Targeted Inhibition in Leukemia

摘要 我们已经在体内和体外发现了第一个Vav3的小分子抑制剂，Vav3是一个过度表达的信号枢纽。 在许多癌症中受到抑制，并且是小GTP酶Rac的激活剂。RAC是肿瘤发生和发展的主要调节因子。 癌症干细胞/祖细胞中的非致癌成瘾。在低剂量时，我们的抑制剂消除了对TKI的耐药性 活体，延长前B-ALL小鼠模型的存活时间，并在 小鼠连续移植模型。它诱导原发费城阳性(Ph)儿科细胞凋亡 Ph样B-ALL和化疗耐药的RAM原代儿科AML细胞免疫表型。它特别是 以白血病细胞为靶点，同时避免体内正常造血，且无毒性。此外，它还处于活动状态 在致癌的RAS异种移植小鼠模型中发现人类实体瘤。考虑到这种广泛的活动和广泛的- Vav3和Rac在人类疾病中的变化，我们的抑制剂很可能对几个人有效 对当前治疗方法产生抗药性的癌症。 尽管我们专注于Ph B细胞急性淋巴细胞白血病(Ph B-ALL)作为一种更简单的癌症模型 验证我们的抑制剂的作用机制，我们将在儿童TKI耐药B细胞模型上测试其疗效。 ALL和AML。尽管推出了ABL酪氨酸激酶抑制剂(TKI)治疗，最近高度... 毒性免疫疗法、Ph B-ALL和AML仍然是预后较差的疾病，特别是在成年人中，原因是 经常复发，对目前的治疗方法耐药。这项赠款申请的长期目标是一种多药 一种由TKI和我们的药物或优化的衍生物组成的方法，作为治疗ALL和AML的新疗法 与当前抢救治疗方法相关的毒性。我们假设多目标方法在 ALL和AML是防止对单药TKI治疗产生耐药性所必需的。 根据我们的初步数据，我们假设我们的药物增加了白血病的死亡率。 门控细胞，并通过靶向Vav3克服TKI抗性。拟议研究的目标是(1)验证 Vav3/Rac信号轴作为我们药物的靶点，使用生化和遗传方法，并确定在 活体意义；(2)利用生物物理、结构和遗传AP确定我们的药物在Vav3上的结合位置。 方法，并使用定点突变验证该站点。最后，(3)我们将利用 CCHMC肿瘤学白血病/实体肿瘤存储库测试我们的药物在PDX化疗模型中的疗效- 耐药的儿童ALL和AML单独使用，并结合现有的TKI方法和验证金属- 作为生物标志物的一种。如果成功，我们希望看到我们的药物或更有效的类似物进入临床前 安全性分析，并可能进入对TKI疗法耐药的ALL和AML的I期临床试验。变构 这里提出的针对Vav3自身抑制构象的靶向可以推广到其他不可用药的情况 蛋白质-蛋白质界面。！

ABSTRACT We have identified the first in vivo and in vitro small molecule inhibitor of Vav3, a signaling hub that is overex- pressed in many cancers and an activator of the small GTPase Rac. Rac is a major mediator of oncogenic and non-oncogenic addiction in cancer stem/progenitor cells. At low dose, our inhibitor eliminates TKI-resistance in vivo, prolongs the survival of a mouse model of pre-B-ALL, and eradicates cancer stem cell propagation in a model of mouse serial transplantation. It induces apoptosis of primary pediatric Philadelphia-positive (Ph+) and Ph-like B-ALL and chemotherapy-resistant RAM immunophenotype primary pediatric AML cells. It specifically targets leukemic cells while sparing normal hematopoiesis in vivo and shows no toxicity. In addition, it is active in oncogenic Ras xenografts mouse models of human solid tumors. Given this broad activity and the wide in- volvement of Vav3 and Rac in human disease, it is likely that our inhibitor will be efficacious in several human cancers resistant to current therapies. Even though we focus on Ph+ B-cell acute lymphoblastic leukemia (Ph+ B-ALL) as a simpler cancer model to validate the mechanism of action of our inhibitor, we will test its efficacy in models of pediatric TKI-resistant B- ALL and AML. Despite the introduction of ABL tyrosine kinase inhibitor (TKI) therapy and more recently highly- toxic immunotherapies, Ph+ B-ALL and AML remain poor prognosis diseases, especially in adults, as a result of frequent relapse and resistance to current therapies. The long-term goal of this grant application is a multidrug approach consisting of a TKI and our drug or an optimized derivative as a new therapy for ALL and AML without the toxicity associated with current salvage therapy approaches. We postulate that multitarget approaches in ALL and AML are necessary to prevent resistance to single-agent TKI therapy. Based on our preliminary data, we hypothesize that our drug increases death of leukemia initiating and propa- gating cells and overcomes TKI-resistance by targeting Vav3. The goal of the proposed research is to (1) validate the Vav3/Rac signaling axis as our drug’s target using biochemical and genetic approaches and determine in vivo implications; (2) to identify our drug’s binding site on Vav3 using biophysical, structural, and genetic ap- proaches, and validate the site using site-directed mutagenesis. Finally, (3) we will take advantage of the CCHMC Oncology Leukemia/Solid Tumor Repository to test our drug’s efficacy in PDX models of chemotherapy- resistant pediatric ALL and AML alone and in combination with existing TKI approaches and validate metallothi- onein as a biomarker. If successful, we would like to see our drug, or a more potent analog, move into pre-clinical safety analysis and potentially into a Phase I clinical trial in ALL and AML resistant to TKI therapies. Allosteric targeting of the Vav3 autoinhibited conformation as proposed here could be generalized to other ‘undruggable’ protein-protein interfaces.!