Targeting CD44 signaling in regulating glioblastoma stemness and immune response

项目摘要 胶质母细胞瘤（GBM）是最常见的脑肿瘤，预后非常差。目前，尽管做了手术， 化疗和放疗作为标准治疗，这些方案的组合不 显著延长患者生存期。溶瘤病毒治疗为实体瘤治疗开辟了一个新时代， 治疗脑瘤HSV-1衍生的溶瘤病毒（oHSV）是溶瘤病毒（OV）的最高级类型。 也是目前美国FDA批准的唯一一种病毒。最近，另一种oHSV（Delytact， 由Daiichi销售）在日本被有条件地批准用于治疗GBM患者。因此，在本发明中， 研究改善针对GBM的oHSV治疗的方法是非常重要的。然而，抗肿瘤功效 溶瘤病毒（OV）的增殖受肿瘤微环境（TME）的限制。CD 44信号转导通过以下途径调节GBM TME： 与细胞外基质（ECM）或免疫细胞的相互作用。透明质酸是一种重要的细胞外基质 GBM中的葡萄糖代谢产物。CD 44在GBM细胞中高度表达，是一种标志物， 胶质母细胞瘤干细胞（GSC）。CD 44还结合其他配体，如骨桥蛋白（OPN）、软骨素（chondroitin）、 和蛋白多糖来调节ECM和免疫细胞功能。 我们正在进行的实验表明：1）GBM中的CD 44表达与患者的不良预后相关， 2)瘤内注射溶瘤单纯疱疹病毒1型（oHSV）增加脑肿瘤中HA的分泌 3）oHSV-CD 44显著下调SOX 2并降低GSC的干性，4） oHSV-CD 44显著增加体内抗肿瘤功效，和5）oHSV-CD 44与AKT的组合 抑制剂或照射可进一步降低GSC中SOX 2和Nestin的表达，降低GSC的干性。 鉴于这些发现，我们假设，在肿瘤微环境（TME）中阻断CD 44信号转导， 分泌CD 44细胞外结构域的溶瘤病毒（oHSV-CD 44）不仅可以使CD 44介导的GBM失活， 生长并导致免疫抑制，但也会抑制GSC的干性，同时使细胞对 化疗和放疗。该假设将通过以下实验来解决， 具体目的：1）研究oHSV-CD 44与AKT抑制剂的组合是否降低GSC干性 并增加抗GBM免疫应答，和2）测试oHSV-CD 44是否增加放射治疗- 介导的抗GBM功效。 这项研究意义重大，对几个研究领域产生了广泛的影响。除了脱落 阐明了HA-CD 44信号在GSC干性、GBM侵袭性和 标准治疗敏感性，这项研究将揭示oHSV-CD 44阻断CD 44信号传导的作用 增强GBM治疗的疗效，包括化疗或放疗。此外，组合 oHSV-CD 44与AKT抑制或放射治疗将为肿瘤复发提供新的治疗策略。

Project summary Glioblastoma (GBM) is the most frequent brain tumor with a very poor prognosis. Currently, despite surgery, chemotherapy, and radiotherapy as the standard-of-care treatments, combination of these regimens does not prolong patient survival significantly. Oncolytic viral therapy opens a new era for solid tumor therapy, especially for brain tumors. HSV-1 derived oncolytic viruses (oHSVs) are the most advanced type of oncolytic virus (OV) and the only type of virus that is currently FDA approved in the USA. Very recently, another oHSV (Delytact, marketed by Daiichi) was granted conditional approval for the treatment of GBM patients in Japan. Thus, investigating ways to improve oHSV therapy against GBM is highly significant. However, the anti-tumor efficacy of oncolytic virus (OV) is limited by tumor microenvironment (TME). CD44 signaling regulates GBM TME through interaction with extracellular matrix (ECM) or immune cells. Hyaluronic acid is one of the key extracellular matrix components derived from glucose metabolites in GBM. CD44 is highly expressed in GBM cells and is a marker for glioblastoma stem cells (GSCs). CD44 also bind to other ligands, such as, osteopontin (OPN), chrondroitin and proteoglycan to regulate ECM and immune cell function. Our ongoing experiments show that: 1) CD44 expression in GBM is correlated with poor prognosis of patients, 2) Oncolytic herpes simplex virus type 1 (oHSV) intratumoral injection increases HA secretion in brain tumor xenografts in vivo, 3) oHSV-CD44 significantly downregulates SOX2 and decreases stemness of GSCs, 4) oHSV-CD44 significantly increases anti-tumor efficacy in vivo, and 5) Combination oHSV-CD44 with an AKT inhibitor or irradiation could further decrease SOX2 and Nestin expression in GSCs and reduce GSC stemness. Given these findings, we hypothesize that blocking CD44 signaling in tumor microenvironment (TME) using a CD44 extracellular domain-secreting oncolytic virus (oHSV-CD44) will not only disable CD44-mediated GBM growth and lead to immunosuppression but will also inhibit GSC stemness while sensitizing the cells to chemotherapy and radiotherapy. The hypothesis will be addressed with the following experiments proposed in the Specific Aims: 1) Investigate whether combination oHSV-CD44 with an AKT inhibitor reduces GSC stemness and increases the anti-GBM immune response, and 2) Test whether oHSV-CD44 increases radiotherapy- mediated anti-GBM efficacy. The proposed study is significant and has a broad impact on several research areas. In addition to shedding light on the fundamental understanding of HA-CD44 signaling in GSCs stemness, GBM invasiveness, and standard-of-care treatment sensitivity, this study will uncover the role of CD44 signaling blockade by oHSV-CD44 in enhanced efficacy of GBM treatment, including chemotherapy or radiotherapy. Furthermore, combination of oHSV-CD44 with an AKT inhibition or radiotherapy will provide novel treatment strategies for tumor relapse.