Ex Vivo Expansion of Phenotypic and Transcriptomic Chronic Myeloid Leukemia Stem Cells

•A means to expand leukemic stem cells from murine CML where they phenocopy their primary HSCs is described.•Both transcriptional and functional similarities to primary LSCs exist that lead to identification of targetable pathways, replicating other's findings and suggesting additional pathways for potential therapeutic intervention.•This approach provides a unique means to obtain significant numbers of CML LSCs and could assist in drug screens and mapping of targetable pathways in the future. Despite decades of research, standard therapies remain ineffective for most leukemias, pushing toward an essential unmet need for targeted drug screens. Moreover, preclinical drug testing is an important consideration for success of clinical trials without affecting non-transformed stem cells. Using the transgenic chronic myeloid leukemia (CML) mouse model, we determine that leukemic stem cells (LSCs) are transcriptionally heterogenous with a preexistent drug-insensitive signature. To test targeting of potentially important pathways, we establish ex vivo expanded LSCs that have long-term engraftment and give rise to multilineage hematopoiesis. Expanded LSCs share transcriptomic signatures with primary LSCs including enrichment in Wnt, JAK-STAT, MAPK, mTOR and transforming growth factor β signaling pathways. Drug testing on expanded LSCs show that transforming growth factor β and Wnt inhibitors had significant effects on the viability of LSCs, but not leukemia-exposed healthy HSCs. This platform allows testing of multiple drugs at the same time to identify vulnerabilities of LSCs.