Abstract
Multiple myeloma (MM) and chronic lymphocytic leukemia (CLL) cells must attach to the bone marrow (BM) microvasculature before lodging in the BM microenvironment. Using intravital microscopy (IVM) of the BM calvariae we demonstrate that the α4β1 integrin is required for MM and CLL cell firm arrest onto the BM microvasculature, while endothelial P-selectin and E-selectin mediate cell rolling. Talin, kindlin-3 and ICAP-1 are β1-integrin-binding partners that regulate β1-mediated cell adhesion. We show that talin and kindlin-3 cooperatively stimulate high affinity and strength of α4β1-dependent MM and CLL cell attachment, whereas ICAP-1 negatively regulates this adhesion. A functional connection between talin/kindlin-3 and Rac1 was found to be required for MM cell attachment mediated by α4β1. Importantly, IVM analyses with talin- and kindlin-3-silenced MM cells indicate that these proteins are needed for cell arrest on the BM microvasculature. Instead, MM cell arrest is repressed by ICAP-1. Moreover, MM cells silenced for talin and kindlin-3, and cultured on α4β1 ligands showed higher susceptibility to bortezomib-mediated cell apoptosis. Our results highlight the requirement of α4β1 and selectins for the in vivo attachment of MM and CLL cells to the BM microvasculature, and indicate that talin, kindlin-3 and ICAP-1 differentially control physiological adhesion by regulating α4β1 activity.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Anderson KC, Carrasco RD . Pathogenesis of myeloma. Annu Rev Pathol 2011; 6: 249–274.
Kuehl WM, Bergsagel PL . Molecular pathogenesis of multiple myeloma and its premalignant precursor. J Clin Invest 2012; 122: 3456–3463.
Chiorazzi N, Rai KR, Ferrarini M . Chronic lymphocytic leukemia. N Engl J Med 2005; 352: 804–815.
Morgan GJ, Walker BA, Davies FE . The genetic architecture of multiple myeloma. Nat Rev Cancer 2012; 12: 335–348.
Damiano JS, Cress AE, Hazlehurst LA, Shtil AA, Dalton WS . Cell adhesion mediated drug resistance (CAM-DR): role of integrins and resistance to apoptosis in human myeloma cell lines. Blood 1999; 93: 1658–1667.
Noborio-Hatano K, Kikuchi J, Takatoku M, Shimizu R, Wada T, Ueda M et al. Bortezomib overcomes cell-adhesion-mediated drug resistance through downregulation of VLA-4 expression in multiple myeloma. Oncogene 2009; 28: 231–242.
Nowakowski GS, Witzig TE, Dingli D, Tracz MJ, Gertz MA, Lacy MQ et al. Circulating plasma cells detected by flow cytometry as a predictor of survival in 302 patients with newly diagnosed multiple myeloma. Blood 2005; 106: 2276–2279.
Ghobrial IM . Myeloma as a model for the process of metastasis: implications for therapy. Blood 2012; 120: 20–30.
Azab AK, Quang P, Azab F, Pitsillides C, Thompson B, Chonghaile T et al. P-selectin glycoprotein ligand regulates the interaction of multiple myeloma cells with the bone marrow microenvironment. Blood 2012; 119: 1468–1478.
Uchiyama H, Barut BA, Chauhan D, Cannistra SA, Anderson KC . Characterization of adhesion molecules on human myeloma cell lines. Blood 1992; 80: 2306–2314.
Lokhorst HM, Lamme T, de Smet M, Klein S, de Weger RA, van Oers R et al. Primary tumor cells of myeloma patients induce interleukin-6 secretion in long-term bone marrow cultures. Blood 1994; 84: 2269–2277.
Sanz-Rodriguez F, Ruiz-Velasco N, Pascual-Salcedo D, Teixido J . Characterization of VLA-4-dependent myeloma cell adhesion to fibronectin and VCAM-1. Br J Haematol 1999; 107: 825–834.
Sanz-Rodriguez F, Hidalgo A, Teixido J . Chemokine stromal cell-derived factor-1alpha modulates VLA-4 integrin-mediated multiple myeloma cell adhesion to CS-1/fibronectin and VCAM-1. Blood 2001; 97: 346–351.
Hideshima T, Chauhan D, Schlossman R, Richardson P, Anderson KC . The role of tumor necrosis factor alpha in the pathophysiology of human multiple myeloma: therapeutic applications. Oncogene 2001; 20: 4519–4527.
Till KJ, Spiller DG, Harris RJ, Chen H, Zuzel M, Cawley JC . CLL, but not normal, B cells are dependent on autocrine VEGF and alpha4beta1 integrin for chemokine-induced motility on and through endothelium. Blood 2005; 105: 4813–4819.
Redondo-Munoz J, Escobar-Diaz E, Samaniego R, Terol MJ, Garcia-Marco JA, Garcia-Pardo A . MMP-9 in B-cell chronic lymphocytic leukemia is up-regulated by alpha4beta1 integrin or CXCR4 engagement via distinct signaling pathways, localizes to podosomes, and is involved in cell invasion and migration. Blood 2006; 108: 3143–3151.
Hartmann TN, Grabovsky V, Wang W, Desch P, Rubenzer G, Wollner S et al. Circulating B-cell chronic lymphocytic leukemia cells display impaired migration to lymph nodes and bone marrow. Cancer Res 2009; 69: 3121–3130.
Brachtl G, Sahakyan K, Denk U, Girbl T, Alinger B, Hofbauer SW et al. Differential bone marrow homing capacity of VLA-4 and CD38 high expressing chronic lymphocytic leukemia cells. PLoS One 2011; 6: e23758.
Binsky I, Lantner F, Grabovsky V, Harpaz N, Shvidel L, Berrebi A et al. TAp63 regulates VLA-4 expression and chronic lymphocytic leukemia cell migration to the bone marrow in a CD74-dependent manner. J Immunol 2010; 184: 4761–4769.
Bailon E, Ugarte-Berzal E, Amigo-Jimenez I, Van den Steen P, Opdenakker G, Garcia-Marco JA et al. Overexpression of progelatinase B/proMMP-9 affects migration regulatory pathways and impairs chronic lymphocytic leukemia cell homing to bone marrow and spleen. J Leukoc Biol 2014; 96: 185–199.
Till KJ, Lin K, Zuzel M, Cawley JC . The chemokine receptor CCR7 and alpha4 integrin are important for migration of chronic lymphocytic leukemia cells into lymph nodes. Blood 2002; 99: 2977–2984.
Bulian P, Shanafelt TD, Fegan C, Zucchetto A, Cro L, Nuckel H et al. CD49d is the strongest flow cytometry-based predictor of overall survival in chronic lymphocytic leukemia. J Clin Oncol 2014; 32: 897–904.
Till KJ, Harris RJ, Linford A, Spiller DG, Zuzel M, Cawley JC . Cell motility in chronic lymphocytic leukemia: defective Rap1 and alphaLbeta2 activation by chemokine. Cancer Res 2008; 68: 8429–8436.
Montresor A, Bolomini-Vittori M, Simon SI, Rigo A, Vinante F, Laudanna C . Comparative analysis of normal versus CLL B-lymphocytes reveals patient-specific variability in signaling mechanisms controlling LFA-1 activation by chemokines. Cancer Res 2009; 69: 9281–9290.
Pye DS, Rubio I, Pusch R, Lin K, Pettitt AR, Till KJ . Chemokine unresponsiveness of chronic lymphocytic leukemia cells results from impaired endosomal recycling of Rap1 and is associated with a distinctive type of immunological anergy. J Immunol 2013; 191: 1496–1504.
Redondo-Munoz J, Ugarte-Berzal E, Terol MJ, Van den Steen PE, Hernandez del Cerro M, Roderfeld M et al. Matrix metalloproteinase-9 promotes chronic lymphocytic leukemia b cell survival through its hemopexin domain. Cancer Cell 2010; 17: 160–172.
Ten Hacken E, Burger JA . Microenvironment dependency in Chronic Lymphocytic Leukemia: The basis for new targeted therapies. Pharmacol Ther 2014; 144: 338–348.
Ley K, Laudanna C, Cybulsky MI, Nourshargh S . Getting to the site of inflammation: the leukocyte adhesion cascade updated. Nat Rev Immunol 2007; 7: 678–689.
Garcia-Bernal D, Redondo-Munoz J, Dios-Esponera A, Chevre R, Bailon E, Garayoa M et al. Sphingosine-1-phosphate activates chemokine-promoted myeloma cell adhesion and migration involving alpha4beta1 integrin function. J Pathol 2013; 229: 36–48.
Azab AK, Azab F, Blotta S, Pitsillides CM, Thompson B, Runnels JM et al. RhoA and Rac1 GTPases play major and differential roles in stromal cell-derived factor-1-induced cell adhesion and chemotaxis in multiple myeloma. Blood 2009; 114: 619–629.
Kim C, Ye F, Ginsberg MH . Regulation of integrin activation. Annu Rev Cell Dev Biol 2011; 27: 321–345.
Moser M, Legate KR, Zent R, Fassler R . The tail of integrins, talin, and kindlins. Science 2009; 324: 895–899.
Calderwood DA, Campbell ID, Critchley DR . Talins and kindlins: partners in integrin-mediated adhesion. Nat Rev Mol Cell Biol 2013; 14: 503–517.
Lefort CT, Rossaint J, Moser M, Petrich BG, Zarbock A, Monkley SJ et al. Distinct roles for talin-1 and kindlin-3 in LFA-1 extension and affinity regulation. Blood 2012; 119: 4275–4282.
Manevich E, Grabovsky V, Feigelson SW, Alon R . Talin 1 and paxillin facilitate distinct steps in rapid VLA-4-mediated adhesion strengthening to vascular cell adhesion molecule 1. J Biol Chem 2007; 282: 25338–25348.
Garcia-Bernal D, Parmo-Cabanas M, Dios-Esponera A, Samaniego R, Hernan PdlOD, Teixido J . Chemokine-induced Zap70 kinase-mediated dissociation of the Vav1-talin complex activates alpha4beta1 integrin for T cell adhesion. Immunity 2009; 31: 953–964.
Manevich-Mendelson E, Grabovsky V, Feigelson SW, Cinamon G, Gore Y, Goverse G et al. Talin1 is required for integrin-dependent B lymphocyte homing to lymph nodes and the bone marrow but not for follicular B-cell maturation in the spleen. Blood 2010; 116: 5907–5918.
Ebisuno Y, Katagiri K, Katakai T, Ueda Y, Nemoto T, Inada H et al. Rap1 controls lymphocyte adhesion cascade and interstitial migration within lymph nodes in RAPL-dependent and -independent manners. Blood 2010; 115: 804–814.
Moser M, Bauer M, Schmid S, Ruppert R, Schmidt S, Sixt M et al. Kindlin-3 is required for beta2 integrin-mediated leukocyte adhesion to endothelial cells. Nat Med 2009; 15: 300–305.
Manevich-Mendelson E, Feigelson SW, Pasvolsky R, Aker M, Grabovsky V, Shulman Z et al. Loss of Kindlin-3 in LAD-III eliminates LFA-1 but not VLA-4 adhesiveness developed under shear flow conditions. Blood 2009; 114: 2344–2353.
Chang DD, Wong C, Smith H, Liu J . ICAP-1, a novel beta1 integrin cytoplasmic domain-associated protein, binds to a conserved and functionally important NPXY sequence motif of beta1 integrin. J Cell Biol 1997; 138: 1149–1157.
Zhang XA, Hemler ME . Interaction of the integrin beta1 cytoplasmic domain with ICAP-1 protein. J Biol Chem 1999; 274: 11–19.
Degani S, Balzac F, Brancaccio M, Guazzone S, Retta SF, Silengo L et al. The integrin cytoplasmic domain-associated protein ICAP-1 binds and regulates Rho family GTPases during cell spreading. J Cell Biol 2002; 156: 377–387.
Bouvard D, Millon-Fremillon A, Dupe-Manet S, Block MR, Albiges-Rizo C . Unraveling ICAP-1 function: toward a new direction? Eur J Cell Biol 2006; 85: 275–282.
Bouvard D, Aszodi A, Kostka G, Block MR, Albiges-Rizo C, Fassler R . Defective osteoblast function in ICAP-1-deficient mice. Development 2007; 134: 2615–2625.
Stacchini A, Aragno M, Vallario A, Alfarano A, Circosta P, Gottardi D et al. MEC1 and MEC2: two new cell lines derived from B-chronic lymphocytic leukaemia in prolymphocytoid transformation. Leuk Res 1999; 23: 127–136.
Garcia-Bernal D, Wright N, Sotillo-Mallo E, Nombela-Arrieta C, Stein JV, Bustelo XR et al. Vav1 and Rac Control Chemokine-promoted T Lymphocyte Adhesion Mediated by the Integrin {alpha}4{beta}1. Mol Biol Cell 2005; 16: 3223–3235.
Garcia-Bernal D, Sotillo-Mallo E, Nombela-Arrieta C, Samaniego R, Fukui Y, Stein JV et al. DOCK2 is required for chemokine-promoted human T lymphocyte adhesion under shear stress mediated by the integrin alpha4beta1. J Immunol 2006; 177: 5215–5225.
Parmo-Cabanas M, Bartolome RA, Wright N, Hidalgo A, Drager AM, Teixido J . Integrin alpha4beta1 involvement in stromal cell-derived factor-1alpha-promoted myeloma cell transendothelial migration and adhesion: role of cAMP and the actin cytoskeleton in adhesion. Exp Cell Res 2004; 294: 571–580.
Hidalgo A, Weiss LA, Frenette PS . Functional selectin ligands mediating human CD34(+) cell interactions with bone marrow endothelium are enhanced postnatally. J Clin Invest 2002; 110: 559–569.
Mazo IB, Gutierrez-Ramos JC, Frenette PS, Hynes RO, Wagner DD, von Andrian UH . Hematopoietic progenitor cell rolling in bone marrow microvessels: parallel contributions by endothelial selectins and vascular cell adhesion molecule 1. J Exp Med 1998; 188: 465–474.
Luque A, Gomez M, Puzon W, Takada Y, Sanchez-Madrid F, Cabanas C . Activated conformations of very late activation integrins detected by a group of antibodies (HUTS) specific for a novel regulatory region (355-425) of the common beta 1 chain. J Biol Chem 1996; 271: 11067–11075.
Uchiyama H, Barut BA, Mohrbacher AF, Chauhan D, Anderson KC . Adhesion of human myeloma-derived cell lines to bone marrow stromal cells stimulates interleukin-6 secretion. Blood 1993; 82: 3712–3720.
Mori Y, Shimizu N, Dallas M, Niewolna M, Story B, Williams PJ et al. Anti-alpha4 integrin antibody suppresses the development of multiple myeloma and associated osteoclastic osteolysis. Blood 2004; 104: 2149–2154.
Olson DL, Burkly LC, Leone DR, Dolinski BM, Lobb RR . Anti-alpha4 integrin monoclonal antibody inhibits multiple myeloma growth in a murine model. Mol Cancer Ther 2005; 4: 91–99.
Katayama Y, Hidalgo A, Furie BC, Vestweber D, Furie B, Frenette PS . PSGL-1 participates in E-selectin-mediated progenitor homing to bone marrow: evidence for cooperation between E-selectin ligands and alpha4 integrin. Blood 2003; 102: 2060–2067.
Alsayed Y, Ngo H, Runnels J, Leleu X, Singha UK, Pitsillides CM et al. Mechanisms of regulation of CXCR4/SDF-1 (CXCL12)-dependent migration and homing in multiple myeloma. Blood 2007; 109: 2708–2717.
Davids MS, Burger JA . Cell Trafficking in Chronic Lymphocytic Leukemia. Open J Hematol 2012; 3: 1–13.
Acknowledgements
We thank the multiple myeloma and chronic lymphocytic leukemia patients who donated samples for this research. We thank Dr Pedro Lastres for his help in flow cytometry. We also thank JM Adrover for movie editing. This work was supported by the following grants from the Ministerio de Economía y Competitividad (Spain): SAF2011-24022 to JT; SAF2012-31613 to AG-P; SAF2012-31142 and SAF2013-49662-EXP to AH; PI13/01454 to PS-M; RD12/0036/0061 to JT and AG-P; RD12/0036/0058 to NCG. The work was also funded by grant P2010/BMD-2314 from the Comunidad de Madrid to AG-P, JT, PS-M and AH, and partially supported by a CRIS foundation grant to fight cancer to JM-L. NCG is also funded by a grant from the Asociación Española Contra el Cáncer (GCB120981SAN), and from the Gerencia Regional de Salud, Junta de Castilla y León (GRS-702/A/11). The CNIC is supported by the Pro-CNIC Foundation.
Author contributions
MM-M, ML and NA-M performed experiments and analyzed data. SS-M, SIV and NA-S performed some experiments, analyzed data and prepared valuable reagents. NG, RM, JM-L, IB and JAG-M collected and provided primary cells from MM and CLL patients. JT, AG-P, AH and PS-M designed the research and wrote the manuscript. All authors reviewed and approved the manuscript.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Competing interests
The authors declare no conflict of interest.
Additional information
Supplementary Information accompanies this paper on the Leukemia website
Supplementary information
Rights and permissions
About this article
Cite this article
Martínez-Moreno, M., Leiva, M., Aguilera-Montilla, N. et al. In vivo adhesion of malignant B cells to bone marrow microvasculature is regulated by α4β1 cytoplasmic-binding proteins. Leukemia 30, 861–872 (2016). https://doi.org/10.1038/leu.2015.332
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/leu.2015.332
This article is cited by
-
Identification of TLN1 as a prognostic biomarker to effect cell proliferation and differentiation in acute myeloid leukemia
BMC Cancer (2022)
-
Next generation proteomics with drug sensitivity screening identifies sub-clones informing therapeutic and drug development strategies for multiple myeloma patients
Scientific Reports (2021)
-
E-selectin ligands recognised by HECA452 induce drug resistance in myeloma, which is overcome by the E-selectin antagonist, GMI-1271
Leukemia (2017)