SRP341204 PRJNA770973 GSE185834 Dormant leukemia stem cells reinforced by chemotherapy drive early treatment failure in acute myeloid leukemia patients [scRNA_PDX] Acute myeloid leukemia is characterized by a minor fraction of primitive leukemia stem cells (LSCs) that sustain disease propagation and may be at the origin of late relapse. Yet, LSC contribution to early therapy resistance and AML regeneration remains controversial. We prospectively identified LSCs in NPM1-mutated AML patients and xenografts by means of a microRNA-126 reporter and single cell RNA sequencing, precisely discriminating LSCs from regenerating hematopoiesis, and assessed their longitudinal response to chemotherapy. We here show that chemotherapy resulted in distinct outcomes within AML subpopulations: while the bulk leukemia proliferated and differentiated with expression of oxidative-phosphorylation signatures, persisting miR-126high LSCs enforced protective stemness and dormancy features, along with a generalized inflammatory and senescence-associated response. miR-126high LSCs were enriched at diagnosis in patients with chemotherapy-refractory AML. We derived a novel miR-126high LSC transcriptional signature, which robustly stratified patients for overall survival in large AML cohorts, shining the spotlight on LSCs as determinants of early therapy resistance. Overall design: We undertook an extensive transcriptional characterization of diagnosis and matched early after treatment acute myeloid leukemia blasts through bulk RNA sequencing of xenograft LSC enriched (GFPlow/miR-126high) and LSC depleted (GFPhigh/miR-126low) blast subpopulations coupled with unbiased single cell RNA sequencing of xenograft and patient samples. Xenograft AML (n=4) from control or treated mice (2 replicates each encompassing 5-7 mice each stained with surface Hashtag antibodies - biolegend TotalSeq B chemistry -) were loaded on 10x genomics 3' Gene expression chips and libraries produced with the corresponding kit following manufacturer's instructions. Representative xenografts were sorted for the bottom 20% GFP expressing AML blast population which was marked with an indipendent hashtag and pooled with the corresponding Live human AML blasts for chip loading and library prepration. GSE185834 parent_bioproject PRJNA771663