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

Program: Oral and Poster Abstracts
Type: Oral
Session: 603. Oncogenes and Tumor Suppressors: Lymphoid Malignancies
Hematology Disease Topics & Pathways:
Biological, ALL, Diseases, antibodies, Leukemia, Follicular Lymphoma, CLL, Lymphoma (any), Marginal Zone Lymphoma, Therapies, Mantle Cell Lymphoma, Biological Processes, Non-Hodgkin Lymphoma, B-Cell Lymphoma, immunotherapy, Lymphoid Malignancies, Clinically relevant, signal transduction
Monday, December 3, 2018: 7:00 AM
Room 10 (San Diego Convention Center)

Kadriye Nehir Cosgun, PhD1*, Gauri Deb, MS, PhD1, Xin Yang, MD2*, Gang Xiao, PhD1*, Teresa Sadras, PhD1*, Franziska Auer, PhD1, Jaewoong Lee, PhD1*, Anthony Abarientos, BSc1*, Maurizio Mangolini, PhD3*, Ali Aghajanirefah, PhD1*, Huimin Geng, PhD2, Hassan Jumaa, PhD4*, Andrew G. Polson, PhD5*, Hans Clevers, MD, PhD6* and Markus Muschen, MD7,8

1Department of Systems Biology, City of Hope Comprehensive Cancer Center, Monrovia, CA
2Department of Laboratory Medicine, University of California San Francisco, San Francisco, CA
3Department of Haematology, Cambridge Institute for Medical Research, Cambridge, United Kingdom
4Max-Planck Institute of Immunobiology and Epigenetics, Freiburg, Germany
5Genentech, Inc., South San Francisco, CA
6Hubrecht Laboratory, Utrecht, NLD
7Department of Systems Biology, City of Hope Comprehensive Cancer Center, Monrovia CA, Monrovia, CA
8Department of Systems Biology, City of Hope Comprehensive Cancer Center, Monrovia CA, San Francisco, CA

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, NRASG12D, MYD88L265P).

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 Lgr5fl/fl mouse models for BCR-ABL1- or NRASG12D-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).

Disclosures: No relevant conflicts of interest to declare.

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