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 migrate down the brain stem and into the spinal cord. 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 migrates down the brain stem to identify and target these invasive tumour cells.

We have built this project on an extraordinary resource – in 2020, we received 10 samples of exceptional quality at varying points along the brain stem of a single child’s DIPG brain tumour. Using the innovative technology of spatial transcriptomics we finally have a chance to analyse how the tumour migrates down the brain stem. This will be the first time that this advanced sequencing method has been applied to DIPG. Moreover, we will also determine whether blocking a leading migration receptor could stop this deadly pathway to prolong survival.

The Sid Faithful Brain Cancer Laboratory is the only group in Australia specifically focussed on the most aggressive forms of both adult and paediatric brain cancers.

Collaboration and the sharing of information and resources is of great importance to QIMR Berghofer and the laboratory. Which is why as part of our project, we will develop our spatial transcriptomics data into a freely available online resource for researchers in the DIPG field. This “Google Earth” style map of the tumour called DIPGmapper will help other researchers worldwide identify new ways to improve the outcomes for DIPG patients.

This unique tumour sample is currently sitting in storage untouched, waiting for funding to conduct this research. This child has, with their life, given us information that could greatly benefit others.

Together we can unlock its answers, provide hope to the children and families suffering from DIPG - and honour the rare and special opportunity this child has given us.

Executive Summary

This project will develop a spatial map of DIPG tumours to identify targetable markers of invasiveness. Furthermore, as proof of principle, we will test whether inhibiting tumour cell migration can potentiate current treatments and prevent tumour spread and migration.

This proposal is built on the following strengths:

1. Access (currently in-hand) to wholly intact, RNA-sequencing quality DIPG specimens (10 unique regions per tumour) that track from the midbrain to the spinal cord.
2. our expertise in newly developed spatial transcriptomic sequencing technologies and
3. our proven track record of translating antibody conjugates targeting Ephs and ephrins to human brain cancer clinical trials.  

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 and no clinical trial has yet led to substantial improvements in survival or quality of life. Hence, designing and testing new strategies to treat this incurable disease are desperately needed.

Valuable DIPG patient-derived resource

We have collected four 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 (OCT) samples spanning the spinal cord to brainstem. We have also generated cell lines from two of the four specimens and successfully engrafted these 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 Transcriptomics

Whole transcriptome spatial profiling provides a readout of gene expression, which unlike bulk-RNA sequencing, retains information about the positioning of cells within their microenvironment at close to single-cell resolution. Moreover, this transformative technology provides for the identification of specific cell populations, and position within a tumour and interaction with the tumour microenvironment [1, 2]. Spatial transcriptomics have not been previously been performed on DIPG tumours.  Sequencing of 10 fresh tissue sections of DIPG tumour from a single patient spanning the initiation site to the leading invasive edge throughout the brainstem will allow us to generate a highly detailed atlas of gene expression gradients across the tumour in its entirety. Upon completion of this work, we will deposit this data, through R Shiny, as a freely available online “Google Earth” style resource, known as DIPGmapper. This DIPGmapper resource can then be used by the field to identify 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, and finally do the phenotypes of DIPG tumour cells change as they infiltrate more distal regions of the brain stem?

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 [3]. 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. To explore this opportunity, we have generated a panel of novel EphB3 antibodies in collaboration with The Walter & Eliza Hall Institute of Medical Research (Melbourne, Australia). Monoclonal antibodies (mAbs) work in different ways to kill a cancer cell or stop it from growing. Various mAb-derived therapies have been approved by the FDA in recent times and used clinically for the treatment of different cancer types. These therapies primarily work by blocking receptor signalling, thereby perturbing tumour growth and survival mechanisms. Alternatively, drugs or radioactive substances can be conjugated directly to mAbs (ADCs), allowing target specific drug delivery to the cancerous cell, reducing off target toxicities [4]. Chief Investigator Day and colleagues have already successfully targeted recurrent glioblastoma (GBM) with an EphA3-targeting drug conjugate. In this approach, the EphA3 mAb IIIA4 was conjugated to the cytotoxic drug maytansine (IIIA4-USAN). In combination with radioimmunotherapy, treatment led to significant cell death both in vitro and in vivo with no observed animal toxicity [5, 6]. This research has led to a world first Australian-based clinical trial (NCT03374943). Of note, imaging data released from ongoing trials, showed that the mAb specific for EphA3 can cross the blood brain barrier (BBB) in glioblastoma patients, which poses a major challenge in the field of brain cancer treatment. Based on this exciting work, we now seek to apply these approaches to EphB3 to target DIPG.

QIMR Berghofer - Experts in the field

QIMR Berghofer is a world-leading translational research institute, where research concepts are developed from the laboratory bench through to the patient’s bedside. For 75 years, QIMR Berghofer researchers have led advances in the understanding, prevention, diagnosis and treatment of a range of diseases.

QIMR Berghofer’s research focuses on the areas of infectious diseases, cancer, chronic disorders and mental health disorders. QIMR Berghofer’s wide-ranging research expertise spans immunology, genetics and genomics, population health, and cellular and molecular biology.

QIMR Berghofer is home to approximately 1000 scientists, support staff and students, and over 70 state-of-the-art laboratories. It is also home to the TGA-approved cell manufacturing facility, Q-Gen Cell Therapeutics, which is approved to manufacture cellular therapies for clinical trials in Australia, the United States and Europe.

QIMR Berghofer researchers collaborate with other research institutions, clinicians, hospitals, governments and the pharmaceutical industry worldwide.

Our track record

QIMR Berghofer has an international reputation for research excellence. In 2018, the Institute published more than 800 scientific papers. Nearly half of QIMR Berghofer’s research projects have moved from the ‘discovery phase’ closer to translation into the clinic. In 2018-2019, the Institute led 22 clinical trials as a result of research undertaken at QIMR Berghofer. During 2018–2019, QIMR Berghofer scientists were involved in 34 further trials that were led by other researchers or clinicians.

Our facilities and services

QIMR Berghofer offers a wide range of specialised facilities and services, which are all located at the Institute’s Herston premises. They make QIMR Berghofer a unique location for conducting and translating medical research, and give the Institute an internationally competitive edge in attracting leading scientists from around the world. We are located next to Queensland’s largest hospital, the Royal Brisbane and Women’s Hospital (RBWH) and have ongoing collaborations with the clinicians and surgeons. The Institute is also a partner in the Herston Imaging Research Facility, a state-of-the-art facility housing hybrid scanners that combine MRI, PET and CT imaging. This allows molecular processes and anatomical brain images to be captured simultaneously.

The majority of these studies will be conducted at QIMR Berghofer, a leading research institute, and the largest incubator of cancer research laboratories in the state of Queensland (Australia). At QIMR Berghofer the investigators have access to world class infrastructure. They will leverage their in-house tumour bank and the highly relevant DIPG disease models that have been developed by CI Day over many years. The DIPG models have been comprehensively characterised and form orthotopic tumours in vivo. In addition to the bioinformatic expertise of AI Harris, a Statistics core team at QIMRB is available for further collaboration and advice. Combined, this is an exceptional team that has integrated their expertise to develop a highly innovative, cross-disciplinary proposal. 

The Team of Investigators

Chief Investigator (CI) Professor Bryan Day will lead this project in conjunction with Associate Investigator (AI) Dr Lachlan Harris. Professor Day is Group Leader of an internationally recognised brain cancer laboratory at QIMR Berghofer in Australia. His laboratory is the only group in Australia specifically focussed on the most aggressive forms of both adult and paediatric brain cancers. Professor Day is a past Director for the Australian Society of Medical Research (ASMR) and a Co-Director of the Children’s Brain Cancer Centre (CBCC). He is a leading brain cancer expert in Australia, among his achievements have been the development of a brain tumour bank at QIMR Berghofer and the characterisation of the receptor EphA3 as a therapeutic target in brain cancer. This research has led to clinical testing of a novel EphA3 therapeutic antibody for aggressive treatment refractory brain cancers. He is a Principal Investigator in an ongoing clinical trial with a focus on recurrent GBM.

Professor Day has >2000 career citations, H index 25 and has published >65 scientific articles directly relating to understanding the biology of and designing and testing new therapies for brain cancer. Professor Day and colleagues have collected >350 primary brain cancer specimens from the Royal Brisbane Hospital, from this tissue >80 primary tumour lines have been generated. In collaboration with Associate Professor Andrew Moore and the Queensland Children's Tumour Bank, Professor Day and his team have established DIPG primary cultures and engrafted these models into immunocompromised mice.

Associate Investigator (AI) Lachlan Harris, PhD is a postdoctoral scientist in the CI’s laboratory [7]. As an early career researcher (4-years Post-PhD), he has already published over 25 peer-reviewed articles and has more than 500 career citations. He has recently returned from the pre-eminent research institute of the United Kingdom, The Francis Crick Institute, where he developed a deep expertise in sequencing technologies. This expertise centred on 10x genomics technology, including single-cell RNA-sequencing and ATAC sequencing modalities. In addition, AI Harris is well versed in spatial transcriptomics, where at The Francis Crick Institute an in-house spatial transcriptomics platform had been set-up by Sam Rodriques the inventor of Slide-Seq. For this project CI Day and AI Harris will use a 10x Genomics platform, known as Visium for the purposes of spatial transcriptomics to strengthen their team they have brought on AI Professor Andrew Mallett. His research is on discovery and translation in the kidney genomics field with a focus on applications of spatial transcriptomics in kidney tissues, both in health and disease. AI Harris and AI Mallett will be responsible for generating the spatial-RNA sequencing data and the two matched single-cell RNA-sequencing datasets from the DIPG tumour core and infiltrative edge, respectively. AI Harris will integrate the single-cell RNA-sequencing data with spatial transcriptomics. Integrating these different sequencing modalities improves gene detection and cell classification. Together, the CI and AIs will deposit these data through an online searchable spatial map, called DIPGmapper.

Description of Research Proposal

Budget

Collaborations and Conflicts of Interest