E3 Ubiquitin Ligase Fbw7 Regulates the Survival of Mature B Cells

During B cell maturation, several checkpoints ensure the integrity of the uniquely generated BCR on an individual B cell. Upon completion, mature B cells populate secondary lymphoid organs on standby with the potential to receive activatory stimuli. Signaling via both BCR isotypes, IgM or IgD, is crucial during the initiation of an immune response, but also in the absence of antigenic stimulation, the BCR delivers survival signals, known as tonic BCR signaling (1). The PI3K signaling pathway is a key pathway supporting tonic BCR signals required for the survival of peripheral mature B cells (2). Tonic BCR signaling appears to be less dependent on canonical NF-κB signaling; however, this pathway is critical for downstream signaling after antigenic BCR activation via the signaling platform consisting of Card11, Bcl10, and Malt1 (3). PI3K signaling is also crucial during BCR activation, thereby controlling tonic signals and the dynamics of antigenic activation. We have recently shown that glycogen synthase kinase 3 (Gsk3), which is inhibited by PI3K signaling, regulates the metabolic needs of B cells (4). The phosphorylation of substrates by Gsk3 often primes these for degradation via the proteasome through functional recognition by the E3 ubiquitin ligase F-box and WD repeat domain-containing 7 (Fbw7) (5). Controlled proteolysis is crucial to maintain cellular homeostasis but also to effectively activate or terminate signaling pathways. The Fbxw7 gene encodes three Fbw7 protein isoforms (α, β, and γ) with different tissue distributions and subcellular localizations, of which Fbw7α is the most ubiquitously expressed and predominantly found in the nucleus (5, 6). Among the substrates of Fbw7 are the transcription factors c-Myc (7, 8), Notch (9), NF-κB2 (10–12), and the prosurvival factor Mcl1 (13), which have fundamental roles in B cells. Expression of c-Myc is crucial in developing B cells and during immune responses for germinal center B cell selection and expansion (14–16). In B cells, Notch signaling appears to primarily depend on Notch2, whereas Notch1 is dispensable; however, enforced expression of active Notch1 can greatly shift B cell fate to marginal zone B differentiation, which is normally attributed to Notch2 (17, 18). Deficiency of NF-κB2 (p100/p52) leads to reduced B cell numbers in the spleen and impaired germinal center formation after immunization (19, 20). Deletion of NF-κB2 in ongoing germinal centers does not affect their progression but impairs plasma cell differentiation (21). Notably, aberrant accumulation of p100 in the absence of Fbw7 induces cell death in multiple myeloma cell lines (12). Early ablation of Mcl1 leads to a drastic block during early B cell development, but Mcl1 is also required for the survival of mature B cells (22, 23).

In immune cells, loss of Fbw7 in hematopoietic stem cells reduces the self-renewal capacity and affects T and B lymphopoiesis (24). T cell–specific deletion of Fbw7 leads to increased proliferation of double-positive thymocytes and, ultimately, malignant transformation (25). However, the role of Fbw7 in B cells has not been elucidated yet, which prompted us to investigate its function during B cell development. In this study, we show that Fbw7 plays an important role in mature B cell survival by regulating NF-κB and PI3K signaling pathways and restricting c-Myc and Bim protein levels to prevent BCR-mediated apoptosis. Regulators of BCR signaling are becoming increasingly important to modulate B cell responses in autoimmune diseases, treat B cell cancers, and inhibit protumorigenic B cells.

Fbxw7^L/L (Jax 017563), Mb1^Cre (Jax 020505), Eμ-BCL-2-22 (Jax 002319), ^LSLYFP (Jax 006148), ^LSLMYC (Jax 020458), Cd19^Cre (Jax 006785), SW_HEL, and hCD20-Cre-ER^T2 were all kept on a C57BL/6 background. A minimum of three (female and/or male) animals were used per experiment. Age-matched Fbxw7^+/+ and Mb1^Cre/+ or cohoused Fbxw7^L/L littermates were used as controls, unless otherwise noted. All mice were kept under specific pathogen-free conditions at the animal facility of Sanford Burnham Prebys. Experimental procedures were in accordance with Institutional Animal Care and Use Committee regulations.

B cells from the spleen were enriched by MACS, collecting the negative fraction of CD43 (Ly48) Microbeads (Miltenyi Biotec). Cells were cultured in RPMI 1640 (Corning) supplemented with 10% FBS (MilliporeSigma), 1× penicillin-streptomycin, 1× MEM Nonessential Amino Acids (Corning), 1 mM sodium pyruvate, 2 mM GlutaMax, and 55 μM 2-ME (Thermo Fisher Scientific). The following concentrations were used for cell stimulation: 10 μg/ml anti-mouse IgM F(ab′)₂ (Jackson ImmunoResearch), 10 ng/ml mouse rBAFF (R&D Systems), 5 μg/ml anti-mouse CD40 (1C10), and 10 ng/ml mouse rIL-4 (Thermo Fisher Scientific). Total bone marrow cells were cultured in IMDM with GlutaMax (Thermo Fisher Scientific) supplemented with 10% FBS (MilliporeSigma), 1× penicillin-streptomycin (Corning), and 55 μM 2-ME (Thermo Fisher Scientific) and in the presence of 10 ng/ml mouse rIL-7 (PeproTech).

Single cell suspensions from tissues were ammonium-chloride-potassium lysed and stained in 1% FBS in PBS containing 0.05% sodium azide. Cells were gated according to size and granularity based on forward light scatter (FSC)-A and side light scatter-A. Doublets were excluded by FSC-A versus FSC-H gating. The following Abs were used: B220 (RA3-6B2) allophycocyanin-eFluor780, CD19 (1D3) PerCP-Cy5.5 or allophycocyanin, IgM (II/41) allophycocyanin or PerCP-eFluor710, IgD (11-26c) allophycocyanin or FITC, CD23 (B3B4) PE, CD21 (8D9) PE-Cy7, CD5 (53-7.3) FITC, CD25 (PC61.5) PE, and Streptavidin PE (Thermo Fisher Scientific). Lysozyme Biotin (GTX82960) (Genetex). CD43 (S7) PE or FITC, FAS (Jo2) PE-Cy7, and GL7 FITC (BD Biosciences) were used. CD5 (53-7.3) PE (BioLegend) was used. Apoptosis assays were performed by using the PE Annexin V Apoptosis Detection Kit I (BD Biosciences) according to the manufacturer’s protocol. Cell proliferation was analyzed using the eBioscience Cell Proliferation Dye eFluor670 (Thermo Fisher Scientific). For Ca²⁺ measurements, B splenocytes were loaded with Indo-1 AM and treated with or without probenecid before acquisition (Thermo Fisher Scientific). Samples were acquired on a FACSCanto or LSRFortessa X-20 (BD Biosciences) and analyzed with FlowJo (Becton Dickinson).

Cells were lysed either in CelLytic M (MilliporeSigma) supplemented with Protease/Phosphatase Inhibitor Cocktail (Cell Signaling Technology) or in 1% SDS buffer. Immunoblotting was performed following standard procedures. PVDF membranes were blocked with 5% milk in TBST and probed with primary Abs overnight on a rotating platform at 4°C. The following Abs were used: phospho-Erk1/2 T202/Y204 (D13.14.4E), phospho-Akt S473 (D9E), phospho-S6 S235/236 (D57.2.2E), phospho-Gsk3α/β S21/9 (no. 9331), phospho-c-Myc T58/S62 (no. 9401), c-Myc (D84C12), Mcl1 (D35A5), Erk1/2 (L34F12), β-actin (13E5), Vinculin (E1E9V), p65 (D14E12), Notch2 (D76A6), cleaved Notch1 (D3B8), NF-κB2 p100/p52 (no. 4882), Bim (C34C5), and IκBα (no. 9242) (Cell Signaling Technology); and Ship1 (Clone32) and Bcl2 (Clone7) (BD Biosciences). The following HRP-coupled Abs were used as secondaries: donkey anti-rabbit IgG and goat anti-mouse IgG (Jackson ImmunoResearch). Protein was detected on a ChemiDoc Imaging System (Bio-Rad Laboratories). For protein detection on the Odyssey system (LI-COR Biosciences), the following products and workflow was used. After transferring onto PVDF, the Revert Total Protein Stain was used to normalize the loading of protein. The membranes were blocked using Odyssey blocking buffer, stained with the primary Ab, followed by staining with an IRDye 800CW conjugated donkey anti-rabbit IgG.

Spleens embedded in Tissue-Tek O.C.T. (Sakura Finetek) were sectioned on a Microtome Cryostat HM 505 E (Microm). Sections were fixed with acetone and blocked with 5% FBS in PBS. Florescent images were acquired on an Axio Imager.M1 (ZEISS) microscope equipped with an Orca-ER (Hamamatsu) camera. SlideBook (3i) was used as the imaging software. GIMP (GNU Image Manipulation Program) was used for image editing. The following Abs were used: B220 (RA3-6B2) allophycocyanin, IgM (II/41) allophycocyanin, IgD (11-26c) PE (Thermo Fisher Scientific), Moma1 Biotin (Abcam), and Streptavidin-Cy3 (Jackson ImmunoResearch). For immunohistochemical analysis, spleens were formalin fixed and paraffin embedded. B220 (RA3-6B2) Biotin (BD Biosciences) and Myc (Y69) (Abcam) were used as primary Abs and HRP-coupled streptavidin or anti-rabbit IgG were used as secondary Abs. TUNEL staining was performed by using the ApopTag Peroxidase In Situ Apoptosis Detection Kit (MilliporeSigma) according to the manufacturer’s protocol.

B splenocytes were purified, and pellets were flash frozen in liquid nitrogen. Cells were lysed in 8 M urea and 50 mM ammonium bicarbonate supplemented with benzonase. After cell lysis, proteins were digested overnight in Trypsin/Lys-C Mix (Promega). Samples were then acidified with formic acid and desalted on an AssayMap Bravo liquid handling platform (Agilent Technologies). Dried samples were reconstituted in 2% acetonitrile and 0.1% formic acid and analyzed by liquid chromatography with tandem mass spectrometry on an EASY nLC system coupled to an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific). Mass spectra were analyzed with MaxQuant (https://maxquant.org) using Mus musculus protein sequence database (https://www.uniprot.org) as reference. Calculated peptide intensities were log₂ transformed and normalized across samples. Protein-level quantification and testing for differential abundance were performed using MSstats (http://bioconductor.org/packages/MSstats). Morpheus (http://software.broadinstitute.org/morpheus) was used to calculate z-scores and visualize expression values as a heatmap.

Acute deletion of Fbw7 was induced with two to four doses of 1 mg tamoxifen in 100 μl olive oil by oral gavage. Mice were immunized i.p. with 5 or 50 μg of either NP₅₃-AECM-Ficoll or NP_0.3-LPS (Biosearch Technologies) in 100 μl PBS. Serum was collected on indicated days, and Ab titers were determined by ELISA on plates coated with NP₂₃-BSA. For germinal center responses, mice were immunized i.p. with 50 μg NP₂₀-CGG (Biosearch Technologies) in 100 μl PBS. Sera was collected on indicated days, and Ab titers were determined on plates coated with NP₄- or NP₂₃-BSA.

RNA was extracted with RNeasy Mini Kit (QIAGEN), cDNA was synthesized with iScript cDNA Synthesis Kit, and reactions were run with iTaq Universal SYBR green (Bio-Rad Laboratories) on a LightCycler 96 (Roche). The following program was used: preincubation at 95°C for 5 min, two-step amplification of 45 cycles at 95°C for 10 s, and 60°C for 40 s. The following primers were used: Actin (forward [Fwd] 5′-GGCTGTATTCCCCTCCATCG-3′; reverse [Rev] 5′-CCAGTTGGTAACAATGCCATGT-3′), Hprt (5′-GGGGGCTATAAGTTCTTTGC-3′; 5′-TCCAACACTTCGAGAGGTCC-3′), Fbxw7 (Fwd 5′-TTCATTCCTGGAACCCAAAG-3′; Rev 5′-TCCATGGGCTGTGTATGAAA-3′), Bcl2a1 (Fwd 5′-TACAGGTACCCGCCTTTGAG-3′; Rev 5′-TCCACGTGAAAGTCATCCAA-3′), Bax (Fwd 5′-TGCAGAGGATGATTGCTGAC-3′; Rev 5′-GATCAGCTCGGGCACTTTAG-3′), and Bcl2l11 (Fwd 5′-GGAGATACGGATTGCACAGG-3′; Rev 5′-TCAATGCCTTCTCCATACCA-3′). Primers for mouse-specific Fbxw7α, Fbxw7β, and Fbxw7γ isoforms were described by Matsumoto et al. (6) before.

Prism (GraphPad Software) was used for statistical analysis, and p values from unpaired two-tailed tests are indicated in the figures: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

During B lymphopoiesis in the bone marrow, lineage differentiation depends on timed expression of key transcription factors and phases of cytokine versus pre-BCR–driven differentiation and expansion. To investigate the functional effects of Fbw7 in the B cell compartment, ablation of Fbw7 (Fbxw7^L/L) (24) was induced in mice during early B cell development (Mb1^Cre) (26). Two major checkpoints in the bone marrow facilitate proper BCR construction in a stepwise manner (27). First, pre–B cells express the successfully rearranged H chain locus of the BCR assembled with surrogate L chains (in the form of a pre-BCR) to undergo clonal expansion; second, the completed BCR is expressed on immature B cells to monitor autoreactivity. Defects in the BCR signaling cascade are therefore frequently evident by developmental blocks at the pre–B cell stage. Fbw7-deficient B cells progressed through early developmental stages and showed no obvious signs of maturation defects in the bone marrow (Fig. 1A). Albeit pre–B cells were increased and proliferated more in response to IL-7 signaling (Supplemental Fig. 1A), differentiation to immature B cells appeared normal compared with control. After immature B cells egress the bone marrow to finalize maturation and populate peripheral lymphoid organs, some return to the bone marrow as mature recirculating B cells, which were nearly absent in Fbw7-deficient mice. Correspondingly, peripheral B cells in the spleen were overall reduced in number and showed specific decreases in follicular B cells, T2, and marginal zone B cells. However, T1 cell numbers were comparable to control (Fig. 1B). Notably, Fbw7-deficient mature B cells downregulate CD23 expression, commonly used to identify B cell subsets in the spleen but could be identified by IgM, CD21, and IgD surface expression, albeit the latter two showed increased expression levels, and marginal downregulation of IgM was detectable (Supplemental Fig. 1B). The expression of BAFFR was normal on splenic B cells. To determine if B cells were localized normally in the periphery, we examined spleens from Fbw7-deficient mice by immunofluorescence for any abnormalities in the splenic architecture. Intact follicles with the presence of marginal zone B cells were detectable (Fig. 1C). However, follicles appeared to be smaller in size by gross analysis of the whole spleen (Fig. 1D). Following the reductions in mature B splenocytes, we determined that the third major mature B cell subset of B1 cells, an innate-like B cell compartment, was affected. Interestingly, B1 cells were drastically reduced in the peritoneal cavity, which affected both B1a and B1b cell subsets (Fig. 1E). In accordance with splenic B cell reductions, B2 cells in the peritoneal cavity were also greatly reduced.

The early absence of Fbw7 drastically reduced the mature B cell population and may have constituted unfavorable conditions preceding the mature stage to engage in functional immune responses. Thus, inducible deletion of Fbw7 in mature B1 and B2 cells (post–B lymphopoiesis) was preferred. It has not been reported if hCD20-Cre-ER^T2 mice (28) have specific Cre expression in mature B1 cells (in addition to B2 cells), but this seemed likely because of CD20 expression on B1 cells (29). To test the efficacy of Cre deletion in mature B cells, we crossed hCD20-Cre-ER^T2 to Fbxw7^L/L animals and subsequently to ^LSLYFP mice (30) as a reporter. After administration of tamoxifen by oral gavage, we analyzed B cells in the bone marrow, spleen, and peritoneal cavity after 3 wk. Notably, YFP⁺ cells were detectable in all mature B cell compartments, including B1 and B2 cells in the peritoneal cavity (Fig. 2A). Compared with control cells, acute deletion of Fbw7 recapitulated the loss of mature B cells in the bone marrow and spleen. However, B1 cells were not as drastically lost as compared with early B cell deletion by Mb1^Cre but trended lower. Using this system, we induced Fbw7 deletion and subsequently immunized mice with T cell–independent type I and II Ags, NP-LPS, and NP-Ficoll, respectively, to test the role of Fbw7 in B1 cell activation in vivo. Fbw7-deficient mice showed comparable early Ab responses on Day 3 for both Ags; however, after 7 d, serum Abs were lower after NP-LPS immunization (Fig. 2C) but not with NP-Ficoll (Fig. 2B).

The reduction of B cells in Fbw7-deficient mice was surprising as Fbw7 has been largely conceptualized as a negative regulator of growth, proliferation, and survival (5). To gain a better understanding of the specific role of Fbw7 in B cells, we analyzed the global proteome of purified B cell population from the spleen of Fbw7-deficient and control animals, which yielded more than 3500 detectable proteins in our sample set. Among the significant perturbations, several notable proteins involved in survival signaling (Bcl2 and Caspase 3) and BCR signaling (Cd79a, Card11, p65, Stim1, and Ship1) were differentially regulated upon Fbw7 deficiency (Fig. 3A). Some proteins were solely detectable in the control samples (potentially due to the limit of detection) but not in the Fbw7-deficient cells, which included Cd23 and Bcl2. Notably, CD23 was already validated by flow cytometry (Supplemental Fig. 1B), and lower protein levels of Bcl2 were confirmed in Fbw7-deficient cells by immunoblot (Fig. 3B). There was also evidence of lower canonical NF-κB signaling due to lower levels of Card11 (also known as Carma1) and p65 (also known as Rela). Card11 acts upstream of the negative regulator IκBα, which sequesters p65 in the cytoplasm (31), so we tested whether p65 was lower and IκBα was more stabilized. Fbw7-deficient cells showed less degraded IκBα protein and lower p65 levels, highlighting a potential insufficient use of this pathway. Further, the lipid phosphatase Ship1 was drastically downregulated in the Fbw7-deficient cells (Fig. 3B). Consistent with the role of Ship1 in antagonizing Akt activity, Akt phosphorylation was heightened in Fbw7-deficient cells (Fig. 3C). Active Akt mediates inhibitory phosphorylation of Gsk3, which then restricts Gsk3-mediated phosphorylation of c-Myc, leading to its stabilization. We found increased phosphorylation of Gsk3α and Gsk3β isoforms and heightened c-Myc protein levels. However, the phosphodegron motif in c-Myc protein was present, highlighting the stabilization of c-Myc predominantly through insufficient protein degradation in the absence of Fbw7.

Two major signaling pathways have been implicated thus far for B cell homeostasis in the spleen, namely BAFFR signaling and tonic BCR signaling (32). Thus, we treated B cells ex vivo in the presence of BAFF or anti-IgM or left without stimulation in media. Notably, the viability of Fbw7-deficient cells was greatly reduced without any stimuli within 1 d (Fig. 4A). The addition of BAFF increased the viability slightly compared with media alone. The addition of BCR stimulation via anti-IgM had drastic consequences on Fbw7-deficient cells, in which the majority of cells underwent apoptosis and were unable to grow (Fig. 4A, Supplemental Fig. 3B). Importantly, this cell death preceded the BCR-induced proliferation, which was also diminished in the remaining viable Fbw7-deficient cells (Supplemental Fig. 3B). The majority of these cells were unable to undergo cell division with only a small fraction of cells dividing in contrast to control cells.

Notably, Fbw7-deficient cells did not proliferate or grow in the presence of BAFF or media alone, which is not mitogenic under normal conditions but may have supported proliferative and growth signals due to higher signaling of Akt in the absence of Fbw7. To rule out that the difference in survival was due to aberrant signaling thresholds in the absence of Fbw7, we examined the induction of apoptosis in the presence of varying concentrations of anti-IgM for 24 h. Deficiency of Fbw7 resulted in high apoptosis despite the concentration of IgM in the cell culture media (Supplemental Fig. 3A). The addition of BAFF, anti-CD40, or IL-4 was able to partially rescue apoptosis in Fbw7-deficient IgM-stimulated cells (Fig. 4A). Early adverse effects were evident in the calcium mobilization, which was diminished in Fbw7-deficient cells (Fig. 4B), although activation with anti-IgM induced comparable phosphorylation of p-Akt as early as 5 min (Fig. 4D, Supplemental Fig. 3E). However, activated Akt signaling and downstream effector S6 was diminished after 1 h poststimulation and persisted lower. Also, canonical NF-κB signaling was defective after BCR stimulation (Fig. 4E); however, noncanonical NF-κB signaling appeared to be normal when stimulated with BAFF (Fig. 4G). The strong induction of c-Myc protein downstream of BCR signaling was further substantiated in Fbw7-deficient cells, although Gsk3 protein was less inhibited by phosphorylation (Fig. 4F). Notably, proapoptotic members of the Bcl2 family Bax and Bim (Bcl2l11) were transcriptionally increased (Fig. 4C), and Bim protein was enhanced in Fbw7-deficient cells (Fig. 4G).

To test if the excessive accumulation of c-Myc can induce apoptosis in B cells, we used mice with B cell–restricted human c-MYC (^LSLMYC) (16) under the control of Cd19^Cre/+ (33). Purified B cells from the spleen of single copy (^LSLMYC;Cd19^Cre/+), double copy (^LSLMYC/MYC;Cd19^Cre/+), or control (^LSLMYC) mice were cultured in the presence of anti-IgM, anti-CD40, a combination of the two, or media alone. After 1 d in culture, the viability was comparable in all conditions, except double copy c-MYC cells had increased apoptosis when left untreated (Fig. 5A). Apoptosis was further enhanced after 3 d without stimulation. Notably, BCR stimulation induced apoptosis in a dose-dependent manner of c-MYC expression, whereas CD40 signaling was comparable and able to rescue some of the survival loss seen with IgM stimulation. Although c-Myc levels are higher in mice carrying double copies of ^LSLMYC compared with single copy, Fbw7-deficient cells had comparable c-Myc protein induction (Fig. 5B). This elevation of c-Myc protein appears to occur in most Fbw7-deficient cells within the splenic follicles (Fig. 5C).

We hypothesized that the decrease of mature B cells in Fbw7-deficient mice may occur because of apoptosis in vivo. Notably, TUNEL staining revealed increased numbers of apoptotic cells in splenic follicles of Fbw7-deficient mice compared with controls (Fig. 6A). Thus, we posited the question if strong prosurvival signals by Bcl2 can overcome the apoptotic fate and deficits of Fbw7-deficient B cells. For this, we introduced a B cell–restricted transgenic human BCL2 (Eμ-BCL-2-22) (34) to Fbw7-deficient animals and analyzed the effects. In the spleen, ectopic BCL2 expression in Fbw7-deficient mice surpassed B cell numbers of wildtype controls, albeit still lower than BCL2 transgene controls; nonetheless, BCL2 efficiently rescued the loss of B splenocytes in the absence of Fbw7 (Fig. 6B). In the peritoneum, the BCL2 transgene increased B1 cells (Fig. 6B) compared with Fbw7 deficiency alone but could not reinstate the typical levels of B1 cells in the peritoneum of control mice. TUNEL staining was very low in the spleen of Fbw7-deficient mice with BCL2 and comparable to control BCL2 mice (Fig. 6C). Interestingly, ex vivo Fbw7-deficient B cell cultures were also more viable in the presence of BCL2 after 1 d post-IgM stimulation (Supplemental Fig. 4B) than Fbw7 deficiency alone (Fig. 4A). Overall, apoptosis appeared to be a major outcome in Fbw7-deficient cells, which could be partially rescued by BCL2.

In this study, we show for the first time, to our knowledge, that Fbw7 regulates the survival of mature B cells. Early ablation of Fbw7 during B lymphopoiesis manifests in reduced mature B cells in the bone marrow, spleen, and peritoneal cavity. Follicles in the spleen show increased apoptosis in the absence of Fbw7. Importantly, Fbw7-deficient B cells undergo heightened apoptosis ex vivo without any stimuli, highlighting their lower fitness compared with wildtype cells (Fig. 4A). Our global proteome analysis revealed deficiencies in the NF-κB pathway and lower Bcl2 levels in the absence of Fbw7 (Fig. 3A, 3B). Importantly, these cells have a significant elevation of c-Myc protein, which is insufficiently degraded in the absence of Fbw7 (Fig. 3C). BCR stimulation further substantiates c-Myc levels in Fbw7-deficient B cells but also increases a strong, albeit lower, induction of c-Myc in the wildtype cells (Fig. 4F). The relative level of c-Myc protein appears to be mirrored in Bim levels (by mRNA and protein), which has been shown to mediate c-Myc–induced apoptosis (35). Importantly, mature B cells that are lost in the spleen and bone marrow of Fbw7-deficient mice are significantly accumulated in Bim-deficient mice (Mb1^Cre), suggesting the mechanistic contribution of Bim to apoptosis in these cell stages (36). Interestingly, increased levels of ectopic c-MYC expression (single or double copy) in B cells induced specifically BCR-dependent apoptosis but not via CD40 signaling, which is integral during the germinal center response (Fig. 5A). Although elevated c-MYC protein induces apoptosis downstream of BCR signaling, this effect occurred later than in Fbw7-deficient cells. The specific makeup of Fbw7-deficient cells based on aberrant pathway signaling, including PI3K, Erk, and NF-κB signaling, suggests that these additional defects are contributing to the apoptotic fate. Fbw7-deficient cells present initially with higher PI3K signaling, probably because of secondary effects and downregulation of the negative regulator Ship1; however, the PI3K pathway is diminished post-BCR stimulation (Fig. 4D). This defect may play a crucial role in the apoptotic fate as excessive c-Myc levels require supplementation by PI3K signaling to enable proliferative and growth potential and to potentially prevent c-Myc–induced apoptosis (37). Overall, the combined accumulation of c-Myc and Bim and the lower levels of Bcl2, PI3K, Erk, and NF-κB signaling induce apoptosis in Fbw7-deficient mature B cells. Further, the partial rescue of apoptosis by BCL2 transgenic expression in the absence of Fbw7 reveals the missing counterbalance to proapoptotic induction (in part by Bim) in Fbw7-deficient B cells.

B1 cells were drastically reduced with early deletion of Fbw7 but less affected after acute deletion. B1 cells are innate-like cells that form mostly during fetal development before conventional B2 cells and bear a restricted BCR repertoire geared to self-antigens and mostly mucosal pathogens (38). Unlike follicular or marginal zone B cells, B1 cells depend on self-renewal and are not continuously replenished, which may explain their drastic disappearance in Fbw7-deficient animals. BCR signaling strength is an important contributor to fate decisions. B1 cells depend on strong BCR signaling during development contrary to follicular and marginal zone B cells (39). The IgM-induced apoptosis in Fbw7-deficient B cells may be indicative for a potential role of Fbw7 regulating BCR signaling in early B1 cells. The strong positive selection during development may lead to the apparent loss in this compartment. Also, the acute deletion of Fbw7 shows less dependencies in the homeostatic role and T cell–independent Ag activation of B1 cells, highlighting potential developmental defects. Notably, BCR transgenic (HEL⁺) B cells are not lost in the absence of Fbw7 (Supplemental Fig. 2A). Compared with wildtype cells, HEL⁺ cells have lower tonic BCR signaling (40) and are nearly absent within the B1 cell pool (41). It is not clear what source of BCR signaling is mediating B cell apoptosis in vivo in the absence of Fbw7, but it is possible that B cells with heightened (Ag-independent) tonic BCR signaling succumb.

In B cell malignancies, missense mutations of FBW7 have been identified in ∼3.4% of acute lymphoblastic leukemia (42), ∼1.6% of diffuse large B cell lymphoma (DLBCL) (43), and ∼2.5% of chronic lymphocytic leukemia (CLL) (44) patients. Notably, we found increased pre–B cell proliferation in the absence of Fbw7, which may reflect some of its roles in their malignant counterparts in acute lymphoblastic leukemia. At this stage, pre–B cell expansion is controlled in part by c-Myc, which must be suppressed to halt proliferation and enable further differentiation (45). Fbw7-deficient pre–B cells potentially are unable to effectively remove c-Myc protein and thereby exit the cell cycle delayed and accumulate in number. DLBCL arise from malignant germinal center B cells (46). Low FBW7 expression in DLBCL patients has shown to correlate with poor survival outcome (47). Surprisingly, the germinal center response was intact upon Fbw7 deficiency and appeared normal, aside from lower IgG1 Ab secretion (Supplemental Fig. 2B). Although, c-Myc is involved in germinal center expansion, the dynamics of germinal center B cells are much more complex and involve a multitude of pathways and selection mechanisms. It appears that BCR stimulation with real Ag can effectively stimulate Fbw7-deficient B cells in vivo, which then are able expand and form functional germinal center B cells. Notably, CD40 and IL-4 signaling (typically provided by T follicular helper cells in the germinal center) can partially rescue the survival defect of Fbw7-defcient cells after IgM stimulation (Fig. 4A). Further, Fbw7-deficient B cells proliferate normally when stimulated with CD40 and IL-4 (data not shown). Thus, the difference in early, mature, and germinal center B cell proliferation upon Fbw7 loss might be due to the distinct activated stimulatory pathways among these cell populations. The cellular origins for CLL remain unclear, but functionally, CLL cells have been ascribed to B1 cells (48). Notably, recent work addressed the mechanistic role of FBW7 in CLL, showing that functional loss of FBW7 elevates NOTCH1 protein levels and its associated signaling pathway (49). Although we found Notch proteins to be induced in Fbw7-deficient B cells (Supplemental Fig. 3D), enhanced marginal zone B cell differentiation or proliferative responses appeared not to be induced. Thus, it would be important to study the molecular roles of Fbw7 in specific B cell subsets with context-dependent constituents (i.e., cooperating mutations and microenvironmental cues) to directly relate these findings to B cell pathogenesis, specifically in patients with FBXW7 mutations.

We thank Michael Reth and Mark Shlomchik for providing the Mb1^Cre and hCD20-Cre-ER^T2 mice, respectively. Further, we thank Robert Brink for providing the SW_HEL mice and discussions on the HEL system. We also thank the Rickert laboratory for discussions and Sanford Burnham Prebys’s cores: Animal facility (Diana Sandoval/Buddy Charbono), Histopathology (Guillermina Garcia), Flow Cytometry (Yoav Altman), and Proteomics (Alexandre Rosa Campos).

The authors have no financial conflicts of interest.