Mechanistic biomarkers to enable Bcl2 inhibitor therapies for neuroblastoma

项目摘要 抑制Bcl2家族生存蛋白的药物有望改变癌症治疗的格局。癌症 在逃避细胞生长限制时，激活BH3结构域蛋白形式的应激信号，如Bim 并入侵恶劣的环境。为了保持生存，肿瘤使用生存蛋白如bcl2、bclx、bclw和mcl1。 通过特定的蛋白质-蛋白质相互作用(PPI)隔离这些BH3蛋白质。这会阻止它们的凋亡 信号，但也使这些肿瘤持续依赖于这一功能。竞争性取代的药物 从存活蛋白中分离出来的BH3蛋白释放出强大的细胞凋亡信号。的确，ventoclax 是一种bcl2抑制剂，已显示出显著的临床疗效，获得了FDA的批准，用于治疗 慢性淋巴细胞白血病和成人髓系白血病。这些肿瘤类型没有bcl2激活突变 但根据经验定义，它们的生存依赖于bcl2。相比之下，许多实体肿瘤都有 他们用来阻断BH3信号的生存蛋白的异质性，以及缺乏预测BH3信号的生物标记物 对这一新兴药物类别的敏感性仍然是一个障碍。我们的目标是识别可预测的生物标志物和 开发诊断工具以利用Bcl2家族抑制剂用于临床。我们创建了一个国家基础设施来 建立致命的儿童肿瘤、神经母细胞瘤的患者来源的异种移植和细胞系模型，并利用 创新的功能分析，以确定他们赖以生存的Bcl2家族蛋白。我们发现 神经母细胞瘤通过PPI中和了内源性激活的BIM，具有单一显性存活 蛋白。这隔离了BIM的凋亡活动，但也编码了一种持续的依赖性，并定义了一个 定义生存所需的生存蛋白的机械性生物标志物。一个意想不到的发现是，这 生存依赖是高度固有和稳定的癌细胞：在患者匹配的原发肿瘤中是一致的 和转移部位，以及诊断和复发。在Bim与Bcl2(Bim：Bcl2 PPI)、Bcl2结合的肿瘤中 像威尼托克拉克斯这样的抑制剂在体外和体内都有很高的活性。在Bim与MCL1(Bim：MCL1 PPI)结合的肿瘤中， Bcl2抑制剂没有活性。此外，我们发现所有带有MAPK通路突变的神经母细胞瘤都是在 Bim：mcl1类。令人惊讶的是，MCL1抑制剂对这一亚群也没有活性，尽管取代了Bim MCL1.在这些肿瘤中，bim被bclx重新隔离，并且所有的肿瘤对mcl1/bclx的结合都非常敏感。 抑制力。这证明了我们的预测性Bim PPI生物标记物的稳定性，我们将利用该生物标记物来识别 神经母细胞瘤中的所有生存依赖类别。我们将定义生物标记物在多大程度上保持 稳定的固有肿瘤特征，预测对Bcl2家族抑制剂的选择性易感性。我们还利用 用于对抗MCL1依赖的治疗性MAPK抑制剂。重要的是，我们寻求开发体外诊断 使用邻近连接分析识别预测性BimPPI和认证基因组MAPK生物标记物的工具 定义MCL1依赖项。总而言之，我们的工作应用精确医学方法来分配Bcl2家族 临床使用的抑制剂，并为临床试验设计提供信息，包括预测合理的联合疗法。

Project Summary Drugs that inhibit Bcl2-family survival proteins promise to change the landscape of cancer care. Cancers activate stress signals in the form of BH3-domain proteins like Bim as they escape cellular growth constraints and invade hostile environments. To remain viable, tumors use survival proteins like Bcl2, Bclx, Bclw and Mcl1 to sequester these BH3 proteins through specific protein-protein interactions (PPIs). This blocks their apoptotic signal but also renders such tumors continually dependent on this function. Drugs that competitively displace BH3 proteins from the survival protein sequestering them unleash a potent apoptotic signal. Indeed, venetoclax is a Bcl2 inhibitor that has demonstrated striking clinical efficacy, garnering FDA-approval for the treatment of chronic lymphocytic and adult myelogenous leukemias.These tumor types do not have Bcl2-activatingmutations but are empirically defined to be dependent on Bcl2 for their survival. In contrast, many solid tumors have heterogeneity in which survival protein they use to block BH3 signals, and the absence of biomarkers that predict sensitivity to this emerging drug class remains a barrier. Our objectives are to identify predictive biomarkers and develop diagnostic tools to leverage Bcl2-family inhibitors for clinical use. We created a national infrastructure to generate patient-derivedxenograftand cell line models of the lethal childhood tumor, neuroblastoma, and utilized innovative functional assays to define the Bcl2-family protein they depend on for survival. We discovered that neuroblastomas have endogenously activated Bim neutralized through a PPI with a single dominant survival protein. This sequesters Bim's apoptotic activity, but also encodes a continual dependency, and defines a mechanistic biomarker defining the survival protein required for viability. An unanticipated finding is that this survival dependency is highly cancer cell intrinsic and stable: consistent in patient-matched tumors from primary and metastatic sites, and at diagnosis and relapse. In tumors with Bim bound by Bcl2 (Bim:Bcl2 PPI), Bcl2 inhibitors like venetoclax are highly active in vitro and in vivo. In tumors with Bim bound by Mcl1 (Bim:Mcl1 PPI), Bcl2 inhibitors have no activity. Further, we find that all neuroblastomas with MAPK pathway mutations are in the Bim:Mcl1 class. Surprisingly, Mcl1 inhibitors also have no activity for this subset, despite displacing Bim from Mcl1. In these tumors, Bim is re-sequestered by Bclx, and all are exquisitely sensitive to combined Mcl1/Bclx inhibition. This demonstrates the robustness of our predictive Bim PPI biomarker that we will exploit to identify all survival dependency classes in neuroblastoma. We will define the extent to which the biomarker remains a stable intrinsic tumor feature that predicts selective vulnerability to Bcl2-family inhibitors. We also leverage therapeutic MAPK inhibitors to antagonize Mcl1 dependency. Importantly, we seek to develop in vitro diagnostic tools to identify predictive BimPPIs using proximity-ligation assays, and to credential genomic MAPK biomarkers to define Mcl1 dependence. Collectively, our work applies precision medicine approaches to assign Bcl2-family inhibitors for clinical use, and inform clinical trial designs, including predicting rational combination therapies.