Advancing RAS pathway targeted therapy in NF1-MPNST: effects of SHP2 and CDK4/6 inhibitors on the tumor and the tumor immune microenvironment

项目概要/摘要 恶性周围神经鞘瘤患者的总生存率几乎没有进步 （MPNST），尽管经过了数十年的研究和许多临床试验，因此需要新的疗法。损失的同时 NF1 GTPase 激活蛋白功能的研究表明，靶向 RAS 可能是一种合理的治疗方法， 目前还没有批准的药物可以有效、直接针对野生型 RAS。新颖的设计 治疗组合需要深入了解多动症调节的信号通路 RAS 和反馈调节对其抑制的反应。 MEK、SHP2 和 CDK4/6 是关键节点 MPNST 肿瘤中的 RAS 效应信号传导，以及针对它们的小分子抑制剂的组合可能 具有协同抗肿瘤活性。此外，响应 RAS 效应通路的适应性信号变化 抑制不仅发生在肿瘤细胞中，而且发生在构成肿瘤免疫微细胞的细胞中。 环境（时间）。因此，我们的 MPNST 小鼠模型中 TIME 的表征及其调节 通过 RAS 通路的小分子抑制剂，将识别导致 瘤内骨髓细胞重新编程，增强内源性免疫反应。 我们提出三个目标： 1. 识别并功能验证 SHP2 获得性抗性机制 MPNST 抑制剂，以开发新的治疗组合。我们将确定肿瘤如何变成 对 SHP2i 以及 SHP2i CDK4/6i 组合具有抗性，并将进行基因操作研究 为了在功能上验证优先级命中。 2. 确定联合小剂量药物的疗效和耐受性 RAS 信号传导的分子抑制剂，特别是 SHP2i CDK4/6i，在体外、患者来源的异种移植物 (PDX) 中， 和具有免疫能力的同基因 Nf1 -/-/Ink4a/Arf -/- 小鼠模型。我们将测试SHP2i的效果和 CDK4/6i 单一药物或组合作为工具来探测发生的生化和生物学变化 通路扰动，并将确定这些组合的机制和抗肿瘤活性， 根据我们先进的初步数据，优先关注 SHP2i CDK4/6i 组合。 3. 重新编程 SHP2i 和 CDK4/6i 联合治疗小鼠瘤内病理性骨髓细胞 MPNST。我们的初步数据表明 MPNST 的景观中肿瘤浸润密集 骨髓细胞。为了评估这些药物及其组合对 TIME 的影响，我们将利用单一药物 细胞转录分析、多参数流式细胞术和多重免疫组织化学来破译 这些药物对构成肿瘤的细胞类型的特定作用，包括免疫浸润和 原发性肿瘤细胞。通过确定这些合理设计的治疗方法的功效和机制 PDX 和免疫活性小鼠模型中的策略，我们的研究将提供基于机制的、 有希望的组合方法可以快速成功地转化为临床，并将为 有效的患者选择策略和针对 MPNST 患者的新型临床试验的开发。

PROJECT SUMMARY/ ABSTRACT There has been little advancement in overall survival for patients with malignant peripheral nerve sheath tumor (MPNST), despite decades of research and many clinical trials, and thus novel therapies are needed. While loss of NF1 GTPase-activating protein function suggests that targeting RAS may be a logical therapeutic approach, there is currently no approved drug that effectively and directly targets wild-type RAS. The design of novel therapeutic combinations requires a deep understanding of the signaling pathways regulated by hyperactive RAS and feedback that conditions the response to their inhibition. MEK, SHP2, and CDK4/6 are critical nodes in RAS effector signaling in MPNST tumors, and combinations of small molecule inhibitors that target them may have synergistic anti-tumor activity. Further, adaptive signaling changes in response to RAS effector pathway inhibition occurs not only in tumor cells but also in cells that comprise the tumor immune micro- environment (TIME). Thus, the characterization of the TIME in our murine model of MPNST, and its modulation via small-molecule inhibitors of the RAS pathway, will identify key immune pathways leading to the reprogramming of intratumoral myeloid cells, enhancing endogenous immune responses. We propose three Aims: 1. Identify and functionally validate mechanisms of acquired resistance to SHP2 inhibitors in MPNST in order to develop novel therapeutic combinations. We will determine how tumors become resistant to SHP2i, as well as to SHP2i +CDK4/6i in combination, and will perform genetic manipulation studies in order to functionally validate priority hits. 2. Determine the efficacy and tolerability of combination small- molecule inhibitors of RAS signaling, particularly SHP2i + CDK4/6i, in in vitro, patient-derived xenografts (PDX), and immune-competent syngeneic Nf1 -/-/Ink4a/Arf -/- mouse models. We will test the effects of SHP2i and CDK4/6i single agents or combinations as tools to probe the biochemical and biological changes that occur upon pathway perturbation, and will determine the mechanism and anti-tumor activity of these combinations, with a priority focus on the SHP2i+CDK4/6i combination, based on our advanced preliminary data. 3. Reprogram the intratumoral pathological myeloid cells via treatment with the combination of SHP2i and CDK4/6i in murine MPNST. Our preliminary data suggest that the landscape of MPNST is densely populated by tumor infiltrating myeloid cells. To evaluate the effects of these agents and their combinations on the TIME, we will utilize single cell transcriptional analysis, multiparameter flow cytometry and multiplex immunohistochemistry to decipher the specific effects of these drugs on cell types that comprise the tumor, including both immune-infiltrating and primary tumor cells. By determining the efficacy and mechanism of these rationally-designed therapeutic strategies in both PDX and immune-competent mouse models, our studies will provide mechanism-based, promising combinatorial approaches that may be rapidly and successfully translated to the clinic, and will inform effective patient selection strategies and the development of novel clinical trials for patients with MPNST.