Proj 2 - Exploiting Therapeutic Vulnerabilities

神经母细胞瘤（NB）是一种典型的儿科癌症遗传，因为点突变是罕见的，但 扩增和节段性染色体拷贝数改变（CNA）是常见的。值得注意的是， MYCN癌基因的扩增和染色体1 p的丢失都与高风险相关。 疾病虽然这些事件通常同时发生，但人们对它们的相互依存关系知之甚少。我们 假设染色体1 p丢失和MYCN扩增都产生了独特的脆弱性 包括免疫疗法MYCN扩增可以在免疫完整的 基因工程小鼠模型（GEMM），但染色体缺失一直是具有挑战性的模型。 使用正常人诱导多能干细胞（iPSC）和CRISPR/Cas9技术，我们产生了 由MYCN驱动的同基因体内NB模型，观察到携带MYCN和MYCN的肿瘤中的转移率增加。 和染色体1 p中的工程化缺失。因此，我们在存在或不存在的情况下对MYCN驱动的NB进行了建模， 没有1 p缺失，使我们能够确定与1 p缺失相关的治疗漏洞。MYCN 通过调节PD-L1等检查点蛋白广泛影响肿瘤免疫应答。我们 已经测试了一系列用于治疗NB的靶向和常规疗法，其中许多针对 MYCN，但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GD 2的dinutuximab单克隆抗体，靶向免疫治疗现在是 NB的标准治疗。使用PI实验室开发的MYCN驱动小鼠模型，我们将表征NB 用CyTOF质谱细胞计数单细胞蛋白质组学测量免疫细胞，肿瘤细胞， 以及同时在肿瘤和宿主细胞中的信号传导，以鉴定和表征小分子抑制剂 在神经母细胞瘤的治疗中与免疫疗法合作。 A1：利用基于人干细胞的神经母细胞瘤体内模型来鉴定治疗性 与染色体1 p缺失相关的漏洞 A2：利用MYCN驱动的NB免疫活性模型来鉴定有效的联合治疗， 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1 p缺失。目标2的成功完成确定了与以下方面合作的靶向治疗： 在治疗MYCN驱动的NB中的免疫疗法。这两个目标都建议对现有的小鼠模型进行表征 NB利用精准医学（免疫疗法和1 p定向疗法）的机会。这 表征为NB儿童的临床试验提供了靶点、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.