CTP and dCTP are elevated, and DNMT1 protein is preserved in HMA-resistant AML cells. (A) DNMT1 depletion observed in parental AML cells treated with HMA was abrogated in HMA-resistant cells. HMA-resistant p53-null AML cells (THP1) emerged from continuous culture in decitabine/5-azacytidine 0.5/5μM and 1.5/5μM. Western blot using 50 μg total protein from each sample. (B) 5-Azacytidine and decitabine, analogs of cytidine and deoxycytidine, respectively, must compete with their endogenous pyrimidine nucleotide counterparts (CTP, dCTP) in order to deplete DNMT1. UCK2 and DCK are the pyrimidine metabolism enzymes that rate control conversion of 5-azacytidine and decitabine into DNMT1-depleting nucleotides. (C) CTP and dCTP were elevated in HMA-resistant AML cells. High-resolution LC-MS (HR-LCMS) analyses of extracts obtained from equal numbers of cells, harvested independently in octuplicate. Values are normalized to taurine levels in the same cells (taurine internal control). Data represent as means ± standard deviation (SD); P values are from unpaired, 2-sided t tests. (D) Key de novo pyrimidine synthesis enzymes. (E) The initial and rate-limiting enzyme in this pathway, CAD, was upregulated in HMA-resistant vs parental AML cells. Western blot (left). Replicates from independent cell harvests. Graph shows CAD intensity normalized to actin in the same sample using ImageJ software, fold change HMA-resistant vs parental THP1. CAD fluorescence intensity measured by flow cytometry in fixed/permeabilized cells and HMA-resistant vs parental cells (∼8900 cells each) (right). P values for mean fluorescence intensity between HMA-resistant vs parental cells were calculated using an unpaired 2-sided t test. (F) CAD expression in TP53-mutated vs wild-type (WT) TP53 AML cells. The Cancer Genome Atlas (TCGA) RNA-sequencing gene-level log₂(x+1) transformed RNA-Seq by expectation-maximization (RSEM) normalized counts, primary AML cells containing WT TP53 (no TP53 mutations or deletions by genomic identification of significant targets in cancer (GISTIC) threshold analyses of copy number; n = 129) or containing mutated-TP53 and complex (≥3) cytogenetic abnormalities (n = 16). P values from unpaired 2-sided t test. (G) Tp53 knockout from GMP (lin^–cKit⁺Sca^–1^–CD16/32⁺CD34⁺) decreases Rrm2b and activates other de novo pyrimidine synthesis enzymes, including Cad, Umps, and Rrm1. GSE285355,³² gene expression counts normalized via log transformation and size-factor adjustment.³² GMP isolated from Vav-cre Tp53fl/fl mice and age-matched WT mice.³²P values, unpaired 2-sided t test. (H) TP53-mutated AML cells display a similar pyrimidine metabolism gene expression pattern. BEAT AML RNA-sequencing, gene-level counts processed by conditional quantile normalization,³³ primary AML cells containing WT TP53 are subcategorized into cases with normal cytogenetics (n = 258) or ≥1 cytogenetic abnormality (n = 270), compared with primary AML cells containing mutated-TP53 and ≥3 cytogenetic abnormalities (n = 56); P values from unpaired 2-sided t test. (I) TP53-mutated pan-cancer cells display a similar pyrimidine metabolism gene expression pattern. TCGA Pan-Cancer, TP53-mutated (n = 3320) vs no TP53 mutations (n = 5760). RNA-sequencing and analyses as per panel F. Aza-C, 5-azacytidine; Aza-dC, decitabine; DEC, decitabine; IgG, immunoglobulin G; max, maximum; min, minimum; mut, mutated; Res, resistant; Veh, vehicle.