Novel Allosteric Kinase Inhibitors Target Imatinib-Resistant GIST

描述(申请人提供)：胃肠道间质瘤(GIST)是最常见的肉瘤，在美国每年约有6,000例新病例。GIST的治疗是建立在推动肉瘤形成的“癌基因成瘾”试剂盒的基础上的。因此，ATP依赖(模拟)试剂盒抑制剂伊马替尼是治疗晚期GIST的一线药物。尽管接受伊马替尼治疗，但疾病的复发/进展仍然频繁，因为GIST对继发性试剂盒突变引起的伊马替尼耐药。这些突变降低了其他三磷酸腺苷模拟物的效力。因此，靶向于激酶的化合物，同时避免了ATP模拟耐药机制，是非常有意义的。我的导师David Cheresh报告了变构激酶抑制剂(AKI)的开发，它可以不依赖于ATP的方式阻止KIT/BRAF/CRAF/PDGFR激酶的激活。虽然目前的三磷酸腺苷模拟物达到了“正确”的目标(S)，Akis可能：1)比三磷酸腺苷模拟物具有更高的特异性；2)避免使三磷酸腺苷模拟物无效的“把关人”突变；以及3)产生强大的抗伊马替尼耐药的GIST活性，而对AKI耐药的担忧较少。我们推测，这种具有试剂盒抑制作用的新型Akis可能用于治疗对伊马替尼耐药的GIST，这种疾病的治疗选择有限。我们的目标是在伊马替尼耐药的GIST的背景下，确定变构激酶抑制的功能/机制作用。在目标1中，我们将使用对伊马替尼敏感和耐药的GIST细胞系来鉴定一系列高效的Aki细胞。我们将以时间/剂量依赖的方式定义AKI对细胞活力、细胞凋亡和细胞周期停滞的影响。在目标2中，我们将进行生化/机械分析，以确定Aki铅对野生型和突变型KIT酶的动力学效应和结合亲和力。在目标3中，我们将利用遗传学和药理学分析来研究KIT(和BRAF/CRAF/PDGFR)Akis如何影响细胞内激酶信号通路，这些信号通路控制着伊马替尼耐药株的肿瘤细胞增殖、存活、凋亡和坏死等基本过程。[我们还将定义AI对试剂盒适配蛋白相互作用的影响。]在目标4中，我们将根据我们的IACUC批准的方案，使用肿瘤异种移植/转基因小鼠模型来研究AIMS 1-3中确定的抑制GIST生长的领先Akis。我们将在体外和体内通过不同的机制研究靶向KIT的新化合物，以靶向伊马替尼耐药的GIST，并寻找创新的抗肿瘤药物(S)。这些研究将作为针对这些有效的激酶靶点的临床检查的基础，在对伊马替尼耐药的GIST患者中。如果在体外和体内确定有效的药物，我们打算寻求额外的资金，以支持以临床试验的形式将这些药物(S)转移到床边。

DESCRIPTION (provided by applicant): Gastrointestinal Stromal Tumor (GIST) is the most common sarcoma with ~6,000 new cases annually in the U.S. GIST therapy is predicated upon KIT "oncogene-addiction" which drives sarcomagenesis. As a result, the ATP-dependent (-mimetic) KIT inhibitor, imatinib, is the first-line treatment for advanced GIST. Despite imatinib therapy, disease recurrence/progression remain frequent because GISTs develop imatinib-resistance caused by secondary KIT mutations. These mutations decrease the efficacy of other ATP-mimetics. Thus, compounds that target kinases, while avoiding ATP-mimetic resistance mechanisms, are of distinct interest. My mentor, David Cheresh, has reported the development of allosteric kinase inhibitors (AKI) that prevent KIT/BRAF/CRAF/PDGFR kinase activation in an ATP-independent manner. While current ATP-mimetics hit the "right" target(s), AKIs may: 1) have higher specificity than ATP-mimetics; 2) avoid "gatekeeper" mutations which render ATP-mimetics inactive; and 3) produce strong activity against imatinib-resistant GIST with fewer concerns for AKI-resistance. We hypothesize that this novel class of AKIs with KIT inhibition may be used to treat imatinib-resistant GIST, a disease for which there are limited therapeutic options. We aim to define the functional/mechanistic roles of allosteric kinase inhibition within the context of imatinib-resistant GIST. In Aim 1, we will characterize a series of highly potent AKIs using imatinib-sensitive and -resistant GIST cell lines. We will define AKI effects on cell viability, apoptosis, and cell cycle arrest in a time-/dose-dependent fashion. In Aim 2, we will perform biochemical/mechanistic analyses to determine the kinetic effects and binding affinities of lead AKIs on wild-type versus mutant KIT enzymes. In Aim 3, we will employ genetic and pharmacologic analyses to investigate how KIT (and BRAF/CRAF/PDGFR) AKIs influence intracellular kinase signaling pathways, which control fundamental processes such as tumor cell proliferation, survival, apoptosis and necrosis in imatinib-resistant lines. [We will also define AI effects upon KIT adapter protein interactions.] In Aim 4, we will investigate the lead AKIs identified in Aims 1-3 for inhibition of GIST growth using tumor xenograft/transgenic mouse models according to our IACUC-approved protocol. We will study novel compounds that target KIT via a distinct mechanism both in vitro and in vivo in order to target imatinib- resistant GIST and identify innovative anti-tumor agent(s). These studies will serve as the basis for clinical examination of AKIs directed against these validated kinase targets in patients with imatinib-resistant GIST. If efficacious agents are identified in vitro and in vivo, we intend to pursue additional funding to support moving these agent(s) to "the bedside" in the form of clinical trials