Multiple myeloma (MM) is an incurable hematological cancer characterized by the uncontrolled proliferation of clonal plasma cells (PC) within the bone marrow (BM).¹ One of the key features of MM is the presence of MM PC at multiple sites throughout the BM, highlighting that dissemination of transformed PC is a critical process during disease development.^1,2 In support of this, circulating MM PC are detectable by flow cytometry in approximately 75% of newly diagnosed MM patients.³ Importantly, the presence of elevated circulating MM PC predicts faster time to progression and poorer overall survival, independent of BM tumor burden.^4-12

The dissemination of MM PC is a multi-step process requiring release from the supportive niche in the BM, intravasation into nearby blood vessels and subsequent extravasation and homing to another BM site. Integrin mediated adhesion of MM PC to BM stromal cells (BMSC), and extracellular matrix components synthesized by BMSC, is well-established to mediate retention of MM PC within the niche.¹³ For example, MM PC express the integrin α4b1 (also known as very late antigen 4, VLA-4) that mediates adhesion to vascular cell–adhesion molecule 1 (VCAM-1) on BMSC and to the extracellular matrix component fibronectin.¹³ Importantly, the C-X-C chemokine ligand CXCL12 (also known as stromal cell-derived factor- 1; SDF-1), abundantly produced by BMSC,¹⁴ enhances adhesion to fibronectin and VCAM-1 through binding to its receptor CXCR4 on the surface of MM PC and inducing rapid conformational changes of the integrin α4b1 complex on MM PC.¹⁵ Notably, plerixafor-mediated inhibition of the CXCL12 receptor CXCR4 on MM PC results in mobilization of MM cells to the peripheral blood (PB) in a preclinical model of MM.¹⁵ These data suggest that CXCL12 is a critical BM retention signal for MM PC and that overcoming the CXCL12/CXCR4 signal may be required for release from the niche during dissemination.

In a previous study by Azab and colleagues, increased hypoxia in the BM was shown to be associated with an increase in circulating MM PC in a preclinical model.¹⁶ Additionally, we have previously identified that overexpression of the hypoxia-inducible factor 2α (HIF-2α) in MM cell lines reduces their response to exogenous CXCL12 in vitro, suggesting that hypoxia may overcome CXCL12-mediated retention. Furthermore, we identified that hypoxia and HIF-2α increased expression of the C-C chemokine receptor CCR1 in human MM cell lines.¹⁷ CCR1 is a seven-transmembrane G-protein coupled receptor and its most potent activator is CCL3 (also known as macrophage inflammatory protein 1α; MIP-1α). Previous literature suggests that MM PC abundantly produce CCL3^18-21 which activates CCR1 expressed on osteoclasts leading to increased osteolysis,¹⁹ with CCR1 antagonists reducing osteolysis in a murine model of MM.^22,23 In addition, CCL3 is a potent inducer of migration of patientderived MM PC and MM cell lines in vitro.^17,19,20,24 In hematopoietic progenitors and natural killer cells, CCL3/CCR1 signaling drives mobilization from the BM, in part by inactivation of CXCL12/CXCR4.^25,26 Similarly, our previous studies showed that either pre-treatment of MM cell lines with CCL3 or elevated CCR1 expression decreased tumor cell migration towards CXCL12 in vitro.¹⁷ Taken together, these data suggest that hypoxia-mediated increases in CCR1 expression may desensitize cells to CXCL12-mediated BM retention and thereby facilitate dissemination. In support of this, we have previously shown that expression of CCR1 in MM PC is associated with poorer prognosis and an increase in the number of circulating MM PC in newly diagnosed MM patients.¹⁷ Here, we further investigated the association between CCR1 expression and poor overall survival rates in MM patients. Furthermore, we investigated the role for CCR1 in the dissemination of MM PC in vivo. Initially, we determined whether CCR1 overexpression can promote tumor dissemination in the syngeneic 5TGM1/KaLwRij murine model of MM. Furthermore, using xenograft models of MM, we assessed whether CCR1 knockout limits the dissemination of MM PC in vivo. Lastly, we investigated whether pharmacological inhibition of CCR1 can be used as a viable therapeutic strategy to limit MM PC dissemination.

MM is characterized by the presence of multiple tumors throughout the skeleton, and in some patients, soft tissues. The dissemination of MM PC is central to the progression of disease and subsequent disease relapse, highlighting the therapeutic potential of targeting key factors that regulate dissemination to delay disease progression and prevent overt relapse. While the inhibition of several factors, including selectins,³⁰ N-cadherin^31,32 and CXCR4³³ have been demonstrated to slow BM homing of MM cells in vivo, very few genes have been demonstrated to play a role in the spontaneous dissemination of MM PC from the BM. For example, overexpression of heparanase, an enzyme that cleaves heparan sulphate chains, has been reported to increase the incidence of spontaneous dissemination of MM cells in a mouse MM xenograft model.³⁴ Additionally, recent data suggests that the transcription factor Twist-1 increases dissemination in an intratibial 5TGM1/KaLwRij model in vivo.³⁵ Furthermore, as far as we are aware, no therapeutic interventions have been described that can inhibit spontaneous dissemination of MM PC in vivo. Here, our findings suggest a novel role for the chemokine receptor CCR1 in regulating the egress of MM PC from the BM to the circulation during dissemination. These findings are consistent with a role for CCR1 in metastasis in other cancer settings, with a study showing that short hairpin RNAknockdown of CCR1 decreased migration of hepatocellular carcinoma cells in vitro and reduced the incidence of lung metastasis in vivo.³⁶

We have previously demonstrated that hypoxia, through induction of HIF-2α, increases the expression of CCR1 in human MM cell lines.¹⁷ This led us to hypothesize that tumor growth in the BM exacerbates BM hypoxia, leading to increased CCR1 expression and tumor dissemination.¹⁷ Consistent with this hypothesis, our flow cytometric analysis suggests that CCR1 expression on BM PC is increased in MM patients compared with MGUS patients. In addition, our analysis suggested that elevated CCR1 expression is an independent predictor of poor overall survival in MM patients. Mechanistically, we have previously demonstrated that CCL3 treatment of human MM cell lines reduces their capacity to migrate towards exogenous CXCL12 or undergo cytoskeletal remodeling in response to CXCL12 treatment.¹⁷ Furthermore, we found that the human MM cell line U266, which does not respond to exogenous CXCL12, produces abundant CCL3, suggesting that endogenous CCL3 expression can suppress response to CXCL12. Notably, migration of U266 cells towards CXCL12 could be restored by either CCR1 KO or treatment with a CCR1 inhibitor.¹⁷ Here, we observed that treatment of OPM2-EV-1 control cells with CCL3 prevented migration towards CXCL12, in accordance with our previous study,¹⁷ whereas, the chemotactic response of OPM2- CCR1-KO-1 cells to exogenous CXCL12 was maintained following CCL3 treatment. These data strongly suggest that CCL3/CCR1 signaling is responsible for blocking migration towards CXCL12 in these cell lines. Notably, CCL3/CCR1 signaling has been shown to drive mobilization of hematopoietic progenitors and natural killer cells from the BM in part through inactivation of CXCR4.^25,26,37 Based on these studies, we postulated that CCL3/CCR1 signaling may inhibit the CXCL12 mediated retention of MM PC in the BM, enabling the egress of MM PC into the circulation and subsequent dissemination. Indeed, we demonstrate here that CCR1 expression increases the 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 mobilization.

In addition to its role in counteracting CXCL12/CXCR4 signaling, CCL3 is known to act as a potent chemoattractant for murine and human MM cell lines and patientderived 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 osteoclasts^19,20 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 therefore likely that autocrine CCL3 production would interfere with the chemoattractant effect of exogenous CCL3. In further 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 significant 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 previous 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 agonistindependent 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 recombinant 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, suggesting that CCR1 KO may affect growth of OPM2 cells in vivo. However, we did not observe an effect of CCR1 overexpression or inhibition on primary MM tumor growth in our other in vivo models, suggesting this effect may be independent of CCR1. Additional studies are required to determine 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 proliferation 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 previous study which reported that treatment with the CCR1 inhibitor CCX721 at high doses had no effect on the proliferation 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 suggests that CCL3/CCR1 inhibitors may affect growth factor production by cells of the BM microenvironment to indirectly affect 5TMM tumor growth.²³ Similar effects were observed with osteoclast ablation using zoledronate, suggesting 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, supporting 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, suggesting 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 previously 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 development 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 pathogenesis, 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 dissemination. Together with previous studies demonstrating that targeting of CCR1 prevents the development of severe osteolytic lesions in vivo, and our data demonstrating that CCR1 is an independent prognostic factor in MM patients, our study suggests that CCR1 is a potential attractive therapeutic target for MM. Future preclinical studies are warranted to investigate whether therapeutic inhibition of CCR1 has efficacy as a maintenance therapy, extending post-therapy remission and preventing overt relapse.