Discovery of compounds targeting human acute myeloid leukemia stem cells via a novel high-throughput screen

Acute myeloid leukemia (AML) is sustained by leukemic stem cells (LSCs), which must be eradicated for durable remission yet remain therapy-resistant. Here, we present a scalable platform to identify compounds that eliminate LSCs by performing the first large high-throughput drug screen directly on human LSC-enriched cells. Using this system, we screened 11 142 compounds and identified 20 inhibitors selective for LSCs. For three candidates, BIO-acetoxime, SJB2-043 and UMxxxxx03, we confirmed anti-LSC activity across multiple primary patient samples and validated efficacy through xenotransplantation assays. Single-cell RNA sequencing uncovered convergent and distinct anti-LSC mechanisms of action, including USP1 inhibition, which suppressed stemness and cell-cycle programs while promoting metabolic and inflammatory stress responses. Collectively, the LSC-enriched fraction was eliminated through apoptosis or differentiation. Together, this work establishes the first platform to directly screen LSCs at scale and identifies novel vulnerabilities and therapeutics capable of eliminating the root of AML.