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. 2018 Oct 16;11(552):eaat2214.
doi: 10.1126/scisignal.aat2214.

Biased antagonism of CXCR4 avoids antagonist tolerance

Ben Hitchinson  1 , Jonathan M Eby  2 , Xianlong Gao  2   3 , Francois Guite-Vinet  4 , Joshua J Ziarek  5   6 , Hazem Abdelkarim  1 , Youngshim Lee  7 , Yukari Okamoto  1 , Sojin Shikano  1 , Matthias Majetschak  2   3 , Nikolaus Heveker  4 , Brian F Volkman  6 , Nadya I Tarasova  8 , Vadim Gaponenko  9
Affiliations

Biased antagonism of CXCR4 avoids antagonist tolerance

Ben Hitchinson et al. Sci Signal. .

Abstract

Repeated dosing of drugs targeting G protein-coupled receptors can stimulate antagonist tolerance, which reduces their efficacy; thus, strategies to avoid tolerance are needed. The efficacy of AMD3100, a competitive antagonist of the chemokine receptor CXCR4 that mobilizes leukemic blasts from the bone marrow into the blood to sensitize them to chemotherapy, is reduced after prolonged treatment. Tolerance to AMD3100 increases the abundance of CXCR4 on the surface of leukemic blasts, which promotes their rehoming to the bone marrow. AMD3100 inhibits both G protein signaling by CXCR4 and β-arrestin1/2-dependent receptor endocytosis. We demonstrated that biased antagonists of G protein-dependent chemotaxis but not β-arrestin1/2 recruitment and subsequent receptor endocytosis avoided tolerance. The peptide antagonist X4-2-6, which is derived from transmembrane helix 2 and extracellular loop 1 of CXCR4, limited chemotaxis and signaling but did not promote CXCR4 accumulation on the cell surface or cause tolerance. The activity of X4-2-6 was due to its distinct mechanism of inhibition of CXCR4. The peptide formed a ternary complex with the receptor and its ligand, the chemokine CXCL12. Within this complex, X4-2-6 released the portion of CXCL12 critical for receptor-mediated activation of G proteins but enabled the rest of the chemokine to recruit β-arrestins to the receptor. In contrast, AMD3100 displaced all components of the chemokine responsible for CXCR4 activation. We further identified a small molecule with similar biased antagonist properties to those of X4-2-6, which may provide a viable alternative to patients when antagonist tolerance prevents drugs from reaching efficacy.

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Conflict of interest statement

Competing interests: N.I.T. is a co-inventor on a patent application filed by the NIH/NCI for the X4-2-6 peptide. B.F.V. has an ownership interest in Protein Foundry LLC. The other authors declare that they have no competing interests.

Figures

Fig. 1.
Fig. 1.. Antagonist tolerance develops after inhibition of endocytosis.
(A) IC50 values calculated from Transwell chemotaxis assays of Jurkat cell migration toward CXCL12 after pre-treatment with AMD3100 or X4-2-6 at the indicated concentrations for the indicated times. Data are means ± SEM of three independent experiments. *P < 0.001 by Student’s t test. (B) Flow cytometry analysis of CXCR4 cell surface expression on Jurkat cells treated with AMD3100 (▲) or X4-2-6 (●) for 72 hours as compared to vehicle-treated cells. Data are means ± SD of three experiments performed in triplicate at each condition. (C) Flow cytometry analysis of CXCR4 endocytosis stimulated by CXCL12 in Jurkat cells treated with AMD3100 (▲) or X4-2-6 (●) as compared to cells stimulated with CXCL12 alone. Data are means ± SD from three experiments performed in triplicate for each condition. (D) Transwell migration assay of Jurkat cell chemotaxis toward CXCL12 after treatment with the indicated concentration of AMD3100 with or without dynasore for 1 hour. Chemotaxis is plotted relative to chemotaxis in the presence of CXCL12 alone. Data are means ± SD from three independent experiments performed in triplicate for each condition. *P < 0.04 by Student’s t test. Flow cytometry analysis of CXCR4 cell surface expression before and after dynasore treatment (upper left inset) is representative of all experiments. IgGκB, immunoglobulin G κB; PE, phycoerythrin.
Fig. 2.
Fig. 2.. BA1/2 recruitment and function downstream of CXCR4 is not substantially affected by X4-2-6.
(A) Confocal microscopy analysis of CXCR4 and BA2 in Jurkat cells pretreated with vehicle, AMD3100, or X4-2-6 and stimulated with CXCL12, as indicated. Arrows indicate CXCR4 (red), BA2 (green), or colocalization (orange). Images are representative of three independent experiments. Scale bars, 10 μM. DAPI, 4′,6-diamidino-2-phenylindole, dihydrochloride. (B) PRESTO-Tango assay analysis of the recruitment of BA1/2 to CXCR4 after treatment with increasing concentrations of CXCL12 in the presence of vehicle (■), AMD3100 (▲), or X4-2-6 (●). Data are means ± SD of three independent experiments performed on six replicates per condition. (C) Western blotting analysis of CXCR4 Ser324/325 and Ser339 phosphorylation in lysates of Jurkat cells treated as indicated. Blots are representative of at least three independent experiments. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (D and E) Western blotting analysis of ERK1/2 Thr202/Tyr204 phosphorylation in the lysates of Jurkat cells treated as indicated. (D) Blots are representative of at least three independent experiments. (E) Quantification of the relative abundance of pERK1/2 normalized to that of total ERK1/2. Data are means ± SD from all experiments. *P < 0.05 and **P < 0.01 by one-way analysis of variance (ANOVA) with post hoc Tukey test.
Fig. 3.
Fig. 3.. X4-2-6 specifically inhibits G protein activation downstream of CXCR4.
(A and B) Western blotting analysis for the GTP loading of Gαi in Jurkat cells pretreated with vehicle, AMD3100, or X4-2-6 and stimulated with CXCL12. (A) Blots are representative of three independent experiments. IP, immuno-precipitation; IB, immunblotting. (B) Quantified data are means ± SD from all experiments. *P < 0.01 by ANOVA. (C) Enzyme-linked immunosorbent assay (ELISA)–based analysis of the intracellular concentrations of cAMP in Jurkat cells treated as indicated. Data are means ± SD of three independent experiments with six replicates per condition. *P < 0.05 and **P < 0.005 by Student’s t test. (D) Transwell migration assay of Jurkat cell chemotaxis toward CXCL12 after treatment with AMD3100 or X4-2-6. Data are means ± SD of three independent experiments each performed in triplicate. (E) Ca2+ flux analysis in THP-1 cells treated with vehicle or X4-2-6 and stimulated with CXCL12 as indicated. Data are means ± SD from 12 biological replicates. *P ≤ 5 × 10−8 by Student’s t test. (F) Ca2+ flux analysis in THP-1 cells treated with vehicle or X4-2-6 and stimulated with CCL2 as indicated. Data are means ± SD from 12 biological replicates. *P ≤ 5 × 10−8 by Student’s t test.
Fig. 4.
Fig. 4.. X4-2-6 forms a ternary complex with CXCL12 and CXCR4 to function as a biased antagonist.
(A to C) NMR spectroscopy analysis of CXCL12 alone (gray) and in the presence of X4-2-6 (blue). Peak superimposition (A) and changes in CXCL12 HSQC signal intensity caused by the addition of X4-2-6 (B) are representative of two independent experiments. ppm, parts per million. (C) Substantially altered residues are mapped onto the NMR structure of CXCL12 (Protein Data Bank ID: 2KEE), and the cartoon models the interaction between the N-loop of CXCL12 and X4-2-6. (D to F) NMR spectroscopy analysis of CXCL12 alone (gray) and with membrane preparations containing CXCR4 (red). Peak superimposition (D) and changes in the signal intensity of CXCL12 residues caused by the addition of CXCR4 (E) are representative of two independent experiments. (F) Substantially altered residues are mapped onto the structure of CXCL12, and the cartoon models the interaction involving insertion of the N terminus of the chemokine into the receptor transmembrane helical bundle. (G to I) NMR spectroscopy analysis of CXCL12 alone (gray) and with CXCR4-containing membranes and X4-2-6 (magenta). Peak superimposition (G) and changes in CXCL12 signal intensity caused by the addition of CXCR4 and X4-2-6(H) are representative of two independent experiments. (I) The most substantial changes are mapped onto the NMR structure of CXCL12, and the cartoon models X4-2-6 binding to CXCR4 and CXCL12 to partially inhibit the binding of the extreme N terminus of the chemokine to the receptor.
Fig. 5.
Fig. 5.. Proposed model for the mechanism of biased antagonism and development of tolerance to unbiased antagonists.
(A) The current paradigm of CXCL12-mediated CXCR4 signaling suggests that the CXCL12 N terminus and N-loop insert into the CXCR4 transmembrane helical bundle, whereas the receptor N terminus binds to the globular domain of the chemokine. This leads to the activation of CXCR4 and subsequent G protein signaling, BA1/2 recruitment, and receptor endocytosis. (B) Our data suggest that X4-2-6 binds to CXCL12 and CXCR4 to form a ternary complex and displaces the extreme N-terminal portion of CXCL12 away from the transmembrane helical bundle of CXCR4. Thus, X4-2-6 functions as a biased antagonist by inhibiting G protein signaling but not BA1/2 recruitment to CXCR4. (C) In contrast, AMD3100 displaces the entire CXCL12 N terminus to inhibit all CXCR4 signaling. Over time, the inhibition of BA1/2 and the subsequent endocytosis result in the accumulation of CXCR4 on the cell surface, CXCL12 binding to the receptor, and the development of tolerance to AMD3100. GDP, guanosine diphosphate.
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