背景:成为四十年来的护理标准,基于顺铂的化疗在治疗生殖细胞肿瘤(GCT)方面非常有效。然而,通常难治性患者存在剩余的(抗性)卵黄囊瘤(YST(-R))成分,由于除化疗和手术外缺乏新的治疗选择,导致预后不良。这项研究的目的是通过破译治疗抵抗的分子机制来确定治疗YST的新靶标。此外,我们筛选了靶向CLDN6(CLDN6-ADC)的新型抗体-药物缀合物的细胞毒性功效,以及专门针对YST的药理学抑制剂。
方法:通过流式细胞术测量推定目标的蛋白质和mRNA水平,免疫组织化学染色,福尔马林固定石蜡包埋组织的质谱,磷酸激酶阵列,或qRT-PCR。细胞活力,使用XTT细胞活力测定或膜联蛋白V/碘化丙啶流式细胞术进行GCT和非癌细胞的凋亡和细胞周期测定,分别。YST(-R)组织的可药物基因组改变通过TrueSight肿瘤学500测定鉴定。
结果:我们证明,与非癌性对照相比,用CLDN6-ADC处理增强了CLDN6GCT细胞中的凋亡诱导。以细胞系依赖的方式,观察到G2/M细胞周期阶段的积累或有丝分裂突变。基于突变和蛋白质组分析,这项研究确定了靶向FGF的药物,VGF,PDGF,mTOR,CHEK1,AURKA,或PARP信号通路作为靶向YST的有希望的方法。Further,我们确定了与MAPK信号相关的因素,翻译起始和RNA结合,细胞外基质相关过程以及氧化应激和免疫反应参与治疗抵抗。
结论:总之,这项研究提供了一种新型的CLDN6-ADC靶向GCT。此外,这项研究提出了新的药物抑制剂阻断FGF,VGF,PDGF,mTOR,CHEK1,AURKA,或PARP信号用于治疗(难治性)YST患者。最后,这项研究揭示了YST的治疗耐药机制。
Being the standard-of-care for four decades, cisplatin-based chemotherapy is highly efficient in treating germ cell tumors (GCT). However, often refractory patients present with a remaining (resistant) yolk-sac tumor (YST(-R)) component, resulting in poor prognosis due to lack of novel treatment options besides chemotherapy and surgery. The aim of this study was to identify novel targets for the treatment of YST by deciphering the molecular mechanisms of therapy resistance. Additionally, we screened the cytotoxic efficacy of a novel antibody-drug-conjugate targeting
CLDN6 (
CLDN6-ADC), as well as pharmacological inhibitors to target specifically YST.
Protein and mRNA levels of putative targets were measured by flow cytometry, immunohistochemical stainings, mass spectrometry of formalin-fixed paraffin-embedded tissues, phospho-kinase arrays, or qRT-PCR. Cell viability, apoptosis and cell cycle assays of GCT and non-cancerous cells were performed using XTT cell viability assays or Annexin V / propidium iodide flow cytometry, respectively. Druggable genomic alterations of YST(-R) tissues were identified by the TrueSight Oncology 500 assay.
We demonstrated that treatment with a
CLDN6-ADC enhanced apoptosis induction specifically in
CLDN6+ GCT cells in comparison with non-cancerous controls. In a cell line-dependent manner, either an accumulation in the G2 / M cell cycle phase or a mitotic catastrophe was observed. Based on mutational and proteome profiling, this study identified drugs targeting the FGF, VGF, PDGF, mTOR, CHEK1, AURKA, or PARP signaling pathways as promising approaches to target YST. Further, we identified factors relevant for MAPK signaling, translational initiation and RNA binding, extracellular matrix-related processes as well as oxidative stress and immune response to be involved in therapy resistance.
In summary, this study offers a novel
CLDN6-ADC to target GCT. Additionally, this study presents novel pharmacological inhibitors blocking FGF, VGF, PDGF, mTOR, CHEK1, AURKA, or PARP signaling for the treatment of (refractory) YST patients. Finally, this study shed light on the mechanisms of therapy resistance in YST.