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

项目总结/摘要 恶性周围神经鞘膜瘤患者的总体生存率几乎没有提高 （MPNST），尽管几十年的研究和许多临床试验，因此需要新的疗法。虽然损失 NF 1 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， 目前还没有有效和直接靶向野生型RAS的批准药物。小说设计 治疗组合需要深入了解由过度活跃的细胞调节的信号通路， RAS和反馈，条件的反应，他们的抑制。MEK、SHP 2和CDK 4/6是关键节点 在MPNST肿瘤的RAS效应子信号传导中，靶向它们的小分子抑制剂的组合可以 具有协同抗肿瘤活性。此外，适应性信号传导响应RAS效应子途径而改变， 抑制不仅发生在肿瘤细胞中，而且发生在包含肿瘤免疫微- 环境（时间）。因此，在我们的MPNST小鼠模型中，TIME的表征及其调节 通过RAS途径的小分子抑制剂，将确定导致免疫功能低下的关键免疫途径。 肿瘤内髓样细胞的重编程，增强内源性免疫应答。 我们提出三个目标：1。鉴定并功能性验证对SHP 2的获得性耐药机制 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 对SHP 2 i以及SHP 2 i + CDK 4/6 i组合具有抗性，并将进行遗传操作研究 以便在功能上验证优先命中。2.确定小剂量联合治疗的疗效和耐受性- RAS信号传导的分子抑制剂，特别是SHP 2 i + CDK 4/6 i，在体外，患者来源的异种移植物（PDX）， 和免疫活性同基因Nf 1-/-/Ink 4a/Arf -/-小鼠模型。我们将测试SHP 2 i的作用， CDK 4/6 i单一试剂或组合作为工具，以探测 途径干扰，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SHP 2 i + CDK 4/6 i组合。3.重新编程 通过用SHP 2 i和CDK 4/6 i的组合治疗在小鼠中的肿瘤内病理性髓样细胞 MPNST。我们的初步数据表明，MPNST的景观是密集的肿瘤浸润， 骨髓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来破译 这些药物对构成肿瘤的细胞类型的特异性作用，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药物的疗效和机制， 在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， 有前途的组合方法，可以迅速和成功地转化为临床，并将告知 有效的患者选择策略和MPNST患者的新型临床试验的发展。

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.