Incidence of childhood leukaemia is increasing and whilst survival rates are high, treatment can lead to long term side effects and early mortality. Although the aetiology is unclear, various genetic aberrations, such as chromosomal translocations, are suggested to be initiating events in leukaemia and have been retrospectively detected at birth, suggesting they occur in utero. Further factors, such as epigenetic modifications, are likely required for disease progression. Epidemiological studies have identified various exposures associated with risk of childhood leukaemia including maternal nutrition, smoking and air pollution. Using a meet-in-the-middle approach comparing DNA methylation signatures between environment and leukaemia, we observed significant overlaps in altered methylation between specific leukaemia subtypes and some exposures, including radiation, alcohol, smoking, and reported colds, whereas methylation associated with maternal folate levels overlap with most leukaemia subtypes. Using monoculture cell line and 3D organoid experimental models that enable controlled manipulation of risk exposures, we examined the influence of environment on childhood leukaemia aetiology and the mechanisms through which they contribute to disease progression.

In our preliminary cell line model we exposed NALM6 B cells to varying concentrations of leukaemia-associated risk exposures; benzene and cotinine (components of cigarette smoking), caffeine and folic acid over 4 days. Reverse transcription PCR (RT-PCR) assays were then used to identify chromosomal translocations commonly found in childhood leukaemia. We then developed a 3D organoid model of the bone marrow niche under varying folate conditions to further investigate the development of leukaemia-initiating events in a more physiologically relevant system. Mesenchymal stem cells (MSCs) were used to create 2D and 3D organoids supporting growth of GM12878 B cells in media containing different amounts and forms of folate, folic acid and 5-methyltetrahydrofolate. Cell growth was evaluated through trypan blue exclusion. Intra- and extra-cellular folate was measured using LC-MS and ELISA assays, with RT-PCR used to monitor cell markers and leukaemia-initiating events.

In our preliminary cell line model, positive translocation events were observed for both TCF3::PBX1 and RUNX1-RUNX1T1 in cells exposed to different concentrations of benzene, cotinine and folic acid, and RUNX1-RUNX1T1 in cells exposed to caffeine. When a selection of translocation-positive conditions was repeated up to 20 times, the presence of translocation events was significantly enriched for all exposures. For GM12878 B cells grown in co-culture with MSCs, B cell growth is increased compared to growing alone. The largest increase in B cell growth was observed in 3D systems compared to growing in a 2D model or alone. A reduction in intracellular folate was observed as early as 2 days following folate depletion.

Our preliminary data suggests environmental exposures associated with childhood leukaemia risk may induce initiating translocations, suggesting a biologically plausible mechanism for epidemiological associations. By optimising a 3D model of exposure, we will be able to investigate the induction of leukaemia-initiators in a more physiologically relevant model. Such knowledge may be useful to influence public health policy during pregnancy with the aim of preventing childhood leukaemia.