Endocytic Trafficking of ADCs in GBM cancer stem-like cells

胶质母细胞瘤（GBM）是一种侵袭性癌症，预后差（中位生存期约15个月）。 尽管抗体-药物缀合物（ADC）具有很大的前景，但这种方法在免疫治疗中出乎意料地无效。 大量的病人。基因组分析已经确定了调节内吞作用的多个基因的改变， 但是，内吞途径的重新配置可能会损害GBM肿瘤中的内吞途径。 ADC治疗的功效尚未被探索。这项研究的目的是检验以下假设： 血管周围癌干细胞样细胞（CSLC）内吞运输的肿瘤相关重构 影响GBM中ADC治疗的结果。为了验证这一假设，我们正在利用一个高度合作的 跨学科的方法和重点ABT-414作为原型ADC。ABT-414是完全人源化的IgG 1 针对EGFRvIII和与单甲基澳瑞他汀F偶联的修饰的wt-EGFR的mAb。是 在约50%的复发性GBM选定患者中有效。我们已经证实，GBM CSLC对 ABT-414，并组装了一组充分表征的GBM患者来源的异种移植物（PDX），其显示了一系列的 在体外和体内对治疗的敏感性。我们还在体外和体内证实， GBM中的血管周围空间使ABT-414内化，并且它通过内吞途径运输到再循环 这些细胞可以被释放到细胞室以及溶酶体中，在那里发生弹头的释放。我们建议操纵两个 基因RBSN和EHD 3与再循环相关，并表现出一定比例的表达失调 GBM肿瘤。初步数据表明，这些基因在CSLC中表达的改变可以降低CSLC的表达。 将ABT-414转移到再循环隔室并增强CSLC杀灭。我们提出两个具体目标 其中我们将确定：（1）在GBM衍生的CSLCs中操纵RBSN对运输的影响 RBSN缺失/下调后ABT-414和CSLC体外存活的命运;内体阻断 使用shVps 39的成熟;和标记的突变体-RBSN的表达。RBSN缺失的影响 下调，以及对ABT-414治疗功效的影响，将使用原位GBM在体内进行测试。 PDX模型。(2)操纵GBM源性CSLCs中的EHD 3对ABT-414转运命运的影响 以及EHD 3缺失或过表达以及突变体EHD 3表达后CSLC的体外存活。同时， 我们将确定EHD 3基因启动子的超甲基化是否抑制GBM活检组织中EHD 3的表达。 EHD 3或突变EHD 3的过表达的作用，以及它们对ABT-414治疗功效的影响， 使用原位GBM PDX模型在体内进行测试。

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.