Journal: bioRxiv

Article Title: Nuclear adenosine metabolism defines a metabolic vulnerability unmasked by TP53 loss

doi: 10.64898/2026.07.06.736733

Figure Lengend Snippet: A) Kaplan–Meier curves showing patient overall survival. Data were retrieved from the TCGA-PANCAN dataset, with patients stratified based on KRAS and TP53 status. B) Schematic representation of the drop-out screen. C) Differentially abundant sgRNAs compared to day 0. Each dot represents the mean of all sgRNAs/gene. ß-scores (essentiality scores) are negative when a gene is more essential. In yellow, all the genes scored more essential in KP A549 cells than K A549 cells. In cyan, top scored purine synthesis genes in KP A549 ; in purple, pyrimidine metabolism-related genes. D) GO terms associated with significantly depleted sgRNAs in KP A549 vs K A549 lung cancer cells. E) KP A549 top-scored purine biosynthesis genes with their corresponding ß-scores. F) Differential essentiality scores from K A549 vs KP A549 . In cyan de novo purine synthesis enzymes, in purple de novo pyrimidine synthesis enzymes. G) Project Achilles cancer cell lines (CCLE dataset) segregated by TP53 activity (Progeny). In cyan top-scored purine synthesis genes. P-value was calculated by two-sided, unpaired Student’s t-test performed for each gene individually and corrected for multiple comparisons using Benjamini-Hochberg (FDR) adjustment. H) Volcano plot showing differentially expressed genes between TP53-mutant and TP53-wild-type groups based on TCGA transcriptomics dataset (only stage III KRAS mutated tumours). Genes were considered significant based on a Benjamini-Hochberg adjusted p-value (FDR < 0.05) and log fold-change thresholds. Significantly upregulated purine biosynthesis genes in patients harbouring concurrent mutations in TP53 and KRAS are highlighted.

Article Snippet: For combination therapy experiments, A549 cells were treated with increasing doses of 6MP in combination with increasing doses of Etoposide (MedChem Express, #HY-13629) or Paclitaxel (MedChem Express, #HY-B0015).

Techniques: Activity Assay, Mutagenesis, Transcriptomics

Journal: bioRxiv

Article Title: Nuclear adenosine metabolism defines a metabolic vulnerability unmasked by TP53 loss

doi: 10.64898/2026.07.06.736733

Figure Lengend Snippet: A) Schematic representation of the treatment administration regime. Relative growth of B) K A549 and KP A549 , C) K 3KT and KP KT cells treated with the indicated concentrations of 6MP or DMSO, and D) K A549 and KP A549 cells treated with DMSO or 6MP and supplemented with the indicated metabolites for 72 hours, following NLS-GFP signal analysis of nuclei count. E) (left) Relative spheroid area growth of K A549 and KP A549 treated with DMSO or 6MP for 7 days with the indicated concentrations, in presence or absence of 100µM of adenosine. (right) Representative images of spheroids, scale bar 200 μm. Data are normalised to the DMSO-treated condition. Data represents N=3 independent experiments. Mean ± SD, P-values ****P ≤ 0.00005; ***P ≤ 0.0005; **P ≤ 0.005; *P ≤ 0.05, pairwise t-test with Bonferroni adjusted.

Article Snippet: For combination therapy experiments, A549 cells were treated with increasing doses of 6MP in combination with increasing doses of Etoposide (MedChem Express, #HY-13629) or Paclitaxel (MedChem Express, #HY-B0015).

Techniques:

Journal: bioRxiv

Article Title: Nuclear adenosine metabolism defines a metabolic vulnerability unmasked by TP53 loss

doi: 10.64898/2026.07.06.736733

Figure Lengend Snippet: K A549 and KP A549 treated with DMSO or 15 µM 6MP for 72 hours in presence or absence of 100 µM Adenosine. A) (left) Quantification of 53BP1 foci, (right) representative images. B) (left) Quantification of γH2AX foci, (right) representative images. N = 1000-4000 cells per condition. Mean ± SD, Bonferroni adjusted one-way ANOVA, ****P ≤ 0.00005. 53BP1 and γH2AX are in ICA_gradient and DAPI marks nuclei in blue. Scale bar = 10 µm. C) (left) Quantification of H3K9me3 nuclear intensity in K A549 and KP A549 , (right) representative images, D) (left) Quantification of H3K27me3 nuclear intensity in K A549 and KP A549 , (right) representative images. N = 1000-4000 cells per condition. Data represents N=2 independent experiments. Mean ± SD, Bonferroni adjusted one-way ANOVA, ****P ≤ 0.00005. H3K9me3 and H3K27me3 are in FIRE_gradient, DAPI marks nuclei in blue; scale bar = 10 µm.

Article Snippet: For combination therapy experiments, A549 cells were treated with increasing doses of 6MP in combination with increasing doses of Etoposide (MedChem Express, #HY-13629) or Paclitaxel (MedChem Express, #HY-B0015).

Techniques:

Journal: bioRxiv

Article Title: Nuclear adenosine metabolism defines a metabolic vulnerability unmasked by TP53 loss

doi: 10.64898/2026.07.06.736733

Figure Lengend Snippet: A,B) ATP-sensor quantification in K A549 and KP A549 , normalised to total expression of the vector for both cytoplasmic ATP (A) and nuclear ATP (B). C) Normalised relative abundance of SAM and SAH in K 3KT and KP 3KT cells with DMSO or 5 µM 6MP. D) Growth of K A549 and KP A549 treated with the indicated concentrations of NepA for 72 hours. Data is represented as mean ± SD, P-values ****P ≤ 0.00005; ***P ≤ 0.0005; **P ≤ 0.005; *P ≤ 0.05, pairwise t-test with Bonferroni adjusted. E) Scheme of the enzymes involved in SAM associated with adenosine and ATP metabolism, highlighting their relative chromatin abundance (pink gradient). F) Quantification of γH2AX foci of KP A549 cells treated with 10 µM NepA or DMSO and combined with or without γ-irradiation at the indicated timepoints. Two-way ANOVA with Holm-adjusted pairwise comparisons, ****P ≤ 0.00005. G) Quantification of high-intensity regions of H3K27me3 of KP A549 cells treated with 10 µM NepA or DMSO and combined with or without γ-irradiation at the indicated timepoints. Two-way ANOVA with Holm-adjusted pairwise comparisons, ****P ≤ 0.00005. H) Representative images of γH2AX in ICA_gradient and H3K27me3 in FIRE_gradient. Scale bar = 10 µm. I) Whole cell and J) nuclear fractions ATP measurements of KP A549 following DMSO or 10µM NepA treatment with or without irradiation. Grey lines show the replicates. Linear modeling with Batch as a covariate, followed by Holm-adjusted pairwise comparisons, **P ≤ 0.005; *P ≤ 0.05.

Article Snippet: For combination therapy experiments, A549 cells were treated with increasing doses of 6MP in combination with increasing doses of Etoposide (MedChem Express, #HY-13629) or Paclitaxel (MedChem Express, #HY-B0015).

Techniques: Expressing, Plasmid Preparation, Irradiation

Journal: bioRxiv

Article Title: Nuclear adenosine metabolism defines a metabolic vulnerability unmasked by TP53 loss

doi: 10.64898/2026.07.06.736733

Figure Lengend Snippet: Co-treatment matrix for K A549 and KP A549 treated with 6MP in combination with A) paclitaxel and B) etoposide at the indicated concentrations. Data are normalised to the DMSO-treated condition, following NLS-GFP signal analysis of nuclei count. C) Batch corrected relative cell growth of K A549 and KP A549 cells transduced with shPFAS, normalised to scrambled (shControl) and analysed by NLS-GFP signal of nuclei count. D) (Top) Quantification of number of K A549 and KP A549 spheroid formation following transduction with shControl or PFAS shRNAs. (Bottom) representative images. Scale bar = 100µm. N=3, Bonferroni adjusted Wilcoxon pairwise comparison. ***P ≤ 0.0005, ****P ≤ 0.00005. In vivo subcutaneous tumour growth into NSG mice measured over 42 days. E) Tumour volume relative to volume after one week of injection for indicated genotypes. Mean ± SEM. ****P ≤ 0.00005; p-values were calculated with Linear Mixed Effect Model with POST-HOC Tukey HSD correction. F) Tumour weight at ethical endpoint. N = 10-12 tumours/genotype. Bonferroni adjusted Wilcoxon pairwise comparison. ***P ≤ 0.0005.

Article Snippet: For combination therapy experiments, A549 cells were treated with increasing doses of 6MP in combination with increasing doses of Etoposide (MedChem Express, #HY-13629) or Paclitaxel (MedChem Express, #HY-B0015).

Techniques: Transduction, Comparison, In Vivo, Injection