Dormant leukemia stem cells reinforced by chemotherapy drive early treatment failure in acute myeloid leukemia patients [bulkRNA]

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. 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 AML, 5-12 mice each) from control or treated mice were sorted for the top and bottom 20% GFP expressing blast subpopulations. GFPlow/miR-126high and GFPhigh/miR-126low blasts from each mouse were then processed for RNA extraction with the miRNEasy Micro Kit (Qiagen) and library production (SMART-Seq v4 Ultra Low Input RNA Kit (Takara Bio USA)) and sequenced on Illumina platforms.