In Vivo Group
 
Group members

Daisuke Kawauchi, PhD (Group Leader “Developmental Neuro-oncology”)
Daniel Haag, PhD (Visiting scientist)
Haibin Wang, PhD (Visiting scientist)
Mikaella Vouri, PhD (postdoctoral Fellow)
Marcus Giese (MD student)
Linda Linke (Technical Assistant)
Laura Sieber (Technical Assistant)

 

 
Introduction
With the advances of molecular genetics in recent years our understanding of genetic alterations in brain tumors has been greatly expanded. This has led to the discovery of tumor-specific recurrent aberrations in genes for which an oncogenic role had already been described in other malignancies but also in genes that have not been implicated in tumorigenic processes previously. With the wealth of new genomic information about pediatric and adult brain tumors at hand it is now of significant importance to distinguish the pathogenetically and thereby therapeutically relevant alterations from so called passenger mutations. A versatile and flexible in vivo validation pipeline is thus an integral part of the process of translational medicine.
Our aim is to investigate these newly identified alterations in an in vivo context and thereby provide new insight into tumor biology and provide models for preclinical testing in order to facilitate the bench-to-bedside translation of these novel findings.
 
Embryonal Brain Tumors (Daisuke Kawauchi)

Medulloblastoma Biology

Medulloblastoma (MB) is one of the most common malignant brain tumors in children. The molecular heterogeneity of this disease has been revealed as classification into 4 subgroups (WNT, SHH, Group3 and Group4). The survival rate of patients with these tumor entities are getting better for decades, but still not 100% curable. Even for cured patients, there can be a risk of side effect caused by current therapies. Thus, more efficient and safer therapeutic methods have been explored. Toward establishment of better therapies, our group is studying etiology of this disease using animal models. Profound understanding of how MB develops would identify critical signaling pathways for tumor initiation and progression, leading to discovery of novel chemotherapeutic approaches.

1. Identification of novel genetic pathways in SHH-subgroup medulloblastomas

The mechanisms underlying initiation of SHH-subgroup medulloblastoma has been relatively well-characterized so far. It has been thought that this subgroup arises from cerebellar granule cell precursors (GNPs) by aberrant SHH signaling activation. Nevertheless, recent intensive genomics studies revealed heterogeneous recurrent mutations of chromatin modifiers in addition to genes regulating SHH signaling pathways, raising the possibility that such chromatin modifiers could be involved in tumor initiation and progression. To understand how these chromatin modifiers are influence GNP proliferation, transformation into malignant tumor cells and drug resistance, we are currently using state-of-art approaches, including in vivo electroporation, mouse genetics, primary neuronal culture, iPSC technology, viral gene transduction, RNA-seq, ChIP-seq and CRISPR technologies.

2. Cell of origin of non-WNT/SHH subgroup medulloblastomas

Non-WNT/SHH subgroups, so-called Group3 and Group4 medulloblastoma have been known as the most aggressive subgroup. Compared to the other two subgroups, the etiology of these subgroups has been relatively less known. Biological questions regarding the cell of origin and oncogenic impacts behind the entities still remains to be defined. With international collaborations, we have screened and identified candidate genes/signaling pathways. With germline and non-germline genetically engineered mouse models, we are developing animal models for these entities and validating function of potential oncogenic hits.

3. Single cell analysis on the developing cerebellum and SHH subgroup medulloblastoma

The cancer is supposed to develop from normal cells via multistep mutations in tumor suppressors and oncogenes. Nevertheless, accumulation of non-oncogenic “passive” mutations hinders discovery of causal factors for cancers. Recent advances of the single cell analysis technology could be a powerful tool to understand how normal cells transform into pre-neoplastic and subsequently malignant tumor cells. We are currently applying the single cell technology to SHH-medulloblastoma murine models and exploring novel markers for tumor-propagating cells as well as specific treatments targeting these cells.

 

 
Glial Brain Tumors (Jan Gronych)
Methods Development
With the purpose of getting a better understanding of the role of certain mutated genes in the pathogenesis of brain tumors and to provide models for preclinical testing that faithfully resemble the situation in human tumors we utilize and further develop methods for in vivo somatic gene transfer. These allow us to investigate oncogenes but also tumor suppressor genes in an in vivo context in a flexible manner. We have therefore established a variety of delivery systems enabling us to overexpress or delete candidate genes after orthotopic injection in neonatal mice. Thereby, we aim to investigate tumorigenic processes and to establish autochthonous disease models that can be utilized for evaluating novel therapeutic approaches for adult and pediatric brain tumors, in particular different subtypes of glioma.
In addition to RNAi-based approaches, for the investigation of loss-of-function candidate genes in addition to RNAi-based approaches we use and optimize targeted nuclease expression systems for the analysis of loss-of-function candidate genes somatic gene transfer approaches that will allow gene knockout or gene targeting in vivo. With this method we hope to circumvent the disadvantages implicated in RNAi-related disadvantages for the investigation of candidate tumor suppressors.
Target Validation, Model Development and Preclinical Studies
We have previously used somatic gene transfer-based approaches for the generation of the first animal model for sporadic pilocytic astrocytoma, the most common brain tumor in children which is exclusively driven by activating aberrations of MAPK/Erk signaling. Using retroviral somatic transfer of a constitutively active form of the BRAF oncogene into neural progenitor cells in vivo we were able to induce tumors that faithfully resemble the biological and clinical characteristics of PAs in patients (Gronych et al. JCI 2011). One interesting feature of these neoplasias is the presence of oncogene-induced senescence (OIS), a permanent cell cycle arrest as a response to excessive growth signaling. Using the PA animal model together with in vitro analyses we want to elucidate the molecular mechanisms underlying this phenomenon.
In addition to pilocytic astrocytomas we are also investigating genetic alterations newly identified in high grade glioma for their oncogenic potential in vivo. Recently, we successfully established a novel mouse model for malignant glioma.
We are utilizing these in vivo model systems in order to test novel therapy approaches with targeted inhibitors. The implementation of different imaging techniques (magnetic resonance imaging, intravital bioluminescence) enables us to assess the efficacy of these targeted therapies intraindividually in a preclinical setting.
 
Contact: Daisuke Kawauchi (This email address is being protected from spambots. You need JavaScript enabled to view it.) and Jan Gronych (This email address is being protected from spambots. You need JavaScript enabled to view it.)
 
Selected publications

Kawauchi D#, Ogg RJ, Liu L, Shih DJ, Finkelstein D, Murphy BL, Rehg JE, Korshunov A, Calabrese C, Zindy F, Phoenix T, Kawaguchi Y, Gronych J, Gilbertson RJ, Lichter P, Gajjar A, Kool M, Northcott PA, Pfister SM and Roussel MF#.  Novel MYC-driven medulloblastoma models from multiple embryonic cerebellar cells. Oncogene. in press (#corresponding authors)

Feng W*, Kawauchi D*#, Körkel-Qu H, Deng H, Serger E, Sieber L, Lieberman JA, Jimeno-González S, Lambo S, Hanna BS, Harim Y, Jansen M, Neuerburg A, Friesen O, Zuckermann M, Rajendran V, Gronych J, Ayrault O, Korshunov A, Jones DT, Kool M, Northcott PA, Lichter P, Cortés-Ledesma F, Pfister SM#, Liu HK#. Chd7 is indispensable for mammalian brain development through activation of a neuronal differentiation programme. Nat Commun. 2017. 8:14758 (*Equal contribution, #corresponding authors)

Zuckermann M, Hovestadt V, Knobbe-Thomsen CB, Zapatka M, Northcott PA, Schramm K, Belic J, Jones DT, Tschida B, Moriarity B, Largaespada D, Roussel MF, Korshunov A, Reifenberger G, Pfister SM, Lichter P, Kawauchi D#, Gronych J#. Somatic CRISPR/Cas9-mediated tumour suppressor disruption enables versatile brain tumour modelling. Nat Commun. 2015. 6:7391. (#corresponding authors)

Kawauchi D, Robinson G, Uziel T, Gibson P, Rehg J, Gao C, Finkelstein D, Qu C, Pounds S, Ellison DW, Gilbertson RJ, Roussel MF. A mouse model of the most aggressive subgroup of human medulloblastoma. Cancer Cell. 2012. 21(2):168-80.