MAPK as target of glioma immunoediting by CD8 T-cells, and predictor of response to immunotherapy

摘要 虽然PD1阻断治疗胶质母细胞瘤(GBM)的随机试验为阴性，但这些患者中的2个亚组 3确定应答的基础将导致免疫治疗有效 用于某些病人。我们最近报告了66例复发的GBM患者，这些患者接受了 PD1阻断1虽然反应是基于成像标准定义的，但有反应的患者表现出 与临床特征或附加治疗无关的显著提高的存活率。30% 反应性患者的肿瘤中BRAF和PTPN11激活突变丰富，从而刺激MAPK 通路信号1因此，对大多数患者的PD1阻断反应的分子指标 仍未确认身份。我们对p38和ERK的两个效应因子p38和ERK进行了免疫组化检测 这条路。这些标记物的阳性程度预示着PD1阻断后的总存活率。的 与我们的患者肿瘤研究相关，我们模拟了CD8T细胞耗尽对肿瘤进展的影响 新发的转基因小鼠胶质瘤。在这种情况下，CD8 T细胞耗尽导致免疫原性 移植后，CD8 T细胞耗尽的受者优先移植的肿瘤 有免疫能力的宿主。在CD8 T细胞缺失的情况下发生的胶质瘤表现为BRAF和 PTPN11及其相关的MAPK激活通过p38和ERK的磷酸化来指示。CD8 T细胞耗竭 在胶质瘤发展过程中，还导致肿瘤相关巨噬细胞/小胶质细胞的强劲增加()，以及 促炎因子的基因表达。MAPK活性与标记在转基因小鼠中的相关性 胶质瘤和人的基底膜，以及几种促进促炎因子的趋化因子。CRISPR敲门- 筛选出颅内胶质瘤的动态组也表明MAPK参与了T细胞对肿瘤细胞的识别。这些 研究结果表明，肿瘤细胞中的MAPK活性(在胶质瘤进展过程中被CD8 T细胞抑制)， 宣扬支持发炎的。我们推测，MAPK信号升高的GBM更容易受到影响 对CD8T细胞的识别，因为这一途径促进了抗肿瘤。因此，具有MAPK的GBM的子集 信号转导对PD1阻断和CAR T细胞免疫治疗敏感。为了调查这件事，我们将 确定脑胶质瘤发生发展过程中CD8T细胞耗竭对表型的影响。SA 2)确定 MAPK信号通路是否促进抗脑胶质瘤。SA 3)确定对胶质瘤的调节作用 PD1阻断和CAR T细胞治疗反应中的MAPK信号转导。我们将使用转基因模型 肿瘤从头开始发展，具有不同水平的MAPK激活的细胞系，以及遗传MAPK操纵 作为临床上可用的药物，抑制和/或促进MAPK信号通过血脑屏障。成功 这些研究的实施将确立MAPK信号在胶质瘤中的新的免疫相关作用。我们会 确定肿瘤细胞的MAPK信号是否影响对PD1阻断和CAR T细胞治疗的反应。

SUMMARY Whereas randomized trials for PD1 blockade in glioblastoma (GBM) are negative,2 a subset of these patients do respond to immunotherapy.3 Determining the basis of response will result in immunotherapy being effectively used for some patients. We recently reported the analysis of 66 recurrent GBM patients that were treated with PD1 blockade.1 Whereas response was defined based on imaging criteria, responsive patients exhibited significant increased survival that was independent of clinical characteristics or additional treatments. 30% of tumors of responsive patients had enrichment of BRAF and PTPN11 activating mutations, which stimulate MAPK pathway signaling.1 Therefore, molecular indicators of response to PD1 blockade for the majority of patients remain unidentified. We performed immunohistochemistry for phosphorylation of p38 and ERK, two effectors of this pathway. Extent of positivity for these markers was predictive of overall survival following PD1 blockade. Of relevance to our patient tumor studies, we modeled the effect of CD8 T-cell depletion on tumor progression on mouse transgenic gliomas that develop de novo. In this setting, CD8 T-cell depletion resulted in immunogenic tumors that upon transplantation, preferentially engrafted in recipients with CD8 T-cell depletion as opposed to immunocompetent hosts. Gliomas generated in the absence of CD8 T-cells showed upregulation of Braf and Ptpn11, and associated MAPK activation indicated by phosphorylation of p38 and Erk. CD8 T-cell depletion during glioma development also led to robust increase in tumor associated macrophages/microglia (TAM), and gene expression of pro-inflammatory TAM. MAPK activity correlated with TAM markers in mouse transgenic gliomas and human GBM, and with several chemokines that promote pro-inflammatory TAM. A CRISPR knock- out screen for the kinome on intracranial gliomas also implicated MAPK in T-cell recognition of tumor cells. These findings suggest that MAPK activity in tumor cells (that is suppressed by CD8 T-cells during glioma progression), promotes pro-inflammatory TAM. We hypothesize that GBM with elevated MAPK signaling, are more susceptible to CD8 T-cell recognition, as this pathway promotes anti-tumoral TAM. Thus, the subset of GBM that have MAPK signaling are susceptible to PD1 blockade and CAR T-cell immunotherapy. To investigate this, we will SA1) Determine the effects of CD8 T-cell depletion during glioma development on TAM phenotype. SA 2) Determine whether MAPK signaling promotes anti-tumoral glioma TAM. SA 3) Determine the effect of modulating glioma MAPK signaling on response to PD1 blockade and CAR T-cell therapy. We will use transgenic models in which tumors develop de novo, cell lines with varying levels of MAPK activation, genetic MAPK manipulation as well as clinically available drugs inhibit and/or promote MAPK signaling that cross the blood-brain barrier. Successful execution of these studies will establish a novel immune-related role of MAPK signaling in glioma. We will determine whether MAPK signaling by tumor cells influences response to PD1 blockade, and CAR T-cell therapy.