Enhanced Immunotherapy by Targeting the Pros1:Macrophage Mer Axis

项目总结/摘要 肿瘤可以利用正常生理免疫调节机制来抑制肿瘤特异性免疫应答。 免疫反应针对免疫检查点的靶向免疫疗法，如PD-1/PD-L1和 CTLA 4改善了患者的预后，但仅在一部分癌症中有效。持久的反应 检查点靶向治疗导致的一些患者的死亡，引发了人们对识别和 针对其他先天性和适应性免疫检查点。 最近的研究已经将受体酪氨酸激酶的Tyro 3/Axl/Mer（TAM）家族鉴定为新的酪氨酸激酶。 先天免疫检查点Ubil等人（JCI，2018）表明，肿瘤分泌Pros 1，一种Tyro 3/Mer配体， 抑制宿主巨噬细胞的促炎M1反应。Pros 1结合诱导了 p38和PTP 1b到Mer，阻止p38的核转位和随后的M1相关蛋白的表达。 基因.巨噬细胞Mer或PTP 1b的基因缺失在Pros 1存在下恢复M1极化。 通过靶向Pros 1：巨噬细胞Mer信号传导， 免疫疗法为了更好地理解和预防Mer介导的免疫抑制， 目的：1）确定Mer激酶活性和衔接蛋白募集在M1中的作用 抑制，2）确定Pros 1分泌肿瘤的可变免疫抑制的基础和3）确定 PTP 1b抑制作为单一或联合治疗是否可以增加肿瘤内免疫浸润， 提高生存率。 为了确定Mer激酶活性的重要性，CRISPR将用于基因消融Mer ATP， 结合位点和RNAseq用于监测Pros 1诱导的M1抑制期间的总体转录变化。 生物信息学分析将确定受Mer激酶影响的关键途径，并进行生物化学验证。 免疫沉淀和蛋白质组学分析将用于确定Pros 1诱导的细胞凋亡的重要性。 动态衔接蛋白募集和新的蛋白质：蛋白质关联。细微的基因改变会导致 Pros 1蛋白活性降低。肿瘤Pros 1序列的变化将与抑制肿瘤细胞增殖的能力相关。 M1极化和构建的预测模型，以确定哪些肿瘤对Pros 1：Mer敏感 靶向治疗药理学PTP 1b抑制（单药和联合治疗）增加 在原发性和转移性肿瘤模型中确定肿瘤免疫浸润和存活。 总之，拟议的研究可以确定新的治疗靶点来预防Pros 1：Mer介导的 免疫抑制，产生关于哪些患者可能从Pros 1：Mer靶向治疗中获益的预测标准 以及药物PTP 1b抑制是否是改善对癌症的免疫反应的可行策略。

PROJECT SUMMARY/ABSTRACT Tumors can utilize mechanisms of normal physiological immune regulation to suppress the tumor-specific immune response. Targeted immunotherapies directed against immune checkpoints, like PD-1/PD-L1 and CTLA4, have improved patient outcomes but are only effective in a subset of cancers. The durable responses of some patients, resulting from checkpoint-targeted therapy, has sparked great interest in identifying and targeting other innate and adaptive immune checkpoints. Recent studies have identified the Tyro3/Axl/Mer (TAM) family of receptor tyrosine kinases as a novel innate immune checkpoint. Ubil et al. (JCI, 2018) show that tumors secrete Pros1, a Tyro3/Mer ligand, which suppresses the pro-inflammatory M1 response of host macrophages. Pros1 binding induces the recruitment of p38 and PTP1b to Mer, preventing nuclear translocation of p38 and subsequent expression of M1 associated genes. Genetic deletion of macrophage Mer or PTP1b restores M1 polarization in the presence of Pros1. By targeting Pros1:macrophage Mer signaling it may be possible to enhance cancer immunotherapy. To better understand and prevent Mer mediated immune suppression the following specific aims are proposed: 1) Determine the roles of Mer kinase activity and adapter protein recruitment in M1 suppression, 2) Identify the basis for variable immune suppression by Pros1 secreting tumors and 3) Ascertain whether PTP1b inhibition as mono- or combination therapy can increase intra-tumoral immune infiltrate and improve survival. To establish the importance of Mer kinase activity, CRISPR will be used to genetically ablate the Mer ATP binding site and RNAseq used to monitor global transcriptional changes during Pros1 induced M1 suppression. Bioinformatics analysis will identify key pathways affected by Mer kinase, which will be validated biochemically. Immunoprecipitation and proteomic analysis will be used to determine the importance of Pros1-induced dynamic adapter protein recruitment and novel protein:protein associations. Subtle genetic alterations can lead to reduced Pros1 protein activity. Changes in tumor Pros1 sequence will be correlated with ability to suppress M1 polarization and a predictive model constructed to identify which tumors will be susceptible to Pros1:Mer targeted therapy. Efficacy of pharmacological PTP1b inhibition (mono- and combination therapy) to increase tumor immune infiltrate and survival will be determined in primary and metastatic tumor models. Together, the proposed study may identify novel therapeutic targets to prevent Pros1:Mer mediated immune suppression, yield predictive criteria as to which patients may benefit from Pros1:Mer targeted therapy and whether pharmacological PTP1b inhibition is a viable strategy to improve the immune response to cancer.