Functional Analysis of the Subclonal Architecture of B-Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL) at a Single Cell Level

Introduction: Transforming events in B-lineage (ALL) occur primarily at the level of committed progenitor cells, but the phenotype, frequency and hierarchical organization of leukemia-initiating cells (LICs) are controversial. Pronounced clonal heterogeneity in ALL and the lack of preclinical models impede functional analysis of LICs and dissecting the clonal architecture of B-cell precursor acute lymphoblastic leukemia (BCP-ALL).

Aims and methods: To identify cellular features associated with cell fate and decisions, engraftment potential, growth kinetics and resistance to therapy of BCP-ALL LICs at the clonal level, we generated multiple single-cell-derived clones from patient derived-long-term cultures (PD-LTCs) of human BCP-ALL. This in vitro ALL-LTC model allows prolonged serum-, cytokine-, and stroma-free culture of patient-derived BCP-ALL cells that are largely stable in terms of cytogenetic and immunophenotypic features for up to 6 months [Nijmeijer B et. al, Exp Hematol. 2009;37:376; Badura S et al., PLoS One. 2013; 8:e80070] Three ALL-LTCs were selected on the basis of different patterns of surface marker expression and cytogenetics, i.e. Ph+ ALL (LTCs PH and BV; common-ALL) and BCR-ABL negative ALL (LTC CR; pre B-ALL), respectively.

Results: Clonogenic growth in semisolid medium ranged from 0.25% to 8% for these ALL-LTCs (CR 8%, PH 0.45% and BV 0.25%), consistent with functional diversity within the bulk as well as the immunophenotypically defined subsets. To analyze these subpopulations at the clonal level, we isolated leukemic cell subsets by cell sorting on the basis of CD20, CD34 and CD38 expression, and then generated hundreds of clones by expanding single-cell sorted subpopulations. All sorted cell fractions from all three ALL-LTCs were capable of initiating long-term growth in vitro. In limiting dilution cultures, 1% to 5% of single sorted cells derived from these cell fractions gave rise to prolonged leukemic cell growth. To evaluate the leukemia-initiating capacity of the clonal ALL subpopulations in vivo, we examined engraftment and outgrowth kinetics of different ALL clones in a xenograft model with sublethally irradiated NSG mice. Individual clones derived from different sorted subpopulations displayed pronounced differences in engraftment potential and aggressiveness based on analyses of blood, bone marrow and spleen. This ranged from rapid appearance of ALL and death within 70-80 days (clone PH 20), to complete lack of leukemic outgrowth 155 days after transplantation (clone PH clone 14). The other four clones derived from this ALL (PH) displayed intermediate engraftment and outgrowth kinetics. Notably, leukemogenic properties were not associated with a distinct surface marker profile based in CD20, CD34 and CD38 expression. A similar heterogeneity of leukemogenic potential was determined with clonal subpopulations from the LTC BV.

To assess the hierarchical relationship between different clones and the association with LIC capacity, we examined the pattern of immunoglobulin VDJ-rearrangement of different clones. The Ig rearrangement patterns confirm that the clones represent distinct clonal populations derived from the original polyclonal LTC, but we observed no hierarchical relationship with respect to the biological properties of different clones. Further functional evaluation of these subclones was performed by videomicroscopy-based single cell tracking that allows analysis of individual cells and their progeny over many cell divisions. We observed concordance between cell cycle length and cell death events as determined by single cell tracking and the engraftment potential and aggressiveness of LTCs derived single cell clones in vivo.

To elucidate the molecular basis for the biologic differences between the clonal ALL populations, we compared highly and poorly leukemogenic clones by SILAC based proteomics. Only 5% of 6500 proteins measured in clones differing in their leukemia-initiating potential were differentially regulated, and these proteins could be assigned to a limited number of pathways.

In conclusion, comparative evaluation of clonal lymphoblast populations generated from patient-derived ALL long-term cultures by combined in vitro analysis, single cell videomicroscopy, xenotransplantation and proteomics is a promising approach to identify specific markers and therapeutic strategies that target LIC in ALL.