Up-Regulation; Validation; Western Blotting; Yeasts; antigen binding; assay development; base; chemotherapeutic agent; crosslink; cytotoxicity; design; dimer; expectation; experimental study; glycosylation; imaging modality; immunoglobulin B; inhibitor/antagonist; innovation; killings; leukemia; macrophage; novel; oncology; particle; peptidomimetics; pre-B cell receptor; prevent; protein protein interaction; public health relevance; receptor; surrogate light chain; therapeutic target; uptake; young adult

 DESCRIPTION (provided by applicant): There is strong evidence to support 'tonic signaling' as central feature in the developmental process leading from pre-B cells to mature, fully competent B cells. These signals are thought to be mediated through the pre-B Cell Receptor (pre-BCR) and essential for pre-B cell protection from apoptosis. This proposal is based upon the central hypothesis that B Cell Precursor Acute Lympoblastic Leukemia (BCP-ALL) blasts also require this pre-BCR survival mechanism, which may contribute to resistance to standard chemotherapeutic challenges and modulate proliferative rates. Moreover, tonic signaling from the pre-BCR is proposed to occur through 1) transient dimerization that occurs through self-association of the l5 component of the surrogate light chain and 2) galectin-mediated crosslinking of pre-BCR, forming more stable pre-BCR dimers and potentially higher- order oligomers. To test these concepts, this study will incorporate state-of-the-art imaging methods, including single particle tracking, FRAP and hyperspectral imaging. This pre-BCR dimerization initiates lyn-mediated phosphorylation of Immunoreceptor Tyrosine-based Activation Motifs (ITAMs) on the cytoplasmic tails of pre- BCR signaling components (Igα and Igβ). ITAM phosphorylation leads to recruitment of spleen tyrosine kinase (Syk) and activation of downstream signaling cascades involved in cell fate decisions. Readouts of these key events will be based on co-immunoprecipitation, anti-PY western blotting and proximity ligation. Another major goal of this proposal is to develop peptidomimetic inhibitors to block pre-BCR dimerization and tonic signaling. This phase of the project will use molecular dynamic approaches to design inhibitory peptides using a stringent scoring criteria, followed by experiments to optimize binding and to test for disruption of the pre-BCR self- association and/or galectin-binding interfaces using the advanced imaging technologies and cell signaling assays. Later, the generated peptides will be used to screen phage and yeast libraries to develop scFv that can bind to the pre-BCR subcomponents with high affinity. The overall goal is to develop monovalent, biologic agents (peptidomimetics, scFv-Fc) that bind with high-affinity to the pre-BCR and block prosurvival signaling. The scFv-Fc will be evaluated for the potential to recruit NK cells and macrophages for antibody-mediated cytotoxicity (ADCC) or phagocytosis (ADCP), a critical first step for development of anti-pre-BCR therapeutic antibodies.

 描述（由适用提供）：有强有力的证据可以支持“滋补信号”作为从前B细胞到成熟，完全竞争的B细胞的发展过程中的主要特征。这些信号被认为是通过前B细胞受体（前BCR）介导的，对于pre-b细胞免受凋亡至关重要。该提议基于以下中心假设：B细胞前体急性淋巴细胞白血病（BCP-ALL）爆炸也需要这种前BCR生存机制，这可能有助于抵抗标准的化学治疗挑战并调节增殖速率。此外，提出了来自前BCR的强直信号传导通过1）瞬时二聚化发生，该二聚化是通过对替代光链的L5分量的自我关联以及2）甲状腺蛋白介导的前BCR的交联，形成更稳定的前BCR二聚体和潜在的高阶寡聚物。为了测试这些概念，本研究将结合最新的成像方法，包括单个粒子跟踪，FRAP和高光谱成像。该前BCR二聚化启动了LYN介导的基于酪氨酸酪氨酸的活化基序（ITAMS）的磷酸化（ITAMS）在前BCR信号成分（IGα和IGβ）的细胞质尾部上。 ITAM磷酸化导致脾脏酪氨酸激酶（SYK）的募集以及参与细胞脂肪决策的下游信号传导级联激活。这些关键事件的读数将基于共免疫沉淀，抗PY Western印迹和接近连接。该提案的另一个主要目标是开发肽映射抑制剂，以阻止BCR前二聚体和滋补信号传导。该项目的这一阶段将使用严格的评分标准使用分子动力学方法来设计抑制性肽，然后进行实验，以优化结合并使用先进的成像技术和细胞信号传导测定法测试前BCR自相关和/或半乳糖素结合接口的破坏。后来，生成的肽将用于筛选噬菌体和酵母菌文库，以开发可与具有高亲和力的前BCR亚组件结合的SCFV。总体目标是开发与亲密关系与BR-BR结合并阻止Prosurvival信号传导的一价生物学剂（肽仪，SCFV-FC）。将评估SCFV-FC的潜力，以募集NK细胞和巨噬细胞的抗体介导的细胞毒性（ADCC）或吞噬作用（ADCP），这是抗Pre-BCR热抗体发展的关键第一步。