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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.

The migratory properties of leukemic cells are commonly associated with their pathological potential and can significantly affect the disease progression. While the research in immunopathology mostly employed powerful indirect methods such as flow cytometry, these cells were rarely observed directly using live imaging microscopy. This is especially true for the malignant cells of the B-cell lineage, such as those originating from chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL). In this study, we employed open-source image analysis tools to automatically and quantitatively describe the amoeboid migration of four B-cell leukemic and lymphoma cell lines and primary CLL cells. To avoid the effect of the shear stress of the medium on these usually non-adherent cells, we have confined the cells using a modified under-agarose assay. Surprisingly, the behavior of tested cell lines differed substantially in terms of basal motility or response to chemokines and VCAM1 stimulation. Since casein kinase 1 (CK1) was reported as a regulator of B-cell migration and a promoter of CLL, we looked at the effects of CK1 inhibition in more detail. Migration analysis revealed that CK1 inhibition induced rapid negative effects on the migratory polarity of these cells, which was quantitatively and morphologically distinct from the effect of ROCK inhibition. We have set up an assay that visualizes endocytic vesicles in the uropod and facilitates morphological analysis. This assay hints that the effect of CK1 inhibition might be connected to defects in polarized intracellular transport. In summary, 1) we introduce and validate a pipeline for the imaging and quantitative assessment of the amoeboid migration of CLL/MCL cells, 2) we provide evidence that the assay is sensitive enough to mechanistically study migration defects identified by the transwell assay, and 3) we describe the polarity defects induced by inhibition or deletion of CK1ε.

word count: 168 words 26 Main text word count: 4183 words 27 Number of Figures: 6 28 Number of tables: 2 29 Running title: Lyn controls chemotaxis and motility of CLL cells via phosphorylation of ROR1.30 Abstract 33 Chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) are malignancies 34 characterized by the dependence on B-cell receptor (BCR) signaling and by the high 35 expression of the cell surface receptor ROR1. Both, BCR and ROR1 are therapeutic targets 36 in these diseases and the understanding of their mutual cross talk is thus of direct 37 therapeutic relevance. In this study we analyzed the role of Lyn, a kinase from the Src 38 family, as a mediator of the BCR-ROR1 crosstalk. We confirm the functional interaction 39 between Lyn and ROR1 and demonstrate that Lyn kinase efficiently phosphorylates ROR1 40 in its kinase domain and aids the recruitment of an E3 ligase c-CBL. The absence of Lyn in 41 Lyn KO Maver-1 cells produced by CRISPR-Cas9 resulted in the increased ROR1 cell 42 surface levels and deregulated migratory properties. Similar correlations between ROR1 43 surface dynamics, levels of active Lyn and chemotactic properties were confirmed in primary 44 CLL samples. Our data establish Lyn-mediated phosphorylation of ROR1 as a point of 45 crosstalk between BCR and ROR1 signaling pathways. 46 47 48 49 50 51 52 53 54 55 56 57 58 59 3 Introduction 60The ROR protein family comprises of ROR1 and ROR2, which are both type 1 61 transmembrane receptors. Upon discovery, ROR proteins were referred to as orphan 62 receptors on account of the lack of identity of their ligands. However, subsequent studies 63 identified their ligands to be the Wnt proteins, mostly Wnt-5a protein 1,2 . Wnt-5a/ROR 64 pathway is an essential signaling pathway that controls cell polarity and migration during 65 embryonic development and tissue homeostasis 3,4 . During embryonic development, RORs 66 are highly and uniformly expressed, most prominently in the skeletal and neural tissues, but 67 postnatally their expression becomes highly restricted 5 . 68Interestingly, ROR1 or ROR2 upregulation has been observed in many cancers: 69ROR1 is upregulated in solid tumors or hematologic malignancies while ROR2 is 70 overexpressed in osteosarcomas or renal cell carcinomas 6 . High expression of ROR1 is 71 typical for some B-cell lymphomas such as mantle cell lymphoma (MCL) 7 and chronic 72 lymphocytic leukemia(CLL) 8,9 . CLL is a form of hematologic cancer which is manifested as a 73 steady accumulation of mature CD5 + B-cells in the bone marrow, lymphoid tissues and 74 peripheral blood. It is the most common form of adult leukemia in the western hemisphere, 75with an incidence of 5 per 100 000 each year and an average median age of on-set around 76 70 years. Most of the CLL cases remain asymptomatic for a long time, in which case 77 therapeutic intervention is not necessary. However, part of the CLL cases progress rapidly, 78 require treatment and their overall life expectancy is decreased 10 . 79CLL cells are in most cases hig...