Novel Allosteric Kinase Inhibitors Target Imatinib-Resistant GIST

描述（由申请人提供）：胃肠道间质瘤（GIST）是最常见的肉瘤，在美国每年约有6，000例新发病例。GIST治疗基于KIT“癌基因成瘾”，其驱动肉瘤发生。因此，ATP依赖性（-模拟）KIT抑制剂伊马替尼是晚期GIST的一线治疗药物。尽管伊马替尼治疗，疾病复发/进展仍然频繁，因为GIST发展由继发性KIT突变引起的伊马替尼耐药。这些突变降低了其他ATP模拟物的功效。因此，靶向激酶的化合物，同时避免ATP模拟耐药机制，是独特的兴趣。我的导师大卫切雷什（David Cheresh）报道了变构激酶抑制剂（阿基）的开发，该抑制剂以ATP非依赖性方式阻止KIT/BRAF/CRAF/PDGFR激酶激活。虽然目前的ATP-模拟物击中了“正确”靶标，但AKI可能：1）具有比ATP-模拟物更高的特异性; 2）避免使ATP-模拟物失活的“看门人”突变;和3）产生针对伊马替尼耐药GIST的强活性，而对AKI耐药的担忧较少。我们假设这种新型的KIT抑制AKI可用于治疗伊马替尼耐药GIST，这种疾病的治疗选择有限。我们的目的是确定在伊马替尼耐药GIST的背景下，变构激酶抑制的功能/机制作用。在目标1中，我们将使用伊马替尼敏感和耐药GIST细胞系表征一系列高效AKI。我们将以时间/剂量依赖性方式定义阿基对细胞活力、凋亡和细胞周期阻滞的影响。在目标2中，我们将进行生化/机制分析，以确定野生型与突变型KIT酶的动力学效应和结合亲和力的铅AKI。在目标3中，我们将采用遗传学和药理学分析来研究KIT（和BRAF/CRAF/PDGFR）AKI如何影响细胞内激酶信号通路，这些通路控制伊马替尼耐药株中肿瘤细胞增殖、存活、凋亡和坏死等基本过程。[We也将定义AI对KIT衔接蛋白相互作用的影响。在目标4中，我们将根据IACUC批准的方案，使用肿瘤异种移植物/转基因小鼠模型研究目标1-3中鉴定的主要AKI抑制GIST生长。我们将研究通过体外和体内不同机制靶向KIT的新型化合物，以靶向伊马替尼耐药GIST并鉴定创新的抗肿瘤药物。这些研究将作为伊马替尼耐药GIST患者中针对这些经验证激酶靶点的AKI临床检查的基础。如果在体外和体内发现有效的药物，我们打算寻求额外的资金，以支持将这些药物以临床试验的形式转移到“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