Proj 2 - Exploiting Therapeutic Vulnerabilities

神经母细胞瘤（NB）在遗传学上代表了一种典型的儿科癌症，因为点突变很少见，但 扩增和片段染色体拷贝数改变（CNA）经常发生。值得注意的是， MYCN 癌基因扩增和 1p 染色体丢失均与高风险显着相关 疾病。虽然这些事件通常同时发生，但人们对它们之间的相互依赖性知之甚少。我们 假设 1p 染色体丢失和 MYCN 扩增都会造成独特的漏洞 治疗，包括免疫治疗。 MYCN 扩增可以在免疫完整状态下准确建模 基因工程小鼠模型（GEMM），但染色体缺失的建模一直具有挑战性。 利用正常人诱导多能干细胞（iPSC）和CRISPR/Cas9技术，我们生成了 由 MYCN 驱动的同基因体内 NB 模型，观察到携带 MYCN 的肿瘤的外显率增加 并在 1p 染色体上进行了工程缺失。因此，我们在存在或 缺乏 1p 缺失，使我们能够识别与 1p 丢失相关的治疗漏洞。 MYCN 通过调节 PD-L1 等检查点蛋白广泛影响肿瘤免疫反应。我们 已经测试了一系列治疗 NB 的靶向和常规疗法，其中许多针对 MYCN，但人们对这些对免疫系统的影响知之甚少。最近 FDA 批准了 针对表面标志物GD2的dinutuximab单克隆抗体，现已成为靶向免疫疗法 NB 的护理标准。使用 PI 实验室开发的 MYCN 驱动小鼠模型，我们将表征 NB 使用 CyTOF 质谱流式单细胞蛋白质组学来测量肿瘤免疫细胞、肿瘤细胞、 同时检测肿瘤和宿主细胞中的信号传导，以识别和表征小分子抑制剂 与免疫疗法配合治疗神经母细胞瘤。 A1：利用基于人类干细胞的神经母细胞瘤体内模型来确定治疗方法 与染色体 1p 缺失相关的漏洞 A2：利用 MYCN 驱动的 NB 免疫活性模型来确定有效的联合疗法， 专注于免疫治疗。 目标 1 的成功完成确定了由 MYCN 驱动的高风险 NB 的治疗漏洞 扩增和1p缺失。目标 2 的成功完成确定了与 免疫疗法治疗 MYCN 驱动的 NB。这两个目标都提出表征现有的小鼠模型 NB 利用精准医学（免疫疗法和 1p 定向疗法）的机会。这个 表征为 NB 儿童的临床试验提供了目标、生物标志物和可行的治疗方法。

Neuroblastoma (NB) represents a prototypical pediatric cancer genetically, in that point mutations are rare, but amplifications and segmental chromosomal copy number alterations (CNAs) occur commonly. Notably, amplification of the MYCN oncogene and loss of chromosome 1p are both prominently associated with highrisk disease. While these events commonly co-occur, their interdependence is poorly understood. We hypothesize that both loss of chromosome 1p and amplification of MYCN create unique vulnerabilities to therapy, including immunotherapy. MYCN amplification can be accurately modeled in immune-intact genetically engineered mouse models (GEMM), but chromosome deletions have been challenging to model. Using normal human induced pluripotent stem cells (iPSC) and CRISPR/Cas9 technology, we generated isogenic in-vivo NB models driven by MYCN, observing increased penetrance in tumors carrying both MYCN and engineered deletions in chromosome 1p. We thus have modeled MYCN-driven NB in the presence or absence of 1p deletions, enabling us to identify therapeutic vulnerabilities associated with 1p loss. MYCN broadly impacts the tumor immune response through regulation of checkpoint proteins such as PD-L1. We have tested an array of targeted and conventional therapies for the treatment of NB, many directed against MYCN, but the effects of these on the immune system are poorly understood. With recent FDA approval of dinutuximab monoclonal antibody directed against the surface marker GD2, targeted immunotherapy is now standard-of-care for NB. Using a MYCN driven mouse model developed in the PI’s lab, we will characterize NB tumors with CyTOF mass cytometric single cell proteomics to measure markers of immune cells, tumor cells, and signaling in both tumor and host cells simultaneously to identify and characterize small molecule inhibitors that cooperate with immunotherapy in the treatment of neuroblastoma. A1: Utilize human stem-cell based in-vivo models of neuroblastoma to identify therapeutic vulnerabilities associated with chromosome 1p deletion A2: Leverage MYCN-driven immunocompetent models of NB to identify effective combination therapy, focusing on immunotherapy. Successful completion of Aim 1 identifies therapeutic vulnerabilities in high-risk NB driven by both MYCN amplification and 1p deletion. Successful completion of Aim 2 identifies targeted therapies that cooperate with immunotherapy in the treatment of MYCN-driven NB. Both aims propose characterizing existing mouse models of NB to leverage opportunities in precision medicine (immunotherapy and 1p-directed therapies). This characterization provides targets, biomarkers, and actionable therapies for clinical trials in children with NB.