Reprograming Macrophages and Targeting Glioma Stem Cells in Glioblastoma

项目摘要 胶质母细胞瘤（GBM）是最致命的原发性脑肿瘤，预后差。GBM包含异质 包括胶质瘤干细胞（GSC）在内的癌细胞和大量的肿瘤相关巨噬细胞 （TAM）。因为大多数TAM是促进肿瘤的巨噬细胞（pTAM，M2）， 生长和增强免疫抑制，将pTAM重编程为肿瘤抑制性TAM（sTAM，M1）， 激活肿瘤细胞的巨噬细胞吞噬作用代表了一种有吸引力的治疗策略。作为pTAM 与GSC积极相互作用，促进肿瘤生长和治疗抗性，将pTAM重定向为sTAM 可以与靶向GSC协同作用以抑制GBM生长。去发现可以重新编程的小分子 pTAMs转化为sTAMs以刺激巨噬细胞吞噬胶质瘤细胞，我们设计了一种吞噬 使用GFP标记的人iPSC衍生的巨噬细胞和表达tdTomato的 包括GSC在内的神经胶质瘤细胞，以鉴定候选药物和潜在的分子靶点。为此我们 鉴定了BACE 1（β-位点淀粉样前体蛋白裂解酶1）的几种抑制剂作为最佳候选物， 并因此将BACE 1定义为将pTAM重定向为sTAM的分子靶标。我们的研究表明， BACE 1优先由人GBM中的pTAM表达，并且是维持pTAM极化所需的。 通过其抑制剂MK-8931抑制BACE 1，有效地将pTAMs重编程为sTAMs，并促进巨噬细胞 吞噬胶质瘤细胞以抑制GBM生长。此外，我们发现，低剂量辐射（IR） TAM对GBM的浸润作用明显增强，与MK-8931联合作用有协同作用。MK-8931最初 MK-8931是为阿尔茨海默病开发的，已被证明在临床试验中对患者是安全的，MK-8931可以用于治疗阿尔茨海默病。 潜在地简化了基于巨噬细胞的肿瘤治疗。此外，我们以前发现，非- 受体酪氨酸激酶BMX通过介导STAT 3超活化维持GSC致瘤潜力， 证明用伊鲁替尼靶向BMX有效地抑制GBM生长并损害辐射抗性。 因为伊鲁替尼和MK-8931都能穿透血脑屏障（BBB）或血肿瘤屏障（BTB）， 很好，重新利用伊鲁替尼和MK-8931治疗GBM应该是直接的， 潜力基于这些研究，我们假设MK-9831将pTAM重定向为sTAM， 通过伊鲁替尼与靶向GSC协同作用以抑制恶性生长，从而改善GBM 治疗我们将通过以下目标来实现我们的目标：（1）我们将评估 将pTAM重编程为sTAM对GBM中的细胞因子谱、GSC和GSC衍生的周细胞的影响;以及（2）我们 将评估将pTAM重编程为sTAM和靶向GSC用于GBM治疗的治疗影响。 拟定临床前研究的结果将确定是否协同重编程TAM 靶向GSC可以作为一种新的治疗策略，有效地改善GBM治疗， 为未来的临床试验提供信息。

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.