Single-cell molecular analysis defines therapy-insensitive stem cell subpopulations in chronic myeloid leukemia
Rebecca Warfvinge*1, Linda Geironson Ulfsson*1, Mikael N.E. Sommarin*1, Stefan Lang, Christine Karlsson, Teona Roshupkina, Leif Stenke, Jesper Stentoft, Ulla Olsson-Strömberg, Henrik Hjorth-Hansen, Satu Mustjoki, Shamit Soneji, Johan Richter, and Göran Karlsson1
1Lund Stem Cell Center, Lund University, Sweden
A groundbreaking example of molecular therapy of malignant disease is the development of tyrosine kinase inhibitors (TKI) that specifically target the BCR/ABL fusion protein responsible for leukemic transformation in CML. However, a subset of leukemic stem cells (LSCs) are insensitive to TKIs as demonstrated by relapses following TKI cessation in patients with prolonged complete molecular responses. Currently no protocols to capture these TKI-insensitive LSCs exist. Here we combine and correlate targeted single-cell molecular analysis with cell-surface marker screens in LSCs from CML patients at diagnosis and following three months of TKI therapy. Using this approach we can discriminate leukemic and normal HSCs by BCR/ABL expression. Additionally the approach offers a broad dynamic range, no amplification bias and high sensitivity for few mRNA copies, allowing for dissecting the heterogeneity of the CML LSC population. Analysis of 2151 single cells revealed that LSC subpopulations with myeloid and proliferative molecular signatures were proportionally reduced at a higher extent compared to subfractions displaying primitive and quiescent signatures. Furthermore immunophenotypic characterization demonstrated that established CML stem cell markers were highly expressed on all subpopulations at diagnosis, but downregulated and unevenly distributed across subpopulations in response to TKI-treatment. The most TKI-insensitive cells of the LSC-compartment could be captured within a Lin-CD38-CD34+CD45RA-CD26+cKIT- subpopulation. Collectively our data visualize the direct effect of TKIs on LSC heterogeneity and provide a protocol for prospective isolation of the most TKI-insensitive LSC subpopulation.
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