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

项目摘要 神经母细胞瘤（NBL）是一种儿科实体瘤，源于交感神经系统发育失调， 神经系统高风险NBL患者的五年生存率仅为40%，无复发生存率为20%。 极其罕见这强调了需要确定生物学相关和临床可行的目标， 患有高风险疾病的儿童。在高风险NBL中发现的一种基因组畸变是谱系扩增- 特异性癌基因MYCN。一旦扩增，MYCN转录因子可以侵入其他谱系的增强子， 癌基因，并建立一个核心调控电路（CRC），从而这些转录因子自动调节 他们自己和彼此保持电路的表达。由于NBL的发展性质，这些 肿瘤显示出独特的细胞表面分子，并且可能对免疫疗法敏感。基于抗体的 靶向鞘糖脂分子GD2的治疗（dinutuximab）延长了 侵袭性高风险NBL患者。然而，GD2也存在于周围神经系统的细胞中 而用dinutuximab治疗会引起剧烈疼痛。为了避免这些靶向/肿瘤外副作用，我们的目标是 为了鉴定受CRC调节并与NBL肿瘤相关的NBL肿瘤特异性表面分子， 未分化的恶性NBL。我们对CRC结合基因的初步分析显示， 已知的免疫靶点，包括ALK和L1CAM。另一种CRC调节的细胞表面蛋白 在我们的分析中鉴定的是δ样典型缺口配体3（DLL3）。DLL3具有NBL特异性细胞表面 在其他细胞类型中，它被称为Notch信号传导的抑制剂。DLL3靶向的临床试验 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尚未完全阐明。 肿瘤类型，在NBL，是不确定的，了解甚少。在本提案中，我们还旨在揭示 在NBL中DLL3的相关生物学，以证明DLL3作为这种儿科疾病的可行免疫靶点。 恶性肿瘤我们的初步数据显示，DLL3的shRNA缺失降低了NBL细胞中的细胞活力， DLL3高表达的品系模型。此外，DLL3的CRISPR-Cas9基因组缺失导致DLL3的缺失。 增加典型Notch靶基因HES1的表达。因此，我们假设DLL3抑制Notch， CRC通过转录调节DLL3来促进NBL细胞的持续存活， 过度表达本计画将结合联合收割机整合生物资讯学与分子生物学的方法， 描述由CRC驱动的差异表达的细胞表面靶点，以及2）确定DLL3在 NBL的恶性状态。我们还预计，我们可以将我们的CRC计算工作流程应用于其他 具有既定转录回路的癌症。这NRSA F31将提供深入了解NBL生物学和 NBL中候选免疫学靶点的调节，这两者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.