Multiomic single-cell analysis identifies von Willebrand factor and TIM3-expressingBCR-ABL1⁺CML stem cells

Tyrosine kinase inhibitors (TKI) only rarely eradicate leukemic stem cells (LSC) in chronic myeloid leukemia (CML) which commonly necessitates life-long therapy and monitoring of patients. Understanding details of leukemic hematopoiesis in CML may identify targetable pathways for sustained LSC elimination. This study utilized multiomic single-cell characterization of the CD14 - CD34 + hematopoietic stem and progenitor cell (HSPC) compartment in CML. Combined proteo-transcriptomic profiling of 597 genes and 51 proteins (CITE-seq) was performed along with parallel detection of BCR-ABL1 transcripts in 70,000 HSPC from 16 chronic phase patients and five healthy controls. CD14 - CD34 + HSPC from diagnosis samples displayed distinct myeloid cell bias with cells mainly annotated as LSC, lympho-myeloid progenitors (LMP)-II, erythrocyte and megakaryocyte progenitors, while few hematopoietic stem cells (HSC), LMP-I, dendritic cell or B cell progenitors were detected. In-depth analysis of the immature CD14 - CD34 + CD38 -/low compartment revealed two distinct populations of BCR-ABL1 -expressing CML LSC (denoted LSC-I and LSC-II), where LSC-I showed features of quiescence and CD45RA - cKIT - CD26 + TKI therapy-resistant phenotype. These subtypes of immature LSC showed high surface expression of TIM3 and transcription of the von Willebrand factor gene ( VWF ). Our findings imply that expression of VWF and TIM3 distinguish LSC from HSC and may be linked to aberrant myeloid-biased hematopoiesis in CML. Additionally, the results identify TIM3 as a conceivable target for sustained elimination of immature LSC in CML. Key points We present a method to detect BCR-ABL1 expression at the single-cell level that is compatible with high-throughput CITE-seq The most immature BCR-ABL1 -expressing LSC population in primary CML shows enhanced expression of von Willebrand factor and TIM3