Dependent lineage regulatory programs in TP53-mutated acute myeloid leukemia revealed through deep single-cell multi-omic profiling of patient samples

TP53 -mutated ( TP53 m) Acute myeloid leukemia (AML) represents a clinically intractable and biologically distinct disease. To study the molecular basis of this disease through hemopoiesis, we integrated single-cell RNA-seq (10x and long-read Oxford Nanopore sequencing) with open chromatin profiles (scATAC-seq), whole-genome (WGS) to capture structural variants and copy number alterations, and deep panel DNA sequencing from bone marrow samples taken from a cohort of 49 TP53 m AML patients, 7 TP53 wild type (WT) AML patients, and 4 healthy donors. Phased genotyping plus WGS revealed biallelic loss to be associated with intrachromosomal breakage, whereas monoallelic loss favored numerical chromosomal alterations. TP53 m clones pervaded stem/progenitors and myeloerythroid lineages but were proportionally depleted in mature lymphocytes. While co-occurring AML driver mutations influence the lineage biases of TP53m clones, we observed expansion of HSC/MPP, LMPP, and late erythroid compartments. In HSC/MPP dominant disease, TP53m cells displayed suppression of translational and mitochondrial-respiratory programs, with enrichment of a “p53-LSC” signature linked to chronic inflammatory stress. This suggests adaptation through metabolic quiescence, conferring persistence under inflammatory pressure. In LMPP-dominant expansion, we observed enriched MYC/E2F targets. Transcriptomic neighborhood analysis with pseudotime showed the erythroid compartment to be heterogeneous, with less mature basophilic cells enriched for TP53 m clones, whereas more mature orthochromatic cells were enriched for TP53 wt clones, nominating this transition as a tipping point for differentiation delay/arrest. Consistently, in erythroid dominant patients, late-stage erythroid cells demonstrated impaired GATA-1 and KLF1 (erythroid TFs) activities, and TP53 m clones showed heightened GATA2, SPI1, and CEBPD regulon activity. TP53 m versus TP53 wt clones in erythroid differentiation showed heightened transcriptional activity of the EIF2AK1(HRI)→eIF2alpha mediated integrated stress response (ISR). Differential expression analysis within the aforementioned erythroid compartment showed ISR activation in TP53m clones; SESN2 , DDIT4 , and DDIT3 /CHOP significantly up, with ATF3 , and XBP1 , up-trending, together with increased GATA2 . Replication/mitochondrial-handling components ( GINS2 , RPA3 , ABCB8 ) were significantly downregulated. These data together support a model in which deficient GATA-1/KLF1 function underpins the intra erythroid arrest of TP53 m clones possibly due to activated HRI-ISR circuitry. These detailed molecular differentiation stage specific analyses of TP53 m AML provide mechanistic insights and a platform for functional and synthetic lethal studies to specifically target TP53 m leukemic cells.