Novel Allosteric Kinase Inhibitors Target Imatinib-Resistant GIST

描述（由申请人提供）：胃肠道间质瘤（GIST）是美国最常见的肉瘤，每年约有6000例新发病例。GIST的治疗基于KIT“癌基因成瘾”，它驱动了肉瘤的形成。因此，atp依赖性（模拟）KIT抑制剂伊马替尼是晚期GIST的一线治疗方法。尽管有伊马替尼治疗，但由于继发性KIT突变导致gist对伊马替尼产生耐药性，因此疾病复发/进展仍然频繁。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模拟抗性机制，是非常有趣的。我的导师David Cheresh报道了变链激酶抑制剂（AKI）的发展，该抑制剂以不依赖atp的方式阻止KIT/BRAF/CRAF/PDGFR激酶激活。虽然目前的atp模拟物击中了“正确的”目标，但AKIs可能：1)比atp模拟物具有更高的特异性；2)避免使atp模拟物失活的“守门人”突变；3)对伊马替尼耐药GIST具有较强的活性，对aki耐药的担忧较少。我们假设这类具有KIT抑制的新型AKIs可用于治疗伊马替尼耐药GIST，这是一种治疗选择有限的疾病。我们的目的是确定变构激酶抑制在伊马替尼耐药GIST中的功能/机制作用。在Aim 1中，我们将使用伊马替尼敏感和耐药的GIST细胞系来表征一系列高效AKIs。我们将以时间/剂量依赖的方式定义AKI对细胞活力、凋亡和细胞周期阻滞的影响。在Aim 2中，我们将进行生化/机制分析，以确定AKIs铅对野生型和突变型KIT酶的动力学效应和结合亲和力。在Aim 3中，我们将采用遗传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系中肿瘤细胞增殖、存活、凋亡和坏死等基本过程。我们还将定义AI对KIT适配器蛋白相互作用的影响。在Aims 4中，我们将根据iacuc批准的方案，使用肿瘤异种移植/转基因小鼠模型，研究Aims 1-3中鉴定的先导AKIs抑制GIST生长的作用。我们将研究通过体外和体内不同机制靶向KIT的新化合物，以靶向伊马替尼耐药GIST并确定创新的抗肿瘤药物。这些研究将作为针对伊马替尼耐药GIST患者中这些已证实的激酶靶点的AKIs临床检查的基础。如果在体外和体内鉴定出有效的药物，我们打算寻求额外的资金支持，以临床试验的形式将这些药物转移到“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