- Title
- Molecular characterisation of oncogenic signalling networks to develop treatment strategies for diffuse intrinsic pontine glioma
- Creator
- Jackson, Evangeline R.
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2023
- Description
- Research Doctorate - Doctor of Philosophy (PhD)
- Description
- Diffuse midline glioma (DMG) is a uniformly lethal childhood brain cancer, responsible for more childhood cancer deaths than any other. Tragically, patients only survive <12 months from diagnosis, with <10% of patients surviving 2 years and no long-term survivors. DMG initiates along the midline structures of the brain, primarily in the brainstem (including the pons, termed diffuse intrinsic pontine glioma, DIPG). Currently, the only approved therapy is palliative radiotherapy (RT), which provides some temporary relief from neurological symptoms, and in some cases, delivers a transient survival benefit (~3 months). Today, patients diagnosed with DMG are provided with the same treatments as patients diagnosed 60 years ago, highlighting the desperate imperative and clinical importance of developing therapies and treatment strategies that improve outcomes. DMG is characterised by dominant-negative hypomethylation of histone tails on both canonical (HIST1H3B/C) and non-canonical (H3F3A) H3 proteins. Loss of trimethylation on lysine 27 of H3 is an epigenetic mark of open chromatin and active transcription. This occurs either through a lysine 27 to methionine gain-of-function mutation (H3.1K27M and H3.3K27M), or for a small population of DMG patients, overexpression of the enhancer of zeste homolog inhibitory protein (EZHIP), which acts in a very similar biochemical way to H3K27M mutations and drives global hypomethylation of H3K27. Intriguingly, even though these histone alterations are disease instigating, they are insufficient to drive malignant tumour growth. H3K27-altered, DMG co-occurs with mutations and amplifications in type III receptor tyrosine kinases (PDGFR, KIT, VEGFR), type I receptor serine/threonine kinase (ACVR1), intracellular kinases (PI3K, AKT, MTOR), the proto-oncogene (MYC) and tumour suppressor genes (TP53, PTEN, BCOR). In this body of work, we seek to address the knowledge gap behind the functional consequences of these genomic aberrations and the oncogenic signalling pathways that drive DMG lethal prognosis. In Chapter 2, I provide of a summary of the known signalling pathways that underpin the gliomagenesis of DMG. Additionally, in the absence of drugs targeting loss-of-function (LoF) mutations or brain penetrant therapies that address the gain-of-function mutations, we summarise drugs that target commonly amplified pathways in DMG. In the first data chapter of this thesis (Chapter 3), I investigated the small molecule brain penetrant oral therapy ONC201 (dordaviprone) for the treatment of DIPG. We sought to uncover mechanisms of response and resistance to ONC201, as a means to identify combination strategies that address the complex genetic and epigenetic milieu of DMG. Here, we further confirm that ONC201 is a potent agonist of the mitochondrial protease, ClpP, resulting in the proteolysis of mitochondrial proteins, and the release of reactive oxygen species (ROS) as a consequence of electron transport chain protein degradation. Using next-generation sequencing, and CRISPR-Cas9 gene knockdown and knockout we identified that patient-derived DIPG cell lines harbouring LoF TP53 mutations were less sensitive to ONC201. However, in the absence of therapies targeting this LoF mutation, we employed a proteomic profiling approach to identify pathways activated in response to ONC201 treatment, revealing redox-activated and increased PI3K/AKT signalling, that we targeted with the brain penetrant PI3K/AKT inhibitor, paxalisib. The combination of ONC201 and paxalisib synergistically decreased in vitro proliferation and provided a significant survival benefit in orthogonal DIPG mouse models (n=3). Finally, case study compassionate access of the first two DIPG patients to receive the combination of ONC201 and paxalisib, one at diagnosis and one at progression, showed promising clinical benefits, resulting in tumour reduction by 68.2% compared to post-RT, and 34% compared to regression, respectively. This research underpins the preclinical data for the establishment of the international adaptive combination therapies Phase II clinical trial, PNOC022 (NCT05009992). In Chapter 4, we investigated a pooled CRIPSR-Cas9 gene deletion (LoF) dataset provided to us by our collaborators, Dr Jason Cain, and Prof Ron Firestein. This confirmed our suspicions that PIK3CA and MTOR are genetic dependencies -across DMGs. My supervisor Prof Dun had discovered the PI3K/AKT/mTOR inhibitor paxalisib for the treatment of DMG in 2018; therefore, our lab was already studying its preclinical potential, however, these LoF data further fuelled our research into the preclinical/clinical potential of exploiting this genetic dependency. My work was aimed at increasing efficacy and to identify novel combinations using paxalisib. Early-stage clinical trials testing paxalisib in DMG identified drug-related DLTs, including hyperglycaemia. Therefore, to reduce side effects and increase compliance, we altered dosing, and explored the mouse equivalent half-MTD, twice daily, instead of once daily MTD, confirming good brain penetration and clinical potential. Further, through the combination of paxalisib with the anti-hyperglycaemic, metformin, frequently used to maintain glucose homeostasis, we further promoted the preclinical benefit of paxalisib. To identify therapeutic targets upregulated in response to paxalisib treatment we used multiomic profiling and identified calcium (Ca2+) activated PKC signalling, counteracted using the blood-brain barrier (BBB)-permeable PKC inhibitor, enzastaurin. The combination of paxalisib and enzastaurin synergistically suppressed cell proliferation in vitro, providing a synergistic survival benefit in orthotopic DMG patient derived xenograft mouse models (n=3). During my studies, the Phase III randomised placebo controlled clinical trial, ONC201 in H3 K27M-mutant DMG following RT (the ACTION Study) opened internationally. Hence, my supervisor was invited by Neuro-Oncology to provide the field with an in-depth literature review summarising the mechanisms of action of ONC201 in DMG in support of the Phase III trial. Here in Chapter 5, I highlighted combination therapies which have concurrently undergone preclinical testing, including ONC201 and paxalisib. Additionally, I begin to investigate the potential role the tumour immune microenvironment (TIME) plays in its efficacy, a currently underexplored role for ONC201. During my PhD, I have performed preclinical work that has supported the establishment of international clinical trials and is underpinning the development of new ones. In this body of work, I have helped to highlight the importance of detailed mechanistic studies providing new understanding of the unique biology of DMG and identifying the potential of exciting new therapies and treatment strategies, that go some way to addressing this biology. Studying the fundamentals of DMG biology using patient derived samples, isogenic models, patient derived xenograft (PDX) and syngeneic models has helped our understanding of the complex functional consequences of genomic and epigenetic alterations that currently remain untargetable in DMG. Therefore, through my systems-wide research I outline several novel combination treatment strategies that I hope will go some way to improving the survival of patients diagnosed with DMG/DIPG.
- Subject
- diffuse midline glioma; diffuse intrinsic pontine glioma; proteomics; phosphoproteomics; PI3K; MTOR; PKC; thesis by publication; DMG; DIPG; ONC201; dordaviprone; paxalisib; enzastaurin; ClpP agonist; epigenetics
- Identifier
- http://hdl.handle.net/1959.13/1495888
- Identifier
- uon:54081
- Rights
- Copyright 2023 Evangeline R. Jackson
- Language
- eng
- Full Text
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