Novel therapeutic strategies for the treatment of paediatric brain cancer

Paediatric brain cancer remains the leading cause of cancer-related mortality in childhood. Medulloblastoma (MB) is the most frequent malignant brain tumour to occur in children. It comprises four distinct molecular subtypes: Wingless (WNT), Sonic Hedgehog (SHH), Group 3 and Group 4. Although overall survival (OS) rates for the patients have reached 70-80%, these children often develop cognitive, neurological and behavioural disorders because of radiotherapy. In addition, survivors suffer from long-term adverse sequelae due to high-dose radiation given at an early age. In order to prevent these patients suffering from long-term adverse sequelae, more effective and less toxic therapies for paediatric MB are desperately needed. Diffuse intrinsic pontine glioma (DIPG) is a highly aggressive malignant childhood brain cancer, which grows diffusely in the ventral pons, making surgical resection impossible. This rare paediatric brain cancer responds very poorly to the current standards of care and OS is less than two years from diagnosis. Identifying novel therapeutic targets and effective pharmacological therapies are urgently needed to treat this devastating disease.Paediatric brain cancer model generation and characterisationPatient-derived cell lines represent a more reliable depiction of the original tumour genomic profile than immortalised cell lines. Patient-derived orthotopic xenograft (PDX) mouse models are generated by engrafting dissociated tumour cells directly into immune- compromised mouse brain. They have a strong predictive response value. This study describes a standardised and reproducible method for establishing and propagating paediatric brain cancer PDX models and cell cultures grown as serum-free monolayers and three-dimensional spheroid cultures. Three MB primary models and two DIPG primary models were generated and characterised as part of this thesis chapter. MB-R203 patient tumour tissue was classified as Group 4 subtype VI, MB-R302 patient tumour tissue and patient-derived cell lines were classified as Group 3 subtype II. DIPG1.1 and DIPG1.2 were characterised as diffuse midline glioma, H3K27 mutant tumours. Models were also assessed for growth and therapy response to radiation and chemotherapy treatment. These models are highly valuable and will be useful to the scientific community for undertaking faithful pre-clinical in vitro and in vivo studies into the future.Eph receptor tyrosine kinases as therapeutic targets for medulloblastomaEph receptors are transmembrane receptor tyrosine kinases (RTK) that function normally during embryonic development, where they regulate cell adhesion and repulsion during tissue boundary formation. Typically, these family of receptors are lowly or discreetly expressed in healthy adult tissues. It is now well-established that Eph and ephrins are re- expressed and functional in a wide variety of human tumours including brain cancer. Increasing evidence points to a key role for Eph/ephrin signalling in cancer development and progression, making these proteins potential targets for anti-cancer therapies. This study has identified overexpression of EphB2 and EphB6 in MB. EphB2 is highly elevated in Group 3 and Group 4 tumours, while EphB6 is overexpressed in WNT-driven MB. This study, for the first time, revealed EphB2 and EphB6 can be co-immunoprecipitated in MB cells, providing further evidence of receptor crosstalk and kinase-independent functions. The EphB2 high-affinity ligand ephrin B1 effectively initiates EphB2 receptor activation and downstream signalling in vitro leading to reduced MB proliferation and invasion. An EphB2 small peptide antagonist inhibited EphB2 receptor phosphorylation and rescued cell proliferation upon ephrin B1-Fc treatment. Proteome profiler analysis demonstrated several downstream signalling pathways were activated in response to receptor activation, including PI3K/AKT/mTOR signalling pathway and MAPK/ERK signalling pathway. Phosphorylation of proteins which were associated with the AKT signalling pathway was abolished or inverted after knockdown of EphB2. Knockdown of EphB2 or EphB6 led to reduced cell proliferation and orthotopic tumour formation in vivo, implying EphB2 and EphB6 play a key role in MB progression and are excellent actionable candidates for future therapeutic targeting in MB.Evaluating Olig2 inhibitor anti-tumour activity in paediatric brain cancersOlig2 is a basic helix-loop-helix transcription factor that is expressed in neural progenitor cells during embryonic development. Olig2 has been shown to be re-expressed in almost all high-grade gliomas and is critical for tumour formation. Single-cell RNA sequencing analysis in MB revealed a developmental hierarchy of progenitor pools in SHH-MB, and identified Olig2-expressing glial progenitors as transit amplifying cells during initiation of tumourigenesis. Evidence suggests these cells also present as a stem-like population in MB tumours. Thus, anti-Olig2 therapy has the potential to target this population and increase response rates. In this thesis, a panel of paediatric cell lines and PDX tumour tissues were assessed for Olig2 expression. The novel small molecule Olig2 inhibitor CT179 was examined, CT179 functions via altering Olig2 conformation, sequentially preventing transcription. This study, for the first time, demonstrates anti-tumour efficacy of CT179 in paediatric cancer cells. Tumour cells after treatment accumulated in G2/M phase and underwent significant apoptosis which correlated with a significant drop in Olig2 protein levels post CT179 treatment. Further findings also revealed a gradual degradation of cyclin B1 and phospho-CDK1 after CT179 treatment which caused cells to exit mitosis without dividing chromosomes in anaphase. Consistent with western blot results, immunofluorescence staining also showed tetraploid Daoy cells with satellite micronuclei and ancillary nuclear lobe formation, indicating a mitotic slippage response and cells underwent apoptosis in the end. Moreover, CT179 had additive anti-tumour effect when used in combination with radiotherapy in vitro and in vivo. Our finding suggests that inhibition or eliminating Olig2-positive cancer stem cells in combination with radiotherapy significantly extends latency in MB mouse models, could potentially improve the outcomes of patients and minimize the onset of therapeutic resistance.In summary, this thesis highlights three main areas of MB research: 1) the characterisation and development of highly relevant pre-clinical MB models. These models are critical to drive both our knowledge of this aggressive disease and test novel therapeutic approaches to improve OS. 2) Eph receptors are the largest family of RTKs, an in-depth expression analysis revealed elevated tumour-specific expression of EphB2 and EphB6. Detailed functional analysis of EphB2 and EphB6 in MB was conducted and revealed oncogenic roles in MB progression and maintenance. Findings from this chapter pave the way for further development of EphB2 and EphB6 therapeutic targeting in MB. 3) Lastly, expression analysis and functional targeting of the Olig2 transcription factor in MB was conducted. Results show the novel small molecule Olig2 inhibitor CT179 effectively disrupts Olig2 and cooperates synergistically with radiotherapy to delay MB tumour formation in vivo. This study highlights the significant translational potential of CT179 and importantly lays the ground work for clinical testing of this novel therapy in MB