Expression of functional sphingosine-1 phosphate receptor-1 is reduced by B cell receptor signaling and increased by inhibition of PI3 kinase δ but not SYK or BTK in chronic lymphocytic leukemia cells

Abstract

BCR signaling pathway inhibitors such as ibrutinib, idelalisib, and fostamatinib (respective inhibitors of Bruton's tyrosine kinase, PI3Kδ, and spleen tyrosine kinase) represent a significant therapeutic advance in B cell malignancies, including chronic lymphocytic leukemia (CLL). These drugs are distinctive in increasing blood lymphocytes while simultaneously shrinking enlarged lymph nodes, suggesting anatomical redistribution of CLL cells from lymph nodes into the blood. However, the mechanisms underlying this phenomenon are incompletely understood. In this study, we showed that the egress receptor, sphingosine-1-phosphate (S1P) receptor 1 (S1PR1), was expressed at low levels in normal germinal centers and CLL lymph nodes in vivo but became upregulated on normal B cells and, to a variable and lesser extent, CLL cells following in vitro incubation in S1P-free medium. Spontaneous recovery of S1PR1 expression on normal B and CLL cells was prevented by BCR cross-linking, whereas treatment of CLL cells with idelalisib increased S1PR1 expression and migration toward S1P, the greatest increase occurring in cases with unmutated IgH V region genes. Intriguingly, ibrutinib and fostamatinib had no effect on S1PR1 expression or function. Conversely, chemokine-induced migration, which requires integrin activation and is essential for the entry of lymphocytes into lymph nodes as well as their retention, was blocked by ibrutinib and fostamatinib, but not idelalisib. In summary, our results suggest that different BCR signaling inhibitors redistribute CLL cells from lymph nodes into the blood through distinct mechanisms: idelalisib actively promotes egress by upregulating S1PR1, whereas fostamatinib and ibrutinib may reduce CLL cell entry and retention by suppressing chemokine-induced integrin activation.

FIGURE 1.

Expression of S1PR1 in normal and CLL node. (A and B) Normal lymph node. The cells of the germinal center (GC) are identified by staining for CD23, which is mainly expressed on activated B cells. Staining of the adjacent section for S1PR1 shows that CD23⁺ cells do not express S1PR1, whereas the cells within the outer follicle (OF) stain positively. (C–F) Sequential section of CLL lymph nodes stained for S1PR1 and CD20. In (C) and (D), it can be clearly seen that the endothelial cells lining the sinus (S) and other endothelial cells express S1PR1, whereas CD20⁺ CLL cells lack the receptor. (E) and (F) show a representative proliferation center in a CLL lymph node. CD20⁺ CLL cells do not express S1PR1. Parallel staining with the relevant isotypic control Ab is shown in Supplemental Fig. 2A. Scale bar, 50 μM; original magnification ×20.

FIGURE 2.

S1PR1 expression on normal and CLL B cells cultured in the absence of S1P. (A) Normal B cells from six healthy individuals were cultured for 16 h in medium lacking S1P and examined for S1PR1 expression by flow cytometry. An increase in S1PR1 was observed between 2 and 4 h and reached a peak at 8–16 h. (B) Normal B cells from six donors were cultured in S1P-free medium for 16 h in the presence or absence of anti-IgM and/or idelalisib (1 μM). S1PR1 expression was measured by flow cytometry. IgM cross-linking prevented the spontaneous increase in S1PR1 expression (p = 0.0039). Idelalisib had little effect on S1PR1 expression on normal B cells in the absence of IgM (p = 0.177); however, treatment reversed the anti-IgM–mediated suppression of S1PR1 expression (p = 0.014) with levels returning to those of untreated cells at 16 h (p = 0.177). (C) CLL cells from 20 patients were cultured for 16 h in S1P-free medium and examined for S1PR1 levels by flow cytometry. There was an overall increase in S1PR1 expression (p < 0.002), but the increase was variable, delayed, and generally of lower magnitude compared with that observed in normal B cells. (D) Comparison of S1PR1 upregulation in normal and CLL B cells at 8 and 16 h. The increase in expression was significantly greater in normal B cells at both time points. In the box-and-whiskers plot, the bar indicates the median of the mean fluorescence intensity values for S1PR1 expression, whereas an asterisk (*) identifies outlying data points that do not fall within the interquartile range. (E) Seven of the CLL samples showing most spontaneous increase in S1PR1 expression [highlighted in gray in (C)] were cultured in S1P-free medium in the presence or absence of anti-IgM and examined for S1PR1 expression by flow cytometry. Upregulation of S1PR1 was prevented by BCR cross-linking in all cases. (F) Pooled analysis of the seven cases of CLL described in (E) showing near-complete abrogation of spontaneous upregulation of S1PR1 expression (p = 0.73).

FIGURE 3.

Effect of idelalisib on S1PR1 expression in CLL cells. (A) CLL cells from 20 cases were cultured in the presence or absence of idelalisib (1 μM) and examined by flow cytometry for S1PR1 levels. The chart shows the fold increase in mean fluorescence intensity compared with cells cultured for the same amount of time in the absence of idelalisib. The increase in S1PR1 expression was statistically significant at both time points (p < 0.002). (B) Box-and-whiskers plot of idelalisib-induced upregulation of S1PR1 at 16 h. Idelalisib induced an increase in S1PR1 expression in both IGHV-mutated (n = 11) and unmutated (n = 8) CLL samples, but the effect was greater in the latter cases (p = 0.0397). The bar represents the grand median of all samples. (C) CLL cells displaying little or no spontaneous recovery of S1PR1 expression (n = 4) were cultured in the presence or absence of idelalisib on different cell monolayers and examined by flow cytometry for S1PR1 levels. A representative example (patient 2141) is shown (data from individual cases are shown in Supplemental Fig. 2H). Idelalisib (1 μM) produced a marked increase in S1PR1 expression at 8 h irrespective of whether the CLL cells were cultured on endothelial cells (HUVEC), stromal cells (HS-5), or CD154-expressing fibroblasts.

FIGURE 4.

Effect of BCR inhibitors on TEM toward S1P and CCL21. (A) CLL cells from five patients were incubated for 16 h in the presence or absence of idelalisib (1 μM) and then examined for migration toward S1P using HUVEC-coated transwells. The numbers above bars indicate mean fluorescence intensity values for S1PR1 staining at the end of the incubation period. Idelalisib increased TEM toward S1P in all cases, the amount of migration correlating with levels of S1PR1. Untreated CLL cells underwent little or no migration. (B) CLL cells from three patients were cultured in the absence or presence of idelalisib, fostamatinib, or ibrutinib (all at 1 μM) and examined for migration toward CCL21 using HUVEC-coated transwells. Untreated CLL cells underwent TEM toward CCL21. Migration was reduced by fostamatinib and ibrutinib, but not idelalisib.