Molecular characterization of the CXCR4 / CXCR7 axis in germ cell tumors and its targetability using nanobody-drug-conjugates.

Being stimulated by the chemokine CXCL12, the CXCR4 / CXCR7 cascade is involved in tumor proliferation, migration, and metastasis. The interaction between CXCL12, secreted by cells from the microenvironment, and its receptors is complex and has been ascribed to promote chemotherapy resistance. However, the role of this signaling axis and its targetability in germ cell tumors (GCT) is not fully understood. Thus, this study investigated the therapeutic efficacy of a nanobody-drug-conjugate targeting CXCR4 (CXCR4-NDC) and functionally characterized this signaling pathway in GCT using small molecule inhibitors and nanobodies. As shown by diminished cell viability, enhanced apoptosis induction, and detection of mitotic catastrophes, we confirmed the cytotoxic efficacy of the CXCR4-NDC in CXCR4+-GCT cells (i.e. seminoma and yolk-sac tumor), while non-malignant CXCR4--fibroblasts, remained largely unaffected. Stimulation of CXCR4+ / CXCR7+-GCT cells with CXCL12 resulted in an enhanced proliferative and migratory capacity, while this effect could be reverted using CXCR4 inhibitors or a CXCR7-nanobody. Molecularly, the CXCR4 / CXCR7-signaling cascade could be activated independently of MAPK (ERK1 / 2)-phosphorylation. Although, in CXCR4- / CXCR7--embryonal carcinoma cells, CXCR7-expression was re-induced upon inhibition of ERK1 / 2-signaling. This study identified a nanobody-drug-conjugate targeting CXCR4 as a putative therapeutic option for GCT, i.e. seminoma and yolk-sac tumors. Furthermore, this study shed light on the functional role of the CXCR4 / CXCR7 / CXCL12-signaling cascade in GCT, demonstrating an important influence on proliferation and migration.

Targeting CLDN6 in germ cell tumors by an antibody-drug-conjugate and studying therapy resistance of yolk-sac tumors to identify and screen specific therapeutic options.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

[Current view on testicular tumors from a developmental biological perspective : Important biomarkers and molecular pathological investigations]. / Aktuelle Betrachtung der Hodentumoren aus entwicklungsbiologischer Sicht : Wichtige Biomarker und molekularpathologische Untersuchungen.

BACKGROUND: Germ cell tumors (GCTs) are the most common type of cancer in Germany in young men between 15 and 44 years of age. The routinely performed diagnostic procedures are essential for the patient's treatment, but can be difficult due to heterogenous histologies. Additionally, the molecular mechanisms of the development of the special forms growing teratoma syndrome (GTS) and testicular tumors with malignant somatic transformation (MST) as well as of therapy resistance are not fully understood. OBJECTIVES: Updated understanding of the molecular processes underlying GCT development and their special forms as well as recommendations for new and useful biomarkers. RESULTS: The development of GCTs is a dynamic process largely influenced by the microenvironment. Seminomas (SEs) in particular seem to posses a higher cellular plasticity than previously assumed, allowing SEs to be reprogrammed into an embryonal carcinoma (EC) or differentiate into extra-embryonal tissues (yolk sac tumors [YSTs], trophoblastic differentiation). Novel serological (mi371a-3p) and pathological (FOXA2) biomarkers are well suited to early detect GCTs and YSTs, respectively. For more aggressive tumors and special cases (GTS, MST), there are still no reliable diagnostics or specific/tailored therapies available. CONCLUSION: The ability of SEs to transit into EC or YSTs should be considered during therapy. Future research should focus on deciphering the special forms GTS and MST as well as the early recognition of YSTs, since their development seems to be an escape mechanism to chemotherapy.

The pioneer and differentiation factor FOXA2 is a key driver of yolk-sac tumour formation and a new biomarker for paediatric and adult yolk-sac tumours.

Yolk-sac tumours (YSTs), a germ cell tumour subtype, occur in newborns and infants as well as in young adults of age 14-44 years. In clinics, adult patients with YSTs face a poor prognosis, as these tumours are often therapy-resistant and count for many germ cell tumour related deaths. So far, the molecular and (epi)genetic mechanisms that control development of YST are far from being understood. We deciphered the molecular and (epi)genetic mechanisms regulating YST formation by meta-analysing high-throughput data of gene and microRNA expression, DNA methylation and mutational burden. We validated our findings by qRT-PCR and immunohistochemical analyses of paediatric and adult YSTs. On a molecular level, paediatric and adult YSTs were nearly indistinguishable, but were considerably different from embryonal carcinomas, the stem cell precursor of YSTs. We identified FOXA2 as a putative key driver of YST formation, subsequently inducing AFP, GPC3, APOA1/APOB, ALB and GATA3/4/6 expression. In YSTs, WNT-, BMP- and MAPK signalling-related genes were up-regulated, while pluripotency- and (primordial) germ cell-associated genes were down-regulated. Expression of FOXA2 and related key factors seems to be regulated by DNA methylation, histone methylation / acetylation and microRNAs. Additionally, our results highlight FOXA2 as a promising new biomarker for paediatric and adult YSTs.