Mutant TP53 allelic states in human hematopoietic stem and progenitor cells

In human hematopoietic stem and progenitor cells (HSPCs), TP53 mutations characterize clonal hematopoiesis of indeterminate potential (CHIP) and are recognized as a high-risk factor for the development of non-neoplastic and neoplastic disorders, particularly myelodysplastic syndromes (MDS) and acute myeloid leukemia [ 1, 2 ]. During leukemic transformation, the remaining TP53 wild-type (WT) allele is frequently affected, most commonly through loss-of-heterozygosity (LOH), copy-neutral LOH, or a second concurrent mutation. The TP53 allelic state—reflecting whether one or both alleles are affected—remains a subject of debate regarding its prognostic relevance in patients with MDS [ 3 ]. Recently, the biological consequences of monoallelic versus biallelic aberrations were investigated using a Trp53 mouse model [ 4 ]. Here, we address this question in human HSPCs by introducing defined TP53 aberrations via CRISPR/Cas9-mediated genome editing. Material and methods are described in detail in the Supplementary information. Enriched, umbilical cord blood (UCB) derived CD34 + HSPCs were targeted at exon 2 of the TP53 gene using a CRISPR/Cas9 and recombinant adeno-associated virus serotype 6 mediated knock-in strategy (Fig. 1A and Supplementary Figs. S1A, B and S2 ) [ 5 ]. By targeting exon 2, we were able to generate WT and aberrant TP53 alleles within the same genomic context while maintaining the endogenous regulatory control. Integration of the donor template at exon 2 disrupts the endogenous TP53 gene, ensuring that only the donor-encoded cDNA sequences are expressed from the endogenous TP53 promoter. Mutant TP53 cDNA—carrying either the variant of interest or a stop codon—or WT cDNA, each linked to a fluorescent reporter (FR) expression cassette, was introduced via homology-directed repair to generate the following monoallelic and biallelic genotypes: TP53 R175H/WT, TP53 R273H/WT, TP53 KO/WT and TP53 R175H/KO, TP53 R273H/KO, TP53 KO/KO. The p.R175H and p.R273H aberrations are TP53 hot-spot mutations with reported gain-of-function and dominant-negative/loss-of-function properties, respectively (Supplementary Table S1 ) [ 6, 7, 8 ]. FR inserted at the A AVS1 safe-harbor locus served as TP53 WT/WT control. Stable, biallelic integration of TP53 cDNAs was achieved in 0.5–10.0% of HSPCs (mean, 2.23 ± 1.76 SD) and enriched by flow cytometry (Supplementary Fig. 1C ). Site-specific integration was confirmed by PCR and each engineered TP53 genotype was validated by Sanger sequencing of both genomic DNA and complementary DNA (Fig. 1B and Supplementary Fig. 3A, B ), p53 protein expression was confirmed by immunocytochemistry (Supplementary Fig. 3C ). Functionally, the introduced TP53 aberrations resulted in reduced or absent expression of the downstream targets p21 and MDM2 (Supplementary Fig. S4 ). Fig. 1: The TP53 allelic state differentially modulates human HSPC growth and self-renewal. Full size image A CRISPR-mediated knock-in of wild-type (WT) or mutant (MUT) TP53 cDNA into cord blood-derived CD34⁺ HSPCs. B Representative genomic DNA chromatograms displaying monoallelic (R175H/WT, R273H/WT, KO/WT) and biallelic (R175H/KO, R273H/KO, KO/KO) TP53 genotypes. C CFUs from different genotypes 14 days after seeding into methylcellulose. D Morphological classification of colonies as BFU-E (red), monocyte (M, gray) or granulocyte/macrophage (GM, white) from primary plating after 14 days. E CFUs from different genotypes upon replating. C – E Data are presented as mean ± s.e.m. of two pooled samples plated in triplicates. Statistical comparisons to AAVS1 control by one-way ( C ) or two-way ( D ) ANOVA. ns not significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. HSPCs hematopoietic stem and progenitor cells, CFUs colony-forming units, BFU-E burst-forming unit erythroid. We initially investigated whether distinct TP53 mutant allelic states affect colony growth and self-renewal using methylcellulose-based colony-forming unit (CFU) assays. Reporter-positive, genome-edited HSPCs with biallelic TP53 aberrations derived from pooled UCB samples generated significantly more colonies than cells with monoallelic aberrations and AAVS1 controls (Fig. 1C, D ). Both CFU-GM and BFU-E colony numbers increased, with erythroid colonies exhibiting a particularly pronounced rise. PCR and Sanger sequencing of colonies showed complete retention of the initially introduced mutations in HSPCs (Supplementary Fig. S5 ). Serial replating CFU assays demonstrated that HSPCs with WT TP53 or monoallelic aberrations lost their colony-forming capacity after three passages, whereas cells with biallelic aberrations displayed elevated clonogenic potential (Fig. 1E ). Cell cycle analysis revealed a non-significant increase in the proportion of cells with biallelic TP53 aberrations in the S-phase of the cell cycle (Supplementary Fig. S6 ). TP53 aberrations are closely linked to structural chromosomal abnormalities (CAs). Using single-cell template strand sequencing of genome-edited HSPCs derived from single UCB donors, we analyzed CAs induced by TP53 aberrations [ 9 ]. Cells were treated with either doxorubicin, a potent cellular stressor, or DMSO as control (Supplementary Figs. S7, S8 ). Upon doxorubicin treatment, cells with TP53 monoallelic aberrations already exhibited a significantly higher frequency of CAs compared to AAVS1 -targeted controls. In addition, a spontaneous and significant increase in CAs was observed in HSPCs carrying the TP53 R175H mutation (Fig. 2A, Supplementary Fig. S9 ). In HSPCs harboring the TP53 R175H and R273H mutations, chromosomes 3, 5, 7 and 17 were among the most frequently affected. Aberrations of chromosomes 5 and 7 are also common in patients with TP53 mutated myeloid neoplasms. Consistent with the clinical situation, we observed terminal losses (64% of all CAs affecting chromosomes 5 and 7), terminal gains (14%), arm gains (7%), whole chromosome gains (7%) and complex aberrations (8%) in our e