Reprograming Macrophages and Targeting Glioma Stem Cells in Glioblastoma

项目摘要 胶质母细胞瘤(GBM)是最致命的原发脑肿瘤，预后差。GBM包含异质 包括胶质瘤干细胞(GSCs)在内的癌细胞含有丰富的肿瘤相关巨噬细胞 (TAMS)。因为大多数TAM是促进肿瘤的巨噬细胞(pTAM，M2)，支持恶性肿瘤 生长并增强免疫抑制，将pTAM重新编程为肿瘤抑制TAM(STAM，M1)以 激活肿瘤细胞的巨噬细胞吞噬功能是一种有吸引力的治疗策略。作为pTAM 积极与GSCs相互作用，促进肿瘤生长和治疗耐药，将pTAM重定向为STAM 可能与靶向GSC协同作用以抑制GBM生长。发现可以重新编程的小分子 为了刺激巨噬细胞吞噬胶质瘤细胞，我们设计了一种吞噬细胞 利用GFP标记的人iPSC来源的巨噬细胞和表达td番茄的荧光筛选试验 胶质瘤细胞包括GSCs，以确定候选药物和潜在的分子靶点。为此，我们 确定了几种BACE1(β-Site淀粉样前体蛋白裂解酶1)的抑制剂为首选候选药物， 并因此将BACE1定义为将pTAM重定向为STAM的分子靶点。我们的研究表明 BACE1在人GBM中优先由pTAM表达，是维持PTAM极化所必需的。 抑制BACE1的MK-8931可有效地将pTAM重新编程为STAM并促进巨噬细胞 吞噬胶质瘤细胞以抑制基底膜的生长。此外，我们还发现，低剂量辐射(IR) 显著增加对基底膜的渗透，并与MK-8931治疗有协同作用。作为MK-8931，最初 为阿尔茨海默氏症开发的，已被临床试验证明对患者是安全的，MK-8931可以 对于基于巨噬细胞的肿瘤治疗具有潜在的流线型。此外，我们之前发现，非 受体酪氨酸激酶BMX通过介导STAT3的过度激活来维持GSC的致瘤潜能，并且 结果显示，伊布鲁替尼靶向BMX可以有效地抑制GBM的生长并削弱其辐射抗性。 因为伊布鲁替尼和MK-8931都能穿透血脑屏障(BBB)或血肿瘤屏障(BTB) 很好，将伊布鲁替尼和MK-8931重新用于GBM治疗应该是简单而有前途的 潜力。在这些研究的基础上，我们假设MK-9831将pTAM重定向为STAM 与伊布鲁替尼靶向GSCs协同抑制恶性生长，从而改善GBM 治疗。我们将通过以下目标实现我们的目标：(1)评估 在GBM中将pTAM重新编程为细胞因子谱、GSC和GSC来源的周细胞上的STAM；以及(2)我们 将评估将pTAM重新编程为STAM和靶向GSCs治疗GBM的治疗效果。 拟议的临床前研究的结果将决定是否协同重新编程TAMs 靶向GSCs可以作为一种新的治疗策略，有效地改善GBM的治疗，这将 通知未来的临床试验。

Project Summary Glioblastoma (GBM) is the most lethal primary brain tumor with poor prognosis. GBM contains heterogeneous cancer cells including glioma stem cells (GSCs) and harbors abundant tumor-associated macrophages (TAMs). Because the majority of TAMs are tumor-promoting macrophages (pTAMs, M2) that support malignant growth and augment immune suppression, reprograming pTAMs into tumor-suppressive TAMs (sTAMs, M1) to activate macrophage phagocytosis of tumor cells represents an attractive therapeutic strategy. As pTAMs actively interact with GSCs to promote tumor growth and therapeutic resistance, redirecting pTAMs into sTAMs may synergize with targeting GSCs to suppress GBM growth. To discover small molecules that can reprogram pTAMs into sTAMs to stimulate macrophage phagocytosis of glioma cells, we designed a phagocytosis fluorescent screening assay, using GFP-labeled human iPSC-derived macrophages and tdTomato-expressing glioma cells including GSCs to identify drug candidates and the potential molecular targets. To this end, we identified several inhibitors of BACE1 (β-site amyloid precursor protein cleaving enzyme 1) as top candidates, and thus defined BACE1 as a molecular target to redirect pTAMs into sTAMs. Our studies demonstrated that BACE1 is preferentially expressed by pTAMs in human GBMs and required for maintaining pTAM polarization. Inhibiting BACE1 by its inhibitor MK-8931 potently reprogramed pTAMs into sTAMs and promoted macrophage phagocytosis of glioma cells to suppress GBM growth. Moreover, we found that low-dose radiation (IR) markedly enhanced TAM infiltration into GBM and synergized with MK-8931 treatment. As MK-8931, initially developed for Alzheimer's disease, has been shown to be safe for patients in clinical trials, MK-8931 can be potentially streamlined for the macrophage-based tumor therapy. In addition, we previously found that the non- receptor tyrosine kinase BMX maintains GSC tumorigenic potential by mediating STAT3 hyper-activation, and demonstrated that targeting BMX with ibrutinib potently suppressed GBM growth and impaired radioresistance. Because both ibrutinib and MK-8931 penetrate the blood-brain barrier (BBB) or the blood-tumor barrier (BTB) very well, repurposing ibrutinib and MK-8931 for GBM treatment should be straightforward and have promising potential. Based on these studies, we hypothesize that redirecting pTAMs into sTAMs by MK-9831 synergizes with targeting GSCs by ibrutinib to suppress malignant growth and thus improves GBM treatment. We will accomplish our objectives through the following Aims: (1) We will assess the effect of reprograming pTAMs into sTAMs on cytokine profile, GSCs, and GSC-derived pericytes in GBM; and (2) We will evaluate the therapeutic impact of reprograming pTAMs to sTAMs and targeting GSCs for GBM treatment. The outcomes from the proposed pre-clinical studies will determine whether synergistically reprograming TAMs and targeting GSCs can serve as a novel therapeutic strategy to effectively improve GBM treatment, which will inform future clinical trials.