Journal: bioRxiv
Article Title: hENT Inhibition Prevents Pyrimidine-Driven Resistance to DHODH Inhibition in Malignant Rhabdoid Tumors
doi: 10.64898/2026.01.25.701565
Figure Lengend Snippet: A) Schematic overview of de novo nucleotide biosynthesis and nucleotide salvage pathways. Nucleosides and nucleobases are imported into the cell via hENT 1 and 2 and subsequently phosphorylated to their corresponding nucleotide monophosphates (NMPs). In parallel, nucleotides can be synthesized de novo from metabolic precursors, resulting in the formation of NMPs. Key enzymes involved in these pathways, including uridine-cytidine kinase (UCK), DHODH, and carbamoyl-phosphate synthetase 2/ aspartate transcarbamylase/ dihydroorotase (CAD), are highlighted. Both pathways can be subjected to pharmacological inhibition by agents targeting hENT1/2 (e.g., dipyridamole) and DHODH (e.g., BAY-2402234 and GTX-196). B) Dose-response curves for ecMRT tumoroid models 60T and 103T treated with BAY-2402234 for 120 hours in Plasmax™ medium, with or without dipyridamole (500 nM). ecMRT tumoroids treated with BAY-2402234 in KOM medium served as a reference. Data represent mean ± SD from three independent experiments, each performed with technical duplicates. Data are normalized to DMSO vehicle in KOM (100%). The grey dotted horizontal line represents a viability of 50% (IC 50 ). C-D) Dose-response curves for ecMRT tumoroid model 103T treated with BAY-2402234 (panel C ) or GTX-196 (panel D) for 120 hours in KOM supplemented with 30 μM uridine, with or without different concentrations of dipyridamole. Data represent mean ± SD from three independent drug matrix experiments. Data are normalized to DMSO vehicle (100%). The grey dotted horizontal line represents a viability of 50% (IC 50 ). E-F) ZIP synergy landscapes for BAY-2402234 + dipyridamole (E) and GTX-196 + dipyridamole (F) , corresponding to panels C–D. ZIP scores >10 indicate synergistic interactions, scores between -10 and 10 represent additive effects, and scores <10 indicate antagonism. Regions outlined in white represent the most synergistic concentration combinations. Synergy landscapes were generated using SynergyFinder software . G) Bar graph depicting the average cell viability (%) of ecMRT tumoroid models following 120-hour treatment with 5 nM GTX-196, 10 μM dipyridamole, or their combination, in the presence or absence of nucleoside supplementation. Data represent the mean ± standard deviation (SD) of n = 3 independent experiments, each performed in technical triplicates. Viability values were normalized to the DMSO vehicle control without nucleoside supplementation (set to 100%). H) Relative isotopologue distribution (fractional labeling) of CTP and UTP in two ecMRT models following 24-hour incubation with [U- 13 C 6 ]-glucose upon 48-hour treatment with DMSO vehicle or 10 μM DP, in the presence or absence of 30⍰μM uridine. Statistical testing was performed within each uridine condition. I) Isotopic labeling pattern of CTP and UTP in two ecMRT tumoroid models following 24-hour incubation with [U- 13 C 6 ]-glucose upon 48-hour treatment with DMSO vehicle, 5 nM GTX-196, 10 μM DP, or the combination thereof, in the presence or absence of 30⍰μM uridine. DMSO and GTX-196 reference data originate from the same [U- 13 C 6 ]-glucose tracing experiment as previously presented in , but are shown again to provide direct comparison across all treatment conditions. Unless specified otherwise, all conditions were statistically compared to the DMSO ctrl within each uridine condition. J) Isotopic labeling pattern of CTP and UTP in two ecMRT tumoroid models following 24-hour incubation with 30⍰μM [ 13 C 9 - 15 N 2 ]-uridine upon 48-hour treatment with DMSO vehicle, 5 nM GTX-196, 10 μM DP, or the combination thereof. DMSO and GTX-196 reference data originate from the same [ 13 C 9 - 15 N 2 ]-uridine tracing experiment as previously presented in , but are shown again to provide direct comparison across all treatment conditions. Unless specified otherwise, all conditions were statistically compared to the DMSO ctrl. (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001).
Article Snippet: Single cells were plated at a density of 2.000 cells/μL in 5 μL 75% BME droplets topped with KOM PL and Plasmax (CancerTools, 156371) (see exact formulations in ) in a pre-warmed flat-bottom 96-well plate (Greiner, 655-160).
Techniques: Synthesized, Inhibition, Concentration Assay, Generated, Software, Standard Deviation, Control, Labeling, Incubation, Isotopic Labeling, Comparison
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![A) Overview of the concentrations of nucleosides and nucleobases in Advanced DMEM/F12 (base medium for KOM) and <t>Plasmax™</t> according to the manufacturer. “NA” indicates that the respective compound is absent from the medium formulation. Shades of red represent relative concentrations of each component. B) Dose-response curves for ecMRT tumoroid model 60T treated with BAY or GTX-196 for 120 hours in standard KOM, KOM supplemented with 3 μM uridine, or Plasmax. Data represent mean ± SD from three independent experiments. Data are normalized to DMSO vehicle in KOM (100%). The grey dotted horizontal line represents a viability of 50% (IC 50 ). C) Relative abundance of different isotopologues in ADP, ATP, GDP, and GTP in two different ecMRT tumoroid models following 24-hour incubation with [U- 13 C 6 ]-glucose in the presence of 0⍰μM, 3⍰μM, or 30⍰μM uridine. All conditions were statistically compared to the 0⍰μM uridine condition. D) Total abundance and isotopic labeling pattern of CDP and UDP in two ecMRT tumoroid models following 24-hour incubation with 3⍰μM or 30⍰μM [ 13 C 9 - 15 N 2 ]-uridine. Statistical testing was performed within each uridine condition. E) Isotopic labeling pattern of CTP and UTP in two ecMRT tumoroid models following 24-hour incubation with 3⍰μM or 30⍰μM [ 13 C 9 - 15 N 2 ]-uridine upon 48-hour treatment with 5 nM GTX-196 or DMSO vehicle. Unless specified otherwise, all conditions were statistically compared to the DMSO ctrl within each uridine condition. (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001).](https://bio-rxiv-images-cdn.bioz.com/dois_ending_with_65/10__64898_slash_2026__01__25__701565/10__64898_slash_2026__01__25__701565___F6.large.jpg)
![A-B) Bar graphs depicting the average cell viability (%) of ecMRT tumoroid models following 120-hour treatment with 1 μM BAY-2402234 (BAY) (panel A ) or 1 μM GTX-196 (panel B ) in the presence of standard KOM or Plasmax™ medium. Data represent the mean ± standard deviation (SD) of n = 3 independent experiments, each performed in technical duplicates. Viability values were normalized to the DMSO vehicle control in KOM (set to 100%). C) Schematic overview illustrating the incorporation of carbon atoms from uniformly labeled [U- 13 C 6 ]-glucose (red dots) and [U- 13 C 9 ]-uridine (yellow dots) into pyrimidine nucleotide UTP via the de novo nucleotide biosynthesis and nucleotide salvage pathways. In the case of [U- 13 C 6 ]-glucose, the ribose moiety (in orange) is synthesized via the pentose phosphate pathway (PPP), resulting in UTP labeled at five carbons ([ 13 C 5 ]). Additional carbons are contributed by aspartate (ASP) to the pyrimidine ring (in purple), generating UTP isotopologues with up to [ 13 C 8 ] labeling. In contrast, [ 13 C 9 - 15 N 2 ]-uridine is taken up through the salvage pathway as an intact molecule, yielding fully labeled UTP ([ 13 C 9 - 15 N 2 ]). Partial catabolism of [ 13 C 9 - 15 N 2 ]-uridine can lead to differential labeling patterns: UTP [ 13 C 5 - 15 N 0 ] indicates salvage of the labeled ribose with replacement of the pyrimidine nucleobase via de novo synthesis, while UMP [ 13 C 4 - 15 N 2 ] reflects incorporation of a labeled uracil base with an unlabeled ribose, suggesting base salvage following uridine breakdown. D-E) Dose-response curves for ecMRT tumoroid models treated with BAY (panel D ) or GTX-196 (panel E ) for 120 hours in standard KOM, or KOM supplemented with 3 μM uridine or 30 μM uridine. Data represent mean ± SD from three independent experiments, each consisting of technical quadruplicates. Data are normalized to DMSO vehicle in KOM (100%). The grey dotted horizontal line represents a viability of 50% (IC 50 ). F) Total abundance and isotopic labeling pattern of CTP and UTP in two ecMRT tumoroid models following 24-hour incubation with [U- 13 C 6 ]-glucose in the presence of 0⍰μM, 3⍰μM, or 30⍰μM uridine upon 48-hour treatment with DMSO vehicle or 5 nM GTX-196. Unless specified otherwise, statistical comparisons were performed within each uridine condition. G) Fraction of unlabeled ([ 13 C 0 ]) CDP, CTP, UDP, and UTP in two different ecMRT tumoroid models following 24-hour incubation with [U- 13 C 6 ]-glucose in the presence of 0⍰μM, 3⍰μM, or 30⍰μM uridine. All conditions were statistically compared to the 0⍰μM uridine condition. H) Total abundance and isotopic labeling pattern of CTP and UTP in two ecMRT tumoroid models following 24-hour incubation with 3⍰μM or 30⍰μM [ 13 C 9 - 15 N 2 ]-uridine upon 48-hour treatment with DMSO vehicle or 5 nM GTX-196. Unless specified otherwise, statistical comparisons were performed within each uridine condition. I) Fraction of [ 13 C 9 - 15 N 2 ]-labelled CDP, CTP, UDP, and UTP in two different ecMRT tumoroid models following 24-hour incubation with 3⍰μM or 30⍰μM [ 13 C 9 - 15 N 2 ]-uridine. (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001).](https://bio-rxiv-images-cdn.bioz.com/dois_ending_with_65/10__64898_slash_2026__01__25__701565/10__64898_slash_2026__01__25__701565___F2.large.jpg)
![A) Schematic overview of de novo nucleotide biosynthesis and nucleotide salvage pathways. Nucleosides and nucleobases are imported into the cell via hENT 1 and 2 and subsequently phosphorylated to their corresponding nucleotide monophosphates (NMPs). In parallel, nucleotides can be synthesized de novo from metabolic precursors, resulting in the formation of NMPs. Key enzymes involved in these pathways, including uridine-cytidine kinase (UCK), DHODH, and carbamoyl-phosphate synthetase 2/ aspartate transcarbamylase/ dihydroorotase (CAD), are highlighted. Both pathways can be subjected to pharmacological inhibition by agents targeting hENT1/2 (e.g., dipyridamole) and DHODH (e.g., BAY-2402234 and GTX-196). B) Dose-response curves for ecMRT tumoroid models 60T and 103T treated with BAY-2402234 for 120 hours in Plasmax™ medium, with or without dipyridamole (500 nM). ecMRT tumoroids treated with BAY-2402234 in KOM medium served as a reference. Data represent mean ± SD from three independent experiments, each performed with technical duplicates. Data are normalized to DMSO vehicle in KOM (100%). The grey dotted horizontal line represents a viability of 50% (IC 50 ). C-D) Dose-response curves for ecMRT tumoroid model 103T treated with BAY-2402234 (panel C ) or GTX-196 (panel D) for 120 hours in KOM supplemented with 30 μM uridine, with or without different concentrations of dipyridamole. Data represent mean ± SD from three independent drug matrix experiments. Data are normalized to DMSO vehicle (100%). The grey dotted horizontal line represents a viability of 50% (IC 50 ). E-F) ZIP synergy landscapes for BAY-2402234 + dipyridamole (E) and GTX-196 + dipyridamole (F) , corresponding to panels C–D. ZIP scores >10 indicate synergistic interactions, scores between -10 and 10 represent additive effects, and scores <10 indicate antagonism. Regions outlined in white represent the most synergistic concentration combinations. Synergy landscapes were generated using SynergyFinder software . G) Bar graph depicting the average cell viability (%) of ecMRT tumoroid models following 120-hour treatment with 5 nM GTX-196, 10 μM dipyridamole, or their combination, in the presence or absence of nucleoside supplementation. Data represent the mean ± standard deviation (SD) of n = 3 independent experiments, each performed in technical triplicates. Viability values were normalized to the DMSO vehicle control without nucleoside supplementation (set to 100%). H) Relative isotopologue distribution (fractional labeling) of CTP and UTP in two ecMRT models following 24-hour incubation with [U- 13 C 6 ]-glucose upon 48-hour treatment with DMSO vehicle or 10 μM DP, in the presence or absence of 30⍰μM uridine. Statistical testing was performed within each uridine condition. I) Isotopic labeling pattern of CTP and UTP in two ecMRT tumoroid models following 24-hour incubation with [U- 13 C 6 ]-glucose upon 48-hour treatment with DMSO vehicle, 5 nM GTX-196, 10 μM DP, or the combination thereof, in the presence or absence of 30⍰μM uridine. DMSO and GTX-196 reference data originate from the same [U- 13 C 6 ]-glucose tracing experiment as previously presented in , but are shown again to provide direct comparison across all treatment conditions. Unless specified otherwise, all conditions were statistically compared to the DMSO ctrl within each uridine condition. J) Isotopic labeling pattern of CTP and UTP in two ecMRT tumoroid models following 24-hour incubation with 30⍰μM [ 13 C 9 - 15 N 2 ]-uridine upon 48-hour treatment with DMSO vehicle, 5 nM GTX-196, 10 μM DP, or the combination thereof. DMSO and GTX-196 reference data originate from the same [ 13 C 9 - 15 N 2 ]-uridine tracing experiment as previously presented in , but are shown again to provide direct comparison across all treatment conditions. Unless specified otherwise, all conditions were statistically compared to the DMSO ctrl. (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001).](https://bio-rxiv-images-cdn.bioz.com/dois_ending_with_65/10__64898_slash_2026__01__25__701565/10__64898_slash_2026__01__25__701565___F4.large.jpg)
![A) Bar graph depicting the average viability (%) of ecMRT tumoroids cultured in KOM supplemented with 30 μM uridine following 120-hour treatment with various concentrations of dipyridamole. Data represent the mean ± SD of n = 3 independent experiments, each consisting of technical triplicates. Viability values were normalized to the DMSO vehicle control (100%). B) Dose-response curves for ecMRT tumoroid models 60T and 78T treated with GTX-196 for 120 hours in Plasmax™ medium, with or without the hENT1/2 inhibitor dipyridamole (500 nM). ecMRT tumoroids treated with GTX-196 in KOM medium served as a reference. Data represent mean ± SD from three independent experiments, each performed with technical duplicates. Data are normalized to DMSO vehicle in KOM (100%). The grey dotted horizontal line represents a viability of 50% (IC 50 ). C-D) Dose-response curves for ecMRT tumoroid models 60T treated with BAY-2402234 (panel C ) and 103T treated with GTX-196 (panel D) for 120 hours in KOM supplemented with 3 μM uridine, with or without different concentrations of hENT1/2 inhibitor dipyridamole. Data represent mean ± SD from three independent drug matrix experiments. Data are normalized to DMSO vehicle (100%). The grey dotted horizontal line represents a viability of 50% (IC 50 ). E-F) Two-dimensional (2D) synergy landscapes visualized as contour plots, displaying ZIP (Zero Interaction Potency) synergy scores across concentration matrices for BAY-2402234 combined with dipyridamole (panel E ) and GTX-196 combined with dipyridamole (panel F ) (corresponding to figure panels C and D ). ZIP scores >10 indicate synergistic interactions, scores between -10 and 10 represent additive effects, and scores <–10 indicate antagonism. Regions outlined in white represent the most synergistic concentration combinations. Synergy landscapes were generated using SynergyFinder software . G , I) Dose-response curves for ecMRT tumoroid model 103T treated with GTX-196 for 120 hours in KOM supplemented with 30 μM uridine, with or without different concentrations of hENT inhibitors draflazine (panel G ) or nitrobenzylthioinosine (NBMPR; panel I). Data represent mean ± SD from three independent drug matrix experiments. Data are normalized to DMSO vehicle (100%). The grey dotted horizontal line represents a viability of 50% (IC 50 ). H , J ) Two-dimensional (2D) synergy landscapes visualized as contour plots, displaying ZIP (Zero Interaction Potency) synergy scores across concentration matrices for GTX-196 combined with draflazine (panel H ) and GTX-196 combined with NBMPR (panel J ) (corresponding to figure panels G and I ). ZIP scores >10 indicate synergistic interactions, scores between -10 and 10 represent additive effects, and scores <–10 indicate antagonism. Regions outlined in white represent the most synergistic concentration combinations. Synergy landscapes were generated using SynergyFinder software . K) Isotopic labeling pattern of extracellular uridine levels measured in the medium of BME (no cells) and two different ecMRT tumoroids following 24-hour incubation with 3⍰μM or 30⍰μM [ 13 C 9 - 15 N 2 ]-uridine upon 48-hour treatment with DMSO vehicle, 5 nM GTX-196, 10 μM DP, or the combination thereof. All conditions were statistically compared to the BME (no cells) reference group. (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001).](https://bio-rxiv-images-cdn.bioz.com/dois_ending_with_65/10__64898_slash_2026__01__25__701565/10__64898_slash_2026__01__25__701565___F8.large.jpg)