Lenalidomide Suppression of Multiple Myeloma Cell Proliferation Is Associated with Downregulation of LEF/TCF Activity

Abstract 5014

Lenalidomide (Rev) is frequently used to treat multiple myeloma (MM). We reported that Rev promotes Dkk expression in MM cells. A recent study reported that resistance to Rev was associated with induction of Wnt/β-catenin signaling by increased β-catenin transcription and its decreased destruction (Bjorklund, JBC 2011). In this study, we evaluated whether these reported effects represent selection of pre-existing cell by exposure to Rev or regulation of the canonical Wnt signaling pathway by Rev, and whether the Wnt signaling pathway is associated with Rev's direct effect on MM cell survival.

To test the effect on Rev on proliferation of MM cells lines, the six MM cell lines H929, INA6, MM144, OPM-1, RPMI 8226, and U266 were cultured in growth media containing serial concentrations (0 to 1000 μM) of the drug for 24, 48 and 72 hours, and effect on proliferation measured by MTT assay. Rev diminished proliferation of these cell lines at concentrations between 50 to 1000 μM at 24 hours, and maximal inhibition occurred at 72 hours. Rev had little effect on the proliferation of the five MM cell lines at levels lower than 50 μM. Treatment with ≥5 μM Rev for 24, 48 and 72 hours resulted in increased DKK1 mRNA and Dkk1 protein levels as determined by qRT-PCR and by ELISA, respectively, in a dose dependent fashion, even at concentration that did not inhibit cell proliferation. These data suggest that Rev diminish MM proliferation is independent of its effects on Dkk1. We next examined the effect of Rev on β-catenin protein in cells treated with serial concentrations (0 to 1000 μM) of Rev for 6 hours. Immunoblotting analysis showed increased total β-catenin protein in 8226, OPM-2, H929, MM144 and U266 exposed to ≤100 μM, and no further increase in β-catenin levels when these cells were exposed to Rev concentrations higher than 100 μM. Rev did not affect changes in β-catenin levels in INA6. To determine the effects on Rev concentrations on TCF transcriptional activity, we infected cell lines with lentiviral particles containing the TCF reporter or with empty vector. Rev increased TCF activity at lower concentrations (10–20 μM) in all cells. As Rev concentration increased, TCF transcriptional activity gradually decreased, and was strongly inhibited (over 80%) at concentrations from 125 μM to 1000 μM, depending on the cell line; in this range, Rev suppressed MM proliferation.

These results suggest that at cytotoxic concentrations, Rev regulation of TCF transcriptional activity is independent of its effect on total β-catenin levels. It remains to be determined if Rev-mediated inhibition of TCF activity is the cause of the drug's cytotoxic effect, and the mechanism of the concentration dependent effects of Rev on TCF activity.