BackgroundAcute leukemias represent deadly malignancies that require better treatment. As a challenge, treatment is counteracted by a microenvironment protecting dormant leukemia stem cells.MethodsTo identify responsible surface proteins, we performed deep proteome profiling on minute numbers of dormant patient-derived xenograft (PDX) leukemia stem cells isolated from mice. Candidates were functionally screened by establishing a comprehensive CRISPR‒Cas9 pipeline in PDX models in vivo.ResultsA disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) was identified as an essential vulnerability required for the survival and growth of different types of acute leukemias in vivo, and reconstitution assays in PDX models confirmed the relevance of its sheddase activity. Of translational importance, molecular or pharmacological targeting of ADAM10 reduced PDX leukemia burden, cell homing to the murine bone marrow and stem cell frequency, and increased leukemia response to conventional chemotherapy in vivo.ConclusionsThese findings identify ADAM10 as an attractive therapeutic target for the future treatment of acute leukemias.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12943-023-01803-0.