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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 3 - Medulloblastoma (Molecularly defined models of human childhood brain tumors)

Teaser

Summary

The two main challenges in the clinical management of paediatric brain tumours are improving survival and reducing long-term detriments due to treatment toxicities, especially from craniospinal radiotherapy. The overall aim of this project is to improve survival of medulloblastoma, the most common paediatric brain malignancy, through better understanding of the biology underlying tumour initiation and progression.

Medulloblastoma is suggested to originate from specific cells in the small brain, cerebellum, and can be classified into four defined subgroups called: WNT, SHH, Group 3 and Group 4. MYC proteins such as MYC and MYCN are transcription factors that are mis-regulated in more than half of all types of human cancer including medulloblastoma. We have generated sophisticated regulatable transgenic animal models that overexpress MYC proteins. These models carefully resemble at least two of the four defined subgroups of human medulloblastoma. In this proposal we intend to use the models to identify the specific cell type these brain tumours originates from. We also aim to refine our medulloblastoma models and develop novel models to define and study cells involved in brain metastasis and tumour recurrence, the main causes of death in brain tumour patients.

We have managed to culture normal human cerebellar stem cells and we next plan to model human medulloblastoma development in these cells by overexpressing oncogenes, or silencing suppressor genes, that are defined as clinically relevant medulloblastoma drivers. We will use a forward genetics screen to identify novel drivers and specifiers of subtypes of medulloblastoma. We hope these combined efforts will help us better understand human medulloblastoma formation and we expect to generate tumours that correlate well, both pathologically and molecularly, with primary cell cultures derived from medulloblastoma patients. Our data will subsequently be used to provide novel targets for therapeutic intervention of the disease.

Work performed

The main aim of this project is divided into two specific aims:

I. Characterise cells of origin and cells driving recurrence in MYCN-driven medulloblastoma.
II. Develop new models for the four medulloblastoma subgroups using human hindbrain stem cells.

Main results so far under Aim I:
We previously showed that stabilisation of MYCN is essential for brain tumour 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. SOX9 is a transcription factor and a neural stem cell marker implicated in paediatric brain tumour formation and in the mechanisms of relapse that we have overexpressed and studied together with FBW7 in cell lines derived from various medulloblastoma patients.

The cell of origin of Group 3 medulloblastoma is not known. When crossing our Group 3 animal model with FBW7 knock out mice, there is no tumour formation suggesting we could study this phenotype to localise cells of origin. Currently, we are comparing apoptotic cell status during embryonic and postnatal brain development in MYCN-driven FBW7 homozygous knock outs (in where tumour formation is blocked) as compared to FBW7 heterozygous animals (that generate a large number of brain tumours). As MYCN and Luciferase is expressed under the same promoter in these animals we can compare doubly-positive apoptotic (Cl.Casp 3)/luciferase (Luc) positive cells in order to identify regions and cells of tumour origin. Careful analysis of defined cellular markers using immunofluorescence staining is ongoing.

Main results so far under Aim II:
We have now generated a new humanized mouse models for medulloblastoma by overexpressing MYCN using lentiviral infection of defined human embryonic stem cells/neuroectodermal stem (NES) cells and from iPS cell-derived NES cells. We are currently evaluating what subtype of medulloblastoma these tumours could be categorised under. We are using RNA-Seq and immunohistochemistry in order to determine this correctly.

We have set up a new Piggy Back transposon system for our forward genetic screen that will be used to identify collaborators of MYCN during brain tumour formation. By using electroporation into NES cells we can transfer Piggy Back vectors containing MYC, MYCN and OTX2 to single cells. Transposons that integrate into the genome will cause insertional mutagenesis and drive tumour formation. Currently, most NES cells are dying from MYCN overexpression as expected. Whenever transformation occurs we can identify MYC collaborators that might be involved in the anti-apoptotic pathway.

Finally, we are expanding our recurrence model of medulloblastoma. We now generated more than 20 recurrent tumours that are significantly located in a distant metastatic location as expected during medulloblastoma recurrence. Interestingly, several tumours show an increased metastatic potential with drop metastases and dissemination down the spinal cord. RNA-Seq analysis of primary versus metastatic relapsed tumours identifies markers of recurrence, including both PDGF ligands and PDGF receptors. This suggests that PDGF signalling is activated by autocrine loops and that PDGFR activation inhibition, using PDGF/tyrosine kinase inhibitors, would be a promising new approach to target these tumours.

Publications so far:
We recently published a report in EMBO Journal (see Suryo Rahmanto et al. 2016 under Publications) in where we identify that FBW7 is regulating SOX9 stability. Increased SOX9 stability due to FBW7 suppression increases tumour cell migration and metastasis. By suppressing the mTOR/PI3K/AKT pathway we can obstruct the SOX9 stabilization. Further characterisation of SOX9-positive tumour cells will help us understand the mechanisms behind metastatic medulloblastoma re

Final results

Identifying the true cell of origin of medulloblastoma has a huge impact for brain tumour biology and cancer research. Hopefully, our work on brain tumour formation in this ERC project will reveal the nature of this cell type.

Recurrence is what kills children with brain tumours. Good models for tumour recurrence are rare and we have generated the first genetically modified animal model for this. This will improve our understanding of cancer recurrence mechanisms which can identify novel drug targets for better cancer treatment for these patients.