Paper Details

ABSTRACT

Acute myeloid leukemia (AML) is sustained by leukemia stem cells (LSCs) that drive treatment failure and relapse, but phenotypic heterogeneity has limited robust prospective isolation across genotypes. Although epithelial cell adhesion molecule (EpCAM; CD326) has been linked to AML chemoresistance and tumor initiation, its relationship to functionally defined LSC states, CD25, and the underlying regulatory circuitry remains unresolved. We combined genetically engineered mice carrying Flt3^ITD/ITD with Dnmt3a^R878H and/or Npm1^cA/+ to produce three leukemias of differing latency (median survival: FD, CMML-like, 77 days; FN, AML, 20.5 days; FDN, AML, 32.5 days). All three shared a hypomethylation-encoded program enriched for leukemia and hematopoietic stem-cell pathways, confirming hallmark features of human AML. We integrated these mouse myeloid leukemia models with cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq), epigenomics, transplantation, chemotherapy challenge, and genetic perturbation. Across genotypes, leukemia converged on shared primitive progenitor-like states enriched for stemness programs; an unbiased CITE-seq screen of 46,719 leukemia cells using 195 antibody-derived tags ranked CD25 and EpCAM first and second for distinguishing primitive LSC-like cells (n=6,369) from mature leukemia (n=36,449; log₂ fold change=2.15 and 1.23; both adjusted P<0.001). CD25 ranked first in every genotype and EpCAM remained among the top 4; together they marked <10% of CD117⁺ leukemia cells and localized to multipotent/multilineage progenitor-like states. Whole-genome bisulfite sequencing and ChromLinker single-cell GRN modeling revealed abnormal transcription factor (TF) activity for Hoxa9/Hoxa10, Mecom, Mycn, non-canonical NF-κB and retinoid receptors, and unique expression and activity of Tcf7. Epcam expression correlated with focal hypomethylation and composite TF elements with Foxf2, Hoxa9, Mecom and Mycn stem-progenitor developmental program. In contrast, Il2ra (encoding CD25) expression was correlated with Tcf7-driven composite elements and NF-κB activity. Abnormal activity of TCF7 was validated in human AML subsets with abnormal lymphoid gene expression.

Each sorted CD25⁺, EpCAM⁺, and double-positive fraction regenerated phenotypically heterogeneous leukemia. Collectively, these fractions contained one LSC in 588 cells versus one in 3,951 CD117⁺ bulk cells (6.7-fold enrichment; P=0.007). Single-cell index sorting of 1,363 cells identified 134 clonogenic cells and refined a CD11b^-/CD11c^-/Ly-6C^-/CD27⁺/CD117⁺/CD25⁺/EpCAM⁺/Sca-1⁺ phenotype containing at least one LSC in 13 cells. CD25/EpCAM-enriched cells were selectively retained after cytarabine/doxorubicin and venetoclax/azacitidine; within this compartment, primitive states increased from 36.7% at baseline to 60.1% after venetoclax/azacitidine. In a 1:1 in vivo competition against Rosa26-targeting controls, dual-gRNA CRISPR disruption reduced Epcam edited cells from 50% at input to 19-24% across blood, marrow, and spleen by day 27 (P=0.027-0.039). Persisting edited cells shifted toward mature CD11b⁺Ly6G⁺ output (66.9% versus 34.1%; P=0.041), indicating reduced competitive fitness and partial release of differentiation blockade.

We next analyzed 186,844 cells from 27 human AML samples with xenograft-defined LSC; spanning primitive LSC (p-LSC; n=12), mixed primitive/monocytic LSC (n=7), and monocytic LSC (n=8) phenotypes. EPCAM-high and IL2RA-high cells mapped to lymphoid-primed multipotent and multilineage granulocyte-monocyte progenitor-like states. Their distributions were heterogeneous but overlapping, so combining the markers captured more primitive LSCs than either alone. EPCAM and IL2RA expression differed across functional LSC classes (P=0.002 and P<0.0001), with highest expression in p-LSC AML. Most importantly, EpCAM⁺ (but not EpCAM^-) fractions from primary human AML engrafted NSG-S recipients (P=0.020). Thus, CD25 and EpCAM identify complementary, mechanistically distinct, chemotherapy-persistent LSC states; prospective human transplantation supports EpCAM as an LSC marker, while genetic perturbation identifies Epcam as a mouse AML stem-cell fitness dependency.