TP53-mutated Acute Myeloid Leukemia through a single-cell lens Free

Introduction

TP53 variants occur more commonly in older patients with therapy-related clonal hematopoiesis, myelodysplastic syndromes and acute myeloid leukemia (AML). In AML, TP53 variants are more commonly multi-hit and associated with complex copy number changes. More recently, single-cell methods have identified monoallelic TP53 mutations within mature NK-/T-cells (Li, medRvix 2025). In the current study, we sought to characterize the zygosity, clonal dynamics and lineage associations of TP53 mutated (TP53m) AML using a multiomic single-cell DNA (scDNA) and protein sequencing approach.

Methods

Seventeen bone marrow mononuclear (BMM) samples from 9 patients with TP53m AML, including 6 patients with serial samples were analyzed by bulk next-generation sequencing and single-cell DNA and protein sequencing (Tapestri v3). For the latter, BMM cells were stained with a panel of antibody conjugated oligonucleotides (AOC) (TotalSeq^TM-D 24-AOC custom or scMRD AOC panel), cells encapsulated into single cell droplets, barcoded and sequenced using a 30-45 gene myeloid panel. In 4 cases, BMM cells were CD34/117+ enriched and flow through spiked in to enrich for progenitor cells. Analysis was performed using the Mosaic python package.

Key findings

Multi-hit TP53 abnormalities were predominantly found in AML blasts By single cell analysis of TP53m AML, all 9 cases had multi-hit TP53m abnormalities identified in the CD34+ and/or CD117+ blast compartment. Multi-hit TP53m abnormalities were either loss of heterozygosity (LOH, n=8) or homozygous (same TP53m at each locus with no apparent LOH with conventional assessment; n=1) in nature. Intriguingly, multi-hit TP53 abnormalities were not identified in mature hematopoietic populations (T-, B-, NK-cells). In contrast, in each of the 9 cases, a putative minor ancestral TP53m heterozygous (TP53m HET) population representing 0.8-4% of cells wasfound in theblast compartment, accompanied by a larger and dominant multi-hit TP53m population. TP53m HETcells were also found in mature myeloid and lymphoid cells (T- and NK-cells). In one case with diagnostic, remission and relapse samples available, a dominant TP53m multi-hit clone with deletion 17p (TP53 p.Trp146*) was present in AML blasts at diagnosis. In remission, there was regression of this TP53m multi-hit (LOH) clone, with persistence of a TP53m HET (TP53 p.Trp146*) population in progenitor, myeloid and T-cells, along with 5 TP53m HET clones (p.Val272Ala, VAF 0.87%; p.Arg175His, 0.3%; p.Cys238Tyr, 0.5%; Ser127Phe, 1.5%; Pro177_Cys172del, 1.8%) arising in parallel within the mature lymphoid compartment. At relapse, the TP53m p.Trp146* multi-hit clone re-emerged in concert with the resurgence of AML blasts.

Complex copy number changes were restricted to AML blasts with multi-hit TP53m abnormalities In 5 cases where copy number changes and phenotype could be assessed with single-cell resolution, the complex copy number changes were exclusively found in TP53m multi-hit CD34+/117+ blasts, but not WT progenitor cells.

Venetoclax (VEN)-based therapy led to expansion of TP53m multi-hit clones

In 5 patients who received VEN therapy with serial sampling, single-cell studies revealed selective expansion of the dominant TP53m multi-hit clone in 3/5 cases, which included 2 cases with a stable interval bone marrow blast level. In the same cases where a minor TP53m HET clone was present, VEN therapy did not result in an appreciable change in clone size.

Mutant IDH1 could sensitize multi-hit TP53 blasts to venetoclax-based therapy In 2 cases with co-present TP53m multi-hit abnormalities and IDH1 mutation within AML blasts, the mutant IDH1 was not found in association with either TP53m HET or TP53 WT cells. The TP53 multi-hit/IDH1 mutant population was cleared by VEN-based therapy in 1 of the 2 cases.

Conclusions

Using single-cell multiomic technology, we provide insights into the clonal structure and dynamic response of TP53m AML at the single-cell level. We show a striking association between TP53 multi-hit abnormalities and complex copy number changes that was restricted to AML blasts, whereas TP53m heterozygous clones were typically found in mature clonal hematopoietic cells or as a minor ancestral population within the AML blast compartment. In the presence of VEN-based therapy, TP53 multi-hit abnormalities promoted resistance, whereas co-occurring sub-clonal IDH1 variants could function as a sensitizer to VEN.