Targeted Inhibition in Leukemia

摘要 我们已经鉴定了Vav 3的第一个体内和体外小分子抑制剂，Vav 3是一个过度表达的信号中枢， 在许多癌症中被压制，并且是小GT3 Rac的激活剂。Rac是一种主要的致癌介质， 癌症干/祖细胞中的非致癌成瘾。在低剂量下，我们的抑制剂消除了TKI耐药性， 在体内，它可以降低前B-ALL小鼠模型的存活率，并在体内根除癌症干细胞的繁殖。 小鼠连续移植模型。它诱导原发性儿科费城阳性（Ph+）细胞凋亡， Ph样B-ALL和化疗耐药RAM免疫表型原代儿科AML细胞。它特别 靶向白血病细胞，同时保留体内正常的造血作用，并且没有毒性。此外，它还积极与 在人实体瘤的致癌Ras异种移植物小鼠模型中。鉴于这一广泛的活动和广泛的- 鉴于Vav 3和Rac在人类疾病中的作用，我们的抑制剂可能在几种人类疾病中有效， 对现有疗法有抵抗力的癌症。 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， 为了验证我们的抑制剂的作用机制，我们将在儿童TKI耐药B- ALL和AML。尽管引入了ABL酪氨酸激酶抑制剂（TKI）疗法，并且最近高度- 毒性免疫疗法、Ph+ B-ALL和AML仍然是预后不良的疾病，特别是在成人中， 频繁复发和对当前疗法的抵抗。这项拨款申请的长期目标是一个多药 由TKI和我们的药物或优化的衍生物组成的方法，作为ALL和AML的新疗法， 与当前挽救治疗方法相关的毒性。我们假设，多目标方法， ALL和AML是防止对单药TKI治疗耐药的必要条件。 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， 门控细胞并通过靶向Vav 3克服TKI抗性。本研究的目的是（1）验证 Vav 3/Rac信号传导轴作为我们药物的靶点，使用生物化学和遗传学方法， 体内影响;（2）使用生物物理，结构和遗传学方法鉴定我们的药物在Vav 3上的结合位点， 使用定点诱变来接近并验证该位点。最后，（3）我们将利用 CCHMC肿瘤白血病/实体瘤库，以测试我们的药物在PDX化疗模型中的疗效- 单用耐药儿童ALL和AML以及与现有TKI方法联合使用，并验证金属治疗， onein作为生物标志物。如果成功，我们希望看到我们的药物，或更有效的类似物，进入临床前阶段。 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 本文提出的Vav 3自抑制构象的靶向可以推广到其他“不可用药的”构象。 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.!