Fig 2: Capacity for glycolysis from uridine is governed by lineage and transcriptional control of UPP1/UPP2 gene expression.a, Schematic of the PRISM screen with 482 cancer cells lines grown for 6 d in sugar-free medium complemented with 10 mM glucose or uridine (n = 2). b, Lineage analysis (n = 22 lineages) highlighting growth on uridine as compared to glucose. False discovery rates (FDRs) were calculated using a Benjamini–Hochberg algorithm correcting for multiple comparisons13. c,d, Correlation between uridine growth and expression of transcripts (n = 8,123; c) and proteins (n = 3,216; d) across cancer cell lines. e, Correlation between gene copy number (n = 5,950) and growth on uridine across the cell lines, highlighting chromosome 7p. UPP1 is encoded on Chr7p12.3. f, Cell growth assay in sugar-free medium complemented with 10 mM glucose or uridine of a panel of melanoma (n = 9) and non-melanoma (n = 3, 293T, K562 and HeLa) cell lines. Data are shown as the mean ± s.e.m. (n = 4). MDA, MDA-MB-435S. g, Cell growth assay of melanoma UACC-257 wild-type (UPP1WT) and knock-out (UPP1KO) clones in sugar-free medium complemented with 10 mM of glucose or uridine. Data are shown as the mean ± s.e.m. (n = 3, P < 7.4 × 10-6, P < 2.2 × 10-4, P < 5.3 × 10-6) with two-sided t-test relative to UPP1WT cells in the same medium. h, 13C5-uridine tracer analysis reporting representative intracellular metabolites from the PPP, glycolysis and the TCA cycle in UACC-257 wild-type (UPP1WT) and two knock-out (UPP1KO) clones after 5 h (n = 4, P < 1.2 × 10-9, P < 2.2 × 10-12, P < 2.6 × 10-8, P < 6.1 × 10-9, P < 8.7 × 10-9, P < 8.2 × 10-8). i–k, Expression of UPP1 (Upp1) and IL1B (Il1b) in human THP1 cells (n = 4, P < 1.7 × 10-3, P < 1.9 × 10-6, P < 2.2 × 10-6, P < 1.4 × 10-7; i), human M-CSF-matured PBMCs (n = 4 donors, P < 1.5 × 10-2, P < 5.5 × 10-5, P < 1.2 × 10-3, P < 8.4 × 10-5; j) and BMDMs (n = 3 mice, P < 1.6 × 10-2, P < 5.9 × 10-2, P < 2.5 × 10-3, P < 2.0 × 10-2, P < 1.9 × 10-3, P < 4.4 × 10-4; k) after treatment with 100 nM phorbol myristate acetate (PMA) for 48 h (THP1), 100 ng ml-1 lipopolysaccharides (LPS; THP1, BMDMs), 1 mg ml-1 purified yeast RNA (THP1, PBMCs, BMDMs) or 5 µg ml-1 of TLR7/TLR8 agonist (R848) for 24 h and as determined by quantitative PCR (qPCR). l, 13C5-uridine tracer analysis reporting incorporation in media lactate from BMDMs treated for 24 h with 5 µg ml-1 R848 and further grown for 16 h in glucose-free DMEM containing 5 mM 13C5-uridine and 5 µg ml-1 R848 (n = 3 mice, P < 1.4 × 10-3, P < 3.6 × 10-5, P < 6.8 × 10-6). Data are shown as the mean ± s.e.m. with two-sided t-test relative to untreated cells.Source data

Fig 3: Additional analysis of the ORF screen.(a) Replicate plot and Pearson correlation (R) of n = 2 replicate ORF screens in glucose and galactose highlighting UPP1 and UPP2 ORFs shown as log2 TPM + 1, or log2 fold of day 0. (b) Representation of all six UPP ORFs, expressed as read per million in the global population of glucose or galactose-grown cells and as a function of time (n = 2). TRCN0000470579 encodes a splice variant of UPP1 that is N-terminal truncated and lacks the uridine binding site (NM_001287428.2). (c) Top 10 ontologies associated with the ORFs enriched and depleted in galactose relative to glucose. The complete gene ontology analysis is reported in the Supplementary Data Table 1. (d) Protein immunoblot of K562 cells expressing UPP1-FLAG grown for 16 h in sugar-free media supplemented with 10 mM of glucose or uridine and immunolabelled with antibodies to total S6 ribosomal protein and phosphorylated S6 ribosomal protein (p-S6). Representative of n = 2 experiments. Total S6 loading control was performed on the same gel. All growth assays and screens included 4mM L-glutamine and 10% dialyzed FBS. Source data

Fig 4: Additional metabolomics analysis.(a) Steady-state abundance of representative intracellular metabolites from the pentose phosphate pathway (PPP) and glycolysis in sugar-free media complemented with 10 mM glucose, 10 mM galactose or 10 mM uridine, in the presence of 4mM L-glutamine and 10% dialyzed FBS (n = 3 replicate wells, P < 2.2 × 10-5, P < 8.1 × 10-4, P < 4.7 × 10-7, P < 1.4 × 10-4). Data are shown as mean ± SEM with two-sided t-test relative to control cells. (b) 13C5-uridine tracer analysis of liver and blood uridine 30 min after intraperitoneal injection in fed or overnight fasted mice with 0.4 g/kg 13C5-uridine (n = 3 replicate wells, P < 2.3 × 10-4, P < 2.9 × 10-4, P < 1.4 × 10-2, P < 9.3 × 10-5). Data are shown as mean ± SEM (c) 13C5-uridine tracer analysis of liver ribose-phosphate (ribose-P) and circulating lactate and glucose 30 min after intraperitoneal injection in overnight fasted and (d) in fed animals with 0.4 g/kg 13C5-uridine. Data are shown as mean ± SEM and are corrected for natural isotope abundance (n = 4 mice in each group). (e) 13C5-uridine tracer analysis of liver ribose-phosphate, blood lactate and blood glucose 30 min after intraperitoneal injection in fed mice with 0.4 g/kg 13C5-uridine shown as the percentage of 13C-labeled intermediate compared to the total pool. Data are shown as mean ± SEM and are corrected for natural isotope abundance (n = 4 mice). See also (f) qPCR determination in the liver of ad libitum fed mice, or fasted for 12 h or 24 h, with probes to Upp1, Upp2 and Hmgc2. Hmgc2 transcripts are expected to increase with fasting. Data are shown as mean ± SEM (n = 3 mice in each group). Source data

Fig 5: Uridine phosphorylase activity supports growth on uridine or RNA.a, Schematic overview of the ORF proliferation screen. b, Volcano plot representation of the screen hits after 21 d of growth in medium containing 25 mM glucose or 25 mM galactose, 0.2 mM uridine and 1 mM sodium pyruvate (n = 2). LFC, log2 (fold change). P values were calculated using a two-sided Student’s t-test. Statistics were not adjusted for multiple comparisons. c, Reaction catalysed by UPP1 and UPP2 proteins. d–f, Cell growth assays of K562 control cells and K562 cells expressing UPP1-FLAG or UPP2-FLAG in pyruvate-free media in the presence of: 25 mM glucose or 25 mM galactose or 0.2 mM uridine (±U; n = 3 replicate wells, P < 1.1 × 10-4 and P < 4.7 × 10-5; d), 10 mM of either glucose, galactose or uridine (n = 3, P < 2.1 × 10-5; e) or 5 mM of the indicated nucleosides (n = 3, P < 2.0 × 10-7; f). Data are shown as the mean ± s.e.m. with two-sided t-test relative to control cells. g, Schematic of RNA highlighting its ribose groups. h, Intracellular abundance of the four nucleoside precursors of RNA in control or UPP1-FLAG-expressing K562 cells grown in sugar-free medium supplemented with 0.5 mg ml-1 purified yeast RNA after 24 h. Data are expressed as fold changes of sugar-free medium (n = 4, P < 1.2 × 10-6) and shown as the mean ± s.e.m. with two-sided t-test relative to control. i, Cell growth assays of control or UPP1-FLAG-expressing K562 cells in sugar-free medium supplemented with 0.5 mg ml-1 of purified yeast RNA (n = 3, P < 2.6 × 10-5). Data are shown as the mean ± s.e.m. with two-sided t-test relative to control cells. All growth assays, metabolomics and screens included 4 mM l-glutamine and 10% dialysed FBS.Source data