Chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) are malignancies characterized by the dependence on B-cell receptor (BCR) signaling and by the high expression of ROR1, the cell surface receptor for Wnt-5a. Both, BCR and ROR1 are therapeutic targets in these diseases and the understanding of their mutual cross talk is thus of direct therapeutic relevance. In this study we analyzed the role of Lyn, a kinase from the Src family participating in BCR signaling, as a mediator of the BCR-ROR1 crosstalk. We confirm the functional interaction between Lyn and ROR1 and demonstrate that Lyn kinase efficiently phosphorylates ROR1 in its kinase domain and aids the recruitment of the E3 ligase c-CBL. We show that ROR1 surface dynamics in migrating primary CLL cells as well as chemotactic properties of CLL cells were inhibited by Lyn inhibitor dasatinib. Our data establish Lyn-mediated phosphorylation of ROR1 as a point of crosstalk between BCR and ROR1 signaling pathways.

Central European Institute of Technology Masaryk University Brno Czech Republic

Department of Biology Faculty of Medicine Masaryk University Brno Czech Republic

Department of Cytokinetics Institute of Biophysics Academy of Sciences of the Czech Republic v v i Brno Czech Republic

Department of Experimental Biology Faculty of Science Masaryk University Brno Czech Republic

Department of Internal Medicine Hematology and Oncology University Hospital Brno and Faculty of Medicine Masaryk University Brno Czech Republic

International Clinical Research Center St Anne's University Hospital Brno Czech Republic

National Centre for Biomolecular Research Faculty of Science Masaryk University Brno Czech Republic

Zobrazit více v PubMed

Akbarzadeh S., Wheldon L. M., Sweet S. M. M., Talma S., Mardakheh F. K., Heath J. K. (2008). The Deleted in Brachydactyly B Domain of ROR2 Is Required for Receptor Activation by Recruitment of Src. PLoS One. 3 (3), e1873. 10.1371/journal.pone.0001873 PubMed DOI PMC

Al-Shawi R., Ashton S. V., Underwood C., Simons J. P. (2001). Expression of the Ror1 and Ror2 Receptor Tyrosine Kinase Genes during Mouse Development. Development Genes Evol. 211 (4), 161–171. 10.1007/s004270100140 PubMed DOI

Barna G., Mihalik R., Timár B., Tömböl J., Csende Z., Sebestyén A., et al. (2011). ROR1 Expression Is Not a Unique Marker of CLL. Hematol. Oncol. 29 (1), 17–21. 10.1002/hon.948 PubMed DOI

Baskar S., Kwong K. Y., Hofer T., Levy J. M., Kennedy M. G., Lee E., et al. (2008). Unique Cell Surface Expression of Receptor Tyrosine Kinase ROR1 in Human B-Cell Chronic Lymphocytic Leukemia. Clin. Cancer Res. 14 (2), 396–404. 10.1158/1078-0432.ccr-07-1823 PubMed DOI

Baskar S., Wiestner A., Wilson W. H., Pastan I., Rader C. (2012). Targeting Malignant B Cells with an Immunotoxin against ROR1. MAbs. 4 (3), 349–361. 10.4161/mabs.19870 PubMed DOI PMC

Brown M. T., Cooper J. A. (1996). Regulation, Substrates and Functions of Src. Biochim. Biophys. Acta. 1287 (2–3), 121–149. 10.1016/0304-419x(96)00003-0 PubMed DOI

Burger J. A., Chiorazzi N. (2013). B Cell Receptor Signaling in Chronic Lymphocytic Leukemia. Trends Immunol. 34 (12), 592–601. 10.1016/j.it.2013.07.002 PubMed DOI PMC

Choi M. Y., Widhopf G. F., Wu C. C. N., Cui B., Lao F., Sadarangani A., et al. (2015). Pre-Clinical Specificity and Safety of UC-961, a First-In-Class Monoclonal Antibody Targeting ROR1. Clin. Lymphoma Myeloma Leuk. 15, S167. 10.1016/j.clml.2015.02.010 PubMed DOI PMC

Contri A., Brunati A. M., Trentin L., Cabrelle A., Miorin M., Cesaro L., et al. (2005). Chronic Lymphocytic Leukemia B Cells Contain Anomalous Lyn Tyrosine Kinase, a Putative Contribution to Defective Apoptosis. J. Clin. Invest. 115 (2), 369–378. 10.1172/jci200522094 PubMed DOI PMC

DaneshManesh A. H., Mikaelsson E., Jeddi‐Tehrani M., Bayat A. A., Ghods R., Ostadkarampour M., et al. (2008). Ror1, a Cell Surface Receptor Tyrosine Kinase Is Expressed in Chronic Lymphocytic Leukemia and May Serve as a Putative Target for Therapy. Int. J. Cancer. 123 (5), 1190–1195. 10.1002/ijc.23587 PubMed DOI

Das J., Chen P., Norris D., Padmanabha R., Lin J., Moquin R. V., et al. (2006). 2-Aminothiazole as a Novel Kinase Inhibitor Template. Structure−Activity Relationship Studies toward the Discovery of N-(2-Chloro-6-methylphenyl)-2-[[6-[4-(2-hydroxyethyl)-1- Piperazinyl)]-2-Methyl-4-Pyrimidinyl]amino)]-1,3-Thiazole-5-Carboxamide (Dasatinib, BMS-354825) as a Potent Pan-Src Kinase Inhibitor. J. Med. Chem. 49 (23), 6819–6832. 10.1021/jm060727j PubMed DOI

Dürig J., Nückel H., Cremer M., Führer A., Halfmeyer K., Fandrey J., et al. (2003). ZAP-70 Expression Is a Prognostic Factor in Chronic Lymphocytic Leukemia. Leukemia. 17 (12), 2426–2434. 10.1038/sj.leu.2403147 PubMed DOI

Fukuda T., Chen L., Endo T., Tang L., Lu D., Castro J. E., et al. (2008). Antisera Induced by Infusions of Autologous Ad-Cd154-Leukemia B Cells Identify ROR1 as an Oncofetal Antigen and Receptor for Wnt5a. Proc. Natl. Acad. Sci. 105 (8), 3047–3052. 10.1073/pnas.0712148105 PubMed DOI PMC

Gentile A., Lazzari L., Benvenuti S., Trusolino L., Comoglio P. M. (2014). The ROR1 Pseudokinase Diversifies Signaling Outputs in MET-Addicted Cancer Cells. Int. J. Cancer. 135 (10), 2305–2316. 10.1002/ijc.28879 PubMed DOI

Ghosh A. K., Secreto C., Boysen J., Sassoon T., Shanafelt T. D., Mukhopadhyay D., et al. (2011). The Novel Receptor Tyrosine Kinase Axl Is Constitutively Active in B-Cell Chronic Lymphocytic Leukemia and Acts as a Docking Site of Nonreceptor Kinases: Implications for Therapy. Blood. 117 (6), 1928–1937. 10.1182/blood-2010-09-305649 PubMed DOI PMC

Hallek M. (2019). Chronic Lymphocytic Leukemia: 2020 Update on Diagnosis, Risk Stratification and Treatment. Am. J. Hematol. 94 (11), 1266–1287. 10.1002/ajh.25595 PubMed DOI

Hasan M. K., Rassenti L., Widhopf G. F., Yu J., Kipps T. J. (2019). Wnt5a Causes ROR1 to Complex and Activate Cortactin to Enhance Migration of Chronic Lymphocytic Leukemia Cells. Leukemia. 33 (3), 653–661. 10.1038/s41375-018-0306-7 PubMed DOI PMC

Hasan M. K., Yu J., Chen L., Cui B., Widhopf II G. F., Rassenti L., et al. (2017). Wnt5a Induces ROR1 to Complex with HS1 to Enhance Migration of Chronic Lymphocytic Leukemia Cells. Leukemia. 31 (12), 2615–2622. 10.1038/leu.2017.133 PubMed DOI PMC

He F., Xiong W., Yu X., Espinoza-Lewis R., Liu C., Gu S., et al. (2008). Wnt5a Regulates Directional Cell Migration and Cell Proliferation via Ror2-Mediated Noncanonical Pathway in Mammalian Palate Development. Development. 135 (23), 3871–3879. 10.1242/dev.025767 PubMed DOI PMC

Ho H.-Y. H., Susman M. W., Bikoff J. B., Ryu Y. K., Jonas A. M., Hu L., et al. (2012). Wnt5a-Ror-Dishevelled Signaling Constitutes a Core Developmental Pathway that Controls Tissue Morphogenesis. Proc. Natl. Acad. Sci. 109 (11), 4044–4051. 10.1073/pnas.1200421109 PubMed DOI PMC

Hudecek M., Schmitt T. M., Baskar S., Lupo-Stanghellini M. T., Nishida T., Yamamoto T. N., et al. (2010). The B-Cell Tumor-Associated Antigen ROR1 Can Be Targeted with T Cells Modified to Express a ROR1-specific Chimeric Antigen Receptor. Blood. 116 (22), 4532–4541. 10.1182/blood-2010-05-283309 PubMed DOI PMC

Hunter T. (2014). The Genesis of Tyrosine Phosphorylation. Cold Spring Harbor Perspect. Biol. 6 (5), a020644. 10.1101/cshperspect.a020644 PubMed DOI PMC

Janovska P., Verner J., Kohoutek J., Bryjova L., Gregorova M., Dzimkova M., et al. (2018). Casein Kinase 1 Is a Therapeutic Target in Chronic Lymphocytic Leukemia. Blood. 131 (11), 1311206–1311218. 10.1182/blood-2017-05-786947 PubMed DOI

Janovska P., Poppova L., Plevova K., Plesingerova H., Behal M., Kaucka M., et al. (2016). Autocrine Signaling by Wnt-5a Deregulates Chemotaxis of Leukemic Cells and Predicts Clinical Outcome in Chronic Lymphocytic Leukemia. Clin. Cancer Res. 22 (2), 459–469. 10.1158/1078-0432.ccr-15-0154 PubMed DOI PMC

Kaabeche K., Lemonnier J., Le Mée S., Caverzasio J., Marie P. J. (2004). Cbl-mediated Degradation of Lyn and Fyn Induced by Constitutive Fibroblast Growth Factor Receptor-2 Activation Supports Osteoblast Differentiation. J. Biol. Chem. 279 (35), 36259–36267. 10.1074/jbc.m402469200 PubMed DOI

Karvonen H., Chiron D., Niininen W., Ek S., Jerkeman M., Moradi E., et al. (2017). Crosstalk between ROR1 and BCR Pathways Defines Novel Treatment Strategies in Mantle Cell Lymphoma. Blood Adv. 1 (24), 2257–2268. 10.1182/bloodadvances.2017010215 PubMed DOI PMC

Karvonen H., Niininen W., Murumägi A., Ungureanu D. (2017). Targeting ROR1 Identifies New Treatment Strategies in Hematological Cancers. Biochem. Soc. Trans. 45, 457–464. 10.1042/bst20160272 PubMed DOI

Karvonen H., Summala K., Niininen W., Barker H. R., Ungureanu D. (2018). Interaction between ROR1 and MuSK Activation Complex in Myogenic Cells. FEBS Lett. 592 (3), 434–445. 10.1002/1873-3468.12966 PubMed DOI

Kasahara K., Nakayama Y., Ikeda K., Fukushima Y., Matsuda D., Horimoto S., et al. (2004). Trafficking of Lyn through the Golgi Caveolin Involves the Charged Residues on αE and αI Helices in the Kinase Domain. J. Cell Biol. 165 (5), 641–652. 10.1083/jcb.200403011 PubMed DOI PMC

Kaucká M., Plevová K., Pavlová Š., Janovská P., Mishra A., Verner J., et al. (2013). The Planar Cell Polarity Pathway Drives Pathogenesis of Chronic Lymphocytic Leukemia by the Regulation of B-Lymphocyte Migration. Cancer Res. 73 (5), 1491–1501. 10.1158/0008-5472.can-12-1752 PubMed DOI

Mankai A., Eveillard J.-R., Buhe V., Le Ster K., Loisel S., Ghedira I., et al. (2007). Is the C-Cbl Proto-Oncogene Involved in Chronic Lymphocytic Leukemia? Ann. New York Acad. Sci. 1107 (1), 193–205. 10.1196/annals.1381.021 PubMed DOI

Martini V., Gattazzo C., Frezzato F., Trimarco V., Pizzi M., Chiodin G., et al. (2017). Cortactin, a Lyn Substrate, Is a Checkpoint Molecule at the Intersection of BCR and CXCR4 Signalling Pathway in Chronic Lymphocytic Leukaemia Cells. Br. J. Haematol. 178 (1), 81–93. 10.1111/bjh.14642 PubMed DOI

Mohapatra B., Ahmad G., Nadeau S., Zutshi N., An W., Scheffe S., et al. (2013). Protein Tyrosine Kinase Regulation by Ubiquitination: Critical Roles of Cbl-Family Ubiquitin Ligases. Biochim. Biophys. Acta (Bba) - Mol. Cell Res. 1833 (1), 122–139. 10.1016/j.bbamcr.2012.10.010 PubMed DOI PMC

Nguyen P.-H., Fedorchenko O., Rosen N., Koch M., Barthel R., Winarski T., et al. (2016). LYN Kinase in the Tumor Microenvironment Is Essential for the Progression of Chronic Lymphocytic Leukemia. Cancer Cell. 30 (4), 610–622. 10.1016/j.ccell.2016.09.007 PubMed DOI

Oishi I., Suzuki H., Onishi N., Takada R., Kani S., Ohkawara B., et al. (2003). The Receptor Tyrosine Kinase Ror2 Is Involved in Non-canonical Wnt5a/JNK Signalling Pathway. Genes to Cells. 8 (7), 645–654. 10.1046/j.1365-2443.2003.00662.x PubMed DOI

Petrelli A., Gilestro G. F., Lanzardo S., Comoglio P. M., Migone N., Giordano S. (2002). The Endophilin-CIN85-Cbl Complex Mediates Ligand-dependent Downregulation of C-Met. Nature. 416 (6877), 187–190. 10.1038/416187a PubMed DOI

Qi J., Li X., Peng H., Cook E. M., Dadashian E. L., Wiestner A., et al. (2018). Potent and Selective Antitumor Activity of a T Cell-Engaging Bispecific Antibody Targeting a Membrane-Proximal Epitope of ROR1. Proc. Natl. Acad. Sci. USA. 115 (24), E5467–E5476. 10.1073/pnas.1719905115 PubMed DOI PMC

Rebagay G., Yan S., Liu C., Cheung N. K. (2012). ROR1 and ROR2 in Human Malignancies: Potentials for Targeted Therapy. Front. Oncol. 2 (34), 34–38. 10.3389/fonc.2012.00034 PubMed DOI PMC

Scielzo C., Bertilaccio M. T. S., Simonetti G., Dagklis A., ten Hacken E., Fazi C., et al. (2010). HS1 Has a central Role in the Trafficking and Homing of Leukemic B Cells. Blood. 116 (18), 3537–3546. 10.1182/blood-2009-12-258814 PubMed DOI

Ten Hacken E., Gounari M., Ghia P., Burger J. A. (2019). The Importance of B Cell Receptor Isotypes and Stereotypes in Chronic Lymphocytic Leukemia. Leukemia. 33 (2), 287–298. 10.1038/s41375-018-0303-x PubMed DOI PMC

Ten Hacken E., Scielzo C., Bertilaccio M. T. S., Scarfò L., Apollonio B., Barbaglio F., et al. (2013). Targeting the LYN/HS1 Signaling axis in Chronic Lymphocytic Leukemia. Blood. 121 (12), 2264–2273. 10.1182/blood-2012-09-457119 PubMed DOI

Tezuka T., Umemori H., Fusaki N., Yagi T., Takata M., Kurosaki T., et al. (1996). Physical and Functional Association of the Cbl Protooncogen Product with an Src-Family Protein Tyrosine Kinase, P53/56lyn, in the B Cell Antigen Receptor-Mediated Signaling. J. Exp. Med. 183, 675–680. 10.1084/jem.183.2.675 PubMed DOI PMC

Xu W., Doshi A., Lei M., Eck M. J., Harrison S. C. (1999). Crystal Structures of C-Src Reveal Features of its Autoinhibitory Mechanism. Mol. Cell. 3 (5), 629–638. 10.1016/s1097-2765(00)80356-1 PubMed DOI

Xu Y., Harder K., Huntington N., Hibbs M., Tarlinton D. (2005). Lyn Tyrosine KinaseAccentuating the Positive and the Negative. Immunity. 22 (1), 9–18. 10.1016/s1074-7613(04)00381-4 PubMed DOI

Yamaguchi T., Yanagisawa K., Sugiyama R., Hosono Y., Shimada Y., Arima C., et al. (2012). NKX2-1/TITF1/TTF-1-Induced ROR1 Is Required to Sustain EGFR Survival Signaling in Lung Adenocarcinoma. Cancer Cell. 21 (3), 348–361. 10.1016/j.ccr.2012.02.008 PubMed DOI

Yu J., Chen L., Chen Y., Hasan M. K., Ghia E. M., Zhang L., et al. (2017a). Wnt5a Induces ROR1 to Associate with 14-3-3ζ for Enhanced Chemotaxis and Proliferation of Chronic Lymphocytic Leukemia Cells. Leukemia. 31 (12), 2608–2614. 10.1038/leu.2017.132 PubMed DOI PMC

Yu J., Chen L., Cui B., Wu C., Choi M. Y., Chen Y., et al. (2017). Cirmtuzumab Inhibits Wnt5a-Induced Rac1 Activation in Chronic Lymphocytic Leukemia Treated with Ibrutinib. Leukemia. 31 (6), 1333–1339. 10.1038/leu.2016.368 PubMed DOI PMC

Zhang Q., Wang H. Y., Liu X., Nunez-Cruz S., Jillab M., Melnikov O., et al. (2019). Cutting Edge: ROR1/CD19 Receptor Complex Promotes Growth of Mantle Cell Lymphoma Cells Independently of the B Cell Receptor-BTK Signaling Pathway. J Immunol. 203 (8), 2043–2048. 10.4049/jimmunol.1801327 PubMed DOI PMC