Credentialing Delta-like 3 (DLL3) as an oncoprotein and immunotherapeutic target in neuroblastoma

项目摘要 神经母细胞瘤(NBL)是一种儿童实体瘤，其起源于交感神经的松弛发育。 神经系统。高危NBL患者的五年生存率仅为40%，无复发生存率为 极其罕见。这突显了确定与生物相关和临床可操作的靶点的必要性 患有高危疾病的儿童。在高危NBL中发现的一个基因组异常是一个谱系的扩增- 特异性癌基因，MYCN。一旦被扩增，MYCN转录因子就可以入侵其他谱系的增强子 并建立核心调控回路(CRC)，从而使这些转录因子自动调节 他们自己和彼此保持着环路的表情。由于NBL的发育性质，这些 肿瘤显示独特的细胞表面分子，可能对免疫治疗敏感。一种基于抗体的 针对神经鞘糖脂分子GD2的治疗(地诺昔单抗)延长了患者的5年存活率 患有侵袭性、高危NBL的患者。然而，在周围神经系统的细胞上也发现了GD2 而使用地诺昔单抗治疗会引起剧痛。为了避免这些靶点上/肿瘤外的副作用，我们的目标是 鉴定受CRC调控的NBL肿瘤特异性表面分子并与 未分化、恶性的NBL。我们对CRC结合基因的初步分析揭示了新的和 已知的免疫治疗靶点，包括ALK和L1CAM。另一种CRC调控的细胞表面蛋白 在我们的分析中确定了Delta样正则缺口配体3(DLL3)。DLL3具有NBL特有的细胞表面 在其他类型的细胞中被认为是Notch信号的抑制因子。靶向DLL3的临床试验 成人癌症的免疫治疗目前正在进行中，然而，DLL3在这些癌症中的致癌机制 在NBL中，肿瘤类型尚不明确，也知之甚少。在这项提案中，我们还旨在揭示 DLL3在NBL中的相关生物学，以证明DLL3是该儿童可行的免疫治疗靶点 恶毒。我们的初步数据显示，DLL3的shRNA缺失降低了NBL细胞的细胞活力 DLL3高表达的细胞系模型。此外，DLL3的CRISPR-Cas9基因组缺失导致 规范的Notch靶基因HES1的表达增加。因此，我们推测DLL3抑制Notch 促进持续NBL细胞存活的信号，以及CRC转录调节DLL3导致 过度表达。该项目将结合生物信息学和分子生物学的综合方法来1) 描述由CRC驱动的差异表达的细胞表面靶点和2)确定DLL3在 NBL的恶性状态。我们还期望将CRC计算工作流应用于其他 具有已建立转录回路的癌症。NRSA F31将让您深入了解NBL生物学和 NBL候选免疫治疗靶点的调节，这两项都将为临床前和临床研究提供信息 有针对性的免疫疗法。

Project Summary Neuroblastoma (NBL) is a pediatric solid tumor that arises from deregulated development of the sympathetic nervous system. The five-year survival of patients with high-risk NBL is only 40% and relapse-free survival is extremely rare. This underscores the need to identify biologically-relevant and clinically-actionable targets for children with high-risk disease. One genomic aberration found in high-risk NBL is amplification of a lineage- specific oncogene, MYCN. Once amplified, the MYCN transcription factor can invade enhancers of other lineage oncogenes and establish a core regulatory circuit (CRC), whereby these transcription factors autoregulate themselves and each other to maintain expression of the circuit. Due to the developmental nature of NBL, these tumors display unique cell-surface molecules and may be susceptible to immunotherapy. An antibody-based therapy (dinutuximab) targeting the glycosphingolipid molecule, GD2, extends the 5-year survival rates of patients with aggressive, high-risk NBL. However, GD2 is also found on cells of the peripheral nervous system and treatment with dinutuximab causes agonizing pain. To avoid these on-target / off-tumor side effects, we aim to identify NBL tumor-specific surface molecules that are regulated by the CRC and associated with the undifferentiated, malignant state of NBL. Our preliminary analysis of CRC-bound genes revealed both novel and known immunotherapeutic targets, including ALK and L1CAM. Another CRC-regulated cell-surface protein identified in our analysis is Delta-like canonical notch ligand 3 (DLL3). DLL3 has NBL-specific cell-surface expression, and is known as an inhibitor of Notch signaling in other cell types. Clinical trials with DLL3-targeted immunotherapies in adult cancers are currently ongoing, however, the oncogenic mechanism of DLL3 in these tumor-types, and in NBL, is uncharacterized and poorly understood. In this proposal, we also aim to uncover the relevant biology of DLL3 in NBL in order to credential DLL3 as a viable immunotherapeutic target for this pediatric malignancy. Our preliminary data shows that shRNA-depletion of DLL3 reduces the cellular viability in NBL cell line models with high expression of DLL3. Furthermore, CRISPR-Cas9 genomic depletion of DLL3 results in increased expression of a canonical Notch target gene, HES1. Therefore, we postulate that DLL3 inhibits Notch signaling to promote persistent NBL cell survival, and that the CRC transcriptionally regulates DLL3 to cause overexpression. This project will combine integrative approaches in bioinformatics and molecular biology to 1) describe differentially expressed, cell-surface targets driven by the CRC and 2) determine the role of DLL3 in the malignant state of NBL. We also anticipate that we can apply our CRC computational workflow to other cancers with established transcriptional circuits. This NRSA F31 will provide insight into NBL biology and the regulation of candidate immunotherapeutic targets in NBL, both of which will inform preclinical and clinical studies with targeted immunotherapies.