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The use of single-cell profiling of phenotypes is suggested to inform about chemoresistance and lack of treatment response in cancer. Mass cytometry by time-of-flight (CyTOF) allows high throughput multiparametric analysis at the single-cell level to perform for in-depth characterisation of heterogeneity in leukemia. However, computational identification of cell populations from CyTOF, and utilisation of single-cell data for biomarker discoveries is challenging. Here, we deployed a machine learning-based framework that enables automatic cell population annotation, and systematic exploration of interactions between signalling proteins in a CyTOF antibody panel. We applied the developed framework to analyse a cohort of 45 leukemia patients. We investigated associations between the cellular composition and clinicopathological and genetic features, and reported salient signalling interactions of Multipotent Progenitor-like leukemia cells that were sufficient to predict short-term survival at time of diagnosis. Our findings confirmed that targeting cell type-specific signalling interactions in leukemia might improve existing patient stratification methods with the potential to inform early about more precise treatment options.

SUMMARYAcute myeloid leukaemia (AML) is a highly heterogeneous disease. Here, we decipher the disease composition of a single AML patient through longitudinal sampling scrutinized by high-resolution genetic and phenotypic approaches, including sequencing, immunophenotyping, ex vivo drug sensitivity testing and establishment of patient-derived xenograft models. Throughout the disease course we identified patterns of both divergent and convergent molecular evolution within the leukemic compartment. We identified at least six discrete leukaemia initiating cell populations, of which five were characterised by known recurrent mutations in AML. These populations partly correlated with immunophenotypically defined cell subsets, drug sensitivity profiles and population-specific potential for engraftment in immunodeficient mice. Our results indicate that the genetic and phenotypic development are closely intertwined, and that diversity in the leukaemic gene-environment likely influences disease trajectories.SIGNIFICANCENovel therapeutic approaches in AML are characterised by targeting molecular mechanisms thought to drive leukemogenesis, but emergent evidence suggests that intra-leukemic composition may be more diverse than previously appreciated. Through in-depth genetic and phenotypic characterization of the disease course of a single AML patient, we demonstrate a high degree of inter-individual complexity that exceeds the prevailing disease conception. The temporal molecular landscape of this patient suggests that leukemogenic transitions may not be categorically monoclonal. Patterns of converging molecular evolution further imply that higher levels of biological organisation than the molecular machinery of single cells may influence leukemogenic trajectories. Disease dynamics, relational properties and causal contribution from several levels of biological organization comes into conflict with the linear monocausal explanatory model on which precision oncology is largely built. This may have implications for current precision oncology oriented prectices, including molecular categorization, molecular therapeutic targeting and predictive models.

Summary statementThis study describes the effects of tyrosine kinase inhibitors on tunneling nanotube formation via increased adhesion through β-integrin in chronic myeloid leukemia cells.AbstractThe actin-containing cell-to-cell communicator tunneling nanotube (TNT) is involved in regulation of cell death threshold of leukemic cells, while the mechanism of TNT regulation is mostly unknown. We have investigated TNT formation and its response to treatment in chronic myeloid leukemia (CML) cells with the pathognomonic chimeric fusion kinase BCR-ABL1 after treatment with the tyrosine kinase inhibitor nilotinib and interferon-α. Bone marrow cells of chronic phase CML patients and the CML cell line Kcl-22 formed few or no TNTs. Nilotinib and interferon-α treatment induced TNT formation in Kcl-22 cells and were found to be linked to increased adherence to fibronectin coated surfaces by restoration of β1-integrin function. This suggests modulation of TNT cell-cell communication in CML as a novel mechanism in kinase inhibitor therapy of CML.