GMP-like and MLP-like Subpopulations of Hematopoietic Stem and Progenitor Cells Harboring Mutated EZH2 and TP53 at Diagnosis Promote Acute Myeloid Leukemia Relapse: Data of Combined Molecular, Functional, and Genomic Single-Stem-Cell Analyses.

Acute myeloid leukemia (AML) is associated with unfavorable patient outcomes primarily related to disease relapse. Since specific types of leukemic hematopoietic stem and progenitor cells (HSPCs) are suggested to contribute to AML propagation, this study aimed to identify and explore relapse-initiating HSPC subpopulations present at diagnosis, using single-cell analysis (SCA). We developed unique high-resolution techniques capable of tracking single-HSPC-derived subclones during AML evolution. Each subclone was evaluated for chemo-resistance, in vivo leukemogenic potential, mutational profile, and the cell of origin. In BM samples of 15 AML patients, GMP-like and MLP-like HSPC subpopulations were identified as prevalent at relapse, exhibiting chemo-resistance to commonly used chemotherapy agents cytosine arabinoside (Ara-C) and daunorubicin. Reconstruction of phylogenetic lineage trees combined with genetic analysis of single HSPCs and single-HSPC-derived subclones demonstrated two distinct clusters, originating from MLP-like or GMP-like subpopulations, observed both at diagnosis and relapse. These subpopulations induced leukemia development ex vivo and in vivo. Genetic SCA showed that these relapse-related subpopulations harbored mutated EZH2 and TP53, detected already at diagnosis. This study, using combined molecular, functional, and genomic analyses at the level of single cells, identified patient-specific chemo-resistant HSPC subpopulations at the time of diagnosis, promoting AML relapse.