Characterization of MYCN effects in retinoblastoma progression and targeting MYCN for personalized retinoblastoma treatment (MYtargetRB)

视网膜母细胞瘤是儿童最常见的眼部肿瘤。这是一种由肿瘤抑制基因RB1的双等位基因失活启动的肿瘤范例。额外的遗传改变是罕见的，但转录因子MYCN的放大是常见的，并且MYCN mRNA的表达超过了在其他肿瘤实体中观察到的水平。解决MYCN的放松管制被认为是一种很有前途的跨实体治疗方法。在MYTarget RB中，我们假设，对视网膜母细胞瘤中MYCN调节的功能后果和分子机制的更完整了解将确定可能受益于MYCN定向治疗的患者目标群体。我们的实验方法包括利用MYCN指导的shRNA在视网膜母细胞瘤细胞系中进行MYCN表达的基因靶向，以在体外和体内实验中表征MYCN对增殖、凋亡、细胞周期、肿瘤细胞侵袭和转移潜能的影响。MYTarget RB的第二部分致力于研究MYCN作为视网膜母细胞瘤创新靶向治疗候选药物的潜力。初步结果显示，无论是针对MYCN的表观遗传调控还是针对MYCN蛋白的稳定性，都具有令人鼓舞的抗肿瘤作用。针对MYCN的几个小分子将在体外和在视网膜母细胞瘤模型中进行评估，其对下游靶基因表达的影响将通过RNA-Seq进行量化。基于基因表达谱的特征，我们将MYCN信号定义为能够选择对MYCN定向治疗最有效的分子肿瘤亚群的预测性生物标记物。这一特征用于对12个原发视网膜母细胞瘤进行分类，并确定每个亚组的临床参数。本项目的主要目的是研究MYCN在视网膜母细胞瘤发生中的作用，并为MYCN靶向治疗视网膜母细胞瘤提供初步数据。需要一种新的治疗药物来提高转移性视网膜母细胞瘤患者的存活率，并减少幸存者的长期后遗症。

Retinoblastoma is the most common eye tumor in childhood. It is the paradigm of a tumor that is initiated by biallelic inactivation of the tumor suppressor gene RB1. Additional genetic alterations are rare, but amplification of the transcription factor MYCN is common and MYCN mRNA expression exceeds levels observed in other tumor entities. Addressing MYCN deregulation is considered a promising therapeutic approach across entities. In MYtargetRB, we hypothesize that a more complete understanding of the functional consequences and molecular mechanisms of MYCN modulation in retinoblastoma will define a patient target group who may benefit from MYCN-directed therapies. Our experimental approaches include genetic targeting of MYCN expression in retinoblastoma cell lines using MYCN-directed shRNA to characterize the effect of MYCN on proliferation, apoptosis, cell cycle, tumor cell invasiveness and metastatic potential in vitro and in in ovo assays. The second part of MYtargetRB is dedicated to investigate the potential of MYCN as a candidate for innovative targeted therapies of retinoblastoma. Preliminary results show an encouraging anti-tumor effect targeting either epigenetic regulation of MYCN or the stability of MYCN protein. The effect of several small molecules targeting MYCN will be assessed in in vitro and in ovo retinoblastoma models and the effect on gene expression of downstream targets will be quantified with RNA-Seq. Based on the characteristics in gene expression profiles, we will define a MYCN signature as a predictive biomarker capable of selecting the molecular tumor subgroup that best responds to MYCN directed therapies. This signature serves to then classify twelve primary retinoblastomas and to define the clinical parameters of each subgroup. The main goal of this project is to characterize the role of MYCN in retinoblastoma tumorigenesis and to provide first data for the therapeutic potential of MYCN targeting in retinoblastoma. A new therapeutic agent is needed to improve survival rates in patients with the devastating prognosis of metastasized retinoblastoma and to reduce long-term sequelae in survivors.