Altered dNTP pools accelerate tumor formation in mice

Abstract

Alterations in deoxyribonucleoside triphosphate (dNTP) pools have been linked to increased mutation rates and genome instability in unicellular organisms and cell cultures. However, the role of dNTP pool changes in tumor development in mammals remains unclear. In this study, we present a mouse model with a point mutation at the allosteric specificity site of ribonucleotide reductase, RRM1-Y285A. This mutation reduced ribonucleotide reductase activity, impairing the synthesis of deoxyadenosine triphosphate (dATP) and deoxyguanosine triphosphate (dGTP). Heterozygous Rrm1+/Y285A mice exhibited distinct alterations in dNTP pools across various organs, shorter lifespans and earlier tumor onset compared with wild-type controls. Mutational spectrum analysis of tumors revealed two distinct signatures, one resembling a signature extracted from a human cancer harboring a mutation of the same amino acid residue in ribonucleotide reductase, RRM1Y285C. Our findings suggest that mutations in enzymes involved in dNTP metabolism can serve as drivers of cancer development.

Graphical Abstract

Figure 1.

Activity and subunit composition of hRRM1 and hRRM1-Y285A in the presence of different allosteric effectors. (A) Relative CDP-reducing activity of hRRM1 and hRRM1-Y285A proteins in the presence of hRRM2 with 2 mM ATP, with or without 20 μM dATP, 40 μM dTTP or 40 μM dGTP. Activity of hRRM1 in the presence of ATP is normalized to 1. (B–E) Activity of hRRM1, hRRM1-Y285A and a 1:1 mixture of hRRM1/hRRM1-Y285A protein mixture in the presence of hRRM2 with four substrates (200 μM each of CDP, UDP, GDP and ADP) and four allosteric effectors at physiological concentrations (2 mM ATP, 30 μM dTTP, 14 μM dATP and 6 μM dGTP) over a 30-min time course. (F) Mass photometry analysis of the subunit composition of hRRM1 and hRRM1-Y285A proteins in the presence or absence of positive allosteric effectors (2 mM ATP, 100 μM dTTP and 100 μM dGTP).

Figure 2.

dNTP pools in various organs of heterozygous Rrm1^+/Y285A mice. (A) E13.5 embryos, n = 6 for WT, n = 12 for Rrm1^+/Y285A. (B) Thymus, n = 6. (C) Heart, n = 4. (D) Liver, n = 4. (E) Kidney, n = 9 for WT, n = 13 for Rrm1^+/Y285A. (F) Spleen, n = 10. dNTPs were normalized to total NTPs. Data are presented as mean ± standard error of the mean. Statistical significance was determined using Mann–Whitney U-tests, with *** indicating P < 0.001, ** indicating P < 0.01, * indicating P < 0.05 and ‘ns’ indicating non-significant.

Figure 3.

Altered dNTP pools lead to a shortened lifespan and earlier onset of cancers. (A) Kaplan–Meier survival curve spanning 150 weeks, compiled from 27 WT and 26 Rrm1^+/Y285A mice. Statistical analysis was performed using the Mantel–Cox test. (B) Diagram depicting observed abnormalities in mice during the lifespan experiment plotted against the ages at which the abnormalities were detected. Each color represents an individual mouse. (C) Representative hematoxylin and eosin (HE) stained sections of indicated tumors. (D) Cumulative numbers of mice with lymphomas and glandular tumors (adenomas and adenocarcinomas) compiled from 12 Rrm1^+/Y285A and 13 age-matched WT mice euthanized during the lifespan study. (E) The number of 1-year old mice with lymphoma at one or multiple locations, compiled from 8 WT and 9 Rrm1^+/Y285A mice. (F) Representative HE-stained sections of the intestine, lung, liver and spleen of mice in (E). Arrows indicate lymphocytic infiltrates. Scale bar: 200 μm. Note the distinct borders between the white and red pulps in the WT spleen section, which are diminished in the Rrm1^+/Y285A spleen section.

Figure 4.

Mutation analysis of mouse and human tumors with mutations in RRM1 at position 285. (A) Mutation burden comparison between WT and Rrm1^+/Y285A mice. (B) Unsupervised hierarchical clustering of mutation signatures from WT and Rrm1^+/Y285A mouse tumors. SBSA and SBSB: single base substitutions A and B. (C) Mutational signatures observed in WT and Rrm1+/Y285A mouse tumors, alongside a mutation spectrum from a human patient (TCGA-DD-AAEI, The Cancer Genome Atlas) with an RRM1-Y285C mutation.