Uncovering treatment targets for peripheral nerve sheath tumor progression in NF1

项目摘要/摘要 1型神经纤维瘤病(NF1)综合征是一种常染色体显性遗传的癌症易感综合征 全球每3,500人中有1人患有良性丛状和/或真皮病变 神经纤维瘤。丛状神经纤维瘤是毁容、发病率和死亡率的终生来源， 并有可能转化为恶性周围神经鞘瘤(MPNST)，一种侵袭性较强的软组织 组织肉瘤。事实上，大约15%的NF1患者出现预后不良的MPNSTs，通常在 生命的第二或第三个十年。MPNSTs的治疗选择仅限于复杂的外科手术 经典的化疗，到目前为止，分子靶向治疗的效果有限。我们 迫切需要新的MPNST治疗方案和方法来防止它们的发展。它 最近发现丛状神经纤维瘤通过中间的“非典型”进展为MPNST。 神经纤维瘤(ANF)，70%的肿瘤显示CDKN2A基因杂合性或纯合性丢失 编码p16INK4a和p14ARF(Beert等人，2011年；Pemov等人，2018年)。我们的提案将解决严重未满足的问题 这一领域的需要，包括更好的ANF体外和体内模型以及确定关键脆弱性 这些细胞。为了提供一个临床前测试和预防ANF到MPNST发展的模型，我们 结合沙漠刺猬(DHH)-Cre驱动的NF1双等位基因缺失和CDKN2a杂合性丢失， 创建了一种在原有神经纤维瘤内发展的可移植ANF的独特模型(Chaney等人， 已呈交)。我们还结合了DHH-CRE驱动的NF1和Pten的双等位基因缺失，产生了快速发展的 围产期ANF样损害(Keng等人，2012年)。DHH-Cre；Nf1fl/fl；CDKN2a+/-小鼠移植的ANF 皮下进入免疫功能低下的宿主生长，经过一段时间的延迟，提供了更快、更易驯服的、 转型体系。我们计划在这些模型中进行完整的转录组和外显子组分析(目标1a)，以及 通过鉴定和验证小鼠体内改变的细胞群体和标记进一步研究该模型 利用一种新的图像分析技术在未受干扰的组织切片中检测人的PNF、ANF和MPNST 这种方法称为通过索引进行一氧化碳检测(目标1b)。免疫环境的调节作用正在增强 用于治疗。因此，我们将定义神经微环境和免疫系统的影响 关于从ANF到MPNST的进展(目标1c)。为了识别ANF漏洞，我们已经完成了药物和 基于CRISPR的NF1等基因永生化人雪旺细胞的遗传合成致死性筛选 使用基因编辑使野生型或纯合子的NF1功能突变丧失。候选药物 抑制PP2A和其他药物筛选工作的新靶点将在ANF中测试它们的效果。 类似于体外培养的细胞(目标2a)，当成功时，在我们独特的GEMM中(目标2b)。同样，我们的基因 筛选工作将用于确定在体外(Aim 2c)和体内(Aim 2d)测试的其他脆弱性。

Project Summary/Abstract Neurofibromatosis type 1 (NF1) syndrome is an autosomal dominant cancer-predisposing syndrome afflicting ~1 in every 3,500 persons worldwide with the majority of patients developing benign plexiform and/or dermal neurofibromas. Plexiform neurofibromas constitute a lifelong source of disfigurement, morbidity and mortality, and have the potential to transform to a malignant peripheral nerve sheath tumor (MPNST), an aggressive soft tissue sarcoma. In fact, approximately 15% of NF1 patients develop poor prognosis MPNSTs, often in the second or third decade of life. Treatment options for MPNSTs are limited to complicated surgical procedures and classical chemotherapy and, so far, molecular targeted therapies have demonstrated limited efficacy. We desperately need new treatment options for the MPNSTs and methods to prevent them from development. It was recently found that plexiform neurofibromas progress to MPNST via an intermediate, “atypical” neurofibroma (ANF) that in 70% of tumors shows heterozygous or homozygous loss of CDKN2A the gene encoding p16INK4a and p14ARF (Beert et al., 2011; Pemov et al., 2018). Our proposal will address critical unmet needs in this field, including better in vitro and in vivo models of ANF and identification of critical vulnerabilities of these cells. To provide a model for preclinical testing and prevention of ANF to MPNST development, we combined Desert hedgehog (Dhh)-Cre driven biallelic deletion of Nf1 with heterozygous loss of Cdkn2a, creating a unique model of transplantable ANF developing within pre-existing neurofibroma (Chaney et al., submitted). We also combined Dhh-Cre driven biallelic deletion of Nf1 and Pten, generating rapidly developing perinatal ANF-like lesions (Keng et al., 2012). ANF from Dhh-Cre;Nf1fl/fl;Cdkn2a+/- mice grafted subcutaneously into immunocompromised hosts grew, after a delay, providing a more rapid, tractable, transformation system. We plan a complete transcriptome and exome analysis in these models (Aim 1a), and further investigate the model by identifying and validating cell populations and markers altered in mouse and human PNF, ANF, and MPNST in unperturbed tissue sections using a new image analysis method called CO-Detection by IndEXing (Aim 1b). Modulation of the immune environment is increasing used therapeutically. We will therefore define the influence of the nerve microenvironment and immune system on progression from ANF to MPNST (Aim 1c). To identify ANF vulnerabilities, we have completed drug and CRISPR-based genetic synthetic lethality screens in isogenic immortalized human Schwann cells that are NF1 wildtype or were made homozygous for NF1 loss of function mutations using gene editing. Candidate drugs that inhibit PP2A, and other novel targets from the drug screening effort, will be tested for their effects in ANF- like cells in vitro (Aim 2a) and, when successful, in our unique GEMMs (Aim 2b). Similarly, our genetic screening effort will be used to define additional vulnerabilities tested in vitro (Aim 2c) and in vivo (Aim 2d).