Lgr5 Enables Positive B-Cell Selection and Tumor-Initiation in B-Cell Malignancies

Background and rationale. Leukemia-initiating cells (LIC) were extensively studied in AML (Bonnet 1997) and CML (Graham 2002), while LIC populations in B-ALL remained elusive. In the absence of a functional definition, targeted approaches for LIC-eradication are not feasible in B-ALL. Some studies suggested immunophenotypes for B-ALL LIC-populations (Cox 2004, Castor 2005, Wang 2007). However, other groups demonstrated that tumor-initiation in B-ALL is not limited to rare populations or developmental hierarchy (Kelly 2007; Le Viseur 2008; Rehe 2013; Aoki 2015).

Concept: We hypothesize that self-renewal in the B-cell lineage is induced by positive selection and antigen-receptor (BCR) signaling, i.e. encounter of cognate antigen. Self-renewal at this stage leads to clonal expansion and survival. Unlike stemness in AML and CML, which is determined by a developmental hierarchy, we propose that self-renewal in the B-cell lineage is transient and driven by environmental antigen and the ability of BCRs to bind with high affinity. In B-cell malignancies, positive B-cell selection events, resulting in Lgr5 surface expression, are mimicked by transforming oncogenes (e.g. BCR-ABL1, NRAS^G12D, MYD88^L265P).

Results: Combining flow cytometry and genetic approaches, we identified surface expression of the leucine-rich repeat containing G-protein coupled receptor 5 (Lgr5) as new biomarker of positively selected pre-B cells in the bone marrow. Conversely, conditional ablation of Lgr5 during earliest stages of B-cell development resulted in near-complete failure to develop a mature B cell pool (reduced by 2-3 log orders). Lgr5 is a Wnt target gene and an established cancer stem cell marker for epithelial cancers (e.g. colorectal cancer and mammary tumors), however, a role for Lgr5 in normal and malignant hematopoiesis is not known. Importantly, Lgr5 represents a previously unrecognized predictor of poor clinical outcome in children and adults with pre-B ALL, including worse overall survival and higher risks of drug-resistance and relapse. Limiting dilution transplant experiments showed that Lgr5-overexpression increased LIC-frequencies in NSG recipient mice. Inducible activation of Cre in Lgr5^fl/fl mouse models for BCR-ABL1- or NRAS^G12D-driven B-ALL resulted in cell cycle arrest, abolished colony forming capacity and compromised the ability of leukemia cells to initiate fatal disease in NSG transplant recipients. Deletion of Lgr5 in pre-B ALL cells caused massive accumulation of nuclear β-catenin and increased expression of β-catenin target genes. Phosphoproteomic analyses revealed increased levels of β-catenin S675-phosphorylation, which increases β-catenin transcriptional activity (Taurin 2006; Hino 2015). Inducible activation of a gain-of-function mutant of β-catenin revealed that pre-B ALL cells are extremely sensitive to β-catenin activation. Thus, Lgr5 enables positive selection and self-renewal of B-ALL cells by curbing β-catenin activity.

Therapeutic implication: To assess Lgr5 surface expression on B-ALL as a target for antibody-drug conjugate (ADC), we treated refractory B-ALL PDX with the Lgr5-MMAE ADC. Single-agent treatment with Lgr5-MMAE significantly reduced B-ALL leukemia burden. Treatment with dexamethasone not only enforced persistent surface expression of Lgr5, but also potentiated efficacy of by Lgr5-MMAE.

Conclusion: Unlike self-renewal in myeloid leukemia that is determined by a developmental hierarchy, our results here show that self-renewal in the B-cell lineage is transient and driven by the ability of BCRs to bind antigen with high affinity. Lgr5 is a biomarker of this selection event, critical for the initiation of B-ALL and other B-cell malignancies in transplant recipients. Given that positive B-cell selection events, resulting in Lgr5 surface expression are mimicked by transforming oncogenes, Lgr5 also represents a promising target for ADC therapy for instance Lgr5-MMAE (Genentech).