Inhibition of de novo pyrimidine biosynthesis in AZA-R cells showing reduced expression of UCK2. (A) AZA-R cells (red) derived from TL-Om1 and MT-2 cells, and each parental cell line (blue) were treated for 96 hours with BAY2402234, a DHIODH-specific inhibitor. Cell growth was measured using a CCK-8 kit. Absorbance of nontreated cells was set as 100%. The results are expressed as the mean ± SD of 3 independent experiments. (B) Fifty percent inhibitory concentration (IC) values of BAY2402234. Data are expressed as the mean ± SD of 3 independent experiments. ∗P < .05; ∗∗P < .01. (C) An extracellular flux assay was used to measure the OCR in AZA-R cells (red) derived from TL-Om1 cells, as well as parental cells (blue), cultivated under normal growth conditions. Oligomycin, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone, rotenone, and antimycin A were added to the assay. (D) AZA-R cells derived from TL-Om1 and parental cell line were treated for 96 hours with dipyridamole alone (blue) or in the presence of IC20 (yellow; 1.2 nM for parental cells and 0.75 nM for AZA-R cells) and IC 40 (red; 1.6 nM for parental cells and 1 nM for AZA-R cells) of BAY2402234. Cell growth was measured using a CCK-8 kit. Absorbance of nontreated cells was set as 100%. The results are expressed as the mean ± SD of 3 independent experiments. (E) Graphical schema showing reprogramming of pyrimidine biosynthesis in normal T cells upon T-cell activation (upper panel), and in malignant T cells upon downregulation of UCK2 (which induces resistance to AZA) (bottom panel).