In vivo adhesion of malignant B cells to bone marrow microvasculature is regulated by [alpha]4[beta]1 cytoplasmic-binding proteins

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 [alpha]4[beta]1 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 [beta]1-integrin-binding partners that regulate [beta]1-mediated cell adhesion. We show that talin and kindlin-3 cooperatively stimulate high affinity and strength of [alpha]4[beta]1-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 [alpha]4[beta]1. 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 [alpha]4[beta]1 ligands showed higher susceptibility to bortezomib-mediated cell apoptosis. Our results highlight the requirement of [alpha]4[beta]1 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 [alpha]4[beta]1 activity.