Lgr5 Functions As Negative Regulator of Wnt Signaling in B Cells and Is Critical for Self-Renewal of Normal and Transformed B Cells

Background and significance: The identification of leukemia-initiating cells (LIC) in myeloid leukemias allowed for focused therapeutic approaches to prevent drug-resistance and relapse. In B cell leukemia and lymphoma, LIC occur at a high frequency (Rehe 2013), are phenotypically diverse (Aoki 2015) and can arise from any stage of B cell development (Le Viseur 2008). While self-renewal of HSCs and other organ-stem cells is regulated by a developmental hierarchy, self-renewal in the B cell lineage is induced by positive selection events, i.e. induced by antigen-receptor (BCR) signaling when B cells bind to their cognate antigen.

Leucine-rich repeat containing G-protein coupled receptor 5 (Lgr5) is a Wnt target gene and through binding to its ligand R-spondin, Lgr5 modulates Wnt signaling strength. Lgr5 is widely used as stem cell marker in multiple organs, however its expression levels are very low in hematopoietic cells.

Results: Unexpectedly, B cells upregulate Lgr5-expression by >20-fold, (i) when they express the pre-BCR for the first time and (ii) when they encounter antigen for the first time in germinal centers. Engagement of BCR signaling on B cell lymphomas strongly increased LGR5 expression, which was sensitive to inhibition of SYK and BTK kinases in the BCR pathway.

In addition, in patients with pediatric or adult ALL, or B cell chronic lymphocytic leukemia (CLL), higher than median mRNA levels of LGR5 at the time of diagnosis were associated with poor clinical outcome and higher likelihood of drug-resistance and relapse. For these reasons, we studied the function of Lgr5 during normal B cell development and malignant transformation. Conditional ablation of Lgr5^fl/fl alleles during earliest stages of B cell development (Mb1-Cre) resulted in a >100-fold reduction of absolute B cell numbers. In addition, inducible deletion of Lgr5 in cultured pre-B and mature B cells caused rapid cell death, demonstrating that Lgr5 function is an essential requirement for B cell development. Most studies in epithelial cells suggest a role of Lgr5 as potentiator of WNT-signaling. However, Cre-mediated deletion of Lgr5 in B cells caused cell death in parallel with massive accumulation of nuclear b-catenin. Our genetic experiments in leukemia models demonstrated that LGR5 is critical for leukemia-initiation and survival. Cre-mediated deletion of Lgr5 and b-catenin accumulation abolished colony forming capacity and the ability of leukemia cells to initiate fatal disease in transplant recipients. Likewise, inducible activation of a gain-of-function mutant of b-catenin resulted in rapid cell death in B cells.

Conclusion: Lgr5 is widely established as a marker of organ stem cells with self-renewal capacity. In the absence of the developmental stem cell hierarchy in B cells, we conclude that Lgr5 expression is a biomarker of positive selection in B cells. Lgr5-expression and positive selection is induced by BCR-engagement (e.g. by antigen) or oncogenic mimics of BCR signaling in B cell malignancies (e.g. transforming oncogenes that engage the BCR pathway). Unlike in epithelial cells, LGR5 expression in B cells restricts the levels of nuclear b-catenin and enables B cell survival through negative regulation of Wnt-signaling.