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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

MYCN gene region with aligned sequencing reads

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 and therapeutic potential of the IGF2BP1-MYCN oncogenic network in neuroblastoma

Aiming to identify potentially targetable oncogenic networks promoting high-risk neuroblastoma, we conducted a thorough analysis of neuroblastoma transcriptomes and chromosomal aberrations including RNA-seq, small RNA-seq, and shallow whole genome sequencing (sWGS) of 100 primary tumor samples. Findings were correlated with a pan-cancer survey of publicly available loss-of-function CRISPR screen data, including MYCN-amplified neuroblastoma cell lines. These comprehensive investigations revealed a significant number of essential genes on chromosome 17q, frequently gained in high-risk neuroblastoma. 
Among these genes, we identified IGF2BP1, an oncofetal RNA-binding protein, acting as a pro-oncogenic driver in neuroblastoma. Studies in cellulo, xenograft as well as transgenic neuroblastoma mouse models indicate that IGF2BP1 promotes an aggressive, high-risk neuroblastoma phenotype by fostering MYCNdriven gene expression. Preliminary analyses of MYCN/IGF2BP1-driven effectors suggest various targetable proteins, e.g. Survivin (BIRC5) located on chromosome 17q. In preliminary studies, we observed that BIRC5 inhibition is IGF2BP1-dependent. Accordingly, we expect that the concomitant pharmacological impairment of MYCN/IGF2BP1 and their effectors has therapeutic benefit. Within this project, we wish to:

1) characterize the role and therapeutic target potential of MYCN/IGF2BP1-driven gene expression in high-risk neuroblastoma and

2) will evaluate novel therapeutic strategies aiming to target the MYCN/IGF2BP1-driven expression of oncogenic factors. These pre-clinical efforts ultimately aim to improve the therapy and outcome of high-risk neuroblastoma patients.

This project is being pursued in collaboration with Prof. Stefan Hüttelmaier and his team at the Martin-Luther-University Halle-Wittenberg.


Minimal Residual Disease Detection for Neuroblastoma Diagnostics

Scheme of neuroblastoma-MRD detection process by analysis of a patient-specific MYCN breakpoint

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.