Lgr5 Functions As a Critical Negative Regulator of Wnt/β-Catenin Signaling and Is Essential for B-Lymphopoiesis and Malignant B-Cell Transformation

Introduction: When early B-cell precursors have successfully undergone V(D)J rearrangement of immunoglobulin genes, they receive a strong positive selection signal, which initiates clonal expansion and the first wave of B-lymphopoiesis. Interestingly, this expansion following successful V(D)J recombination is marked by massive up-regulation of Leucine-rich repeat containing G-protein coupled receptor 5 (Lgr5).

Results: To test the function of Lgr5 at this particular stage and later in B-cell development, we crossed Lgr5^fl/fl mice with Mb1^cre and CD21^cre mice respectively. While Lgr5 deletion at the time of V(D)J recombination resulted in near complete ablation of the B cell lineage in the bone marrow and periphery, deletion at later stages of B-cell development had no significant impact on B-cell survival and proliferation. Here, we identified Lgr5 as a B-cell specific negative regulator of Wnt/β-catenin signaling, which is essential for normal B-lymphopoiesis but also for initiation of B-cell lineage acute lymphoblastic leukemia (B-ALL). Indeed, in children with B-ALL, higher than median mRNA levels of LGR5 at the time of diagnosis was identified as a predictor of poor clinical outcome (COG P9906, n=207, p=0.008). LGR5 over-expression conferred growth advantage to B-ALL cells, enhanced serial plating capacity and the ability to initiate leukemia in NSG mice. Limiting dilution transplant experiments showed that LGR5 overexpression increased LIC-frequencies by 4-fold.

Inducible deletion of Lgr5 in BCR-ABL1- or NRAS^G12D-transformed B-ALL cell lines led to growth arrest, abolished colony forming capacity and compromised the ability of leukemia cells to initiate fatal disease in NSG recipients. Lgr5 is thought to function as a potentiator of Wnt/β-catenin signaling in epithelial cells. Upon deletion of Lgr5 in B-ALL cells, however, we observed massive accumulation of nuclear β-catenin and increased expression of β-catenin target genes, which suggests that Lgr5 acts as an essential negative regulator of β-catenin in the B-cell context. Phosphoproteomic approaches identified β-catenin to be differentially phosphorylated on S675 upon Lgr5-deletion, which enhances nuclear translocation and transcriptional activity of β-catenin. Deletion of LGR5 in human colon cancer cell lines did not result in β-catenin accumulation, which indicates a lineage specific role of Lgr5 in B-lymphocytes.

Studying activating CTNNB1 mutations in 89,325 samples from 23 types of cancer, we found 7,777 activating mutations (8.7%). However, among 2,375 B-cell tumor samples, we did not observe a single activating mutation. Indeed, compared to solid tumors, B-ALL and B-cell lymphomas have very low amounts of nuclear β-catenin. B-ALL cells are extremely sensitive to β-catenin activation, resulting in cell cycle arrest and loss of colony forming ability, which parallels the phenotype observed in the absence of Lgr5. CRISPR-Cas9 mediated deletion of Ctnnb1 in Lgr5 knock-out B-ALL cells strikingly rescued proliferation and colony formation capacity, further corroborating the role of Lgr5 in promoting B-cell leukemogenesis by opposing aberrant activation of β-catenin.

Therapeutic implication: Given that Lgr5 is required for leukemia-initiation and marks LICs, we tested an Lgr5-MMAE antibody-drug conjugate (ADC) in the presence and absence of dexamethasone. Combination treatment with dexamethasone not only enforced persistently high surface expression of LGR5, but also potentiated efficacy of by Lgr5-MMAE in B-ALL eradication.

Conclusion: Lgr5 is critical for normal B cell development and tumor initiation in B-ALL. Lgr5, restricts the levels of nuclear β-catenin and enables B cell survival through negative regulation of Wnt/β-catenin signaling. Moreover, LGR5 represents a promising target for eradicating leukemia initiating cells.