AMD3100 is a potent antagonist at CXCR4(R334X) , a hyperfunctional mutant chemokine receptor and cause of WHIM syndrome

Abstract

WHIM is an acronym for a rare immunodeficiency syndrome (OMIM #193670) caused by autosomal dominant mutations truncating the C-terminus of the chemokine receptor CXC chemokine receptor 4 (CXCR4). WHIM mutations may potentiate CXCR4 signalling, suggesting that the United States Food and Drug Administration (FDA)-approved CXCR4 antagonist AnorMED3100 (AMD3100) (also known as Plerixafor) may be beneficial in WHIM syndrome. We have tested this at the preclinical level by comparing Chinese hamster ovary (CHO) and K562 cell lines matched for expression of recombinant wild-type CXCR4 (CXCR4(WT)) and the most common WHIM variant of CXCR4 (CXCR4(R334X)), as well as leucocytes from a WHIM patient with the CXCR4(R334X) mutation versus healthy controls. We found that CXCR4(R334X) mediated modestly increased signalling (~2-fold) in all functional assays tested, but strongly resisted ligand-dependent down-regulation. AMD3100 was equipotent and equieffective as an antagonist at CXCR4(R334X) and CXCR4(WT) . Together, our data provide further evidence that CXCR4(R334X) is a gain-of-function mutation, and support clinical evaluation of AMD3100 as mechanism-based treatment in patients with WHIM syndrome.

Fig 1

Construction of CHO and K562 cell lines expressing equivalent plasma membrane levels of wild-type CXCR4 or WHIM mutant CXCR4^R334X. Shown are representative flow cytometry histograms of the indicated cell lines stained with anti-CXCR4 mAb 12G5. Cells were mock-transfected (filled histogram) or transfected with plasmids encoding wild-type CXCR4 (CXCR4^WT, thin lines) or CXCR4^R334X (thick line). The x-axis displays fluorescence intensity and y-axis number of events. These cell lines were used for functional comparisons.

Fig 2

WHIM mutation CXCR4^R334X increases CXCL12-induced calcium flux response, but allows homologous desensitization: analysis of transfected cells. (A) Representative real-time sequential stimulation experiment for K562 cells expressing CXCR4^WT or CXCR4^R334X. The cell line, receptor and CXCL12 doses are indicated at the top left and right of each panel. For each tracing, the same dose of CXCL12, specified by the colour code, was added at the times indicated by the arrows. (B) Peak response to the first stimulus of CXCL12. (C) Duration of signalling after first CXCL12 stimulus was estimated as the time from peak response to time of half peak response. (D) Homologous desensitization was quantitated as 1 – (peak response to stimulus 2/peak response to stimulus 1). *P < 0.05 for comparison between CXCR4^WT- and CXCR4^R334X-transfected cells. Data for (B)–(D) are the mean ± S.E.M. and plotted as summary data from three experiments, each done with three replicates.

Fig 3

WHIM mutation CXCR4^R334X enhances CXCR4-induced phosphorylation of ERK and Akt: analysis of transfected cells. The indicated cells and receptors at the top of each panel were stimulated with 10 nM CXCL12 for the indicated times and the percentage of phosphorylated substrate relative to total substrate was quantified (ERK) or percentage of the positive cells quantified (Akt). (A) ERK. (B) Akt. *P < 0.05 for comparison between CXCR4^WT- and CXCR4^R334X-transfected cells. Data shown are the mean ± S.E.M. and plotted as summary data from three experiments, each done with three replicates.

Fig 4

WHIM mutation CXCR4^R334X impairs CXCR4 internalization after CXCL12 stimulation. (A) Dose–response. K562 cells (upper panels) and CHO cells (lower panels) were stimulated for 1 hr at 37°C with the indicated concentration of CXCL12. Representative FACS histograms are shown at the left, and the corresponding summary data at the right. IC: isotype control antibody; MFI: mean fluorescence intensity. (B) Time course. Graphs display expression of the indicated CXCR4 form in CHO cells (left) and K562 cells (right) after 10 nM CXCL12 stimulation for the indicated time at 37°C. Data are summarized from three experiments as the mean ± S.E.M. *P < 0.05 for comparison between CXCR4^WT- and CXCR4^R334X-transfected cells.

Fig 5

PBMCs from a patient with WHIM mutation CXCR4^R334X exhibit enhanced functional responses to CXCL12. HD: healthy donor; P1: WHIM patient described in text. (A) Patient PBMCs include two populations of cells distinguished by low and high expression levels of CXCR4. A zebra plot of size gated lymphocytes is shown. Comparisons are displayed of healthy donors versus WHIM patient P1 for CXCL12-induced peak calcium flux (B), CXCR4 internalization (C), ERK phosphorylation (D and E), and Akt phosphorylation (F). Assays were performed as for cell lines in Figures 2–4 and as described in ‘Materials and methods’. For summary data, HD1–3 represent the mean ± S.E.M. of values for three healthy donors tested on the same day as P1, 2 replicates/condition, with peak calcium flux normalized for relative receptor expression. *P < 0.05 for comparison of PBMC responses from HD and P1. For FACS plots in (E) and (F), the time of stimulation is indicated above the corresponding column in which it is found and the source of the sample is indicated to the right of the row in which it is found. In (F), the filled histogram represents stimulation with buffer alone. In (B) and (D–F), the stimulus is 10 nM CXCL12.

Fig 6

AMD3100 is an equipotent and equieffective antagonist at wild-type CXCR4 and WHIM mutant CXCR4^R334X. (A–C) Calcium flux responses mediated by the receptor forms in the cell types indicated at the top of each panel. In (C), HD denotes healthy donors and P1 denotes the WHIM patient described in the text. (A) Real-time sequential stimulation at the times indicated by the arrows first with varying doses of AMD3100 (colour coded key in right panel) followed by 10 nM CXCL12. (B) and (C) Peak response to 10 nM CXCL12 added after 3 min. incubation with indicated concentration of AMD3100. (D) ERK phosphorylation in K562 cells expressing the receptor listed at the right of the corresponding row after pre-treatment for 10 min. with the concentration of AMD3100 indicated at the top of the corresponding column and stimulation for 2 min. with 10 nM CXCL12. The percentage of total ERK that is phosphorylated is given in the upper right quadrant of each panel. The experiment is representative of three experiments performed in duplicate. (E) Neutrophil chemotaxis to indicated concentrations of CXCL12. (F) Neutrophil chemotaxis stimulated by 30 nM of CXCL12 can be inhibited by indicated concentration of AMD3100 when added to both the upper and lower chamber.