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GRASP55 Is Dispensable for Normal Hematopoiesis but Necessary for Myc-Dependent Leukemic Growth

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GRASP55 Is Dispensable for Normal Hematopoiesis but Necessary for Myc-Dependent Leukemic Growth GRASP55 Is Dispensable for Normal Hematopoiesis but Necessary for Myc-Dependent Leukemic Growth This information is current as Anne-Laure Bailly, Julien M. P. Grenier, Amandine of October 3, 2021. Cartier-Michaud, Florence Bardin, Marielle Balzano, Armelle Goubard, Jean-Claude Lissitzky, Maria De Grandis, Stéphane J. C. Mancini, Arnauld Serge and Michel Aurrand-Lions J Immunol published online 30 March 2020 http://www.jimmunol.org/content/early/2020/03/27/jimmun Downloaded from ol.1901124 Supplementary http://www.jimmunol.org/content/suppl/2020/03/27/jimmunol.190112 http://www.jimmunol.org/ Material 4.DCSupplemental Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists by guest on October 3, 2021 • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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 © 2020 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published March 30, 2020, doi:10.4049/jimmunol.1901124 The Journal of Immunology GRASP55 Is Dispensable for Normal Hematopoiesis but Necessary for Myc-Dependent Leukemic Growth Anne-Laure Bailly,* Julien M. P. Grenier,* Amandine Cartier-Michaud,* Florence Bardin,* Marielle Balzano,* Armelle Goubard,* Jean-Claude Lissitzky,* Maria De Grandis,† Ste´phane J. C. Mancini,* Arnauld Serge,* and Michel Aurrand-Lions* Grasp55 is a ubiquitous Golgi stacking protein involved in autophagy, protein trafficking, and glucose deprivation sensing. The function of Grasp55 in protein trafficking has been attributed to its PDZ-mediated interaction with the C-terminal PDZ-binding motifs of protein cargos. We have recently shown that such an interaction occurs between Grasp55 and the adhesion molecule Jam-C, which plays a central role in stemness maintenance of hematopoietic and spermatogenic cells. Accordingly, we have found that Grasp55-deficient mice suffer from spermatogenesis defects similar to Jam-C knockout mice. However, whether Grasp55 is involved in the maintenance of immunohematopoietic homeostasis through regulation of protein transport and Jam-C expression Downloaded from remains unknown. In this study, we show that Grasp55 deficiency does not affect hematopoietic stem cell differentiation, engraft- ment, or mobilization, which are known to depend on expression of Grasp55-dependent protein cargos. In contrast, using an Myc- dependent leukemic model addicted to autophagy, we show that knockdown of Grasp55 in leukemic cells reduces spleen and bone marrow tumor burden upon i.v. leukemic engraftment. This is not due to reduced homing of Grasp55-deficient cells to these organs but to increased spontaneous apoptosis of Grasp55-deficient leukemic cells correlated with increased sensitivity of the cells to glucose deprivation. These results show that Grasp55 plays a role in Myc-transformed hematopoietic cells but not in normal http://www.jimmunol.org/ hematopoietic cells in vivo. The Journal of Immunology, 2020, 204: 000–000. ematopoiesis and spermatogenesis are two differentia- MPPs (7). In agreement with its function in HSC maintenance, tion processes in which adult stem cells produce their JAM-C is downregulated in MPPs during steady-state hemato- H progeny in a sequentially ordered manner. It has been poiesis (2). JAM-C expression by HSC is also rapidly down- shown previously that JAM-C is expressed by hematopoietic stem regulated after HSC mobilization using cyclophosphamide and cells (HSC) and by male germ cells (1–3). In both biological G-CSF (8), suggesting that JAM-C may play a role in the switch models, JAM-C plays a role in the maintenance of tissue ho- from steady-state to emergency hematopoiesis. However, down- meostasis through adhesion regulation between developing cells regulation of JAM-C is not unique to G-CSF–induced mobiliza- by guest on October 3, 2021 and surrounding stromal cells expressing JAM-B, the high-affinity tion because downregulation of other bone marrow retention ligand of JAM-C (4, 5). During spermatogenesis, JAM-C regulates signals such as VCAM-1/VLA-4, CXCL12/CXCR4, or SCF/Kit polarization of developing spermatids, a critical step in sperma- have also been documented (9–11). tozoa development (3). During hematopoiesis, JAM-C interaction We have recently identified the Golgi reassembly–stacking with JAM-B plays a critical role in maintenance of HSC, which are protein of 55 kDa (Grasp55) as a PDZ domain–containing protein at the apex of hematopoietic hierarchy (6). HSC differentiate into interacting with the C-terminal PDZ-binding motif of Jam-B and more mature multipotent progenitors (MPPs), which have decreased Jam-C in mouse (12). Grasp55, and the related protein Grasp65, self-renewal capacity and progressively engage in megakaryocyte are highly conserved throughout evolution and play complemen- (MPP2), myeloid-biased (MPP3), or lymphoid-biased (MPP4) tary roles in Golgi cisternal stacking through oligomerization and *Equipe Labellise´e Ligue contre le Cancer, Aix Marseille University, CNRS, experiments. J.-C.L. performed lentiviral transductions. S.J.C.M. designed the INSERM, Institut Paoli-Calmettes, Cancerology Research Center of Marseille, Ma- flow cytometry panels and wrote the manuscript. A.S. performed imaging anal- rseille 13009, France; and †Etablissement Franc¸ais du Sang PACA Corse, Biologie ysis. M.A.-L. supervised the study, analyzed results, discussed data, and wrote the des Groupes Sanguins, UMR 7268, Aix Marseille Universite´, CNRS, Marseille manuscript. All authors provided valuable inputs on the manuscript. 13005, France Address correspondence and reprint requests to Michel Aurrand-Lions, Cancerology ORCIDs: 0000-0003-4141-1132 (J.-C.L.); 0000-0001-9255-4606 (S.J.C.M.); 0000- Research Center of Marseille, Aix Marseille University, CNRS, INSERM, Institut 0003-4271-3706 (A.S.); 0000-0002-8361-3034 (M.A.-L.). Paoli-Calmettes, 27 Boulevard Leı¨ Roure CS3005913273 Cedex 09, Marseille, Bou- ches du Rhone 13009, France. E-mail address: [email protected] Received for publication September 24, 2019. Accepted for publication March 3, 2020. The online version of this article contains supplemental material. This work was supported in part by grants from the French National Research Agency Abbreviations used in this article: BMDM, bone marrow–derived macrophage; Cy7, (ANR-BSV1-019-02 and A*MIDEX project ANR-11-IDEX-0001-02); Cance´ropoˆle cyanine 7; D35, day 35; 5-FU, 5-fluorouracil; Grasp55, Golgi reassembly–stacking D PACA; the Fondation ARC pour la Recherche sur le Cancer (PJA20141201990 [to protein of 55 kDa; Grasp55 Hem, hematopoietic-specific, Grasp55-deficient M.A.-L.], PJA20131200298 [to S.J.C.M.], and PJA20131200238 [to A.S.]); and the mice; GRASP55KD, GRASP55 knockdown; Grasp55WT, GRASP55 wild-type; FEDER, funded by the “Investissements d’Avenir” French Government Program and HSC, hematopoietic stem cell; HSPC, hematopoietic stem and progenitor cell; managed by the French National Research Agency. A.-L.B. and M.B. were respective LSK, LinNegScaPosKitPos; MEF, mouse embryonic fibroblast; MPP, multipotent Ph.D. student recipients of grants from Ligue contre le Cancer (TDQR15906) and progenitor; shRNA, short hairpin RNA; UPR, unfolded protein response. Fondation pour la Recherche Me´dicale (FDT20150532380). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the Copyright Ó 2020 by The American Association of Immunologists, Inc. 0022-1767/20/$37.50 manuscript. A.-L.B. and J.M.P.G. designed and performed experiments, analyzed results, and wrote the manuscript. A.C.-M., F.B., M.D.G., M.B., and A.G. performed www.jimmunol.org/cgi/doi/10.4049/jimmunol.1901124 2 GRASP55 IS DISPENSABLE FOR NORMAL HEMATOPOIESIS PDZ-mediated interactions with proteins of the golgin family, in Cancerology Research Center of Marseille Animal Facility (agree- such as Golgin45, GM130, or p24 (13–17). In addition, GRASP55 ment D13 055 04). Experimental protocols were approved by the depletion results in accelerated protein trafficking through the Ethical Committee no. 14 under the declaration number 02294.01. Golgi apparatus and has striking negative effects on protein gly- Genotyping cosylation and sorting (18). More recently, O-GlcNAcylation Genotyping of floxed Grasp55 line was achieved by PCR of genomic DNA has been shown to restrict the function of GRASP55 to Golgi extracted from mouse tails using the same primers and conditions as stacking, whereas de–O-GlcNAcylation switches its function as an constitutive Gorasp2 mice. However, resulting amplification of the 544-bp autophagosome–lysosome-tethering molecule (19). However, the fragment for the wild-type allele and the 700-bp fragment for the floxed function of GRASP55 is not limited to Golgi stacking and autophagy allele were both obtained using heterozygous mice. Genotyping of Mx1- cre and Vav-cre mice was achieved using the generic Cre and the iVav-Cre regulation because several studies have shown that GRASP55 is in- genotyping
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