Increased Palmitoylation Modulates Cytoskeletal Regulators To Enhance Cxcr4-Mediated Motility Of Malignant B Cells

Abstract The development of B-cell precursors and malignant counterparts requires environmental factors that are secreted in bone marrow niches, with stromal cell-derived factor 1 (SDF-1, CXCL12) and the very late antigen-4 integrin (CD49d/CD29) being predominant players. Further downstream, the actin cytoskeleton is regulated by Rho and Rac signals. This pathway can be modulated by palmitoylation of heterotrimeric G proteins as well as Rho family GTPases, which results in their targeting to membrane proximal sites. Acyl-protein thioesterase-1 (APT1) mediates de-palmitoylation of different substrate proteins. We recently observed that APT1 is significantly upregulated in B cell malignancies compared to healthy counterparts. Consequently, we knocked down APT1 in two malignant B-cell acute lymphoblastic leukemia (B-ALL) and other B cell lines, thereby modifying the extent of palmitoylation in these cells. We then determined the ability of the malignant B cells to interact with CXCL12 bearing substrates, mimicking the bone marrow environment in vitro . Using time-lapse videomicroscopy and chemotaxis assays, we assessed the degree of motility, the migrated distance and the velocity of migration of the cells. The increased palmitoylation potentiated CXCL12 induced motility and chemotaxis of APT1 knockout compared to control B cells, with a strong increase in CXCL12 induced microvillar collapse and spreading of the APT1 knockout cells on fibronectin/CXCL12 substrates, as also assessed by electron microscopy. We addressed whether this increased migratory behaviour is coupled to reduced adhesive capacity using real-time videomicroscopy under shear stress. We found that the adhesive capacity of APT1 deficient malignant B cells to the VLA-4 ligand VCAM-1 co-immobilized with CXCL12 was diminished. We also observed that RhoA but not Rac1 activity was attenuated in APT1 knockout cells, consistent with the observed phenotype in light of the known importance of the RhoA/Rac1 balance for adhesion versus migration. Further downstream, the activity of mitogen-activated kinase cascades was increased. To study the direct molecular interaction of APT1 with relevant components of chemokine receptor signalling, we used fluorescence lifetime imaging microscopy forster resonance energy transfer (FLIM-FRET). The analysis revealed a close association of APT1 with GRK6 and s-arrestin-2, suggesting alterations in receptor desensitisation. Together, our data suggest that APT1 is a modulator of GRK6, s-arrestin-2 and RhoA activity downstream of chemokine receptor signalling in malignant B lymphocytes, thereby contributing to the pathophysiology of B-cell malignancies by shaping their protective interactions within the bone marrow microenvironment. Disclosures Greil: Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel support, Research Funding; Novartis, Celgene: Research Funding; Takeda: Honoraria, Research Funding; BMS, Amgen: Honoraria. Hallek: AbbVie: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; F. Hoffmann-LaRoche: Consultancy, Honoraria, Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Janssen-Cilag: Consultancy, Honoraria, Research Funding; Mundipharma: Consultancy, Honoraria, Research Funding. Frenzel: Abbvie: Consultancy; Roche: Research Funding; Gilead: Research Funding.