HIF-2α Upregulates CCR1 to Promote Dissemination of Plasma Cells in Multiple Myeloma

Introduction: Multiple myeloma (MM) disease progression and relapse is dependent on the ability of the MM plasma cells (PC) to leave the bone marrow, re-enter the circulation and disseminate to other sites throughout the skeleton. Recent data suggests that increased bone marrow hypoxia is associated with increased recirculation of MM PC (Azab et. al. Blood 2012), suggesting that hypoxia may act as a stimulus for dissemination of MM PC. However, the mechanisms responsible for this phenomenon remain unclear. In this study, we identified a potential driver of hypoxia-mediated dissemination of MM PC.

Methods and results: To identify which genes were regulated by hypoxia in MM PC, we utilised microarrays to assess the effects of HIF-2α overexpression on gene expression in the human MM cell line, LP-1. The most highly upregulated gene was the C-C chemokine receptor 1 (CCR1) (6.5-fold upregulated), the receptor for C-C chemokine ligand 3 (CCL3; also known as macrophage inflammatory protein-1α [MIP-1α]), a potent inducer of MM PC migration in vitro . In addition, overexpression of HIF-2α in the human MM cell line RPMI-8226 resulted in an almost three-fold increase in migration towards CCL3, indicating that upregulation of CCR1 in response to HIF-2α confers an increase in the migratory response to CCL3.

Previous studies have indicated that over two-thirds of newly diagnosed MM patients have circulating MM PC, with increased PC numbers associated with a poorer prognosis (Nowakowski et. al. Blood 2005). CCR1 expression on peripheral blood MM PC, as assessed by flow cytometry, positively correlated with the number of circulating MM PC. Moreover, in silico analysis of CCR1 expression in publically-available microarray data from newly diagnosed MM patients found that above median CCR1 expression was associated with poor prognosis in these patients in an independent dataset (p=0.025; HR=2.3 [95% CI: 1.1-4.9]; n=142 patients; E-TABM-1138).

To identify the potential role of CCR1 in driving MM PC dissemination, the migration and proliferation of MM PC expressing CCR1 was assessed in vitro and in vivo . The role of CCR1 in tumour growth and dissemination of MM PC was investigated using the C57BL/KalwRijHsd mouse model of MM. In this model, luciferase-expressing mouse 5TGM1 MM PC cells were injected into the tibia, where they establish in the bone marrow and initiate systemic MM disease. Using flow cytometry, our studies show that C57BL/KalwRijHsd mice bearing 5TGM1 CCR1 overexpressing cells (5TGM1-CCR1) have increased numbers of circulating tumour cells, when normalized to tumour burden, compared with mice bearing control 5TGM1 cells. These data suggest that expression of CCR1 drives the egress of MM PC from the bone marrow into the circulation in vivo .

Conclusion: This study identified CCR1 as a novel driver for hypoxia-driven MM PC migration both in vitro and in vivo . Furthermore, this study highlights an association between increased CCR1 expression, poorer prognosis and an increase in circulating tumour cells in newly diagnosed MM patients. Importantly, these findings suggest that therapies targeting CCR1 may inhibit MM PC egress from the bone marrow and limit disease progression and relapse.