Folliculin Interacting Protein 1 Maintains Metabolic Homeostasis during B Cell Development by Modulating AMPK, mTORC1, and TFE3 This information is current as of September 24, 2021. Julita A. Ramírez, Terri Iwata, Heon Park, Mark Tsang, Janella Kang, Katy Cui, Winnie Kwong, Richard G. James, Masaya Baba, Laura S. Schmidt and Brian M. Iritani J Immunol 2019; 203:2899-2908; Prepublished online 1 November 2019; Downloaded from doi: 10.4049/jimmunol.1900395 http://www.jimmunol.org/content/203/11/2899 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2019/10/31/jimmunol.190039 Material 5.DCSupplemental References This article cites 46 articles, 20 of which you can access for free at: http://www.jimmunol.org/content/203/11/2899.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2019 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Folliculin Interacting Protein 1 Maintains Metabolic Homeostasis during B Cell Development by Modulating AMPK, mTORC1, and TFE3

Julita A. Ramı´rez,*,1 Terri Iwata,* Heon Park,* Mark Tsang,*,2 Janella Kang,* Katy Cui,* Winnie Kwong,* Richard G. James,† Masaya Baba,‡,3 Laura S. Schmidt,‡,x and Brian M. Iritani*

Folliculin interacting protein 1 (Fnip1) is a cytoplasmic protein originally discovered through its interaction with the master met- abolic sensor 59 AMP-activated protein kinase (AMPK) and Folliculin, a protein mutated in individuals with Birt-Hogg-Dube´ Syndrome. In response to low energy, AMPK stimulates catabolic pathways such as autophagy to enhance energy production while inhibiting anabolic pathways regulated by the mechanistic target of rapamycin complex 1 (mTORC1). We previously found Downloaded from that constitutive disruption of Fnip1 in mice resulted in a lack of peripheral B cells because of a block in B cell development at the pre–B cell stage. Both AMPK and mTORC1 were activated in Fnip1-deficient B cell progenitors. In this study, we found inappropriate mTOR localization at the lysosome under nutrient-depleted conditions. Ex vivo lysine or arginine depletion resulted in increased apoptosis. Genetic inhibition of AMPK, inhibition of mTORC1, or restoration of cell viability with a Bcl-xL transgene failed to rescue B cell development in Fnip1-deficient mice. Fnip1-deficient B cell progenitors exhibited increased nuclear local- ization of transcription factor binding to IgHM enhancer 3 (TFE3) in developing B cells, which correlated with an increased http://www.jimmunol.org/ expression of TFE3-target genes, increased lysosome numbers and function, and increased autophagic flux. These results indicate that Fnip1 modulates autophagy and energy response pathways in part through the regulation of AMPK, mTORC1, and TFE3 in B cell progenitors. The Journal of Immunology, 2019, 203: 2899–2908.

eveloping B cells must pass through well-defined Emerging evidence suggests that B cells may also be regulated checkpoints, which ensure they are capable of produc- by metabolic checkpoints, which ensure that mature B cells are ing functional Ab molecules with minimal self-reactivity. appropriately equipped with the molecular machinery to fuel the

D by guest on September 24, 2021 cellular growth required for clonal expansion and Ab production. For example, B cell–specific disruption of Raptor, a positive regu- *Department of Comparative Medicine, University of Washington, Seattle, WA 98195; †Seattle Children’s Research Institute, Seattle, WA 98101; ‡Urologic Oncol- lator of mTORC1, inhibits pre–B cell metabolism and triggers a ogy Branch, Center for Cancer Research, National Cancer Institute, National Insti- complete block in B cell development at the pre–B cell stage, tutes of Health, Bethesda, MD 20892; and xBasic Sciences Program, Frederick National Laboratory for Cancer Research, Frederick, MD 21702 suggesting pre–BCR-driven cell growth may act as a checkpoint for 1 testing metabolic capacity (1). Similarly, mice deficient in PI3K and Current address: Pfizer Inc., Pearl River, NY. PLCg1/PLCg2, positive mediators of mTOR signaling, also exhibit 2Current address: Gilead Sciences, Seattle, WA. blocks in B cell development at the pre–B cell stage (2–5). Con- 3Current address: International Research Center for Medical Sciences, Kumamoto, Japan. ditional deletion of Raptor during peripheral B cell development inhibits the generation of plasma cells and germinal center B cells ORCIDs: 0000-0002-6262-1422 (T.I.); 0000-0002-9975-6080 (M.T.); 0000-0002- 6960-7922 (J.K.); 0000-0002-3248-3479 (W.K.); 0000-0002-5308-6683 (M.B.); (see Ref. 6 for review), suggesting that metabolic checkpoints might 0000-0003-1903-7850 (B.M.I.). also regulate peripheral B cell maturation. Received for publication April 5, 2019. Accepted for publication September 30, Despite abundant information about the roles of mTORC1 in the 2019. development of immune cells, the roles of other metabolic path- This work was supported by National Institutes of Health Grants R21AI109020, ways in B cell development remain unclear. Recently, the Folliculin R01AI092093, R56AI092093 (to B.M.I.), K01OD010554 (to J.A.R.), and K01OD21421 (to T.I.). L.S.S. is funded in part with federal funds from the Frederick (Flcn) interacting protein 1 (Fnip1)/Flcn/59 AMP-activated protein National Laboratory for Cancer Research of the National Institutes of Health under kinase (AMPK) complex has emerged as a central mediator in Contract HHSN261200800001E. maintaining metabolic homeostasis during B cell development Address correspondence and reprint requests to Brian M. Iritani, Department of (7, 8). Fnip1 is an evolutionarily conserved cytoplasmic protein Comparative Medicine, University of Washington, I-438 Health Sciences Center, 1959 NE Pacific Street, Building T-140, Seattle, WA 98195-7340. E-mail address: originally discovered through its interaction with Flcn, a protein [email protected] mutated in the rare autosomal dominant disorder Birt-Hogg-Dube´ The online version of this article contains supplemental material. syndrome (BHDS) (9). Patients with BHDS develop benign hair Abbreviations used in this article: AMPK, 59 AMP-activated protein kinase; BHDS, follicle neoplasms and are at high risk for developing lung cysts, Birt-Hogg-Dube´ syndrome; BM, bone marrow; Flcn, Folliculin; Fnip1, Flcn interact- pneumothorax, and renal tumors with a wide variety of histologies ing protein 1; FP, fluorescent protein; IC, intracellular; LC3, microtubule-associated protein 1A/1B–L chain 3; MEF, mouse embryonic fibroblast; NBD, nitrobenzoxa- (reviewed in Ref. 10). Fnip1 interacts in heteromultimeric com- diazole; poly I:C, polyinosinic:polycytidylic acid; PS, phosphatidylserine; S6R, plexes with Flcn, Fnip2, and AMPK, a master regulator of cellular ribosomal S6 protein; TFE3, transcription factor binding to IgHM enhancer 3; metabolism (9). AMPK is phosphorylated during conditions of en- v-ATPase, vacuolar ATPase; WT, wildtype. ergy deprivation and responds by activating energy and nutrient Copyright Ó 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 producing processes such as mitochondrial biogenesis and autophagy www.jimmunol.org/cgi/doi/10.4049/jimmunol.1900395 2900 Fnip1 ENABLES REGULATION OF AMPK, mTORC1, AND TFE3 IN B CELLS while simultaneously inhibiting energy and nutrient consum- phosphatidylserine (PS), nitrobenzoxadiazole (NBD)-PS (Avanti Polar ing pathways controlled by mTORC1. We previously generated Lipids). Briefly, cells were labeled with 5 mM NBD-PS in HBSS (Life Fnip1-deficient mice and showed that a constitutive loss of Fnip1 Technologies) +5.5 mM D-glucose and 20 mM HEPES at 15˚C for 5 min. Labeling was quenched in HBSS +5.5 mM D-glucose, 20 mM HEPES, resulted in a complete block in B cell development at the pre– and 1% lipid-free BSA for 5 min on ice followed by two washes in B cell stage due in part to increased apoptosis (7, 8). Enforced HBSS +5.5 mM D-glucose, 20 mM HEPES, and staining with fluorescent expression of IgH and IgL chain proteins in Fnip1-null pre–B cells Abs for flow cytometric analysis. failed to rescue mature B cell development despite surface IgM Cell labeling using DQ-BSA expression, suggesting an intrinsic defect in cellular growth and differentiation pathways following the disruption of Fnip1. BM cells were stained with Abs for flow cytometry as above, stimulated for 1 h with 10 ng/ml IL-7 in complete media, and then labeled with 20 mg/ml In this study, we took a genetic approach to investigate the DQ-BSA Green (Invitrogen) in complete media at 37˚C for 1 h, followed molecular mechanisms of how Fnip1 controls B cell develop- by two washes with Dulbecco’s PBS +3% FBS before flow cytometric ment. Our results reveal that Fnip1 coordinates multiple metabolic analysis. pathways regulated by AMPK, mTORC1, and transcription factor ImageStream imaging flow cytometry binding to IgHM enhancer 3 (TFE3) to maintain metabolic ho- meostasis necessary for pre–B cell survival and differentiation Cells were cultured ex vivo for 2–3 h or overnight and fixed as described during metabolic stress. above for the detection of IC Ags using rat anti-LAMP2 (Abcam), rabbit anti-mTOR (Cell Signaling Technology), or rabbit anti-TFE3 (Sigma) Abs. Donkey anti-rabbit Alexa Fluor 647 and donkey anti-rat Alexa Fluor 488 Materials and Methods (Life Technologies) secondary Abs were used to detect primary Abs. Data were collected using an ImageStream imaging flow cytometry X Downloaded from Mice (MilliporeSigma), and analyses were performed with IDEAS software 2/2 Tg tm1Tyj fl/fl fl/fl (MilliporeSigma). Fnip1 (7), Bcl-xL , Trp53 , Raptor , Mb1-Cre, Tsc1 , tm1.1Sjm tm1Fwa tm1Wjl Prkaa1 , Rag2 Il2rg ,Em-Myc, and EGFP-microtubule- Mass spectrometry–based proteomics associated protein 1A/1B–L chain 3 (LC3) mice were described previ- Tg 2/2 +/+ ously (11–19). Bcl-xL mice were kindly provided by Tim Behrens, B cells were enriched from total BM from Fnip1 and Fnip1 lit- Mb1-Cre and Em-Myc mice were provided by Robert Eisenman, Tsc1fl/fl termates. Pre– and pro–B cells were enriched via positive selection with

mice were provided by Raymond Yeung, EGFP-LC3 were provided by anti-CD19 Ab-conjugated magnetic beads (EasySep; STEMCELL http://www.jimmunol.org/ Mike Bevan, and ROSA26lox-stop-lox tdTomato mice were provided by Technologies) followed by FACS sorting for IgM negative cells and were R. Palmiter (20). Trp53tm1Tyj and Prkaa1tm1.1Sjm mice were purchased from cultured overnight in rIL-7 (R&D Systems). The 2 3 106 cells from each The Jackson Laboratory, and Rag2tm1FwaIl2rgtm1Wjl mice were purchased replicate were then lysed using 8 M urea. Peptide enrichment and from Taconic Biosciences. Mice were maintained on a C57BL/6J back- chromatography were performed as previously described. Following ground or were backcrossed .10 generations, with the exception of Tsc1fl/fl peptide quantification (Pierce Quantitative Fluorometric Peptide Assay), and Raptorfl/fl crosses, which were on a mixed 129:C57BL/6J background. 1 mg of each sample was analyzed by mass spectrometry as described Cohoused littermates of both sexes were used whenever possible. Animal previously (22). studies were reviewed and approved by the University of Washington Institutional Animal Care and Use Committee. Statistical analyses

Cell viability and proliferation assays Statistical differences between two groups were analyzed by two-tailed by guest on September 24, 2021 unpaired Student t tests with equal variance. Multiple group comparisons To assess apoptosis, cells were stained ex vivo with CellEvent Caspase 3/7 were made using one-way ANOVA with multiple comparisons post hoc (Invitrogen, Carlsbad, CA) and Ghost dye LIVE/DEAD viability stain t tests. GraphPad Prism software (version 6; GraphPad Software, San (Tonbo Biosciences, San Diego, CA) and analyzed according to the man- Diego, CA) was used for analyses, and data were considered significant at ufacturers’ instructions. Analysis of cellular proliferation in vivo was per- *p # 0.05, **p # 0.001, ***p # 0.0001, and ****p , 0.00001. Center formed by i.p. BrdU injection (1 mg; BD Biosciences, San Jose, CA) ∼16 h values shown on graphs represent means, and error bars represent SD. prior to harvest. Intracellular (IC) staining was performed according to the For all graphs, each point plotted represents the analysis of primary cells manufacturer with anti-BrdU PerCP/Cy5.5 (BD Pharmingen, San Jose, CA). derived from a single mouse. Abs and flow cytometry Cells were stained using Abs specific for mouse Ags: CD45R (B220) Results (various fluorochromes) (BD Pharmingen, BioLegend, San Diego, CA and Disruption of Fnip1 results in increased apoptosis of Tonbo Biosciences), IgM (various fluorochromes, Jackson Immuno- B cell progenitors Research, West Grove, PA), CD19 eFluor450, CD25 APC, MHC class II APC(TonboBiosciences),CD43PE,BP-1PE,CD117PE,CD24 To assess whether acute disruption of Fnip1 affects B cell devel- (heat-stable Ag) PE-Cy7, IgD FITC (BD Pharmingen), and CD21 opment in adult animals and not via deletion in embryonic/fetal/ PerCP/Cy5.5 (BioLegend). IC staining was performed with IC Fixation neonatal tissues, we crossed Fnip1fl/fl mice (8) with Mx1-Cre (21) and Permeabilization Buffer (eBioscience). Methanol fixation was per- formed for IC p-S6R detection. Abs used for IC staining were p-ribosomal mice, which express Cre recombinase from the IFN-responsive S6 protein (S6R) S235/236 PE (eBiosciences), p-AMPKa T172, c-Myc Mx1 gene promoter. Cre expression is transiently induced in AF488, and p-Ulk1 S555 (Cell Signaling Technology, Danvers, MA). response to an injection of the dsRNA analogue poly- Donkey anti-rabbit Alexa Fluor 647 (Life Technologies, Carlsbad, CA) inosinic:polycytidylic acid (poly I:C). We found that the secondary Ab was used to detect unlabeled primary Abs. Data were collected percentage of immature and mature B cells [Hardy Fractions using FACS Canto II or LSR II flow cytometers (BD Biosciences), and analyses were performed using FlowJo software (TreeStar, Ashland, OR). E and F (23)] in the BM were markedly reduced 2 wk fol- lowing poly I:C injection (Fig. 1A), whereas the representa- Immunoblotting tion of pro–B/early pre–B cells (Hardy Fractions A–C9)were Immunoblotting was performed on whole cell extracts from cultured im- slightly increased. This likely represents a developmental block as 2 2 mortalized mouse embryonic fibroblasts (MEFs) derived from Fnip1 / Caspase 3/7+ apoptotic pro–B/pre–B cells increased 48 h after mouse embryos (21). Proteins were detected using Abs against LC3B poly I:C administration (Supplemental Fig. 1A). To visualize Fnip1- (D11; Cell Signaling Technology) and GAPDH (loading control; D16H11; fl/fl Cell Signaling Technology). deleted cells, Fnip1 Mx1Cre mice were crossed with tdTomato Cre-reporter mice, which allows Cre-expressing cells to be identi- Cell labeling using nitrobenzoxadiazole-phosphatidylserine fied via the expression of red fluorescent protein (FP). A single + + 2 The phospholipid incorporation assay was performed with Fnip12/2 injection of poly I:C resulted in ∼90% tomato FP B220 IgM or Fnip1+/2 bone marrow (BM) using the fluorescent analogue of pro–B/pre–B cells, ∼80% tomatoFP+ B220loIgM+ immature The Journal of Immunology 2901

FIGURE 1. Disruption of Fnip1 re- sults in increased apoptosis. (A) Repre- sentative flow cytometric analyses of BM cells from Fnip1fl/fl or Fnip1fl/flMx1-Cre mice 13 d after the deletion of Fnip1 via poly I:C (pI:C) injection. Shown are gated lymphocytes (forward scatter/side scatter) following staining for B220 (CD45R), IgM, and CD43 (Ly48). Numbers on the plots represent the frequencies of the gated populations. The scatter plot (right panel) shows frequencies of B220+ BM cells in vari- ous developmental fractions (Hardy fractions). **p , 0.005, t test. (B) p53

deletion and Bcl-xL overexpression Downloaded from fail to rescue B cell development in Fnip1-deficient mice. Scatter plots show frequencies of cleaved Caspase 3/7 substrate in B220+IgM2 BM cells from WT, Fnip12/2, Trp532/2,and Fnip12/2Trp532/2 mice (upper panel) 2/2 or Bcl-xL and Fnip1 Bcl-xL mice http://www.jimmunol.org/ (lower panel). p # 0.0001, one-way ANOVA. (C) Representative flow cyto- metric analyses of BM cells from WT, 2/2 2/2 Fnip1 , Bcl-xL,andFnip1 Bcl-xL mice are shown gated on lymphocytes and stained for B220, IgM, CD43, BP-1 (Ly-51, CD249), and CD24. (D) Scatter plots show developmental (Hardy) frac- tions representing the frequency (bot- tom left panel) and absolute number by guest on September 24, 2021 (bottom right panel) of cells from these mice. Gating for fractions: A, B220+CD43+ CD242BP-12; B, B220+CD43+CD24+ BP-12;C/C9,B220+CD43+CD24+BP-1+; D, B220loCD432 IgM2 ;E,B220lo CD432IgM+, F = B220hiCD432IgM+. *p , 0.05, **p , 0.001, ***p , 0.0001, ****p , 0.00001.

B cells, and ∼50% tomatoFP+ mature B220hiIgM+ B cells by or defective (24). Whereas Fnip12/2 pre–B cells express ICm, 48 h postinjection (Supplemental Fig. 1B). The reduction in indicative of effective recombination, levels of ICm are lower tomatoFP+ cells during the pre–B cell stage to immature B cell than in wildtype (WT) cells (7), possibly indicating a deficiency in stage is consistent with increased apoptosis and selection against survival during V(D)J rearrangement. We therefore asked the deleted allele with maturation. Although poly I:C–induced a/b whether increased apoptosis of Fnip12/2 Bcellprogenitors IFN might synergize with Fnip1 deficiency to mediate cell death, proceeds through a p53-dependent mechanism. Fnip12/2 mice both experimental and control mice were treated with poly I:C. were crossed with p532/2 mice to generate Fnip12/2p532/2 These findings indicate that acute disruption of Fnip1 leads to im- animals. p53 deficiency did not alter levels of apoptosis in paired development at the pre–B cell stage due in part to increased Fnip12/2 B cell progenitors (Fig. 1B) and had no effect on B cell apoptosis. development (Supplemental Fig. 1C). These results suggest that the increase in pro–B/pre–B cell apoptosis following the Increased apoptosis of Fnip1-deficient B cell progenitors disruption of Fnip1 occurs independently of p53. occurs independently of p53 We next investigated the molecular mechanisms leading to in- Bcl-xL overexpression restores cell viability but fails to rescue creased apoptosis of Fnip1-deficient B cell progenitors. V(D)J B cell development in Fnip1-deficient mice recombination activates a p53-dependent checkpoint during B cell We next investigated whether overexpression of a prosurvival development, which induces apoptosis if recombination is prolonged factor could restore normal B cell development. We crossed 2902 Fnip1 ENABLES REGULATION OF AMPK, mTORC1, AND TFE3 IN B CELLS

2/2 Fnip1 mice with transgenic mice overexpressing Bcl-xL,an induction with rapamycin or amino acid restriction but remained anti-apoptotic member of the Bcl-2 family, in B cells (18). Al- higher than Fnip1+/2 cells (Fig. 3B). ImageStream imaging flow though expression of the Bcl-xL transgene markedly reduced ap- cytometry revealed an increased number and size of LC3-GFP 2/2 2/2 optosis in Fnip1 pro– and pre–B cells (Fig. 1B), Bcl-xL only puncta (autophagosomes) in Fnip1 pro– and pre–B cells, which minimally (,10%) rescued the development of early and late pre– appeared largely unchanged during the induction of autophagy B (Fractions B, C/C9, and D) and immature B cells (Fraction E) in with rapamycin, whereas puncta number decreased in control cells the BM and completely failed to restore the development of ma- (Fig. 3B, lower panel). Because altered lysosome number or ture B cells in the BM (Fig. 1C, 1D) and spleen (data not shown). function can help differentiate between autophagy inhibition or An increase in Hardy Fractions B and C is reflective of Bcl-xL maximally increased flux, we measured the number of lysosomes transgenic, which has been shown previously to increase the (number of IC spots of the lysosome marker LAMP2) using number of pro–B cells (18). Our results suggest that Fnip1 de- ImageStream and examined lysosomal activity by evaluating IC ficiency results in impaired B cell development that is only par- fluorescence of proteolyzed DQ-BSA, which requires enzymatic tially dependent on cell survival and that additional mechanisms cleavage to generate a fluorescent product. A higher percentage of contribute to blocked B cell differentiation. Fnip12/2 pro– and pre–B cells had high numbers of lysosomes (Fig. 3C) as well as increased lysosomal activity during the in- Increased c-Myc–mediated cell growth and division fails to duction of autophagy with the Tat-D11 (beclin 1) peptide com- restore B cell development in Fnip1-deficient mice pared with control cells (Supplemental Fig. 1D), which suggests We previously found that constitutive loss of Fnip1 resulted in increased autophagic flux. To investigate whether a defect in lipid enrichment for genes regulated by the Myc transcription factor turnover was responsible for increased autophagosome and lyso- Downloaded from (p = 1.8 3 1027) in microarray analysis of pre–B cells from some numbers, we measured the incorporation of the fluorescent Fnip12/2 compared with WT mice (7). c-Myc is important for phospholipid analogue NBD-phosphatidylserine (NBD-PS) into pre–B cell development (21) and drives cell growth and prolifer- Fnip12/2 pro– and pre–B cells. We found that incorporation of ation while altering the apoptotic threshold of cells (reviewed in NBD-PS into IC membranes was increased in Fnip12/2 cells Ref. 25). Analyses of c-Myc protein levels via IC staining revealed relative to Fnip1+/2 controls (Fig. 3D), indicating lipid turnover 2/2 increased c-Myc in Fnip1 BM late pro–B cells (Fig. 2A, is higher than usual in these cells. We next measured LC3-GFP http://www.jimmunol.org/ Supplemental Fig. 2D), indicating that decreased Myc levels were colocalization with LAMP2 using ImageStream to determine not responsible for blocked B cell development. Because in- if disruption of Fnip1 results in defects in autophagosome– creased c-Myc–driven metabolism might compensate for the ab- lysosome fusion, which could also result in increased organelle sence of Fnip1, we tested whether B cell–specific overexpression number and size. We found that autophagosome–lysosome of c-Myc could further stimulate B cell growth and metabolism colocalization was not different between Fnip12/2 and Fnip1+/2 and drive maturation of Fnip12/2 pre–B cells by generating control cells, and both displayed increased colocalization Fnip12/2 mice that overexpress c-Myc during B cell development following treatment with bafilomycin A1, which blocks autophagy (Em-c-Myc) (14). c-Myc overexpression failed to significantly at the final step of the pathway by preventing the digestion of increase the proliferation of Fnip12/2 B cell progenitors (Fig. 2B) autolysosomes (Fig. 3E). by guest on September 24, 2021 but efficiently increased cell growth, based on an increase in cell To definitively assess the flux of autophagy in Fnip12/2 cells, size (data not shown). However, the c-Myc transgene only enabled we next performed IC staining of LC3-II in late pro–B cells after the development of a small population of pre–B cells and imma- treatment with bafilomycin A1 to block autophagy. Autophagic ture B cells in Fnip12/2 animals and completely failed to rescue flux was then determined by comparing the ratio of LC3-II after the development of circulating mature B cells in the BM (Fig. 2C) bafilomycin treatment to basal levels of LC3-II. Autophagy flux and periphery (data not shown). These results indicate that stimu- was increased in Fnip12/2 late pro–B cells relative to Fnip1+/2 lation of cellular growth, metabolism, and proliferation by over- controls (Fig. 3F). We used immunoblotting to confirm this result, expression of c-Myc is not sufficient to restore B cell development comparing LC3B-I and LC3B-II levels in response to bafilomycin in the absence of Fnip1. in Fnip1+/2 and Fnip12/2 MEFs (Supplemental Fig. 1E). Fnip12/2 MEFs had a much higher basal ratio of membrane- Disruption of Fnip1 increases AMPK activation, autophagy bound:cytoplasmic LC3B (LC3B-II:LC3B-I, lower panel densi- induction, and flux tometry) and, upon starvation, converted LC3-I to LC3-II faster Autophagy is a self-degradation process necessary to generate than control cells, confirming Fnip1 deficiency increases auto- nutrients and energy during development and in response to energy phagic flux. Together, these results indicate that the disruption of stress. Because Fnip1 interacts with AMPK, a primary activator of Fnip1 in B cell progenitors results in more autophagosomes and autophagy, and mTORC1 inhibits autophagy (26), we examined lysosomesaswellasincreasedautophagicfluxcomparedwith whether Fnip1 deficiency impacts autophagy during B cell de- Fnip1+/2 B cell progenitors. velopment. Consistent with our previous report, we found Fnip12/2 pro– and pre–B cells exhibited increased AMPKa mTORC1 signaling is aberrantly increased in Fnip1-deficient phosphorylation at Thr172 as well as increased phosphorylation B cell progenitors of a direct substrate of AMPK, Ulk1 phospho-Ser555, which is mTORC1 is activated by growth factors and sufficient amino acids, thought to initiate autophagy (Fig. 3A, Supplemental Fig. 2D). which are transported to the lysosome, resulting in the activation of Initiation of autophagy may not lead to increased flux through vacuolar ATPases (v-ATPases). Activated v-ATPases then stimu- the pathway if autophagosome/lysosome fusion is blocked. late guanine nucleotide exchange activity of the Regulator complex Therefore, we visualized autophagosomes by generating Fnip12/2 toward the RagA/B/C/D GTPases, which recruit mTORC1 to the LC3-GFP fusion mice. During autophagy, cytosolic LC3-I is lysosome in which mTOR is activated (reviewed in Ref. 27). conjugated to phosphatidylethanolamine to form LC3-II, which Whereas some studies have shown that constitutive Fnip1 or Flcn subsequently becomes incorporated into autophagosomes. Fnip12/2 deficiency in mice is associated with mTORC1 hyperactivation pro– and pre–B cells displayed increased levels of LC3-GFP over- (7, 28–30), other investigations in cell lines have shown that Fnip1 all (LC3-I & II), which decreased in response to autophagy and Flcn positively regulate mTORC1 activity by functioning as The Journal of Immunology 2903

FIGURE 2. Increased c-Myc–mediated cell growth fails to restore B cell devel- opment in Fnip1-deficient mice. Bar graphs show (A) mean fluorescence in- tensity (MFI) of IC c-Myc staining in 2 B220+BP-1+CD25 late pro–B cells from Downloaded from Fnip1+/2 or Fnip12/2 BM and (B)fre- quencies of BrdU+ BM pro– and pre–B cells (B220+IgM2), which represent di- viding cells from WT, Em-Myc, Fnip12/2, and Em-Myc Fnip12/2 mice. (C) Top, Representative flow cytometric analyses of BM cells from WT, Em-Myc, Fnip12/2, and http://www.jimmunol.org/ Em-Myc Fnip12/2 mice are shown gated on lymphocytes and stained for B220, IgM, and CD43. Bottom, Scatter plots show de- velopmental (Hardy) fractions representing the frequency (top panel) and absolute number (bottom panel) of cells from these mice. Fraction A–C9 is gated B220+CD43+. *p , 0.05, **p , 0.001. by guest on September 24, 2021

Rag C/D GTPase activating proteins or Rag A/B guanine nucle- performed whole cell mass spectroscopy–based proteomics on otide exchange factors at the lysosomal surface, thus helping re- sorted pro– and pre–B cells from Fnip12/2 and control litter- cruit and retain mTORC1 at the lysosome (31, 32). To further mates. Consistent with increased mTORC1 activity, we found assess the roles of Fnip1 in mTORC1 activation during B cell multiple ribosomal proteins were more abundant in Fnip12/2 development, we measured the size of BM B cell progenitors relative to Fnip1+/2 control cells (Supplemental Table I). (indicative of cell growth) in Fnip12/2 and Fnip1+/2 mice using Because mTORC1 activity is regulated by recruitment to flow cytometry. Cell size was increased in Fnip12/2 late pro– the lysosomal surface in response to amino acid sufficiency, we B cells relative to Fnip1+/2 control pro–B cells, which correlates examined whether the increase in mTORC1 activation in Fnip12/2 with the stage at which pre–B cells normally decrease in size pre–B cells was associated with altered localization at the lyso- during early B cell development (Fig. 4A). IC p-S6R, a substrate some. We assessed mTOR colocalization with lysosomes in downstream of mTORC1, was increased in Fnip12/2 versus Fnip12/2 versus Fnip1+/2 B cell progenitors using IC staining for Fnip1+/2 pro–B cells (Fig. 4B). Although mTORC1 activity mTOR and LAMP2, followed by visualization with ImageStream decreases in Fnip12/2 pro–B cells in response to amino acid re- imaging flow cytometry. As expected, mTOR/lysosomal colocal- striction, it remains inappropriately high relative to Fnip1+/2 ization decreased in response to amino acid restriction in control controls, both in the presence or absence of amino acids (Fig. 4B). cells (Fig. 4C, left panel). However, Fnip12/2 cells exhibited To address if Fnip1 modulates the pre–B cell proteome, we much higher basal levels of mTOR/lysosomal colocalization, 2904 Fnip1 ENABLES REGULATION OF AMPK, mTORC1, AND TFE3 IN B CELLS

FIGURE 3. Disruption of Fnip1 increases autophagy induction and flux. Bar graphs show mean fluorescence intensities (MFIs) of (A) IC p-AMPK T172 staining (left panel) and IC p-Ulk1 S555 (right panel) on live-gated B220+IgM2 cells. (B) MFIs of EGFP-LC3 and representative images (imaging cytom- etry)ofB220+IgM2GFP+ BM cells from EGFP-LC3 Fnip1+/2 or EGFP-LC3 Fnip12/2 mice without or with rapamycin or arginine and lysine-free media for 24 h as measured by flow cytometry. Scale bars, 7 mm. p , 0.0001, one-way ANOVA. (C)Frequency of B220+IgM2 BM cells from Fnip1+/2 or Fnip12/2 mice with a high number of LAMP2 (lysosomal marker) spots, as mea- sured by ImageStream imaging flow cytom- etry, without or with 2 h of incubation in media without amino acids. p , 0.0001, one- + 2 way ANOVA. (D)MFIofB220IgM BM Downloaded from cells from Fnip1+/2 or Fnip12/2 mice incu- bated with fluorescent phospholipid ana- logue NBD-phosphatidylserine for 1 min. (E) Mean bright detail similarity (BDS) of GFP-LC3 and DyLight 650 LAMP2 from B220+IgM2GFP+ BM cells from EGFP-LC3 2 2 2 Fnip1+/ or EGFP-LC3 Fnip1 / mice cul- http://www.jimmunol.org/ tured 24 h ex vivo in complete media or media without amino acids supplemented with 10 nM bafilomycin A1 (Baf A1) (blocks autophagy through the inhibition of lyso- somal degradation), as measured by imag- ing cytometry. Mean BDS is a measure of colocalization. (F) Relative autophagic flux as measured by IC flow cytometric staining of LC3-II (autophagosome-bound LC3) by guest on September 24, 2021 in B220+IgM2 BM cells from Fnip1+/2 or Fnip12/2 mice. The ratio of LC3-II after 2 h of 100 nM Baf A1 treatment com- pared with basal levels of LC3-II is shown. *p , 0.05, **p , 0.001, ***p , 0.0001, ****p , 0.00001. which decreased upon amino acid starvation but remained aber- mice (1), which disrupts mTORC1 activation during B cell de- rantly high (Fig. 4C). We also observed increased colocalization velopment. Inhibition of mTORC1 did not restore B cell de- of mTOR with lysosomes in MEFs derived from Fnip12/2 mice velopment in Fnip1-andRaptor-double deficient animals using fluorescence microscopy (Fig. 4D). Relative to Fnip1+/2 (Fnip12/2Raptorfl/flMb1Cre+ mice) (Supplemental Fig. 2A–C) control MEFs under nutrient-rich conditions, which should show or Fnip12/2Raptorfl/+Mb1Cre+ mice (data not shown), which the most colocalization, mTOR remained associated with lyso- express attenuated levels of Raptor. These results are consistent somes during amino acid deprivation in Fnip12/2 MEFs, indi- with our previous study showing that prolonged treatment of cating that Fnip1 is necessary for optimal inactivation of mTOR. Fnip12/2 mice with the mTORC1 inhibitor rapamycin was also We next determined whether increased (inappropriate) locali- unable to rescue B cell development (7). zation of mTOR to the lysosome in Fnip12/2 mice might impact cell survival in response to the deprivation of arginine or lysine, Increased mTORC1-mediated cell growth does not inhibit which have been shown to be important for activating mTOR (33). pre–B cell development and fails to restore B cell development We observed decreased numbers of B220+ lymphocytes in Fnip12/2 in Fnip1-deficient mice BM cultured for 24 h in the absence of lysine or arginine com- Because mTORC1 activity is essential for pre–B cell development, pared with control BM, which displayed no significant loss of we next addressed whether the high mTORC1 activity we ob- B cells in response to amino acid deprivation (Fig. 4E). These served in Fnip12/2 pre–B cells could be compensatory for Fnip1 results suggest that Fnip1 may help terminate mTORC1 activation deficiency. Thus, we tested whether further increasing mTORC1 and increase cell survival, in part, by enabling mTOR transloca- activation would restore B cell development in Fnip12/2 mice. tion from the lysosomal surface to the cytosol in response to We disrupted Tsc1 (a negative regulator of mTORC1) by amino acid deprivation. breeding B cell–specific Tsc1 null mice (Tsc1fl/flMb1-Cre)to In light of these findings, we wondered whether genetic inhi- Fnip12/2 mice to generate Tsc1 and Fnip1-double deficient bition of mTORC1 activation could restore B cell development in mice. Fnip12/2Tsc1fl/flMb1-Cre animals develop renal disease Fnip12/2 mice. Fnip12/2 mice were crossed with Raptorfl/flMb1-Cre before 4 wk of age (29), so we transplanted total BM cells from The Journal of Immunology 2905 Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 4. mTORC1 signaling is aberrantly increased in Fnip1-deficient B cell progenitors. (A) Forward scatter (FSC-A, a measure of cell size) of BM B cells as measured by flow cytometry. Summary data from Fnip1+/2 or Fnip12/2 mice. Late pro–B cell: B220+BP-1+CD252; early pre–B cell: B220+BP-1+CD25+; late pre–B cell: B220+BP-12CD25+; immature B cell: B220loIgM+; and mature B cell: B220hiIgM+.(B) Representative dot plots (left panel) or summary mean fluorescence intensity (MFI) flow cytometry data without and with incubation in amino acid–free media (right panel) of IC p-S6R (P-S6R) staining (mTORC1 downstream substrate) from B220+IgM2 BM of Fnip1+/2 or Fnip12/2 mice. p , 0.0001, one-way ANOVA (C) Frequency of cells with colocalization (left panel) or representative images (right panels) of mTOR (red) and LAMP2 (green) IC staining in Fnip1+/2 or Fnip12/2 BM gated B220+IgM2, as measured by imaging flow cytometry, without (left) or with (right) overnight incubation in amino acid–free media. Scale bar, 7 mm. p , 0.0001, one-way ANOVA. (D) Representative immunofluorescent images from Fnip1+/2 or Fnip12/2 MEFs stained with anti-mTOR (red) and anti- LAMP2 (green) Abs (original magnification 3200). Cells were cultured ex vivo in amino acid–free media for 50 min 6 refeeding with amino acids (13 concentration) for 10 min. Pearson coefficient representing colocalization is shown (0 = random distribution, 1 = 100% colocalization). (E) Repre- sentative dot plots of B220+IgM+ cells (left panels) and frequency of B220+ lymphocytes (right panel) in Fnip1+/2 or Fnip12/2 BM after 4 d ex vivo culture in complete media, media without lysine, or media without arginine. *p , 0.05, **p , 0.001, ***p , 0.0001, ****p , 0.00001.

Fnip12/2Tsc1fl/flMb1-Cre animals into irradiated Rag22/2Il2rg2/2 were increased in Tsc1fl/flMb-1Cre and Fnip12/2Tsc1fl/flMb-1Cre or CD45.1 mismatched recipients. In accordance with high levels of animals relative to Fnip12/2 mice (Supplemental Fig. 3A and data mTORC1 signaling, both cell size and IC levels of p-S6R protein not shown). However, Tsc1 deficiency failed to rescue B cell 2906 Fnip1 ENABLES REGULATION OF AMPK, mTORC1, AND TFE3 IN B CELLS development, although a modest increase in B220loCD25+ late pre– conditions (Fig. 4C, 4D). Increased TFE3 nuclear localization also B cells and B220loCD432IgM2 pro– and pre–B cells was noted in correlated with increased transcriptional activation of known TFE3 the BM (Supplemental Fig. 3B). Unlike Raptor deficiency, B cell– target genes, including Gpnmb, Pgc1a, Rragd,andLamp1 (Fig. 5B) specific Tsc1 deficiency does not inhibit B cell development. This (7). These results collectively suggest that in B cell progenitors, evidence combined with the observation that conditional deletion of Fnip1 is required for the retention of TFE3 in the cytosol under Ampka1 in B cells (Ampka1fl/flMb1-Cre) does not rescue B cell nutrient replete conditions when mTORC1 is activated. development in Fnip12/2 mice (Supplemental Fig. 3C) indicate that aberrant mTORC1 or AMPK activation alone are not respon- Discussion sible for the developmental block in Fnip12/2 B cell progenitors. Constitutive disruption of Fnip1 in mice results in a complete absence of mature B cells and Abs because of a block in B cell Fnip1 is necessary for TFE3 inactivation in pro– and development at the pre–B cell stage (7, 8) and decreased survival pre–B cells of B cell progenitors in response to metabolic stressors, such as Both mTORC1 and the Flcn/Fnip1/Fnip2 complex have been nutrient deprivation and pre-BCR cross-linking. In this study, we implicated in inhibiting lysosomal biogenesis through phosphor- found that increased apoptosis occurred independently of p53 and ylation and cytoplasmic retention of TFE3 (34–37), which is that rescuing pre–B cell survival with a Bcl-xL transgene failed to considered a master regulator of lysosome gene expression. Be- efficiently restore mature B cell development. These results sug- cause mTORC1 activity and lysosomal biogenesis were both in- gest that Fnip1 regulates B cell development through mechanisms creased in Fnip12/2 B cell progenitors, we hypothesized that that are only partially dependent on pre–B cell survival. Although disruption of Fnip1 leads to increased TFE3 nuclear localization. our results differ from previously published results showing Downloaded from To investigate this hypothesis, we used imaging flow cytometry to that Bcl-2 expression was able to rescue B cell development in 2/2 +/2 examine the IC localization of TFE3 in Fnip1 and Fnip1 Fnip1-deficient mice (8, 38), Bcl-2 and Bcl-xL are not completely B cell progenitors. TFE3 nuclear localization, based on colocali- functionally redundant; Bcl-xL is more stable than Bcl-2 (39) zation of TFE3 and DAPI staining, increased in response to amino and inhibits both Bax and Bak, direct effectors of mitochondrial 2/2 acid restriction in both control and Fnip1 pro–/pre–B cells as apoptosis, whereas Bcl-2 does not do so. Bcl-XL and Bcl-2 2/2

expected (Fig. 5A). Notably, Fnip1 cells displayed signifi- have different effects on hematopoietic cell fate when ectopi- http://www.jimmunol.org/ cantly higher TFE3 nuclear localization than control cells both in cally expressed in multipotent progenitor cells; Bcl-xL supports the presence and absence of essential amino acids (Fig. 5A), de- erythroid cell fate, whereas Bcl-2 supports myeloid cell fates (40). spite higher levels of lysosome-associated mTORC1 under the same Our studies suggest that the unique functions of Fnip1 in B cells by guest on September 24, 2021

FIGURE 5. Fnip1 regulates TFE3 nuclear localization in B cell progenitors. (A) Median similarity (a measure of image overlap, lower panel) of TFE3 IC staining and nuclear staining (DAPI) and representative images (upper pan- els) of B220+IgM2 cells in Fnip1+/2 or Fnip12/2 BM as measured by imaging flow cytometry. Scale bars 7 mm. p , 0.0001, one- way ANOVA. (B) Expression of selected TFE3 target genes as measured using Illumina cDNA microarray of FACs-sorted B220+IgM2 pro– and pre–B cells, Fnip12/2 versus WT (n =3 mice per group). (C) Schematic model of sig- naling inside Fnip1-deficient B cells compared with normal B cells under nutrient-replete or amino acid–restricted conditions, showing dysregulation of mTORC1 and TFE3 locali- zation in the absence of Fnip1. **p , 0.001, ****p , 0.00001. The Journal of Immunology 2907 include enabling efficient mTORC1 dissociation from the lyso- rescue B cell development in Fnip12/2 mice. In addition, increasing some in response to amino acid deprivation and limiting the mTORC1 activity through B cell–specific disruption of Tsc1 did not translocation of TFE3 to the nucleus. These results highlight recapitulate the block in the early development of Fnip12/2 Bcells. multiple mechanisms whereby Fnip1 maintains metabolic ho- These results indicate that additional factors may contribute to meostasis during B cell development. impaired B cell development in Fnip12/2 mice. Indeed, we found Fnip1 was cloned based on physical interactions with Flcn and all increased nuclear localization of the TFE3 transcription factor in three subunits of AMPK. AMPK phosphorylates both Fnip1 Fnip1-deficient pro– and pre–B cells relative to control cells. Nu- and Flcn, although the consequences of this modification remain clear localization of TFE3 correlated with increased lysosome unclear. In this study, we found that AMPK was activated in numbers and activity, mitochondrial biogenesis, expression of TFE3 Fnip1-deficient pro–B/pre–B cells, which correlated with increased target genes (7), and autophagy. Accordingly, previous studies AMPK-mediated phosphorylation of ULK1 (a positive regulator of showed that inactivation of FLCN in human renal cancer cell lines autophagy), increased induction of autophagy, and increased flux of induced TFE3 transcriptional activity by increasing its nuclear lo- autophagy relative to control cells. Elevated levels of autophagy in calization (36) and that inactivation of FLCN in human fetal lung Fnip12/2 B cell progenitors did not increase further in response to fibroblasts inhibited canonical WNT signaling, which could be amino acid deprivation, consistent with the notion that autophagy completely restored only by silencing TFE3 (45). Other studies flux was near maximum. Previous studies have suggested that have similarly reported that the Flcn/Fnip1/Fnip2 complex is autophagy is also increased in flies lacking the Drosophila Flcn required for mouse embryonic stem cell (35), hematopoietic homolog DBHD (41) as well as in Caenorhabditis elegans and stem cell (46), and osteoclast (37) differentiation by restricting

MEFs lacking Flcn (42). In addition, another group reported that nuclear localization and activity of TFE3. Downloaded from basal autophagy was increased in Fnip12/2 B cell progenitors (38). Previous studies have shown that mTORC1 inhibits TFE3 tran- These previous studies are consistent with our data showing in- scriptional activity by phosphorylating and inhibiting TFE3 nuclear creased autophagic flux in pro– and pre–B cells following the dis- translocation (35). The apparent paradox of high levels of mTORC1 ruption of Fnip1. However, Dunlop et al. (42) found that Flcn and activation concurrent with nuclear TFE3 in Fnip1-deficient pre– Fnip1 positively regulate autophagy in human kidney cells via in- B cells was similarly seen by Wada et al. (34) in Flcn-deficient

teraction with GABARAP, an autophagy component that is crucial murine adipose tissue. In that study, mTORC1-mediated phosphor- http://www.jimmunol.org/ for autophagosome formation and sequestration of cytosolic ylation and cytoplasmic retention of TFE3 was dependent on Flcn, cargo into vesicles. Increased levels of SQSTM-1 and LC3 were whereas phosphorylation of other downstream substrates such as observed in Flcn-deficient MEFs and renal tumor cells from a S6K appeared independent of Flcn function. This may be explained BHDS patient but were interpreted as indicative of impaired by the findings that the Flcn/Fnip complex acts as a GTPase acti- autophagy, whereas autophagic flux was not examined. These vating proteins converting RagC/DGTP to RagC/DGDP (31), and studies highlight the complexity of the interactions between the TFE3 is recruited to the lysosome by binding inactive RagC/DGDP Flcn/Fnip1/Fnip2 complex and autophagic pathways, suggesting (47). Thus, despite increased mTORC1 activity and lysosomal lo- that the impact of Flcn/Fnip deficiency on autophagic flux may calization in the absence of the Fnip1/Flcn complex, TFE3 may not be tissue dependent and/or influenced by the energetic state of be recruited to the lysosome to be phosphorylated by mTORC1. The by guest on September 24, 2021 the tissues examined. resulting aberrantly high levels of TFE3 transcriptional activity may Surprisingly, despite increased autophagy in Fnip12/2 B cell contribute to impaired B cell development as has been observed with progenitors, we also found that mTORC1 activity and lysosomal hematopoietic stem cell and osteoclast differentiation. The ability of localization were aberrantly increased, even under conditions of mTORC1 to phosphorylate and inhibit nuclear localization of TFE3/ AMPK activation and amino acid restriction when mTORC1 Tfeb in a Flcn/Fnip1/Fnip2-dependent manner may be a conserved should be cytosolic and inactive. These results suggest that at mechanism enabling nutrient-sensing pathways to regulate the dif- least in some cell types, Fnip1 may be necessary for AMPK to ferentiation of immune and other cell types. efficiently inactivate mTORC1, which is consistent with findings by others in human cell lines and MEFs. In particular, Nagashima Acknowledgments et al. (43) found that ubiquitination and degradation of Fnip2 led We thank T. Chang for assistance with ImageStream data collection and to the dissociation of Flcn from the lysosome and constitutive analysis and R. Shaw for immortalization of Fnip12/2 MEFs. We thank association and activation of mTOR at the lysosomal surface re- L. Kang, J. Chan, and M. Torres for help with the mouse colony. gardless of nutrient status. Zhang et al. (44) demonstrated that the scaffold protein Axin tethers AMPK to the surface of endosomes Disclosures and lysosomes, where it is activated by the v-ATPase–Regulator The authors have no financial conflicts of interest. complex in response to glucose deprivation. These studies col- lectively suggest that the Fnip1/Fnip2/Flcn complex could serve as a molecular scaffold allowing AMPK to interact with the References lysosomal v-ATPase at the surface of lysosomes, thus modulat- 1. Iwata, T. N., J. A. Ramı´rez, M. Tsang, H. Park, D. H. Margineantu, ing the ability of AMPK to inactivate mTORC1 under nutrient- D. M. Hockenbery, and B. M. Iritani. 2016. Conditional disruption of raptor reveals an essential role for mTORC1 in B cell development, survival, and poor conditions. With both catabolic and anabolic pathways metabolism. J. Immunol. 197: 2250–2260. active concurrently, our results suggest that a significant energy 2. Ramadani, F., D. J. Bolland, F. Garcon, J. L. Emery, B. Vanhaesebroeck, imbalance in Fnip1-deficient B cell progenitors precludes A. E. Corcoran, and K. Okkenhaug. 2010. The PI3K isoforms p110alpha and p110delta are essential for pre-B cell receptor signaling and B cell development. survival during cellular stresses such as amino acid restriction Sci. Signal. 3: ra60. because mTORC1-driven nutrient consumption remains in- 3. Baracho, G. V., M. H. Cato, Z. Zhu, O. R. Jaren, E. Hobeika, M. Reth, and flexibly high. R. C. Rickert. 2014. PDK1 regulates B cell differentiation and homeostasis. Proc. Natl. Acad. Sci. USA 111: 9573–9578. Despite significant elevations in mTORC1 and AMPK activity 4. Yu, M., Y. Chen, H. Zeng, Y. Zheng, G. Fu, W. Zhu, U. Broeckel, P. Aggarwal, following the disruption of Fnip1, we also found that deletion of the A. Turner, G. Neale, et al. 2017. PLCg-dependent mTOR signalling controls IL-7-mediated early B cell development. Nat. Commun. 8: 1457. AMPKa1 subunit or attenuation of mTORC1 activity through the 5. Zeng, H., M. Yu, H. Tan, Y. Li, W. Su, H. Shi, Y. Dhungana, C. Guy, G. Neale, disruption of Raptor or treatment with rapamycin were unable to C. Cloer, et al. 2018. Discrete roles and bifurcation of PTEN signaling and 2908 Fnip1 ENABLES REGULATION OF AMPK, mTORC1, AND TFE3 IN B CELLS

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