Lay Summary

Diffuse Intrinsic Pontine Glioma (DIPG) otherwise known as Diffuse Midline Glioma (DMG) is a fatal brain stem glioma arising in young children. There are no effective therapies for DIPG and children typically succumb to the disease within 2 years. No therapy to-date has improved the survival rate for children with DIPG in a meaningful way.

One of the biggest disease challenges is the tumour’s highly invasive nature. DIPG tumours frequently spread along the brainstem and reach other parts of the brain over far distances, such as the spinal cord or frontal lobe. It is this movement and growth of the tumour that ultimately proves fatal.

Our project offers a new and exceptionally rare opportunity to map how a DIPG tumour progresses along the brainstem to identify and target invasive tumour cells. We have commenced analysis using the innovative technique of spatial transcriptomics. This technique will allow us to generate a highly detailed atlas of gene expression gradients across the tumour in its entirety allowing us to track cells along migration pathways.   

We have now collected intact brainstem DIPG specimens from three patients, in collaboration with the QLD Children’s Tumour Bank (QCTB), 10 DIPG tissue sections were obtained from each patient spanning the tumour initiation site to the leading invasive edge throughout the brainstem. We now plan to extend our studies to perform this analysis on all specimens.

Executive Summary

This project will develop a spatial gene expression map of DIPG tumours to determine cell type specific features as the disease progresses in the spatial context and to identify targetable markers of invasiveness.
No therapy to-date has improved the survival rate for children with DIPG. One of the biggest disease obstacles is the tumour’s highly invasive and infiltrative nature. DIPG tumours frequently migrate along the brainstem down to the spinal cord and reach parts of the brain of far distances.¹ It is this aggressive movement and growth of the tumour that ultimately proves fatal. This proposal offers a new and exceptionally rare opportunity to map how DIPG tumours migrate down the brainstem to identify and target invasive tumour cells. Using the innovative technology of spatial transcriptomics, we now have a chance to analyse how the tumour migrates and invades. To our knowledge, spatial transcriptomics has never before been performed on DIPG tumours.

As proof of principle, we will test whether inhibiting identified target genes can potentiate current treatments and prevent tumour progression. For instance, we will leverage our novel finding regarding the role of the Eph family receptor tyrosine kinase (RTK) EphB3 and whether targeting will hamper tumour spread and migration.           
 

This proposal is built on the following strengths:

1. Access (currently in-hand) to three wholly intact, RNA-sequencing quality DIPG specimens that track from the midbrain to the spinal cord respectively.
2. Expertise in newly developed spatial transcriptomic sequencing technologies and
3. Proven track record of translating antibody conjugates (ADCs) targeting Ephs and ephrins to human brain cancer clinical trials (NCT03374943).  

This work is crucial as around 20 children in Australia, 300 children in the United States and many more worldwide die from DIPG every year. While the field has made significant improvements in understanding the tumour’s biology, DIPG remains incurable. Hence, designing and testing new strategies to treat this incurable disease are desperately needed.

Valuable DIPG patient-derived resource

In collaboration with the QLD Children’s Tumour Bank (QCTB), we have collected three intact DIPG specimens each ranging from the spinal cord to the midbrain, allowing us to track cells along migration trajectories. For each sample, we have formalin fixed paraffin embedded (FFPE) tissue blocks and sections, as well as fresh frozen samples. We have also generated a matching cell line from one of the three specimens and successfully engrafted it into the brains of NOD.Cg-Rag1 mice. This resource allows for comprehensive spatial and single-cell RNA sequencing analysis during tumour progression and importantly for in vitro and in vivo validation studies.

Spatial Transcriptomic Sequencing

Whole transcriptome spatial profiling provides a readout of gene expression, which unlike bulk-RNA sequencing, retains information about the positioning of cells within the micro-environment at close to single-cell resolution.² Moreover, this transformative technology allows for the identification of specific cell populations, and position within a tumour and interaction with the tumour microenvironment.³ This technology will allow for the identification of novel regulators of invasiveness in this devastating cancer, as well as a host of other research questions, for example: do DIPG tumour cells migrate along axonal tracts or along blood vessels? what are the proliferative and most aggressive cell populations in DIPG? do tumour phenotypes alter during migration to more distal regions of the brainstem? As a proof of concept, in collaboration with Prof Quan Nguyen, we have recently conducted spatial transcriptomics on four brainstem regions from a single DIPG patient. We compared two tissue regions close to the tumour initiation site with the two most distal regions (midbrain and spinal cord). Results highlighted unique gene expression clusters underlying tumour heterogeneity.

Furthermore, we have generated preliminary data to assess EPHB3 expression in the four brainstem regions and assessed genes that were most negatively and positively correlated with regions of high EPHB3. Results show that pleiotrophin (PTN), cofillin-2 (CFL2), c-MET (MET) and Rac1 (RAC1) are top genes that are positively associated with EPHB3-high regions. These genes are well known to be involved in the regulation of actin structures and tumour cell invasion programs.^4-6 Interestingly, high PTN expression has recently been described to promote an invasive phenotype in glioma.⁷ Taken together, our preliminary analysis has yielded positive candidates and identified novel links between genes associated with EphB3 and tumour invasion.

Monoclonal antibody therapy against EphB3, a lead candidate of DIPG invasiveness.

As a proof of principle that inhibiting DIPG migration can improve survival, we will test our in-house novel monoclonal antibodies against EphB3. The erythropoietin-producing hepatoma (Eph) receptor and ephrin-ligand RTK subfamily have been shown to exhibit effective anti-cancer potential, including in adult high grade glioma.⁸ These transmembrane receptors have significant potential to function as tumour-specific therapeutic targets and to serve as a novel therapeutic strategy to treat aggressive diseases such as DIPG. PhD candidate Ms Anja Kordowski, in CIAs laboratory, is currently undertaking a study to explore the expression and function of EphB3 in DIPG. Expression analysis showed significant elevated in tumour infiltrate as compared to normal brain. Eph receptors typically show gradient expression which is a critical aspect of normal Eph and ephrin function. We conducted immunofluorescence (IF) on DIPG tumour specimens and found prominent EphB3 gradient expression, which appeared to correlate with infiltrating tumour cells. Through active international and domestic collaborations, we have obtained a panel of primary DIPG cell lines (n=13). Western blot analysis showed high EphB3 protein expression was present in the majority of the lines examined. Taken together, these findings highlight EphB3 as a tumour-specific cell surface receptor expressed in DIPG. To explore EphB3 targeting for the treatment of DIPG, we have generated a panel of novel EphB3 antibodies in collaboration with The Walter & Eliza Hall Institute of Medical Research (Melbourne, Australia). CIA Day has previously targeted adult glioblastoma (GBM) using both radio-immunotherapy and antibody drug conjugate (ADC) approaches coupled to an EphA3-targeting antibody.^9,10 This research has led to a world first Australian-based clinical trial (NCT03374943) in collaboration with Prof Andrew Scott and Prof Hui Gan. Of note, imaging data released from this trial showed that the mAb specific for EphA3 can cross the blood brain barrier (BBB) in recurrent GBM patients, which poses a major challenge in the field of brain cancer treatment. Based on this exciting work, we now seek to apply these pay-loaded approaches to EphB3 to target DIPG.

Description of Research Proposal

Budget

Collaborations and Conflicts of Interest