Validating Syk as a Target for AML Therapy

项目摘要 尽管剂量增加，但急性髓系白血病(AML)的治疗进展甚微 细胞毒性化疗。另一种治疗AML的方法是加入促进分化的药物 将药物转化为标准的化疗方案。为了寻找新的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。我们确认了吉非替尼， 一种表皮生长因子(EGFR)抑制剂，作为AML分化的诱导剂。EGFR未在 在测试的AML细胞系中，因此排除了抑制该激酶作为AML分化机制的可能性。 因为多个EGFR抑制剂诱导表型，我们假设一个共享的非靶点激酶是 AML分化的靶点。为了确定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)被确定为首选候选基因。SYK是一种 非受体酪氨酸激酶，对正常的B细胞分化很重要，与血液学有关 恶性肿瘤。我们假设Syk是AML治疗的目标，失去Syk将导致 AML的分化和/或细胞死亡。我们证实了药物抑制(R406)和遗传 在急性髓系白血病细胞的初步研究中，Syk的丢失诱导分化和/或死亡。我们现在提出更多建议 用以下具体目标广泛地检验这一假设。 具体目的1.研究R406对急性髓系白血病的体内外作用 具体目标2.确定Syk是AML中R406活动目标 明确目标3.确定急性髓系白血病中Syk的下游效应因子 在目标1中，我们将确定Syk抑制作为一种抗AML治疗的广泛潜力。我们将测量内部 一组AML细胞中Syk抑制剂(R406)对分化、细胞生长和凋亡的体外影响。 然后，我们将使用主要的人类急性髓系白血病原位模型将测试扩展到体内研究。接下来，在目标2中，我们 将通过三种平行的方法确认Syk是R406活性的目标：聚合酶链式反应突变筛选 对于挽救R406抗AML活性的Syk突变体；使用RNA干扰的遗传方法， 以及评估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 在使用互补方法的AML中，哪些蛋白质是Syk信号的关键下游效应器： 生化、遗传学、基因组学、药理学和蛋白质组学。Rigel制药化合物，R788， 前药R406已经处于第二阶段测试，最近被证明在 自身免疫性疾病和淋巴瘤。随着第一阶段测试现已完成，并证明了这些产品的有效性 如果有了R788，我们就有能力迅速将R788投入临床试验。这些研究，在五个范围内 这笔赠款的时间框架，将具有直接的翻译相关性，并为发展提供信息 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.