Title

Medulloblastoma: New animal models, preclinical drug testing, and characterising immune infiltrates

Author Identifiers

Courtney George

https://orcid.org/0000-0001-6840-8836

Date of Award

2022

Degree Type

Thesis

Degree Name

Doctor of Philosophy

School

School of Medical and Health Sciences

First Advisor

Elin Gray

Second Advisor

Raelene Endersby

Third Advisor

Mel Ziman

Abstract

Medulloblastoma is the most common malignant brain tumour in children. The current treatment for medulloblastoma consists of surgery, radiation, and chemotherapy. Although these therapies have their merits, the outcome for some patients, particularly those with MYC amplified tumours, is poor, and the damaging nature of these therapies results in morbidities that significantly impact on a patient’s quality of life. To improve outcome and reduce adverse side effects, several strategies have been employed, including expanding the repertoire of accurate disease models, the development of novel therapies and the initiation of clinical trials, and an improvement in the understanding of the disease pathogenesis. The purpose of this thesis was to contribute to the repertoire of animal models, assist in identifying novel therapeutic approaches to treatment, and to advance the knowledge of the medulloblastoma immune microenvironment. We did so by aiming to (1) develop more accurate, immune-competent animal models of MYC- or NMYC-amplified medulloblastoma, (2) testing a novel treatment that combines conventional chemotherapies with a cell cycle checkpoint kinase inhibitor, and (3) investigating the effects of clinically used chemotherapies on immune cell populations in the brains of medulloblastoma-bearing mice.

Currently, the limited availability of preclinical mouse models that accurately represent subgroup-specific medulloblastoma has hindered the development of therapies that succeed in the clinical setting. In addition, the distinct lack of immunologically competent models has prevented the advancement of immunotherapy drugs in treating medulloblastoma. The models of medulloblastoma developed here were designed to aid in preclinical studies, and to contribute specifically to the repertoire of immune competent mouse models for studies to clarify the role of the immune system in medulloblastoma. Here, we utilised mutated human variants of CMYC, NMYC, and P53, to transform mouse neural stem cells into tumour forming cells. Tumours were established in C57Bl/6 mice and characterised by histology and RNAseq analysis. Whilst these animal models could not be confirmed to accurately represent Group 3 or Group 4 medulloblastomas, this study demonstrated that mouse neural stem cells could be transformed using human genes and could generate brain tumours within immune competent hosts. As we were unable to conclusively define the exact nature of the tumours that were produced here, existing mouse models were used for subsequent chapters in this thesis.

Prior to implementing new therapeutic agents into clinical trial, these are evaluated in a pre-clinical setting. The data presented in chapter two contributed to a larger scale pre-clinical testing pipeline that led to the identification and evaluation of multiple kinase inhibitors for the treatment of medulloblastoma. Here, I examined the combination of the cytotoxic agent gemcitabine (GEM) with the cell cycle checkpoint kinase inhibitor (LY2606368, prexasertib) as a novel approach in the treatment of Group 3 medulloblastoma. Combination GEM/LY2606368 treatment improved the survival of mice with aggressive medulloblastoma. Using immunoblotting and flow cytometry I showed that mechanistically LY2606368 enhanced GEM-induced cytotoxicity by impairing DNA damage response pathway activity, which promoted the accumulation of DNA damage leading to increased apoptosis. Together, these data formed part of the preclinical evidence that supported the establishment of the SJ-ELiOT clinical trial, targeted towards improving outcomes for patients who experience recurrent or relapsed disease following standard-of-care therapy.

There is evidence to suggest that inhibiting the DNA damage response pathway can stimulate the immune system, and aid in tumour elimination. This provides strong rationale for implementing immunotherapies for patients who are predicted to have a poor response to conventional treatments. Unfortunately, to date all clinical trials investigating current popular immune-based therapies have failed in medulloblastoma, likely due to a poor understanding of the immune microenvironment in this disease. This presented an opportunity to improve our understanding of the effects of treatment on the immune system in brain. Here I characterised the immune cell populations in the brains of mice with Group 3 medulloblastoma treated with vehicle or two clinically-used chemotherapies – cyclophosphamide (CPA) and GEM. I revealed that CPA and GEM differentially alter immune cells within the brain in a manner similar to that observed outside of the central nervous system. I also demonstrate that the lack of an adaptive immune system (using mice deficient in Rag1) does not influence the anti-cancer effects of these drugs. This information provides a rationale for exploring alternative avenues when considering the use of cancer-targeting immunotherapies in combination with conventional medulloblastoma treatments.

Collectively, these studies demonstrate the complicated nature of modelling high-risk medulloblastoma in the lab. I have improved upon current preclinical tools for medulloblastoma by the development of accurate immune competent models and advanced our knowledge of disease pathogenesis by elucidating the way medulloblastomas interact with the immune system in the brain. Furthermore, I highlight the translational value of preclinical models in the evaluation of new drug combinations ahead of clinical trial. Improving on the current tools available and accurately evaluating new therapies will ideally lead to improved clinical outcomes for patients with medulloblastoma.

Access to this thesis is restricted. Please see the Access Note below for access details.

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