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Griffin, Michaela (2023) CLIC1 and CLIC4 ion channels as therapeutic targets for tumour treating fields in paediatric high grade glioma. PhD thesis, University of Nottingham.

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Abstract

Background: The lack of treatments for paediatric high-grade glioma represents one of the most significant unmet clinical needs in paediatric cancers. pHGGs form a subset of lethal brain tumours with a median overall survival of only 14 months; forming the leading cause of CNS cancer-related death in children. OptuneTM, a non-invasive electrotherapy device that delivers alternating electric fields – coined tumour treating fields (TTFields) – into the tumour. Despite success in adult trials, TTFields treatment is currently not approved in the paediatric population. Increasing evidence show that ion channels not only regulate electrical signalling of excitable cells, but also play a pivotal role in the development and progression of brain tumours. Membrane potential is a crucial biophysical signal that modulates the malignant processes via a plethora of tightly controlled ionic exchange processes. We aim to identify potential targets for TTFields and provide a proof-of-principle to elucidate the role of ion channels for multimodal therapies for paediatric high-grade gliomas.



Methods: Widescale literature and genomic analysis of adult and paediatric HGG tissue was used to characterised CLIC channel expression via multivariate analysis. To understand the functional role of CLIC1 and CLIC4, siRNA depletion, or pharmacological targeting via IAA94/metformin was assessed using cell cycle, clonogenic, invasion and proliferation assays. The potential for multimodal therapies was interrogated by combining in vitro TTFields with CLIC inhibition. Whole cell and cell attached patch clamp protocols, along with Cl- ion tracking was used to assess ion channel activity in pHGG and normal astrocytes. To build a global understanding of TTFields and HGG recurrence, 3’ mRNA sequencing was carried out across three recurrent tumours following TTFields treatment.

Results: We have demonstrated an elevated expression of CLIC1 and CLIC4 in HGG, which is significantly associated with poor overall survival in patient cohorts. siRNA/pharmacological depletion of CLIC1 or CLIC4 propagates a reduction in the proliferation and invasion of pHGG associated with cell cycle arrest. Furthermore, combination of CLIC inhibition and TTFields revealed that CLIC1 and CLIC4 deficiency exacerbated the killing capacity of TTFields, and sufficiently re-sensitised TTFields tolerant cells. Furthermore, electrophysiology experiments reveal increasingly depolarised membrane potential in pHGG compared to astrocytes. RNA sequencing of recurrent GBM tissues revealed that in situ TTFields treatment is indeed associated with decreased CLIC1 and CLIC4 expression. Moreover, in vivo TTFields exposure is associated with a downregulation in DNA repair and increase neurodegenerative pathways.

Conclusions: This study characterised CLIC1 and CLIC4 expression in pHGG, finding they have clear implications in HGG and are prognostic indicators for OS of patients. We provide evidence that CLIC channels provide mechanistic insight into TTFields success. These data provide rationale that genetic, electrical, and pharmacological manipulation of ion channels can reduce the capacity of pHGGs to proliferate and invade and that CLIC channels may be a suitable target for combination therapy to enhance the treatment efficacy of TTFields.

Item Type:	Thesis (University of Nottingham only) (PhD)
Supervisors:	Smith, Stuart Khan, Raheela
Keywords:	Brain tumour; Tumour treating fields; Ion channels
Subjects:	W Medicine and related subjects (NLM Classification) > WL Nervous system
Faculties/Schools:	UK Campuses > Faculty of Medicine and Health Sciences > School of Medicine
Item ID:	76018
Depositing User:	Griffin, Michaela
Date Deposited:	13 Dec 2023 04:40
Last Modified:	13 Dec 2023 04:40
URI:	https://eprints.nottingham.ac.uk/id/eprint/76018

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