Proj 2 - Exploiting Therapeutic Vulnerabilities

神经母细胞瘤(NB)在遗传学上代表典型的儿科癌症，在这一点上突变是罕见的，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1p的丢失都与高风险显著相关 疾病。虽然这些事件通常是同时发生的，但人们对它们之间的相互依存关系知之甚少。我们 假设染色体1p的丢失和MYCN的扩增都会造成独特的脆弱性 治疗，包括免疫治疗。MYCN扩增可以准确地模拟免疫完整 基因工程小鼠模型(GEMM)，但染色体缺失一直是建模的挑战。 使用正常的人类诱导多能干细胞(IPSC)和CRISPR/Cas9技术，我们产生了 MYCN驱动的同源体内NB模型，观察携带两种MYCN的肿瘤外显率增加 以及染色体1p上的人为缺失。因此，我们对存在或存在的MYCN驱动的NB进行建模 没有1p缺失，使我们能够确定与1p缺失相关的治疗漏洞。MYCN 通过调节检查点蛋白，如PD-L1，广泛影响肿瘤免疫反应。我们 已经测试了一系列针对NB治疗的靶向和常规疗法，其中许多是针对 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GD2的地诺昔单抗，目前正在进行靶向免疫治疗 NB的护理标准。使用PI实验室开发的MYCN驱动的小鼠模型，我们将描述NB 用CyTOF质控单细胞蛋白质组学方法检测免疫细胞、肿瘤细胞、 同时在肿瘤细胞和宿主细胞中发出信号以识别和表征小分子抑制物 在神经母细胞瘤的治疗中配合免疫治疗。 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。 AIM 1的成功完成确定了由MYCN和MYCN驱动的高危NB的治疗脆弱性 扩增和1p缺失。成功完成目标2确定了与之合作的靶向治疗 免疫治疗在MYCN相关神经营养不良中的应用这两个目标都建议对现有的老鼠模型进行特征化 利用精准医学(免疫疗法和1P定向疗法)方面的机会。这 特征化为NB儿童的临床试验提供靶点、生物标记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.