Fig 2: Loss of NUDT22 potentiates inhibition of nucleotide metabolism.Dose response curves of ctrl and NUDT22 KO U2OS cells exposed to (A) pyrazofurin, (C) hydroxyurea and (E) after glutamine starvation. Dose response curves of ctrl and NUDT22 KO hTERT-RPE1 cells exposed to (B) pyrazofurin, (D) hydroxyurea and (F) after glutamine starvation. G Expression levels of pyrimidine synthesis genes after 24 h of glutamine starvation relative to β-actin measured by qRT-PCR. Statistical analysis between hTERT-RPE1 and U2OS cells (NUDT22 P = 0.0135; TK1 P = 0.0071; DCK P = 0.0132; DHODH P = ns; UMPS P = 0.004; TYMS P = 0.01; RNR P = 0.0026). P values were calculated by paired t test. Data are presented as the mean values with SD. H Growth rate comparison between U2OS and hTERT-RPE1 cells. All experiments were repeated at least 3 times.

Fig 3: NUDT22 expression is increased in cancer and loss of NUDT22 reduces cancer growth in vivo.A RSEM expected count analysis of NUDT22 expression in the panCancer TCGA and GTEx datasets (P < 0.0001; Mann–Whitney test, mean with SD). B RSEM expected count analysis of pyrimidine metabolism gene expression in breast cancer TCGA and GTEx datasets (P < 0.0001; Mann–Whitney test, mean with SD). C Overall survival (OS) of breast cancer patients with NUDT22 gene alterations (TCGA PanCancer). Altered group is defined as patients with at least one alteration in the NUDT22 gene. D Cell cycle checkpoint activation of ctrl and NUDT22 KO MCF7 cells was rescued by uridine supplementation detected by western blot. E NUDT22 KO MCF7 cells have reduced EdU incorporation, which is further reduced by pyrazofurin exposure quantified by high content microscopy (mean with SD). F Reduced replication fork speed in NUDT22 KO MCF7 cells can be rescued by uridine supplementation (mean with SD). G NUDT22 KO MCF7 cells have increased gH2A.X foci formation, which was further increased with brequinar, leflunomide and pyrazofurin quantified by high content immunofluorescence microscopy (P < 0.0001). H uridine supplementation rescues DNA damage caused by NUDT22KO (P < 0.0001) (mean with SEM). P values were calculated by the Mann–Whitney t test. I Dose-response curves of ctrl and NUDT22 KO MCF7 cells treated with pyrazofurin, (J) brequinar and (K) leflunomide. L In vivo mammary cancer xenograft model with ctrl and NUDT22 KO MCF7 cells. Luc2+ cells were injected into mammary fat pads and imaged weekly by IVIS imaging (P < 0.0023; Mann–Whitney test, mean with SEM). All experiments were repeated at least 3 times.

Fig 4: NUDT22 is a p53 target gene.A The depletion of p53 abolished the cMYC-mediated activation of NUDT22 in BJ-MYCER cells (NUDT22 P = 0.0086; P = 0.0119; P = 0.0046; p53 P = 0.012; P = 0.0146; P = 0.0022), measured by qRT-PCR. B Western blot of stabilized p53 and p21 and increased NUDT22 in BJ-MYCER cells. C Relative luciferase levels of the NUDT22 reporter after stabilization of p53 with Nutlin3a in U2OS cells (NUDT22 P = 0.0003, p53 P = 0.0001). D qRT-PCR for the 2 kb CpG 5ʹ region of the NUDT22 gene after ChIP with a p53(DO1) antibody in U2OS cells. GFP served as a transfection control (P = 0.008). P values were calculated by paired t test. Data are presented as the mean values with SD and all experiments were repeated at least 3 times.

Fig 5: NUDT22 expression is increased in response to stress. A Increased NUDT22 expression after inhibition of HK2 with 2-DG measured by qRT-PCR (NUDT22 P = 0.0254; cMYC P = 0.0001; GRP78 P = 0.0016). cMYC and GRP78 are positive controls. B Increased NUDT22 expression after HK2 depletion with siRNA measured by qRT-PCR (HK2 P = 0.006; NUDT22 P < 0.001). C Elevated NUDT22 expression in four independent HA1EB-cMYC clones measured by qRT-PCR (NUDT22 P = 2.4*10−8; cMYC P = 7.48 *10−7). D Western blot of cMYC-overexpressing cells with increased NUDT22 protein levels. E cMYC-induced NUDT22 expression measured by qRT-PCR (cMYC P = 0.0006; NUDT22 P = 0.0346). F Transient cMYC overexpression in U2OS (NUDT22 P = 0.019) and hTERT-RPE1 (NUDT22 P = 0.0002) cells. G Western blot of cells fractionated in soluble (cytosolic) and insoluble (nuclei, membranes) of U2OS cells transiently transfected with cMYC. P values were calculated by paired t test. Data are presented as the mean values with SD and all experiments were repeated at least 3 times.