Dynamic evolution of TCF3-PBX1 leukemias at the single-cell level under chemotherapy pressure

Abstract

Acute lymphoblastic leukemia (ALL) is the most common childhood cancer. The translocation t(1;19), encoding the TCF3-PBX1 fusion, is associated with intermediate risk and central nervous system (CNS) infiltration at relapse. Using our previously generated TCF3-PBX1 conditional knock-in mice, we established a model to study relapsed clones after in vivo chemotherapy treatment, CNS infiltration, and clonal dynamic evolution of phenotypic diversity at the single cell-level using next-generation sequencing technologies and mass cytometry. Mice transplanted with TCF3-PBX1+ leukemia cells and treated with vehicle succumbed to disease, whereas 40% of treated mice with prednisolone or daunorubicin survived. Bulk and single-cell RNA sequencing of FACS-sorted GFP+ cells from TCF3-PBX1+ leukemias arising after chemotherapy treatment revealed that apoptosis, interleukin-, and TGFβ-signaling pathways were regulated in CNS-infiltrating leukemic cells. Across tissues, upregulation of the MYC signaling pathway was detected in persisting leukemic cells and its downregulation by BRD3/4 inhibition increased sensitivity to chemotherapy. In TCF3-PBX1+ leukemia cells collected after chemotherapy treatment, mass cytometry identified increased phosphorylation of STAT3/5 upon preBCR stimulation, which was susceptible to inhibition by the proteasome inhibitor bortezomib. In summary, we developed a TCF3-PBX1+ ALL mouse model and characterized relapsed disease after in vivo chemotherapy and cell phenotype dependence on microenvironment. Transcriptomics and phospho-proteomics revealed distinct pathways that may underlie chemotherapy resistance and might be suitable for pharmacological interventions in human ALL.