Validating Syk as a Target for AML Therapy

项目摘要 在急性髓性白血病（AML）的治疗中几乎没有进展，尽管使用了剂量强化的化疗。 细胞毒化疗治疗AML的另一种方法是掺入促分化因子。 药物进入标准化疗方案。为了鉴定新的AML分化剂，我们的 实验室开发了一种基于基因表达的小分子筛选方法。我们发现了吉非替尼， 表皮生长因子（EGFR）抑制剂，作为AML分化的诱导剂。EGFR不表达于 所测试的AML细胞系，从而排除了作为AML分化机制的该激酶的抑制。 由于多种EGFR抑制剂诱导表型，我们假设一个共同的脱靶激酶是 AML分化的靶点。为了鉴定AML分化的候选吉非替尼靶点，我们利用 蛋白质组学和遗传学方法。脾酪氨酸激酶（Syk）被确定为最佳候选者。Syk是一个 非受体酪氨酸激酶，在正常B细胞分化中很重要，并与血液学 恶性肿瘤。我们假设Syk是AML治疗的靶点，Syk的缺失将导致 在AML中的分化和/或细胞死亡。我们证实了药理学抑制（R406）和遗传学抑制（R406）。 在AML细胞的初步研究中，Syk的丧失诱导分化和/或死亡。我们现在建议更多 用以下具体目标来广泛地检验这一假设。 具体目标1。表征R406在AML中的体外和体内作用 具体目标2。确定Syk是AML中R406活性的靶点 具体目标3。确定AML中Syk的下游效应物 在目标1中，我们将确定Syk抑制作为抗AML治疗的广泛潜力。我们将测量 Syk抑制剂（R406）在一大组AML细胞中对分化、细胞生长和凋亡的体外作用。 然后，我们将使用原代人AML原位模型将测试扩展到体内研究。在目标2中，我们 将用三种平行方法证实Syk是R406活性的靶标：PCR诱变筛选 对于拯救R406抗AML活性效果的Syk突变体;使用RNA干扰的遗传方法， 以及评价Syk的其他小分子抑制剂的药理学方法。在目标3中，我们将确定 哪些蛋白质是使用互补方法的AML中Syk信号传导的关键下游效应物： 生物化学、遗传学、基因组学、药理学和蛋白质组学。参宿七制药的化合物R788 R406的前体药物，已经在II期试验中，最近被证明具有以下活性： 自身免疫性疾病和淋巴瘤。随着I期试验的完成和疗效的证明，这些 因此，我们将有能力迅速将R788投入临床试验。这些研究中， 这项赠款的一年时间框架，将有直接的翻译相关性，并告知发展 在复发/难治性AML患者中测试Syk抑制的临床试验。

PROJECT ABSTRACT Little progress has been made in the treatment of acute myeloid leukemia (AML) despite dose intensification of cytotoxic chemotherapy. An alternative approach to treating AML is the incorporation of pro-differentiation agents into standard chemotherapy regimens. In order to identify new AML differentiation agents, our laboratory developed a gene expression-based approach to small molecule screening. We identified gefitinib, an epidermal growth factor (EGFR) inhibitor, as an inducer of AML differentiation. EGFR is not expressed in the tested AML cell lines, thus precluding inhibition of this kinase as the mechanism of AML differentiation. Because multiple EGFR inhibitors induce the phenotype, we hypothesize that a shared off-target kinase is the target in AML differentiation. In order to identify candidate gefitinib targets of AML differentiation, we utilized proteomic and genetic approaches. Spleen tyrosine kinase (Syk) was identified as the top candidate. Syk is a nonreceptor tyrosine kinase, important in normal B-cell differentiation, and implicated in hematological malignancies. We hypothesize that Syk is a target for AML therapy and that loss of Syk will result in differentiation and/or cell death in AML. We confirmed with both pharmacological inhibition (R406) and genetic loss of Syk the induction of differentiation and/or death in a pilot study of AML cells. We now propose to more broadly test this hypothesis with the following Specific Aims. Specific Aim 1. Characterize the in vitro and in vivo effects of R406 in AML Specific Aim 2. Establish that Syk is the target of R406 activity in AML Specific Aim 3. Determine the downstream effectors of Syk in AML In Aim 1, we will determine the broad potential of Syk inhibition as an anti-AML therapy. We will measure the in vitro effects of a Syk inhibitor (R406) in a large panel of AML cells on differentiation, cell growth, and apoptosis. We will then extend testing to in vivo studies using primary human AML orthotopic models. Next, in Aim 2, we will confirm that Syk is the target of R406 activity with three parallel approaches: A PCR mutagenesis screen for Syk mutants that rescue the effects of R406 anti-AML activity; a genetic approach using RNA interference, and a pharmacological approach evaluating other small molecule inhibitors of Syk. In Aim 3, we will determine which proteins are critical downstream effectors of Syk signaling in AML using complementary approaches: biochemical, genetic, genomic, pharmacological, and proteomic. The Rigel Pharmaceutical compound, R788, the prodrug of R406, is already in Phase II testing and was recently demonstrated to have activity in autoimmune disease and lymphoma. With Phase I testing now complete and efficacy demonstrated for these diseases, we would be well positioned to rapidly bring R788 to clinical trial. These studies, within the five year time frame of this grant, will have immediate translational relevance and inform the development of a clinical trial testing Syk inhibition in patients with relapsed/refractory AML.