Abstract
Current anti-cancer strategy takes advantage of tumour specific abnormalities in DNA damage response to radio- or chemo-therapy. Inhibition of the ATR/Chk1 pathway has been shown to be synthetically lethal in cells with high levels of oncogene-induced replication stress and in p53- or ATM- deficient cells. In the presented study, we aimed to elucidate molecular mechanisms underlying radiosensitization of T-lymphocyte leukemic MOLT-4 cells by VE-821, a higly potent and specific inhibitor of ATR. We combined multiple approaches: cell biology techniques to reveal the inhibitor-induced phenotypes, and quantitative proteomics, phosphoproteomics, and metabolomics to comprehensively describe drug-induced changes in irradiated cells. VE-821 radiosensitized MOLT-4 cells, and furthermore 10 μM VE-821 significantly affected proliferation of sham-irradiated MOLT-4 cells. We detected 623 differentially regulated phosphorylation sites. We revealed changes not only in DDR-related pathways and kinases, but also in pathways and kinases involved in maintaining cellular metabolism. Notably, we found downregulation of mTOR, the main regulator of cellular metabolism, which was most likely caused by an off-target effect of the inhibitor, and we propose that mTOR inhibition could be one of the factors contributing to the phenotype observed after treating MOLT-4 cells with 10 μM VE-821. In the metabolomic analysis, 206 intermediary metabolites were detected. The data indicated that VE-821 potentiated metabolic disruption induced by irradiation and affected the response to irradiation-induced oxidative stress. Upon irradiation, recovery of damaged deoxynucleotides might be affected by VE-821, hampering DNA repair by their deficiency. Taken together, this is the first study describing a complex scenario of cellular events that might be ATR-dependent or triggered by ATR inhibition in irradiated MOLT-4 cells. Data are available via ProteomeXchange with identifier PXD008925.
Highlights
DNA damage induction by either radio- or chemo-therapy has been the most widely used approach in oncology
The monitored phosphorylation site was upregulated upon irradiation (3 Gy, one hour after irradiation), and it was proportionally inhibited by increasing concentration of VE-821
In response to the inhibitor treatment, we revealed changes in DNA damage response (DDR) related pathways and kinases, and in pathways and kinases involved in maintaining cellular metabolism
Summary
DNA damage induction by either radio- or chemo-therapy has been the most widely used approach in oncology. In a recent study investigating mutational profiles in 3,281 tumours across 12 tumour types [3], genes from the ATM/Chk2/p53 pathway were affected by mutations in almost a half of the investigated cancer cells As this pathway is essential for maintaining the G1/S DNA damage checkpoint after irradiation, the results of this study suggested that targeting the remaining DNA damage checkpoints might be a promising strategy in a considerable proportion of solid tumours conventionally treated using radiotherapy. Another promising strategy is targeting proteins and protein kinases involved in replication stress response. Cancer cells deficient in G1/S checkpoint or with mutations deregulating replication origin firing suffer from premature entry into S-phase, and DNA replication can start before the necessary resources have been generated [4,5]
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