Figures and data
Screening for small compound antagonists acting on the oligomerization site of CXCR4
A) Surface representation of monomeric CXCR4 showing the cavity formed by TMV and TMVI. CXCR4 is shown in grey with TMV in blue and TMVI in pink. The cavity formed by both transmembrane helices includes some of the chemical chains of the participating residues. In green are shown the residues involved in CXCL12 binding. B) Dose-response curve of the selected antagonists in migration experiments of Jurkat cells in response to 12.5 nM CXCL12. Data are shown as percentage of migrating cells (mean ± SD; n = 5; ** ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001). C) Chemical structure of the selected compounds (AGR1.131, AGR1.135 and AGR1.137).
AGR1.135 and AGR1.137 alter CXCL12-mediated CXCR4 dynamics and nanoclustering
Single-particle tracking analysis of JK−/− transiently transfected with CXCR4 (JK−/−-X4) cells treated with DMSO (control), AGR1.131, AGR1.135 or AGR1.137 on fibronectin (FN)- or FN+CXCL12-coated coverslips (DMSO: 581 particles in 59 cells on FN; 1365 in 63 cells on FN+CXCL12; AGR1.131: 1019 particles in 71 cells on FN; 1291 in 69 cells on FN+CXCL12; AGR1.135: 862 particles in 70 cells on FN; 1003 in 77 cells on FN+CXCL12; AGR1.137: 477 particles in 66 cells on FN; 566 in 64 cells on FN+CXCL12) n = 3. A) Frequency of CXCR4-AcGFP particles containing monomers plus dimers (≤2) or nanoclusters (≥3), calculated from mean spot intensity values of each particle as compared with the value of monomeric CD86-AcGFP. B) Intensity distribution (arbitrary units, a.u.) from individual CXCR4-AcGFP trajectories on unstimulated and CXCL12-stimulated JK−/−-X4 cells pretreated or not with the indicated compounds, mean is indicated (red) (n = 3; n.s., not significant; ****p ≤ 0.0001). C) Percentage of mobile and immobile CXCR4-AcGFP particles at the membrane of cells as in (A) untreated or treated with the indicated compounds in basal conditions and after CXCL12 stimulation. D) Diffusion coefficients (D1–4) of mobile single trajectories, with median (black line) corresponding to JK−/−-X4 as in (a). (n.s., not significant, ****p ≤ 0.0001).
AGR1.135 and AGR1.137 treatments do not block CXCL12-mediated signaling pathways
A) Cell surface expression of CXCR4 in Jurkat cells after stimulation with CXCL12 (12.5 nM) at different time points and analyzed by flow cytometry using an anti-CXCR4 antibody in nonpermeabilized cells. Results show mean ± SEM of the percentage of CXCR4 expression at the cell surface (n = 4; n.s. not significant). B) CXCL12-Atto-700 binding on untreated or Jurkat cells pretreated with AGR1.131, AGR1.135 or AGR1.137 and with AMD-3100 as a control, followed by flow cytometry analysis. Results are expressed as mean fluorescence intensity (MFI) values (arbitrary units). Negative corresponds to basal cell fluorescence in the absence of CXCL12-Atto-700. C) Cells untreated or pretreated with the small compounds were stimulated with CXCL12 followed by forskolin. Cells were then lysed and cAMP levels were determined (mean ± SD; n = 3; n.s. not significant; *p ≤ 0.05, **p ≤ 0.01). D) Western blot analysis of phospho (p)Akt and pERK in Jurkat cells pre-treated with DMSO (vehicle), AGR1.131, AGR1.135 or AGR1.137, in response to CXCL12. As a loading control, membranes were re-blotted with an anti-Akt antibody. Representative experiments are shown (n = 4).
AGR1.135 and AGR1.137 treatment alter CXCL12-mediated actin polymerization
A) Actin polymerization in response to CXCL12 as determined by F-actin (phalloidin-TRITC) staining in JK cells untreated or treated with the indicated antagonists. B) Percentage of polarized T cell blasts adhered to fibronectin and treated or not with CXCL12 in the presence of the indicated antagonists, as analyzed by immunostaining with anti-ICAM3-Alexa fluor 488 and phalloidin-TRITC. More than 500 cell analyzed in each condition. Data are presented as percentage of polarized cells (mean +_SD; n=3; n.s. not significant; ****p< 0.0001). C) CD4+ T cells pretreated with AGR1.131, AGR1.135 or AGR1.137 were perfused in flow chambers coated with ICAM-1 co-immobilized with CXCL12, and analyzed for cell contacts with the substrate. Data are presented as percentage of adhered cells (mean ± SD; n = 3; n.s. not significant; ****p ≤ 0.0001). D) Cells in (C) were analyzed for cell migration. Data are presented as percentage of migrating cells (mean ± SD; n = 3; n.s. not significant; ****p ≤ 0.0001).
Ribbon and sticks representation of the CXCR4 modulators bound to the receptor
In the ribbon and sticks representations, TMV helix is colored in blue and TMVI in pink. Residues involved in CXCL12 engagement and initial signal transmission are represented in green and residues mutated are shown in spheres. (A) Superposition of AGR1.135 with carbons in green and AGR1.137 in yellow. (B-D) Show the binding of ligands AGR1.135 (B), AGR1.137 (C) and AGR1.131 (D) represented as sticks with carbon atoms in yellow, oxygens in red, nitrogens in blue and fluorines in green.
The antagonistic behavior of AGR1.135 and AGR1.137 depends on specific residues of CXCR4
A) CXCL12-induced migration of CXCR4-deficient Jurkat cells (JK-/-) transiently transfected with CXCR4wt or its mutants, CXCR4I204K, CXCR4G207I, CXCR4L208K, CXCR4R235L, CXCR4F249L, CXCR4Y256F and CXCR4S260A. Data are shown as the mean percentage (plus SD) of input cells that migrate (n = 3). B) CXCL12-induced migration of AGR1.131-AGR1.135- or AGR1.137-pretreated JK- /- cells transiently transfected with CXCR4wt or the mutants described in (A). Data are shown as the mean percentage ± SD of input cells that migrate (n = 4; n.s. not significant, **p ≤ 0.01, ****p ≤ 0.0001).
AGR1.137 reduces tumorigenesis and metastasis in a zebrafish model
A-D) Migration of HeLa cells treated with vehicle (DMSO) or with the selected small compounds or inhibitor as indicated, on μ-chambers in response to a CXCL12 (A,B) or CXCL2 (C,D) gradient (n = 2, in duplicate, with at least 50 cells tracked in each condition). Figures (A,C) shows representative spider plots with the trajectories of tracked cells migrating along the gradient (black) or moving in the opposite direction (red). Black and red dots in the plots represent the final position of each single tracked cell. B,D) Quantification of the Forward Migration Index of experiments performed in (A) and (C) (mean ± SD; n = 3; n.s. not significant, ****p ≤ 0.0001). E) Representative fluorescent images of DiI-labeled HeLa cells in zebrafish larvae treated with vehicle (DMSO), AGR1.131, AGR1.137 or AMD3100 at 0 or 3 days post-implantation and treatment. Quantitation of the relative tumor size at day 3 compared with that of day 0 is shown for each experimental group (mean ± SD; n = 20; n.s. not significant, **p ≤ 0.01). F) Representative fluorescent images of the caudal hematopoietic plexus of larvae from the same groups as shown in (E) at 3 days post-implantation. Quantitation of the average number of metastasized cells in each group (mean ± SD; n = 20, n.s. not significant, * p ≤ 0.05, *** p ≤ 0.001).
