Multiomic single-cell analysis identifies von Willebrand factor and TIM3-expressing BCR-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 pointsO_LIWe present a method to detect BCR-ABL1 expression at the single-cell level that is compatible with high-throughput CITE-seq C_LIO_LIThe most immature BCR-ABL1-expressing LSC population in primary CML shows enhanced expression of von Willebrand factor and TIM3 C_LI