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CCR1 drives dissemination of multiple myeloma plasma cells
sion of integrin α4 (ITGA4) and integrin b1 (ITGB1), critical adhesion molecules that play a role in MM PC BM reten- tion (Online Supplementary Figure S3).
The CCR1 inhibitor CCX9588 inhibits migration towards CCL3 in vitro
Next, the effects of a selective small molecule CCR1 inhibitor, CCX9588, on MM cells was assessed in vitro. In order to investigate whether the small molecule CCR1 inhibitor CCX9588 effects cell survival and/or proliferation, CCR1-expressing OPM2-EV-1 or RPMI-8226-luc17 cells were cultured with increasing concentrations of CCX9588 or vehicle alone. OPM2-EV-1 cell number (P=0.88, Figure 6A) and viability (P=0.70, Figure 6B) were not affected by treatment with up to 1 mM CCX9588. However, there was a 35% decrease in cell number in RPMI-8226-luc cells treat- ed with 1 mM CCX9588 (P<0.01, Figure 6C), while cell sur- vival was unaffected (P=0.50, Figure 6D), suggesting that high concentrations may decrease proliferation of these cells. Based on these results, concentrations up to 100 nM and 1 mM were used for further characterization in RPMI- 8226 and OPM2 cells, respectively.
In order to confirm the anti-CCR1 function of CCX9588, OPM2-EV-1 or RPMI-8226-luc cells were treated with CCX9588 or vehicle alone and were then stimulated with rhCCL3. Western blot analysis revealed that, in OPM2-EV- 1 cells, CCL3 treatment induced AKT and ERK1/2 phos- phorylation, which was inhibited by 10 nM CCX9588 or higher (Figure 6E). In RPMI-8226-luc cells, AKT phosphory- lation was increased by CCL3 and this was inhibited by 10 nM CCX9588 or higher (Figure 6F). Furthermore, pre-treat-
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ment of OPM2 or RPMI-8226 cells with CCX9588 resulted in a complete inhibition of migration towards rhCCL3 in a transwell assay (OPM2: P<0.001, Figure 6G; RPMI-8226: P<0.01, Figure 6H).
CCX9588 treatment reduces dissemination of multiple myeloma plasma cells in vivo
In order to investigate the effectiveness of CCR1 inhibi- tion in suppressing MM PC dissemination in vivo, the effects of the CCR1 inhibitor CCX9588 were assessed in mice bearing OPM2-EV-1 or RPMI-8226-luc tumors. CCX9588 treatment did not have appreciable adverse effects on the mice, as assessed by body weight (Online Supplementary Figure S4A) or analysis of PB cell counts (Online Supplementary Table S1). Mean trough serum concentration of CCX9588 achieved in vivo was 328 nM (range, 76.8-886 nM; Online Supplementary Figure S4B).
In mice bearing OPM2-EV-1 or RPMI-8226-luc cells, pri- mary tumor burden was unaffected by CCX9588 treatment (OPM2-EV-1: P=0.91, Figure 7A; RPMI-8226-luc: P=0.49, Figure 7B). Consistent with the effect of CCR1 KO in OPM2 cells, we observed a 66% decrease in the mean num- ber of circulating tumor cells in the OPM2-EV-1 model (P<0.0001; Figure 7C); while the decrease in circulating tumor cells in the RPMI-8226-luc model did not reach sta- tistical significance (P=0.09; Figure 7D). CCX9588 treat- ment significantly reduced dissemination to the bone, with a 22% and 70% reduction in mean tumor burden in the BM of the contralateral limb in the OPM2-EV-1 (Figure 7E) and the RPMI-8226-luc models, respectively, compared with controls (P<0.0001; Figure 7F). Furthermore, the degree of
CDE
Figure 4. Knockout of CCR1 in human OPM2 multiple myeloma plasma cells decreases migration towards CCL3 and does not affect proliferation. (A) CRISPR-Cas9- mediated knockout (KO) of CCR1 was confirmed in OPM2-CCR1-KO-1 and OPM2-CCR1-KO-2 cells following staining with an anti-hCCR1 antibody or isotype control. (B) Migration of OPM2-CCR1-KO-1 and OPM2-CCR1-KO-2 cells and empty vector (EV) control cells towards 100 ng/mL rhCCL3, or media alone, was assessed after 18 hours. Migration is expressed relative to no chemoattractant controls. (C) Relative numbers of OPM2-CCR1-KO-1 or OPM2-EV-1 control cells were assessed over 72 hours. (D) Relative numbers of OPM2-CCR1-KO-2 or OPM2-EV-2 control cells were assessed over 72 hours. (E) The effect of 72 hours of treatment with 100 ng/mL rhCCL3 on relative numbers of OPM2-CCR1-KO-1 and OPM2-CCR1-KO-2, and EV-1 and EV-2 control cells. Graphs depict mean ± standard error of the mean of three or more independent experiments (A to E). **P<0.001, two-way ANOVA with Sidak’s multiple comparison test.
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