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CCR1 drives dissemination of multiple myeloma plasma cells
capacity for MM PC dissemination, while CCR1 inhibition or KO decreases mobilization of MM PC to the PB, and the subsequent formation of disseminated tumors in vivo. Our findings support our hypothesis that hypoxia-mediated upregulation of CCR1 may be critical for overcoming CXCL12-mediated BM retention and enabling mobiliza- tion.
In addition to its role in counteracting CXCL12/CXCR4 signaling, CCL3 is known to act as a potent chemoattrac- tant for murine and human MM cell lines and patient- derived MM PC in vitro.17,20,38 In accordance with this, we demonstrated that CCL3 acts as a chemoattractant for OPM2 and RPMI-8226 cells, which could be blocked with CCR1 KO or inhibition. Furthermore, expression of CCR1 in 5TGM1 cells resulted in a chemotactic response to CCL3. However, while CCL3 has been shown to be produced by mesenchymal stem cells20 and osteoclasts19,20 in the BM, the most abundant source of CCL3 in the BM in MM patients is suggested to be the MM PC themselves.18-21,24 It is there- fore likely that autocrine CCL3 production would interfere with the chemoattractant effect of exogenous CCL3. In fur- ther support of this, CCL3 is present at higher levels in the BM than in the PB of MM patients,21,39,40 suggesting that migration towards CCL3 in the PB would not play a signif- icant role in the mobilization of MM PC from the BM. Instead, it is possible that autocrine CCL3 production may increase non-directional migration (chemokinesis), as has been described for chemokines CCL2 and IGF-1 in MM cell lines.41,42 In accordance with this, we found that inhibition of CCR1 in RPMI-8226-luc cells using CCX9588 resulted in a reduction in basal migration. This is consistent with a pre- vious study, whereby the CCR1 inhibitor BX471 prevented basal migration of the human acute monocytic leukemia cell line THP-1.43 Alternatively, CCR1 has been suggested to signal without the presence of ligand and induce agonist- independent migration in some cell types.43 Decreased basal migration or chemokinesis of these cells in the presence of CCR1 inhibitor may, therefore, in part be contribute to the decrease in dissemination of RPMI-8226 and OPM2 cells observed in vivo.
We observed no effect of CCR1 expression or KO on the proliferation of MM cell lines in vitro in either the presence or absence of exogenous CCL3. This was despite the ability of CCL3 to induce AKT and ERK phosphorylation, which are involved in survival/proliferation pathways in MM.24 This contrasts with a previous study suggesting that recom- binant CCL3 increases human MM cell line proliferation in vitro.24 As such, the possibility that the relatively high serum concentration used here could be providing sufficient other growth factors to mask the effects of CCL3 cannot be excluded. Mice injected with OPM2-CCR1-KO cells had lower primary tumor burden compared with controls, sug- gesting that CCR1 KO may affect growth of OPM2 cells in vivo. However, we did not observe an effect of CCR1 over- expression or inhibition on primary MM tumor growth in our other in vivo models, suggesting this effect may be inde- pendent of CCR1. Additional studies are required to deter- mine whether the retention of MM PC in the bone marrow with CCR1 KO may be causing environmental pressures, such as an increase in hypoxia,44 that is slowing their prolif- eration in vivo. In contrast, while CCX9588 treatment had no effect on the proliferation of OPM2 cells in vitro, we observed a decrease in the proliferation of RPMI-8226 cells with 1 mM CCX9588 treatment. This contrasts with a pre- vious study which reported that treatment with the CCR1
inhibitor CCX721 at high doses had no effect on the prolif- eration of RPMI-8226 cells in vitro,23 suggesting that the effects observed at high concentrations of CCX9588 here may be due to off-target effects. In support of this, CCX9588 treatment did not affect OPM2 or RPMI-8226 tumor growth in vivo. Inhibition of CCL3 or CCR1 in the murine 5T2MM and 5TGM1 models has previously been shown to decrease primary BM tumor growth, but not growth of subcutaneous tumors or cells in vitro.22,23 This sug- gests that CCL3/CCR1 inhibitors may affect growth factor production by cells of the BM microenvironment to indi- rectly affect 5TMM tumor growth.23 Similar effects were observed with osteoclast ablation using zoledronate, sug- gesting that these results may be secondary to decreased osteoclast activity/numbers in this model.23 The CCR1 inhibitor MLN3897 has previously been shown to decrease the pro-proliferative effects of osteoclast coculture on a CCR1-negative human MM cell line, at least in part through indirectly decreasing osteoclast IL-6 secretion, sup- porting the idea that effects of CCR1 inhibition on tumor growth in some in vivo models may be due to secondary effects on osteoclasts.19 However, we found no effect of CCR1 inhibition on primary tumor growth in vivo, suggest- ing that inhibition of microenvironmental CCR1 was not contributing to the effects observed here. Notably, we have previously demonstrated that treatment with the CXCR4 inhibitor T140 had no effect on intratibial RPMI-8226 tumor growth, despite dramatic effects on osteolysis and decreased osteoclast numbers, suggesting that inhibition of osteoclasts does not affect primary tumor growth in this model.45
Importantly, we are the first to assess the efficacy of the small molecule CCR1 inhibitor CCX9588 on dissemination in a pre-clinical model of MM. CCX9588 has been previ- ously reported to decrease chemotaxis of T cells towards liver conditioned media in vitro.46 CCX9588 is an analogue of CCX354, which has previously been investigated as a therapeutic for rheumatoid arthritis in a clinical trial,47 and CCX721, which has been shown to have anti-osteolytic activity in an in vivo MM model.23 While we were not able to completely prevent dissemination of MM PC using CCX9588 in OPM2 and RPMI-8226 xenograft models at this dose, these studies suggest that impeding the egress of MM PC from the BM to the PB could slow the develop- ment of disease. Further studies are required to determine whether combination therapy with other anti-myeloma agents, or more intensive treatment regimens, could achieve an enhanced effect on tumor dissemination. Notably, while both of the human MM cell lines used here do not express the alternate CCL3 receptor CCR5, CCR5 is expressed at the mRNA level in up to one third of MM patients (data not shown). Therefore, additional studies are warranted to determine whether CCR1 inhibition alone is sufficient to block dissemination when both CCR1 and CCR5 are expressed.
In summary, our studies have identified a novel role for the chemokine receptor CCR1 in the context of MM patho- genesis, demonstrating that CCR1 is a key driver of MM PC egress from the BM to the circulation during dissemination. Furthermore, we have shown that inhibition of CCR1 via therapeutic targeting or KO can slow MM PC dissemina- tion. Together with previous studies demonstrating that tar- geting of CCR1 prevents the development of severe oste- olytic lesions in vivo, and our data demonstrating that CCR1 is an independent prognostic factor in MM patients, our
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