Uncovering treatment targets for peripheral nerve sheath tumor progression in NF1

项目概要/摘要 1型神经纤维瘤病（NF 1）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人中约有1人，大多数患者发生良性丛状和/或真皮 神经纤维瘤丛状神经纤维瘤构成了终身的毁容，发病率和死亡率的来源， 并有可能转化为恶性外周神经鞘瘤（MPNST），一种侵袭性的软组织肿瘤。 组织肉瘤事实上，大约15%的NF 1患者发展为预后不良的MPNST，通常在 人生的第二或第三个十年。MPNST的治疗选择仅限于复杂的外科手术 传统的化疗和分子靶向治疗已经显示出有限的疗效。我们 他们迫切需要新的治疗方案和方法来防止他们的发展。它 最近发现丛状神经纤维瘤通过中间的“非典型” 神经纤维瘤（ANF），在70%的肿瘤中显示CDKN 2A基因的杂合或纯合缺失， 编码p16 INK 4a和p14 ARF（Beert等人，2011; Pemov等人，2018年）。我们的建议将解决关键的未满足的 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。为了提供临床前检测和预防ANF向MPNST发展的模型，我们 结合沙漠刺猬（Dhh）-Cre驱动的Nf 1双等位基因缺失和Cdkn 2a杂合缺失， 建立了在预先存在的神经纤维瘤内形成的可移植ANF的独特模型（Chaney等， 提交）。我们还结合了Dhh-Cre驱动的Nf 1和Pten的双等位基因缺失， 围产期ANF样病变（Keng等，2012年）。来自Dhh-Cre; Nf 1fl/fl; Cdkn 2a +/-小鼠移植的ANF 皮下移植到免疫功能低下的宿主中，在延迟后，提供了一种更快速，更易处理， 转换系统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（Aim 1a）， 通过鉴定和验证小鼠中改变的细胞群和标记物来进一步研究该模型。 和人类PNF，ANF，和MPNST在未扰动的组织切片中使用新的图像分析 通过IndEXing（目标1b）进行CO检测。免疫环境的调节正在增加 用于治疗。因此，我们将定义神经微环境和免疫系统的影响 从ANF进展为MPNST（目标1c）。为了识别ANF的漏洞，我们已经完成了药物和 基于CRISPR的遗传合成致死性筛选在NF 1的同基因永生化人雪旺细胞中 野生型或使用基因编辑使NF 1功能缺失突变纯合。候选药物 抑制PP 2A和其他来自药物筛选工作的新靶点，将测试它们在ANF中的作用。 如体外细胞（Aim 2a），成功后，在我们独特的GEMM中（Aim 2b）。同样，我们的基因 筛选工作将用于定义体外（目标2c）和体内（目标2d）测试的其他漏洞。

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).