Fredrik Swartling's research on childhood brain tumours

In search for the cellular origin of MYCN-driven medulloblastoma

Anna Borgenvik, Sanna-Maria Hede, Sara Bolin, Holger Weishaupt, Fredrik Swartling
Medulloblastoma is divided into four distinct molecular subtypes (WNT, SHH, Group 3 and Group 4). Group 3 and 4 tumours often show amplifications of MYC and MYCN, respectively, and correlate with poor prognosis. MYC proteins are unstable oncoproteins with short half-lifes. We have shown that MYCN generates Group 3 tumours from a glutamate transporter (GLT1) promoter in a transgenic inducible model (GTML) of medulloblastoma (Swartling et al. Genes & Dev., 2010) and found that tumours originate from a GLT1-positive neural stem cell (NSC).

We have further shown that stabilization of MYCN is essential for brain tumor initiation (Swartling et al. Cancer Cell, 2012). MYCN stability is regulated by the ubiquitin ligase FBW7, which normally targets it for proteasomal degradation. FBW7 is a tumour suppressor gene mutated in various types of cancer including medulloblastoma and we study loss of function of FBW7 in our animal models of medulloblastoma.

We have crossed FBW7 knock-out mice to GTML mice and study how FBW7 loss alters brain tumour formation. Currently we study cellular fate using various brain cell-specific promoters to understand how these tumours develop. We are also isolating putative cells of tumour origin from various brain regions and developmental time windows. Detailed bioinformatic analysis of expression profiles of distinct brain cells is performed in order to reveal the cellular origin for these malignancies.
 

A new model for childhood brain tumour recurrence

Vasil Savov, Sara Bolin, Gabriela Rosén, Anna Borgenvik, Holger Weishaupt, Fredrik Swartling
Tumour recurrence is the main cause of death in children with medulloblastoma. In this project we are studying how MYCN interacts with SOX9, a transcription factor involved in glial fate determination in the brain. Few scattered SOX9-positive cells are found in GTML tumours that are similar to Group 3 or Group 4 human MB.

By using a combination of Tet-ON and Tet-OFF inducible systems we managed to target this rare population of SOX9-positive GTML tumour cells in vivo to show how they were capable of initiating tumour recurrence. The relapsed tumours develop at a distant site in the brain, in line with recent patient data. Further, isolated metastases in Group 3/4 patients had consistently higher SOX9 levels as compared to corresponding primary tumours.

We also showed how FBW7 is regulating SOX9 stability and increases tumour cell migration and metastasis (Suryo Rahmanto et al. EMBOJ, 2016). By suppressing the mTOR/PI3K/AKT pathway we can obstruct this stabilization. Further characterization of SOX9-positive tumour cells will help us understand the mechanisms behind metastatic medulloblastoma recurrence.

Schematic presentation of important signaling pathways involved in the phosphorylation of MYCN
Figure 1. Schematic presentation of important signaling pathways involved in the phosphorylation of MYCN that first leads to its activation/stabilization and then to its degradation by ubiquitin proteasome system. RTKs: Receptor tyrosine kinases; CDKs: Cyclin-Dependent Kinases.

Targeting MYCN through Bromodomains and by using CDK2 inhibitors

Sara Bolin, Anna Borgenvik, Holger Weishaupt, Anders Sundström, Fredrik Swartling
We recently showed that MYCN levels and early proliferation of brain tumours could be reduced by specific inhibition of the bromodomain inhibitor JQ1, which targets MYC proteins epigenetically (Bandopadhayay et al. Clin Can Res., 2014). We also found good efficacy controlling MYCN stabilization by using a CDK2 inhibitor called Milciclib.

Both drugs induced tumour cell senescence or apoptosis in our brain tumour models and also in primary human brain tumour cells. As compared to either drug alone, when combining the two drugs we further reduced MYCN levels and completely abolished brain tumour growth after long-term treatment in vitro.

We are currently evaluating these treatment effects in our models in vivo. Our goal is to understand the underlying mechanisms of this MYCN inhibition and further evaluate the potential of using these promising drugs in the clinic.
 

Using human hindbrain cells and PDX models to study medulloblastoma and DIPG development

Matko Čančer, Sonja Hutter, Anna Borgenvik, Geraldine Giraud, Tobias Bergström, Prathyusha Maturi, Holger Weishaupt, Fredrik Swartling
In this project we are transforming human iPS-derived cells and embryonic hindbrain neural stem cells in order to model the different subgroups of medulloblastoma using lentiviruses carrying clinically relevant cancer driver genes for the distinct tumour subgroups. We have shown that MYCN overexpression can induce SHH-driven tumours in vivo. Depending on cellular origin and reprogramming status tumours of varying prognosis developed.

We are also transforming brain stem-specific cells from humans and mice with MYC in order to model diffuse-intrinsic pontine glioma (DIPG) development. We will evaluate the relevance of using well-defined human hindbrain stem cells to generate these childhood brain tumours and we will compare them to subtype-specific cells similarly cultured from medulloblastoma or DIPG patients. We further study patient-derived xenografts and evaluate their use in drug screens in combination with radiation therapies.

We hope we will understand what actually drives the initiation of medulloblastoma and DIPGs and if various subgroups match certain hindbrain cell types. We currently use genetic and epigenetic analyses to find prognosis markers. We hope to predict how some of these tumours could be treated or if they would be resistant to certain targeted therapies.