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

项目摘要/摘要 幼年性骨髓单核细胞白血病(JMML)是一种致命的儿童骨髓增生性肿瘤，其中髓系 细胞过度生产，造血祖细胞对粒-巨噬细胞集落高度敏感 刺激因子(GM-CSF)。唯一的治愈方法是异基因骨髓移植，但即使是这样 严格的治疗只有50%的无复发5年存活率。大多数患者患有过度活跃的RAS 信号，最常见的突变是PTPN11的体细胞功能增益(GOF)突变， 编码蛋白质酪氨酸磷酸酶Shp2。陈实验室之前的研究表明，PI3K 催化亚基p110δ是AKT和ERK过度激活所必需的，并促进GOF Shp2诱导的GM- 脑脊液超敏和过度增殖，促进了JMML的进展。基于重要的 P110δ在促进GOF Shp2诱导的白血病中的作用，我们研究了可能的酪氨酸激酶可以 与p110δ协同，促进Akt和Erk活化，导致髓系细胞过度增殖。布鲁顿的 酪氨酸激酶(BTK)已被认为是淋巴系统恶性肿瘤和BTK的关键分子。 抑制剂伊布鲁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细胞受体信号转导的背景下研究，已知BTK是在p110δ下游激活的，并且 活性BTK使PI3K(BCAP)的B细胞适配器磷酸化，使磷酸化的BCAP结合到 调节P85α亚基，促进PI3K的激活。基于已知的B细胞，我们假设 BTK和BCAP在GOF Shp2介导的JMML中p110δ信号的过度激活中起关键作用。 为了解决这一假设，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，基因敲除BTK，也进行体外增殖和生化分析 作为在活体内检查p110δ抑制剂治疗的小鼠骨髓增殖性疾病的进展；(2)我们将使用 从缺乏p85α和p85α定点突变构建的小鼠细胞中探索 BCAP和p85α之间的物理相互作用；以及(3)我们将测试BTK(ACP-196)和p110δ的疗效 AcertaPharma提供的(ACP-319)抑制剂对表达Shp2E76K的小鼠的体内作用以及对原代小鼠的作用 JMML患者细胞的体外培养。这些研究是及时和有意义的，因为AcertaPharma抑制剂 目前正在联合测试慢性淋巴细胞白血病患者的安全性和有效性，以及 因此有可能成为JMML患者的一种新的治疗方法。

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.