Research projects in Prof. Johannes Schulte's group
Currently, the Schulte lab focuses on the following research projects:
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Hippo-YAP pathway – driver of relapsed neuroblastoma?
The Hippo-YAP pathway has been described to regulate proliferation and epithelial-mesenchymal transition of cells which allows for a disseminated tumor growth. Metastasis as well as invasive growth and high proliferation rates of tumor cells are characteristic for malignant tumors such as ovarian carcinomas, lung and liver cancers. Indeed, higher activity of Hippo-YAP pathway has been described for those types of cancer.
In neuroblastoma, a bioinformatics analysis of DNA- and RNA-sequencing data from primary and relapsed neuroblastoma samples also revealed a relapse-specific activation of the Hippo-YAP pathway. In this project, we aim to define the role of Hippo-YAP pathway in neuroblastoma pathogenesis and progression. Currently, we are investigating the effect of YAP1 overexpression and downregulation on proliferation, apoptosis, migration and differentiation in human neuroblastoma cell lines in order to gain insights into key functions of this pathway and its mechanisms during tumor progression and metastasis. The overall objective in this project is the identification of new potential target structures for targeted therapy of high-risk neuroblastomas.
The role of circular RNAs in neuroblastoma
Circular RNAs (circRNA) are a class of noncoding RNAs that came recently in the focus of research. They selectively inhibit microRNA (miRNA) function to allow translation of their target mRNAs. We aim to identify circRNAs expressed in pediatric neuroblastoma, and analyze the function of selected candidates in tumor biology. This proof-of-concept study aims to illuminate circuits acting in cells between a candidate circRNA, a target miRNA and key downstream oncogenes. This circuitry is likely to reveal novel control points, which could ultimately improve risk stratification or identify new druggable targets.
Next-generation sequencing approaches for characterising key players in neuroblastoma pathogenesis
What are the (epi-)genomic aberrations that drive physiological neural-crest cells into becoming neuroblastoma cells?
Using high-coverage DNA sequencing, RNA sequencing and epigenomics approaches, we can characterize the genomes, epigenomes and transcriptomes of tumor samples in unprecedented detail at base-pair resolution. In-house generated sequencing data will be integrated with publicly available genomics data from neuroblastoma cell lines and patient samples. Thorough bioinformatics analysis of these data, using published state-of-the-art and custom-developed tools, will provide candidates implicated in important pathogenic mechanisms. These candidates are subsequently followed up with validation experiments in our lab.
Moreover, in order to foster reproducible research in bioinformatics, our custom tools will be published in form of well-documented software packages.
The role of MCPH-genes in embryonal tumors
Abnormal cell proliferation is involved in oncogenesis as well as the etiology of developmental disorders of the brain. While tumors are characterized by increased proliferation, microcephaly is the result of reduced stem cell proliferation. The prototype of microcephaly, autosomal recessive primary microcephaly (MCPH), is genetically heterogeneous and characterized by a reduced brain volume due to a proliferation defect of neural stem cells. The embryonal tumors neuroblastoma and medulloblastoma, on the other hand, arise from an abnormal proliferation of neural stem cells. As especially high-risk cases of these tumors are marked by a poor prognosis, there is need for a better understanding of the tumor biology and the identification of new targets for precision therapies. Our hypothesis is that MCPH gene products can cause both an excess of proliferation as well as reduced proliferation resulting in tumor formations or microcephaly, respectively.
We aim to investigate the role of MCPH genes in medulloblastoma and neuroblastoma. In particular, we aim to identify interaction partners of CDK5RAP2 (MCPH3), assess the MCPH gene expression in tumors, and to characterize the effect of inhibition of MCPH genes in experimental models.
Minimal Residual Disease Detection for Neuroblastoma Diagnostics
Risk stratification for neuroblastoma patients is currently based on several criteria, including MYCN oncogene amplification. Nearly 50% of high-risk cases relapse, which implies the presence of residual cells, referred to as minimal residual disease (MRD). PCR-based assays of MRD status in leukemia patients are already in clinical routine.
In collaboration with NEO New Oncology, we establish a specific 'NEO-NB' hybrid capture-sequencing assay which detects relevant genomic alterations. Breakpoints of the MYCN amplicon, which are highly specific for each individual tumor, are used to design patient-specific PCR assays for assessing MRD. Since the MYCN amplicon is stable during disease progression, these changes will likely persist in all tumor cells. As a proof of concept we want to test the 'NB-MRD' assay for several MYCN breakpoints in neuroblastoma cell lines. Our NB-MRD protocol is feasible for RQ-PCR and digital droplet PCR, and both techniques will be considered.
Eventually we aim to provide analysis guidelines and a standardized pipeline for patient specific NB-MRD detection. The NEO-NB assay will be applied in a prospective MRD diagnostics study using FFPE as well as fresh-frozen primary tumor samples in order to implement these assays in routine diagnostics. Upon validation of the assay, we aim to establish NB-MRD for individual patients and define standardized analysis guidelines.
This project is being pursued in collaboration with PD Dr. Cornelia Eckert, Prof. Dr. Matthias Fischer and NEO New Oncology bearbeitet.
Mouse models of embryonal tumors
We employ genetically engineered mouse models (GEMMs) to investigate the molecular mechanisms of neuroblastoma pathogenesis.
Such models allow for the analysis of both intrinsic cellular processes as well as interactions between the tumor cell and the extra-cellular microenvironment in vivo. Our primary focus is the validation and functional characterization of tumor-initiating factors. For instance, in order to recapitulate the amplification of the neuroblastoma oncogene MYCN, which is observed in about 25% of neuroblastoma patients, our lab developed a Cre/loxP system that allows for the tissue-specific over-expression of MYCN in the neural crest during development. To this end, a genetic cassette carrying the human MYCN cDNA sequence and a loxP-flanked transcriptional stop sequence (LSL) were introduced via homologous recombination into the murine Rosa26 locus. The LSL signal, which prevents expression of the MYCN transgene, can be removed by cross-breeding the LSL-MYCN mice to mouse strains with tissue-specific Cre recombinase expression (Dbh-Cre, Phox2B-Cre, Th-Cre). This generates mice in which ectopic expression of MYCN induces neuroblastomas by cellular transformation of the parasympathetic lineage. LSL-oncogene models provide the opportunity to investigate further candidates genes, e.g. LIN28B, ALK, and BIRC5.
Thus, our experimental in vivo system allows us to analyze and evaluate novel drug targets, supporting pre-clinical drug development and suggesting novel cancer therapy strategies.
Early detection of secondary tumors after retinoblastoma
Retinoblastoma is the most frequent ocular childhood tumor. It is characterized by an inactivation of both alleles of the tumor suppressor gene RB1. Non-hereditary retinoblastomas need to be distinguished from hereditary cases. In patients with non-hereditary retinoblastoma, both alleles are active at birth and become deficient only after birth in the cells of the eye. In hereditary retinoblastoma, on the other hand, one of the two RB1 alleles is already defective at birth and the second one is inactivated later. The general loss of one RB1 allele in every body cell leads to these patients having a higher risk for developing other tumors during their life span. Secondary malignancies represent the greatest challenge in the care of this patient group and early detection of such tumors is of utmost importance for successful Treatment.
Our goal is to develop an assay for early detection of secondary tumors in patients with germline mutations in the RB1 gene. To this aim free circulating DNA in the blood of patients will be analyzed using a targeted sequencing panel. The panel will measure mutations that are common in secondary tumors as well as primary retinoblastomas mutations. Based on the hypothesis that primary retinoblastomas as well as secondary tumors require inactivation of the second RB1 allele at an early stage, we will build an assay system to check circulating free DNA for mutations of the RB1 gene.
This project is kindly supported by the Kinderaugenkrebsstiftung KAKS.