Endocytic Trafficking of ADCs in GBM cancer stem-like cells

摘要：胶质母细胞瘤(GBM)是一种侵袭性癌症，预后差(中位生存期~15mo)。 虽然抗体-药物结合物(Adc)有很大的希望，但这种方法在 病人数量很多。基因组分析已经确定了调节内吞的多个基因的变化。 GBM肿瘤中的通路，但内吞通路的重新配置可能会损害 ADC疗法的疗效尚未被探索。这项拟议的研究的目标是检验这一假设 血管周围肿瘤干细胞(CSLCs)内皮细胞转运的肿瘤相关重组 影响GBM的ADC治疗结果。为了验证这一假设，我们利用了高度协作的 跨学科的方法，并将重点放在ABT-414作为原型ADC。ABT-414是一种完全人性化的IgG1 针对EGFRvIII和与单甲基金黄色素F偶联的扩增的-wt-EGFR的单抗。 在选定的复发性肾小球基底膜患者中，约50%有效。我们已经证实GBM CSLC对 ABT-414，并组装了一组具有良好特征的GBM患者来源的异种移植物(PDX)，显示了一系列 在体外和体内对该疗法的敏感性。我们还在体外和体内验证了CSLCs在 GBM中的血管周围间隙内化ABT-414，并通过内吞途径运输到循环 在弹头释放的地方，弹壳和溶酶体之间也有联系。我们建议操纵两个 与循环相关的基因，RBSN和EHD3，表现出比例失调的表达 基底膜肿瘤。初步数据表明，CSLC中这些基因表达的变化可以减少 将ABT-414转移到回收舱，增强对CSLC的杀灭作用。我们提出了两个具体目标 其中我们将确定：(1)在GBM来源的CSLC中操纵RBSN对贩运的影响 RBSN缺失/下调后ABT-414细胞的命运和CSLC的体外存活；内体阻断 利用shVps39进行成熟培养，并表达标记突变体RBSN。删除RBSN的影响 将使用原位GBM在活体内测试下调表达以及对ABT-414治疗效果的影响 PDX模型。(2)在GBM来源的CSLCs中操纵EHD3对ABT-414细胞转运命运的影响 EHD3缺失或过表达和突变型EHD3表达后CSLC的体外存活。同时， 我们将确定EHD3基因启动子的高甲基化是否抑制GBM活检组织中EHD3的表达。 EHD3或突变型EHD3过表达的影响及其对ABT-414疗效的影响 使用原位GBM PDX模型进行活体测试。

SUMMARY: Glioblastoma (GBM) is an aggressive cancer with a dismal prognosis (median survival ~15 mo). Although antibody-drug conjugates (ADC) hold great promise, this approach is unexpectedly ineffective in a large number of patients. Genomic analyses have identified alterations in multiple genes that regulate endocytic pathways in GBM tumors but the possibility that reconfiguration of the endocytic pathways can compromise the efficacy of ADC therapy has not been explored. The goal of the proposed study is to test the hypothesis that tumor-associated reconfiguration of endocytic trafficking in perivascular cancer stem-like cells (CSLCs) influences the outcome of ADC therapies in GBM. To test this hypothesis, we are utilizing a highly collaborative transdisciplinary approach and focusing on ABT-414 as the prototypic ADC. ABT-414 is a fully humanized IgG1 mAb directed toward EGFRvIII and amplified-wt-EGFR that is conjugated to monomethyl auristatin F. It is effective in ~50% of selected patients with recurrent GBM. We have verified that GBM CSLCs are sensitive to ABT-414 and assembled a well-characterized panel of GBM patient-derived xenografts (PDX) that show a range of sensitivities to the therapy in vitro and in vivo. We also have verified, in vitro and in vivo, that CSLCs in the perivascular space in GBM internalize ABT-414, and that it is trafficked by endocytic pathways to recycling compartments as well as to the lysosome, where release of the warhead occurs. We propose to manipulate two genes, RBSN and EHD3, that are associated with recycling and exhibit dysregulated expression in a proportion of GBM tumors. Preliminary data indicate that alterations in expression of these genes in CSLCs can reduce diversion of ABT-414 to the recycling compartments and enhance CSLC killing. We propose two Specific Aims in which we will determine: (1) The effects of manipulation of RBSN in GBM-derived CSLCs on the trafficking fate of ABT-414 and CSLC survival in vitro after deletion/downregulation of RBSN; blockade of endosome maturation using shVps39; and expression of tagged mutant-RBSN. The effects of RBSN deletion downregulation, and the impact on ABT-414 therapeutic efficacy, will be tested in vivo using an orthotopic GBM PDX model. (2) The effects of manipulation of EHD3 in GBM-derived CSLCs on the trafficking fate of ABT-414 and CSLC survival in vitro after deletion or overexpression of EHD3, and expression of mutant EHD3. In parallel, we will determine if hypermethylation of the EHD3 gene promoter represses EHD3 expression in GBM biopsies. The effects of overexpression of EHD3 or mutant-EHD3, and their impact on ABT-414 therapeutic efficacy, will be tested in vivo using an orthotopic GBM PDX model.