FBXW7 Is Critical for B1 Cell Maintenance and Function
Authors: Ramezani-Rad Parham , Leung Charlotte R , Apgar John R , Rickert Robert C 
Published: October 2019

Abstract
Background: The E3 ubiquitin ligase Fbxw7 is responsible for the proteasomal degradation of several oncogenes involved in cellular proliferation, growth and survival. Notably, Fbxw7 has been characterized as a tumor suppressor in a variety of cancers, including T ALL and CLL. In the absence of Fbxw7, hematopoiesis is defective leading to abnormal development of B and T cells. In these mice, hematopoietic stem cells have impaired self-renewal due to stabilization of c-Myc. Fbxw7 targets several well-characterized molecules (c-Myc, Mcl1, p100, cyclin E, mTOR, SREBP1) with essential functions in B cells. We have recently shown that glycogen synthase kinase 3 (Gsk3), which is negatively regulated by PI3K signaling, controls the metabolic needs of B cells (Jellusova et al., 2017 Nat. Immunol.). A number of Gsk3 substrates are phosphorylated and degraded via the proteasome through recognition of Fbxw7. We sought to investigate the GSK3-FBXW7 axis in B cells and its implications for B cell malignancies.
Results: To elucidate the role of Fbxw7 in B cells, we studied Fbxw7 in a conditional knockout mouse model. We crossed Fbxw7fl/flmice with Mb1Cre mice to ablate Fbxw7 during early B cell development. In the absence of Fbxw7, B cells passed through their developmental stages and showed no significant signs of maturation defects in the bone marrow. However, mature recirculating B cells were nearly absent in Fbxw7-deficient animals. Correspondingly, peripheral B cells in the spleen were reduced and showed specific decreases in follicular B cells. Histological analysis of the spleen architecture showed intact B cell follicles with presence of marginal zone B cells. In B cell malignancies, mutations of FBXW7 have been reported in CLL, for which the normal counterpart are B1 cells. We analyzed the B1 cell compartment in the peritoneal cavity in the absence of Fbxw7. Loss of Fbxw7 resulted in drastic reductions of B1 cells in the peritoneal cavity. Both B1a and B1b cells were mutually affected in these mice. To further characterize this B1 cell defect, we generated a novel acute deletion model for Fbxw7. We crossed Fbxw7fl/flmice with hCD20Cre-ERT2 and administered tamoxifen by oral gavage to induce genetic ablation. Acute deletion of Fbxw7 lead to reduction of B1 cells within a few weeks, indicating the importance of Fbxw7 for the maintenance of B1 cells. When challenged with T cell-independent immunizations the overall antibody responses where reduced. BCR-stimulation of cells in vitro showed diminished growth and signaling potential of knockout cells. These cells have reduced calcium signaling and undergo massive apoptosis. The survival defect could be partially rescued with co-stimulation of IL-4 or ectopic expression of BCL2.
Conclusion: Our findings identify Fbxw7 as a critical regulator of B cell development. Specifically, Fbxw7 regulates B cell signaling, development and maintenance of B1 cells. These findings are directly relevant to the pathogenesis of CLL. Loss of function mutations of FBXW7 in CLL likely occur later during tumor evolution and potentially contribute to the specific microenvironmental niche.

Disclosures
Rickert: Pfizer: Employment.