Assessing CCR1 Antagonists for Chemotaxis Inhibition in a Multiple Myeloma in vitro Model

Abstract

Multiple myeloma (MM) is a plasma B‐cell malignancy characterized by osteolytic bone lesions. MM cells secrete and express CCL3/MIP1α which upregulates osteoclastogenesis. Elevated CCL3 levels display a chemotactic ability on isolated osteoclast precursors. Increased levels of CCL3 in MM patients correlates with a greater disease burden, due to the increase of bone resorption, and is indicative of a worse prognosis when compared to MM patients exhibiting lower CCL3 levels. CCR1, a GPCR chemokine receptor, is endogenously expressed on MM cells, and can bind with CCL3. In a previous study, the RPMI8226 MM cell line, CCL3‐mediated CCR1 chemotaxis was inhibited in a dose‐dependent manner by six CCR1 antagonists (AZD4818, BX471, CCX354, CP481715, MLN3897, PS031291). In this study, we assessed the MM cell line U266 as well as a transfected cell line, U266_CCR1. While U266 cells express lower levels of CCR1 than RPMI8226, U266_CCR1 express higher levels of CCR1. Cells were treated with a serial dilution of the same six CCR1 antagonists and chemotaxis to either supernatant from osteoclast precursor RAW 264.7 cells or Fetal Bovine Serum (FBS) in which a multitude of growth factors and chemokines are present was examined. We hypothesized the six CCR1 antagonists would result in a dose‐dependent inhibition of chemotaxis similar to that seen with RPMI8226. Instead, we found only two of the compounds (AZD4818 and BX471) inhibited chemotaxis of the U266 and U266_CCR1 MM cell lines towards RAW 264.7 supernatant or FBS. For both AZD4818 and BX471 there were differences between the chemotactic response of the U266 and U266_CCR1 cell lines, with the U266_CCR1 cell line having a greater degree of inhibition, suggesting the inhibition is driven by CCR1.