Endocytic Trafficking of ADCs in GBM cancer stem-like cells

GBM 癌症干细胞样细胞中 ADC 的内吞转运

• 批准号: 10596502
• 负责人: Candece L Gladson
• 金额: $ 35.04万
• 依托单位: CLEVELAND CLINIC LERNER COM-CWRU
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-04-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10596502
• 关键词: Affect; Antibodies; Antibody-drug conjugates; Biopsy; Blood Vessels; Cell Death Induction; Cell Survival; Clinical; Clinical Trials; Data; Down-Regulation; Endosomes; Epidermal Growth Factor Receptor; Epigenetic Process; Exhibits; Extracellular Space; Gene Deletion; Gene Expression; Generations; Genes; Genomics; Glioblastoma; Goals; Heterozygote; Hypermethylation; IgG1; Immunoglobulin G; Immunotherapy; In Vitro; Lysosomes; Malignant Neoplasms; Membrane; Modeling; Monoclonal Antibodies; Mutation; Outcome; Parents; Pathway interactions; Patient Selection; Patients; Pharmaceutical Preparations; Phenotype; Predisposition; Prognosis; Proteins; Recurrence; Recycling; Repression; Testing; Therapeutic; Treatment Efficacy; Tubular formation; Xenograft procedure; cell killing; cytotoxicity; epidermal growth factor receptor VIII; expectation; gene product; genotyped patients; improved; in vivo; in vivo evaluation; multidisciplinary; mutant; novel; overexpression; patient derived xenograft model; precision medicine; promoter; protein transport; prototype; stem-like cell; trafficking; tumor

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.

摘要：胶质母细胞瘤（GBM）是一种侵略性癌症，预后较差（中位生存期〜15 mo）。 尽管抗体 - 药物缀合物（ADC）具有巨大的希望，但这种方法在A中意外无效 大量患者。基因组分析已经确定了调节内吞的多个基因的改变 GBM肿瘤中的途径，但是内吞途径重新配置的可能性会损害 尚未探索ADC疗法的功效。拟议的研究的目的是检验以下假设。 血管性癌干细胞（CSLC）中内吞交易的肿瘤相关的重新配置 影响GBM中ADC疗法的结果。为了检验这一假设，我们正在利用高度协作 跨学科的方法，专注于ABT-414作为原型ADC。 ABT-414是完全人性化的IgG1 MAB针对EGFRVIII和与单甲基Auristatin f的Amplied-WT-EGFR。它是 在约50％的重复GBM患者中有效。我们已经验证了GBM CSLC对 ABT-414并组装了一个特征良好的GBM患者衍生异种移植物（PDX），该面板显示了一个范围 在体外和体内对治疗的敏感性。我们还验证了体外和体内的CSLC GBM中的血管周空间内部化ABT-414，并通过内吞途径进行回收途径。 隔离室以及弹性释放的溶酶体。我们建议操纵两个 基因，RBSN和EHD3，与回收利用和表达不足的表达相关。 GBM肿瘤。初步数据表明，这些基因在CSLC中的表达改变可以减少 将ABT-414转移到回收室并增强CSLC杀戮。我们提出了两个具体目标 我们将确定：（1）GBM衍生的CSLC中RBSN操纵对贩运的影响 RBSN的缺失/下调后，ABT-414和CSLC生存的命运；内体的封锁 使用SHVPS39成熟；和标记的突变体-RBSN的表达。 RBSN删除的效果 下调以及对ABT-414治疗功效的影响将在体内使用原位GBM进行测试 PDX模型。 （2）EHD3在GBM衍生的CSLC中操纵对ABT-414的贩运命运的影响 EHD3的缺失或过表达后的体外CSLC存活以及突变EHD3的表达。并联， 我们将确定EHD3基因启动子的高甲基化是否会抑制GBM活检中的EHD3表达。 EHD3或突变体-EHD3的过表达及其对ABT-414治疗功效的影响将 使用原位GBM PDX模型在体内进行测试。