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

项目摘要/摘要 恶性周围神经鞘膜瘤患者的总体存活率几乎没有提高。 尽管进行了数十年的研究和许多临床试验，但仍需要新的治疗方法。当失去的时候 NF1 GTP酶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。 目前还没有批准的有效和直接针对野生型RAS的药物。小说的设计 治疗组合需要对多动调节的信号通路有深入的了解 调节对其抑制的反应的RAS和反馈。MEK、SHP2和CDK4/6是关键节点 在MPNST肿瘤的RAS效应信号中，靶向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，适应性信号对RAS效应通路的响应发生了变化 抑制不仅发生在肿瘤细胞中，而且也发生在组成肿瘤免疫微血管的细胞中。 环境(时间)。因此，我们的MPNST小鼠模型中的时间特征及其调节 通过RAS途径的小分子抑制剂，将识别导致 肿瘤内髓系细胞的重新编程，增强内源性免疫反应。 我们提出了三个目标：1.识别和功能验证对SHP2的获得性抵抗机制 MPNST中的抑制剂，以开发新的治疗组合。我们将确定肿瘤是如何形成的 对SHP2i以及SHP2i CDK4/6i的联合抗性，并将进行基因操作研究 以便在功能上验证优先命中。2.测定小剂量复方制剂的疗效和耐受性 RAS信号的分子抑制剂，特别是SHP2i CDK4/6I，在体外，患者来源的异种移植(PDX)， 和免疫能力强的同基因NF1-/-/Ink4a/Arf-/-小鼠模型。我们将测试SHP2i和 CDK4/6I单一试剂或组合作为工具来探测发生的生化和生物变化 途径扰动，并将确定这些组合的机制和抗肿瘤活性，与一个 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联合CDK4/6I治疗小鼠瘤内病理性髓系细胞 MPNST。我们的初步数据表明，MPNST的景观是密集的肿瘤浸润性病变。 髓系细胞。为了评估这些药物及其组合对时间的影响，我们将使用单一的 细胞转录分析、多参数流式细胞术和多重免疫组织化学破译 这些药物对组成肿瘤的细胞类型的特定影响，包括免疫浸润性和 原代肿瘤细胞。通过确定这些合理设计的治疗方法的疗效和机制 策略在PDX和免疫活性小鼠模型中，我们的研究将提供基于机制的、 有希望的组合方法，可能会迅速并成功地转化为临床，并将告知 针对MPNST患者的有效患者选择策略和新的临床试验的开发。

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.