Lay Summary

Diffuse Intrinsic Pontine Glioma (DIPG) is the most dismal paediatric brain tumour, characterised by universal mortality and an average survival of less than one year. Several factors contribute to the unique challenges in treating DIPG, including localisation, infiltrative pattern of growth and chemoresistance. Treatment options are limited to palliative radiotherapy.

The development of genomics has led to a comprehensive delineation of DIPG heterogenous mutational landscape. This includes several cellular pathways that are all interconnected and can potentially be pursued by drugs targeting cellular copper called chelating agents. These drugs bind to copper and prevent its use in the cell. Copper chelating agents are already clinically approved and used to treat certain genetic disorders. Moreover, they have been proved to accumulate in the brain and have been successfully tested in various cancers, including high-grade gliomas.

Our team have a national and international reputation in researching copper metabolism in paediatric malignancies. We recently made ground-breaking progress in immunotherapy describing how copper can be harnessed to enhance the anti-tumour immune response. Moreover, we have been actively studying how to target DIPG driving pathways by subtracting an essential trace mineral, such as copper, from the cancerous cells, reducing cell growth and inducing apoptosis.

Copper is absorbed into the human body through the digestive tract from various dietary sources and serves as a fundamental structural and metabolic cofactor for numerous proteins and enzymes.  Consequentially, copper is involved in many cellular processes, including immune system activity, brain development and functions and growth of new blood vessels, a key feature of advancing neoplasms. Interestingly, elevated copper levels have been observed in multiple cancer types and several studies have linked dysregulated cellular pathways to copper levels.

In DIPG, specifically, a wide range of such dysregulated signalling mechanisms, central to cell growth, proliferation and survival are enhanced by mutations that alter the expression or function of key enzymes called receptor tyrosine kinases (RTKs). Hence, we hypothesise that targeting copper-dependent enzymes using copper chelating agents will result in DIPG cell death, reduced invasiveness and improved survival of DIPG mouse models. Overall, our final goal is to establish a promising pre-clinical protocol that could rapidly be translated into a clinical trial to improve outcomes for DIPG patients. This will be done by utilising already approved and non-toxic drugs targeting copper, an essential co-factor of cellular pathway that are typically altered in DIPG.

Our preliminary results demonstrate changes in the expression and activation of specific RTKs in response to copper supplementation and copper chelation, as well as activity of copper chelating agents against DIPG cells. In this novel research proposal, the actual mechanisms and effects of this phenomenon will be established. In detail, the first two aims will be focused on studying expression at both RNA and protein levels of key enzymes in response to copper supplementation as well as copper deprivation in DIPG cells.  This will establish the dependence of specific cellular pathways driving DIPG to copper levels. In the third part of the study, we will use a real-time 3D microscope-based technique to visualise and trace DIPG cells while they invade a stem-cell derived “mini-brain”. With this system, we will be able to recapitulate the typical physiological propriety of DIPG invasiveness and then test whether copper targeting drugs are capable to reduce or stop the migration of DIPG cells through the healthy cells. Again, by altering the levels of copper and disrupting the driving malignant signalling, we expect to also interfere with other phenotypical features of DIPG that make it so uniquely aggressive.

Lastly, we will test the efficacy of copper chelators in vivo in xenograft patient derived DIPG mouse models. Given the already existing clinical data in both human and mice, we anticipate that copper chelation therapy will be well-tolerated in animal models. We will compare control mice against those receiving the copper chelating agents in reducing neuropathic symptoms as well as improving survival. At endpoint, comprehensive histological analysis will be conducted to accurately establish the effect of copper chelation on global tumour growth, induction of tumour cell death and invasion in surrounding healthy brain tissue.

We anticipate that a successful pre-clinical study will pave the way to clinical trials testing copper chelating agents in DIPG patients as both single agents and in combination with radiotherapy. In a devastating disease that have not seen any significant improvement to patient treatment for over 30 years, this would be of outmost importance.

Executive Summary

1. Background

Brain cancers represent the second most common paediatric cancer and the leading cause of disease-related death in childhood. Standard treatments utilise 40-year-old cytotoxic drugs and radiation therapy, resulting in devastating late effects. Many brain tumours still have no effective treatments, such as DIPG, the most common paediatric high-grade glioma (HGG). Due to their location within the brainstem they cannot be removed surgically, and until recently could not even be biopsied. They represent one the most aggressive of all cancers, with no active systemic therapies, and most children dying within twelve months.

Unfortunately, these statistics have not changed for decades, despite a multitude of clinical trials. No chemotherapeutic regimen, including patient specific targeted therapies, have been shown to significantly improve outcomes for most patients, emphasizing the need to find novel and effective treatments. One of the principal reasons for this poor survival was our limited knowledge of the biology of DIPG tumours. Thanks to the implementation of post-mortem molecular screenings, we now have a firm idea about DIPG mutational landscape. Recurrent genetic modifications include Histone H3 (H3F3A and HIS1H3B/C) and Phosphoinositide 3-kinase, catalytic, alpha polypeptide (PIK3CA) mutations, TP53 mutations and deletions and Platelet Derived Growth Factor Receptor Alpha (PDGFRα) copy-number gains, among others.

Some of these driving alterations result in the constitutive activation of fundamental cellular signalling, including Ras/MAPK, PI3K/AKT and JAK/STAT pathways. The over-activation of such pathways is strongly associated with cell proliferation, anti-apoptotic mechanisms, mRNA transcription, protein synthesis and migration, all biological hallmarks of cancer. Interestingly, all the enzymes acquiring somatic modifications leading to constitutional pathway activation necessitate co-factors to carry out their signalling function, typically copper. By fluctuating between two states (Cu2+ and Cu+), copper is highly redox active, readily exchanging electrons with such enzymes facilitating the switch between the inactive dephosphorylated form to the active phosphorylated counterpart. Since the common DIPG driving pathways are all interconnected, they can all potentially be targeted at once by copper chelation.

2. Hypothesis

While understanding the biology of DIPG played a fundamental role in enrolling patients in specific targeted therapy clinical trials, these have only contributed to extending overall survival marginally. The next big challenge would be to tackle acquired drug resistance which is inevitably conducting to therapy failure, disease progression and patient demise. Acquired drug resistance typically leads to cancer cells switching between redundant or interchangeable signalling pathway, for example Ras/MAPK and PI3K/AKT.

In this research proposal, we hypothesise that by targeting a common co-factor between all frequently activated driving pathways in DIPG, such as copper, we could turn down all cellular signalling at once, minimising the change of acquiring drug resistance. Overall, by removing intracellular copper using copper chelators, we expect to interfere with the typical DIPG driving pathways simultaneously, leading cancer cells to growth arrest and apoptosis.

Moreover, our study proposes repurposing and utilising already approved drugs with a well-tolerated toxicity profile that are known to cross the blood-brain barrier, making it a highly translational research proposal.

3. Aims

Our study includes four specific aims. Initially, we will conduct in vitro studies to characterise the signalling pathways driving DIPG using patient derived cell lines. This will be done by testing the copper dependence of key enzymes, including PDGFRα, EGFR, PI3K and AKT, by determining their expression levels and phosphorylation status by supplementing (aim 1) or subtracting (aim 2) copper in the culture media, with a variety of molecular biology techniques.

In aim 3, we will evaluate the ability of DIPG cell spheroids to invade through stem-cell derived three-dimensional “mini-brains” in the presence or absence of the copper chelating agents using live cell imaging. With this specific set of experiments, we will determine whether copper chelators are also able to slow down DIPG tumour progression by interfering with their typical invasive pattern of growth.

Aim 4 will focus on in vivo testing of copper chelating agents in a xenografted mouse model of DIPG. In particular, we will determine (i) the safety profile of the drug agents, which is forecasted to be well-tolerated given the wide use of these drugs in genetic disorders, such as Wilson’s Disease, (ii) efficacy of copper chelators through survival study in a cohort of control and treated mice and (iii) their effect on tumour size, induction of tumour cell apoptosis and invasiveness into surrounding healthy brain tissue, using common histopathology techniques.

4. Clinical significance

Despite some encouraging pre-clinical studies, most clinical trials have failed to significantly improve DIPG patients’ survival, compared to standard palliative radiotherapy. This is usually due to drug insufficient brain bioavailability, high toxicity or acquired resistance. Today DIPG is still universally lethal and patients are in desperate need of alternative therapies. We believe that copper chelation could represent one such therapy.

Copper chelators are a consolidated treatment for copper accumulation syndromes, like Wilson’s Disease. As such, copper lowering agents are well known to cross the blood-brain barrier and alleviating neurological symptoms due to copper accumulation while causing no side-effects. We propose to test whether lower copper levels in DIPG cells will be enough to affect all copper dependent cellular signalling which are systematically elevated in DIPG.

Being characterised by expected low toxicity, this approach will allow us to test copper chelation in combination with other chemotherapeutic agents targeting more specific pathways of interest. Similarly, using the same principle, we will be able to also test the depletion of other redox co-factors and transition metals and, in the long run, expand the project to other paediatric high-grade gliomas.

Overall, this project represents a novel way to target DIPG driving pathways, a significant chance to better characterise copper chelation mechanisms in cancer treatment and a unique opportunity to design a clinically significant study with a unique translational potential.

5. Design and methods

Aim 1: DIPG cells will be incubated with appropriate media or copper enriched media (in the form of copper chloride). RNA sequencing will be used to highlight the key molecules that demonstrate a copper-dependent expression profile. Confirmatory reverse quantitative polymerase chain reaction and Western blot will be performed to compare the expression of such molecules between control cells and cells incubated with copper at both RNA and protein level. Surface Sensing of Translation (SUnSET) assay will be used to sort cells based on global protein synthesis. Flow cytometry will be carried out to specifically study the activation status of RTKs, while immunofluorescence will be adopted to verify abundance and localisation intracellular markers.

Aim 2: Similarly, the same set of experiments of aim 1 will be carried out to confirm the copper-dependence of key cellular pathways after treating DIPG cells with copper chelating agents (TEPA and TETA). Together, aim 1 and 2 will establish in detail the relationship between cellular copper levels and DIPG signalling mechanisms, and gain the first insights into copper homeostasis disruption and DIPG cells cytotoxicity.

Aim 3: To study the physiological diffusive nature of DIPG, we will optimise 3D cultures and live imaging. Stem cell-derived cerebral organoids will be co-cultured with DIPG cells and followed in real-time using quantitative fluorescent microscopy to record differences in the ability of DIPG cells to invade through healthy tissue in the presence or absence of copper chelating agents. By inactivating DIPG key drivers, we anticipate to significantly reduce malignant cell spreading.

Aim 4: The ultimate proof of copper chelators’ efficacy will be obtained through in vivo experiments using a DIPG xenograft model in Balb-c/nude mice. DIPG cells will be implanted orthotopically under stereotactic surgical condition. Both control and treated mice will be monitored for neurological symptoms and sacrificed at endpoint. Complete histopathological and survival analysis will be conducted on both cohorts to compare tumour size, apoptosis, invasion and overall benefit in outcome.

6. Scientific Merit

In the past several decades, DIPG patients have participated in hundreds clinical trials involving both single and combination drugs regimens, as well as radiosensitizers. None of these studies have shown any significant benefit in either the likelihood of survival or the median length of survival. Better characterisation of individual DIPG tumour profiling has led to tailored approaches with disappointing results due to poor drug permeability across the blood-brain barrier, unacceptable toxicities or rapid acquisition of resistance.

With this project, we will take advantage of our deep understanding of DIPG biology, not targeting a specific molecular driver of tumour progression, but a common co-factor central to a multitude of enzymes, copper. This will be done utilising copper chelating agents that are known to reach the brain in the presence of an intact blood-brain barrier. Importantly, by impairing multiple cellular pathways simultaneously, we anticipate that DIPG cells will not be able to switch the aberrant signalling to an alternative one, avoiding the acquisition of drug resistance.

Importantly, our project proposes to repurpose already approved, low-cost drugs that have been used in children without any significant toxicities. Lastly, we expect that novel therapeutic targets will emerge from our project while we will strive to adapt it to any other brain malignancy of childhood.

7. Feasibility

The high feasibility of this proposal derives from the location where it will take place and the unique expertise of the team members. Our research group is part of the Children Cancer Institute (CCI), the largest Australian program dedicated to developing new treatments for malignancies of childhood. The institute is clearly already equipped all the techniques and infrastructures to carry out the research proposal. Moreover, substantial biostatistics and bioinformatics support will be made available to us through the in-house CCI data science team. Importantly, we work in close relationship with paediatric oncologists and scientists at the Sydney Children’s Hospitals Network and Kids Cancer Alliance and therefore are well positioned to undertake high-impact translational research. Lastly, we have assembled a motivated team of talented molecular biologists who are not only capable to conduct the proposed project, but deeply driven by the common vision to alleviate patients suffering and succeed in the fight against DIPG.

8. Expertise

Being part of the CCI, we work in close relationship with the Zero Childhood Cancer (ZCC) paediatric precision medicine program that has enrolled >300 children nationally, 40% with malignant brain tumours, providing in-depth, personalised tumour analysis. As such, we already contributed globally to the deep understanding of DIPG genetics and biology. We are now using this wealth of knowledge to map DIPG driving pathways and targeting them using a novel thinking-out-of-the-box approach.

Dr Orazio Vittorio is the team-leader of the Metal-Targeted Therapy (MTTI) and Immunology group. He has a growing reputation in understanding the role of metals, such as copper, in paediatric malignancies progression as well as its targeting through the use of chelating agents.

Dr Federica Saletta is a senior research officer in the MTTI group. She comes not only with a plethora of molecular biology skills, but also with an outstanding track records in histopathology technique development for molecular diagnostics.

Associate Professor Geraldine O’Neill leads the Focal Adhesion Biology (FAB) group of the Children's Cancer Research Unit (Children’s Hospital at Westmead). She is an internationally recognised expert in developing pre-clinical 3D models for brain cancer using the latest advances in tissue engineering and microscopy.

Associate Professor David Ziegler is a senior paediatric oncologist and a world-renowned brain cancer expert. He is leading the Brain Tumour Group at CCI, conducting in vitro drug screening and in vivo personalised xenograft (PDX) brain tumour models.

Description of Research Proposal

Budget

Collaborations and Conflicts of Interest