In the first part of METAREPAIR, I set out to define the effect of one carbon metabolites modulation on DDR through the regulation of histone methylation. To modulate intracellular one carbon metabolism, known to regulate methyltransferases activity, PCa cells were cultured for 24 hr in a modified cell culture media formulation in which methionine was depleted or enriched. Cells were then treated with ionizing radiation (IR) and processed and analyzed to understand how methionine manipulation affected 1) cellular metabolite levels; 2) histone marks known to be involved in DDR, and 3), DDR factors themselves. The work performed to achieve Objective 1 let to the observation that methionine manipulation impacted intracellular levels of metabolites that are involved in methylation processes, and the methylation status of specific histone marks. However, no effect was observed on DNA damage load, and only one DDR factor was affected by methionine manipulation. In the second part of METAREPAIR, we investigated the effect of one carbon metabolites modulation on DDR through the regulation of RNA methylation. We observed a transient decrease in some epitranscriptomic marks in PCa cells deprived of methionine, and this observation correlated with altered expression of one DDR factor after DNA damage. When we genetically or pharmacologically inhibited the levels of this epitranscriptomic mark, the levels of some DDR factors were affected, demonstrating the specificity of the effect. However, no impact on the overall DNA damage load of the cells was observed when PCa cells were subjected to methionine starvation, possibly because of the methionine dependency of cells in general.
Altogether, the results of METAREPAIR provided further evidence that metabolite modulation impacts the epigenetic and epitranscriptomic landscape of cells and that, under some conditions, can influence DDR. However, the link between metabolism and DDR is very complex and is regulated by other mechanisms that were not the object of our investigation. Further studies should be necessary to fully deconvolute these mechanisms and be able to exploit them for therapeutic purposes.
During the course of the action, we regularly presented updates on the progress of the project during meetings held at IFOM. However, many of the results obtained in this project were negative, as we did not observe any significant impact of one carbon metabolism modulation on DDR. For this reason, it was not possible to present our data at conferences or international meetings. Nevertheless, we strongly believe that also negative results are valuable to the scientific community, because they can help other researcher directing their effort to other unexplored directions: therefore, we will try and make our work available to the community.
