Immune priming and immunotherapy for the treatment of childhood brain tumours

Background:
Central nervous system (CNS) tumours are the leading cause of cancer-related deaths in children, with medulloblastoma (MB) being the most common malignancy. Group3 MB show a higher rate of recurrence than other MB subgroups, this pattern of recurrence is almost exclusively metastatic. Recurrent disease is near universally fatal and surgery at time of recurrence is correlated with significant morbidity and distress, therefore, we must advance beyond small molecule inhibitors aimed at mutant oncogenes if we are to improve treatment outcomes. Immunotherapeutic targeting of cell surface molecules using chimeric antigen receptor-expressing T-cells (CARTs) have facilitated extraordinary results in clinical trials for B-cell malignancies and in pre-clinical trials for some CNS malignancies; the lead supervisor has been instrumental in generating powerful pre-clinical data for cerebellar tumours, including MB. Immunotherapeutic strategies are now at the forefront of anti-cancer therapy, however, one mechanism of failure/suboptimal response to therapy is antigen escape, secondary to epigenetic silencing of the target gene. This inter-changeable nature of epigenetic alterations represents an attractive and therapeutically relevant target for MB therapy. Recently published work by the lead supervisor demonstrated the use of immune priming with epigenetic agents in combination with CARTs, as potential therapeutic treatments for cerebellar tumours. To evaluate this mechanism of immune priming, FDA approved azacytidine (AZA) will be used in combination with previously optimized CARTs in metastatic models of MB. Results from this project are critical for direct translation into novel, genetically engineered mouse models of group3 MBs, for the next generation of preclinical trials.

Hypothesis:
High throughput (HT) evaluation of CART in combination with epigenetic modifiers allows for the identification of the remodelling mechanisms for successful immunotherapy of group3 MBs. Two key questions to address:
1. Characterisation of AZA-induced gene expression and how it affects CART function/persistence.
2. Examine how AZA results in upregulation of the CAR target antigen (EPHA2) – upregulation of EPHA2 by AZA is not due to promoter region demethylation but likely via a complicated and indirect mechanism.

Aims/Objectives:
Aim-1: Development of human group3 in vitro models.
Obj-1: Formation of 3D spheroid models for the HT evaluation of grop3 MBs in response to CARTs +/- AZA.
Aim-2: Identification of a candidate gene list of targets and pathways upregulated by AZA and associated with CART therapy.
Obj-2: Co-culture MB cells with AZA +/- CARTs to make a list of gene hits that are upregulated by AZA (‘GroupA’) but not upregulated when CARTs are added to AZA (‘GroupB’), and not downregulated by CARTs alone (‘GroupC’).
Aim-3: CRISPR-Cas9 screen and in vitro target validation.
Obj-3: Gene hits from ‘GroupA’ and ‘GroupB’ will be removed from ‘GroupB’ to give the final ‘hit list’ of up to 50 genes for pooled in vitro CRISPR screens.
Aim-4: Functional characterisation of CRISPR screen hits.
Obj-4: Functional characterisation of validated gene hits in response to CARTs +/- epigenetic modifiers. Methods: Md-1: Optimizing 3D cell culture modelling of MB under defined growth and oxygen tensions,
including plating densities, viability and proliferation assays in response to optimized CARTs +/- AZA. Md-2: Simultaneous purification of DNA and RNA from 3D models of MBs in response to AZA +/- CARTs. GFP+ tumour cells will be flow sorted from CARTs in treatment ‘GroupB’ to allow for target gene discrimination between tumour cells and CARTs. Methylation, RNA-sequencing and down-stream differential expression analyses will be performed using BioMethyl and DESeq2 Bioconductor, respectively, to identify the top gene hits for downstream validation. Md-3: Following CART editing of the tumour in which immune escape variants will be enriched, guide RNAs for the final gene ‘hit list’ will be designed (≥ 5 gRNA/gene), synthesized and cloned into a pooled format for lentiviral transfer and interrogation in MB models +/- AZA/CARTs. PCR and NGS will be used to validate genetic modifications. Md-4: Functional in vitro analyses of gene hits and pathway validation using RT-PCR, Western blot, HT flow cytometry, and IHC in human samples and associated mouse systems.

Collaboration with UofT:
Dr Taylor specialises in functional genomics and elegant mouse modelling of cerebellar brain tumours. Differential gene expression analyses and gRNA development will be conducted in close collaboration during a 6-month placement during the 1st/2nd year of study.

References:
1. Donovan, et al, Nat Med, 2020. https://doi.org/10.1038/s41591-020-0827-2.
2. Vladiou, El-Hamamy, Donovan, et al., Nature, 2019. https://doi.org/10.1038/s41586-019-1158-7.
3. Barton, et al., F1000Research, 2019. https://doi.org/10.12688/f1000research.18209.1.
4. Fousek, et al., Leukemia, 2020. https://doi.org/10.1038/s41375-020-0792-2
5. Topper, et al., Nature Reviews Clinical Oncology. https://doi.org/10.1038/s41571-019-0266-5