Ink4a/ARF/Ink4b locus in Neurofibromatosis Type 1

摘要/摘要 恶性周围神经鞘瘤(MPNST)是慢性阻塞性肺疾病患者的主要死亡原因。 神经纤维瘤病-1(NF1)。MPNSTs出现在良性丛状神经纤维瘤(PNFS)的NF1患者中，但它 不清楚为什么只有30%的PNF转化为MPNST。最近的研究表明，失活的 INK4a/ARF/INK4b基因座(对于INK4a和ARF称为CDKN2A；对于INK4b称为CDKN2B)产生癌前病变 病变称为不典型的神经纤维瘤性肿瘤(ANNUBP)。ANNUBP是一种新的 公认的MPNST的前体，但驱动和分类这种过渡中间体的机制很差 已定义。Ink4a/ARF/INK4b中断是目前与ANNUBPS有关的主要变化，除了NF1损失和 RABL6A上调。在MPNSTs中，基因座在一个、两个或全部三个基因上发生改变，患者的存活率更低 与失去这三个人有关。每个基因(INK4a、ARF和INK4b)编码一个肿瘤抑制因子，但它们的 单独的贡献以及与其他因素在推动癌症中的协同作用仍然不完全清楚。 鉴于INK4a、ARF和INK4b的重要作用，需要更好地了解它们在MPNST开发中的作用， 因为针对该基因的药物要么被批准，要么在其他癌症中表现出希望。 我们的中心假设是p16INK4a、ARF和p15INK4b在多条途径中协同作用 抑制良性PNP和ANNUBP向MPNST的转化。目标1将定义重要的基因 与INK4a、ARF和INK4b失活相一致的蛋白质组学事件 遗传学、分子和组织学分析。结果将与存活率等临床变量相关。这个 一种新开发的液体活检法评估基因座状态和其他肿瘤标志物的预后价值 在NF1患者中进行检测。Aim 2对人类p16INK4a、ARF和/或p15INK4b进行CRISPR编辑 以确定它们在PNF-ANNUBP-MPNST转化中的作用。有针对性地分析 细胞和小鼠模型中的疑似MPNST驱动基因将识别选择性地与 INK4a、ARF或INK4b缺失在体外和体内驱动ANNUBP-MPNST转化。目标3确立了 针对Ink4a/Arf/Ink4b相关通路的药物在PNF-ANNUBP-MPNST治疗中的生物学意义 和预防。获得性耐药的预测媒介将使用分子和 药理学方法。研究将确定靶向治疗相对于MPNST前模型的价值 防止恶性进展。影响：研究将提供对INK4a/ARF/INK4b的新见解，INK4a/ARF/INK4b是 在人类癌症中经常失活的基因座。创新的PNF-ANNUBP-MPNST进展动物模型 将生成并定义导致MPNST的转换机制。临床前研究将 评估以Ink4a/Arf/Ink4b通路为靶点的新的联合疗法在预防恶性进展中的作用。 结果将增进我们对推动MPNST发生的事件的了解，有助于早期诊断和 针对ANNUBP的先发制人干预。

Summary/Abstract Malignant peripheral nerve sheath tumors (MPNSTs) are the leading cause of death in patients with neurofibromatosis-1 (NF1). MPNSTs arise in NF1 patients from benign plexiform neurofibromas (PNFs) but it is unclear why only ~30% of PNFs transform into MPNSTs. Recent studies suggest that inactivation of the INK4a/ARF/INK4b locus (called CDKN2A for INK4a and ARF; CDKN2B for INK4b) generates a pre-malignant lesion called an atypical neurofibromatous neoplasm of uncertain biology (ANNUBP). ANNUBPs are the newly recognized precursor to MPNSTs, but mechanisms that drive and classify this transitional intermediate are poorly defined. INK4a/ARF/INK4b disruption is the main alteration currently linked to ANNUBPs, besides NF1 loss and RABL6A upregulation. In MPNSTs, the locus is altered at one, two or all three genes, with worse patient survival associated with loss of all three. Each gene (INK4a, ARF, and INK4b) encodes a tumor suppressor, but their separate contributions and cooperativity with other factors in driving cancer remain incompletely understood. Given their prominent role, a better understanding of INK4a, ARF, and INK4b in MPNST development is needed, as drugs targeting the locus are either approved or showing promise in other cancers. Our central hypothesis is p16Ink4a, ARF and p15Ink4b act cooperatively in multiple pathways to suppress the transformation of benign PNFs and ANNUBPs to MPNSTs. Aim 1 will define significant genetic and proteomic events coinciding with INK4a, ARF and INK4b inactivation in human ANNUBPs and MPNSTs by genetic, molecular and histologic analyses. Results will be correlated to clinical variables such as survival. The prognostic value of a newly developed liquid biopsy assay evaluating locus status and other tumor markers will be determined in NF1 patients. Aim 2 employs CRISPR editing of p16Ink4a, ARF and/or p15Ink4b in human PNF-derived cells to determine their roles in PNF-ANNUBP-MPNST transformation. Directed analyses of suspected MPNST driver genes in cells and mouse models will identify genes that selectively cooperate with INK4a, ARF, or INK4b loss to drive ANNUBP-MPNST transformation in vitro and in vivo. Aim 3 establishes the biological significance of drugs targeting Ink4a/Arf/Ink4b relevant pathways in PNF-ANNUBP-MPNST therapy and prevention. Predicted mediators of acquired resistance to therapy will be verified using molecular and pharmacologic approaches. Studies will determine the value of targeted therapy against pre-MPNST models to prevent malignant progression. Impact: Studies will provide new insights into INK4a/ARF/INK4b, one of the most frequently inactivated loci in human cancers. Innovative animal models of PNF-ANNUBP-MPNST progression will be generated and mechanisms of transformation leading to MPNST will be defined. Preclinical studies will assess a new combination therapy targeting Ink4a/Arf/Ink4b pathways in preventing malignant progression. Results will advance our understanding of events driving MPNST development, facilitating earlier diagnosis and pre-emptive interventions targeting ANNUBPs.