Activation of the Wnt/β-Catenin Pathway Mediates Lenalidomide Resistance in Multiple Myeloma.

Novel drugs such as the immunomodulatory agent lenalidomide have revolutionized the treatment of multiple myeloma, as evidenced by an increasing overall survival for patients with both newly-diagnosed, and relapsed and/or refractory disease. Despite these improvements, myeloma remains incurable, and is still characterized by a trend for increasing chemoresistance at relapse, with a decreasing duration of benefit from each successive line of therapy. By understanding the mechanisms responsible for the emergence of drug resistance, which have so far not been well characterized in the case of lenalidomide, it may be possible to rationally design novel regimens that could either overcome this resistance, or possibly prevent its emergence altogether.

To improve our understanding of the mechanisms responsible for lenalidomide resistance, we developed cell line models of interleukin (IL)-6-dependent (ANBL-6 and KAS-6/1) and –independent (U266 and MM1.S) lenalidomide-resistant multiple myeloma cells. Starting at a concentration that was 1/10 of the IC₅₀ for lenalidomide's anti-proliferative effects in drug-naïve cells, increasing drug concentrations were used until all the cell lines could proliferate and maintain cell membrane integrity in the presence of 10 μM lenalidomide. These cell lines were then used as an in vitro model of lenalidomide-specific drug resistance, and subjected to further characterization, including with gene expression profiling.

Resistance to lenalidomide was evidenced by a dramatic, 100-1000-fold increase in the IC₅₀ values of these myeloma cells. In the case of ANBL-6 cells, for example, drug-naïve cells showed an IC₅₀ of 0.14 μM using tetrazolium dye-based viability assays, but this increased to >100 μM in the drug-resistant cells, as was the case in U266 and MM1.S cells. This resistance was a stable phenotype, since removal of lenalidomide for seven to ninety days from cell culture conditions did not re-sensitize them when 10 μM lenalidomide was reintroduced. Gene expression profiling followed by pathway analysis to examine changes at the transcript level between wild-type parental and lenalidomide-resistant cell lines identified the Wnt/β-catenin pathway as the most altered across all cell lines. Increased expression was seen in several members of the low-density-lipoprotein receptor related protein family, including LRP1 and 5; members of the wingless-type MMTV integrations site family, including WNT3 and 4; β-catenin; and downstream Wnt/β-catenin targets such as CD44. Similar changes were detected in primary samples from a patient who developed clinically lenalidomide-refractory disease. Reporter assays revealed an up to 5-fold increase in LEF/TCF-dependent transcription both in drug-naïve cells acutely exposed to lenalidomide, and in their chronically exposed, lenalidomide-resistant clones. Western blotting and flow cytometry confirmed that these lenalidomide-resistant cells had increased expression by 2-20 fold of β-catenin and CD44, as well as other LEF/TCF targets, including Cyclin D1 and c-Myc. Comparable changes occurred after lenalidomide exposure in myeloma cells grown in the context of bone marrow stroma. Notably, lenalidomide-resistant cells showed decreased expression of casein kinase 1 and increased phosphorylation of glycogen synthase kinase 3 at Ser21/9, both of which would reduce the phosphorylation of β-catenin needed for its later proteasome-mediated degradation. Stimulation of the Wnt/β-catenin pathway with recombinant human Wnt3a resulted in resistance to lenalidomide in wild-type, drug-naïve cells, as evidenced by a 10-fold increase in the IC₅₀. Conversely, exposure of lenalidomide-resistant cell lines to quercetin, a known antagonist of the β-catenin/TCF interaction, induced a partial re-sensitization to lenalidomide.

These data support the hypothesis that activation of the Wnt/β-catenin pathway represents a mechanism of both acute and chronic resistance to the anti-proliferative effects of lenalidomide in multiple myeloma. Moreover, they support the development of strategies aimed at suppressing Wnt/β-catenin activity to resensitize multiple myeloma to the effects of this immunomodulatory agent in vivo.

No relevant conflicts of interest to declare.