Evaluation of Bruton’s Tyrosine Kinase and p110 delta in Mutant Shp2-Induced JMML

突变型 Shp2 诱导的 JMML 中 Bruton 酪氨酸激酶和 p110 delta 的评估

• 批准号: 9191475
• 负责人: Lisa Deng Yuen
• 金额: $ 3.67万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-01 至 2020-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9191475
• 关键词: Accounting; Address; Agammaglobulinaemia tyrosine kinase; Allogenic; B-Cell Leukemia; B-Lymphocytes; Binding; Biochemical; Blood Cells; Bone Marrow Transplantation; Cancerous; Catalytic Domain; Cells; Childhood; Chronic Lymphocytic Leukemia; Data; Development; Disease; Dominant-Negative Mutation; Evaluation; Feedback; Genetic; Granulocyte-Macrophage Colony-Stimulating Factor; Hematopoietic; Hematopoietic Neoplasms; Hypersensitivity; In Vitro; Juvenile Myelomonocytic Leukemia; Knock-out; Laboratories; Lead; Link; Lipase; Lipids; Malignant Childhood Neoplasm; Malignant lymphoid neoplasm; Mantle Cell Lymphoma; Mediating; Modeling; Molecular Models; Mus; Mutation; Myeloid Cells; Myeloproliferative disease; PLCgamma2; PTPN11 gene; Pathway interactions; Patients; Protein Kinase C; Protein Tyrosine Kinase; Protein Tyrosine Phosphatase; Proteins; Receptor Signaling; Receptors, Antigen, B-Cell; Regulation; Relapse; Role; Safety; Series; Signal Pathway; Signal Transduction; Site; Structure; Testing; Tyrosine Kinase Inhibitor; Waldenstrom Macroglobulinemia; Work; base; curative treatments; efficacy testing; gain of function; gain of function mutation; hyperactive Ras; improved; in vivo; inhibitor/antagonist; leukemia; molecular modeling; mutant; novel; novel strategies; phosphoinositide-3,4,5-triphosphate; progenitor; research study

PROJECT SUMMARY/ABSTRACT Juvenile myelomonocytic leukemia (JMML) is a fatal childhood myeloproliferative neoplasm in which myeloid cells are overproduced and hematopoietic progenitors are hypersensitive to granulocyte-macrophage colony stimulating factor (GM-CSF). The only curative treatment is allogeneic bone marrow transplant, but even this rigorous therapy yields only a 50% relapse-free 5-year survival. A majority of patients have hyperactive Ras signaling, with the most common mutation being somatic gain-of-function (GOF) mutations in PTPN11, which encodes the protein tyrosine phosphatase Shp2. Previous work in the Chan lab has shown that the PI3K catalytic subunit p110δ is needed for both Akt and Erk hyperactivation, and promotes GOF Shp2-induced GM- CSF hypersensitivity and hyperproliferation, contributing to the progression of JMML. Based on the significant role of p110δ in promoting GOF Shp2-induced leukemia, we investigated potential tyrosine kinases that can cooperate with p110δ to promote Akt and Erk activation and lead to hyperproliferation of myeloid cells. Bruton's Tyrosine Kinase (BTK) has been identified as a critical molecule in lymphoid malignancies and the BTK inhibitor, ibrutinib, was recently approved for use in patients with B cell leukemias. As BTK has been well- studied in the context of B cell receptor signaling, it is known that BTK is activated downstream of p110δ and that active BTK phosphorylates B cell adaptor for PI3K (BCAP), allowing phospho-BCAP to bind to the regulatory p85α subunit and promote activation of PI3K. Based on what is known in B cells, we hypothesize that BTK and BCAP are critical players in the GOF Shp2-mediated hyperactivation of p110δ signaling in JMML. To address this hypothesis, we propose three aims: (1) we will use murine cells expressing the GOF Shp2 mutation, E76K, with genetic knockout of BTK to perform proliferation and biochemical analysis in vitro, as well as examine the progression of myeloproliferative disease in p110δ inhibitor-treated mice in vivo; (2) we will use cells from mice lacking expression of p85α along with p85α site-specific mutant constructs to explore the physical interaction between BCAP and p85α; and (3) we will test the efficacy of BTK (ACP-196) and p110δ (ACP-319) inhibitors provided by AcertaPharma on Shp2E76K-expressing mice in vivo, as well as on primary JMML patient cells in vitro. These studies are timely and significant, as the AcertaPharma inhibitors are currently being tested in combination for safety and efficacy in chronic lymphocytic leukemia patients, and therefore have the potential to be a novel treatment in patients with JMML.

项目概要/摘要 幼年型粒单核细胞白血病（JMML）是一种致命的儿童骨髓增生性肿瘤，其中骨髓细胞 细胞过量产生，造血祖细胞对粒细胞-巨噬细胞集落过敏 刺激因子（GM-CSF）。唯一的治疗方法是同种异体骨髓移植，但即使这样 严格的治疗只能实现 50% 的 5 年无复发生存率。大多数患者患有 Ras 过度活跃 信号传导，最常见的突变是 PTPN11 的体细胞功能获得性 (GOF) 突变， 编码蛋白质酪氨酸磷酸酶Shp2。 Chan 实验室之前的工作表明 PI3K Akt 和 Erk 过度激活都需要催化亚基 p110δ，并促进 GOF Shp2 诱导的 GM- CSF 过敏和过度增殖，促进 JMML 的进展。基于显着 为了了解 p110δ 在促进 GOF Shp2 诱导的白血病中的作用，我们研究了潜在的酪氨酸激酶，这些激酶可以 与p110δ协同促进Akt和Erk激活，导致骨髓细胞过度增殖。布鲁顿的 酪氨酸激酶 (BTK) 已被确定为淋巴恶性肿瘤的关键分子，BTK 抑制剂伊布替尼最近被批准用于 B 细胞白血病患者。由于 BTK 一直很好- 在 B 细胞受体信号传导背景下进行的研究表明，BTK 在 p110δ 下游被激活，并且 活性 BTK 磷酸化 PI3K (BCAP) 的 B 细胞接头，使磷酸化 BCAP 与 调节 p85α 亚基并促进 PI3K 的激活。根据 B 细胞的已知情况，我们假设 BTK 和 BCAP 在 JMML 中 GOF Shp2 介导的 p110δ 信号过度激活中发挥着关键作用。 为了解决这个假设，我们提出了三个目标：（1）我们将使用表达 GOF Shp2 的小鼠细胞 突变，E76K，BTK 基因敲除，可在体外进行增殖和生化分析 检查 p110δ 抑制剂治疗小鼠体内骨髓增生性疾病的进展情况； (2) 我们将使用 来自缺乏 p85α 表达的小鼠的细胞以及 p85α 位点特异性突变体构建体，以探索 BCAP 和 p85α 之间的物理相互作用； (3) 我们将测试 BTK (ACP-196) 和 p110δ 的功效 AcertaPharma 为表达 Shp2E76K 的小鼠体内以及原代小鼠提供的 (ACP-319) 抑制剂 JMML 患者细胞体外。这些研究是及时且重要的，因为 AcertaPharma 抑制剂是 目前正在对慢性淋巴细胞白血病患者的安全性和有效性进行联合测试，以及 因此有可能成为 JMML 患者的一种新疗法。