Validating Syk as a Target for AML Therapy

描述（由申请人提供）：尽管细胞毒性化疗剂量增强，但急性髓性白血病（AML）的治疗进展甚微。治疗AML的另一种方法是将促分化剂纳入标准化疗方案。为了鉴定新的AML分化因子，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。我们发现表皮生长因子（EGFR）抑制剂吉非替尼是AML分化的诱导剂。EGFR在测试的AML细胞系中不表达，因此排除了抑制该激酶作为AML分化的机制。由于多种EGFR抑制剂诱导表型，我们假设一个共享的脱靶激酶是AML分化的靶标。为了确定候选吉非替尼的AML分化靶点，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。Syk是一种非受体酪氨酸激酶，在正常b细胞分化中起重要作用，并与血液系统恶性肿瘤有关。我们假设Syk是AML治疗的靶标，Syk的缺失将导致AML的分化和/或细胞死亡。在AML细胞的初步研究中，我们证实了Syk的药理抑制（R406）和遗传缺失诱导分化和/或死亡。我们现在建议以以下具体目标更广泛地检验这一假设。具体目标表征R406在AML特异性Aim 2中的体外和体内作用。确定Syk是AML特异性Aim 3中R406活性的靶标。在Aim 1中，我们将确定Syk抑制作为抗AML治疗的广泛潜力。我们将在大量AML细胞中测量Syk抑制剂（R406）对分化、细胞生长和凋亡的体外影响。然后，我们将使用原发人类AML原位模型将测试扩展到体内研究。接下来，在Aim 2中，我们将通过三种平行方法确认Syk是R406活性的靶标：PCR诱变筛选Syk突变体，恢复R406抗aml活性的作用；使用RNA干扰的遗传方法，以及评估其他Syk小分子抑制剂的药理学方法。在Aim 3中，我们将使用互补的方法确定哪些蛋白质是AML中Syk信号的关键下游效应物：生化、遗传、基因组、药理学和蛋白质组学。Rigel制药公司的化合物R788是R406的前药，已经在II期试验中，最近被证明对自身免疫性疾病和淋巴瘤有活性。随着I期试验的完成和对这些疾病的疗效证明，我们将很好地将R788快速带入临床试验。这些研究在该资助的5年时间框架内，将具有直接的转化相关性，并为复发/难治性AML患者中Syk抑制的临床试验的发展提供信息。公共卫生相关性：本提案中的研究将验证脾脏酪氨酸激酶（Syk）蛋白作为急性髓性白血病（AML）治疗的靶点。我们将在体外和体内AML模型中测试Syk对AML细胞的药理学和遗传抑制作用。由于已经有一种具有抗Syk活性的药物被证明对淋巴瘤和自身免疫性疾病有效，我们预计这些研究将迅速转化为检测Syk抑制AML患者的临床试验。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: The studies in this proposal will validate the protein spleen tyrosine kinase (Syk) as a target for acute myeloid leukemia (AML) therapy. We will test pharmacological and genetic inhibition of Syk in AML cells in vitro and in vivo models of AML. Because there is already a drug with anti-Syk activity with demonstrated efficacy in lymphoma and autoimmune disease, we anticipate the rapid translation of these studies to a clinical trial testing Syk inhibition for patients with AML.