Development of prodrugs targeting DNA repair as novel anti-cancer therapeutics for neuroblastoma

Background
Neuroblastoma is the most common extra-cranial childhood cancer, with more than half of high-risk patients suffering relapse following initial treatment. Novel precision medicines are desperately required. The DNA Damage Response (DDR) plays an important role in neuroblastoma development. Targeting DDR proteins such as ATR offers significant therapeutic potential, but risks toxicity to non-cancerous tissues. To mitigate this, a novel prodrug of a potent ATR inhibitor activated by tumour-specific protease aminopeptidase-N has been developed by the secondary supervisor. This project focuses on evaluation and further development of this lead compound.

Objectives
Evaluation of a series of novel ATR inhibitor prodrugs in neuroblastoma cells, addressing the following:
(1) Quantitative assessment of prodrug activation in neuroblastoma cells/xenografts vs normal cells/tissues ex vivo
(2) Determine efficacy of prodrug in neuroblastoma cells/xenograft models
(3) Examine efficacy of combinations of prodrug with (i) neuroblastoma frontline (induction) chemotherapies, (ii) ionising radiation
(4) Test efficacy of prodrug in combinations with (i) targeted therapies (i.e. ALK, TrKB inhibitors), (ii) minimal residual disease therapies (dinutuximab, isotretinoin)

Novelty
This project is focused on evaluation and development of an entirely new therapeutic class for neuroblastoma - a non-toxic tumour-specific prodrug. Drugs targeting DDR have been trialled in adults for several years, but their potential in paediatric tumours where key defects in DDR are known has only recently been investigated. ATR is an attractive therapeutic target for neuroblastoma.

Timeliness
There is a desperate need for novel therapeutics for neuroblastoma. Targeting ATR could potentially increase survival, but off-target toxicity is a significant risk. A tumour-targeted prodrug will minimise such toxicity. We have had success with this approach using other targets (including a drug scheduled for clinical trial), and to the best of our knowledge this is the first application to therapeutics targeting DDR. This will pave the way towards a non-toxic therapeutic for trials in neuroblastoma patients.

Experimental Approach
Neuroblastoma cell lines will be characterised for expression/activity of aminopeptidase-N using qPCR, Western blotting and established activity assays. Prodrug cytotoxicity versus active drug will be assessed using clonogenic survival assays. Results will be correlated with the extent of DNA damage using comet assays and immunostaining for DDR markers. The mechanism of action will be determined by interrogating predicted active DDR pathways e.g. double-/single-strand break repair. Accumulation of protein-linked DNA breaks will be quantified using CsCl density gradient fractionation followed by immunoblotting. Prodrug/active drug released in tumour/normal tissues ex vivo will be analysed using LCMS.