Targeting the Wnt/β-Catenin Signaling Pathway and CD44-Mediated Adhesion As a Rational Approach to Overcome Lenalidomide Resistance in Multiple Myeloma

Lenalidomide (Len) has been shown to be clinically effective against multiple myeloma (MM) as a single agent and in combination for both newly diagnosed and relapsed/refractory settings, and is increasingly utilized in maintenance therapy. Despite the successful use of Len, some patients display a poor response or become refractory after an initial response. As previously published by our group, hyperactivation of Wnt/β-catenin signaling was identified as a mediating factor for Len-specific resistance in MM cell lines. Additionally, a downstream transcriptional product of Wnt/β-catenin signaling is the extracellular matrix binding protein CD44, which has been implicated in cellular-adhesion-mediated drug-resistance (CAM-DR) in several cancer models, including dexamethasone-resistant MM. These early studies established a rationale to target aberrant Wnt/β-catenin signaling and CD44-mediated CAM-DR to overcome Len resistance.

Wild-type (Wt) and lenalidomide-resistant (LR) MM cell lines were used as models to evaluate the effectiveness of targeting the Wnt/β-catenin signaling pathway and CD44-dependent CAM-DR to overcome Len resistance with Wnt/β-catenin antagonists, shRNA knockdown of CD44, and anti-CD44 neutralization.

All-trans-retinoic acid (ATRA) inactivates β-catenin nuclear signaling by altering β-catenin cellular distribution. Combination treatment with Len and ATRA produced a significant (p<0.05) anti-proliferative and cytotoxic effect on LR cells. ATRA exposure of both Wt and LR cells also effectively reduced β-catenin-dependent T-cell factor/lymphoid enhancer factor (TCF/LEF) promoter activity by as much as 70%, and reversed the apparent increase in activity stimulated by acute Len treatment. Cell surface and total CD44 levels were effectively reduced by as much as 60% when cells were treated with ATRA, even in the presence of Len. Primary patient samples from individuals displaying Len refractory disease were treated ex vivo with Len and ATRA in combination, resulting in a synergistic reduction in cellular viability. We investigated an alternative approach to β-catenin abrogation using FH535, a small molecule β-catenin antagonist that disrupts the transcriptional-dependent association of β-catenin and TCF. Combinations of Len with FH535 resulted in an additive suppression of cellular proliferation on Wt MM cell lines. Similarly, FH535 reduced the viability and enhanced Annexin-V staining on LR KAS-6 and U266 cells by as much as 80% and 55% respectively when used in combination with Len. In addition to selectively targeting β-catenin to overcome LR MM, we evaluated the role of CD44 in LR cell lines. Flow cytometric analysis revealed that cell surface CD44 levels were increased by as much as 11-fold in LR KAS-6 cells when compared to Wt cells. Acute treatment of Wt ANBL-6 and MM1.S cells with Len moderately enhanced the cell surface levels of CD44 by approximately 1.5-fold. The enhanced CD44 surface levels induced by Len on LR ANBL-6 and KAS-6 corresponded to at least a 2-fold increase in cellular adhesion to bone marrow stromal cells, and enhanced adhesion to increasing concentrations of hyaluranon (HA). Pre-incubation with free HA or CD44 neutralizing antibodies reduced the adhesive properties of both Wt and LR ANBL-6 and KAS-6 cell lines. shRNA knockdown of CD44 sensitized LR cells to Len when compared to a non-specific scrambled shRNA transfection. Interestingly, extensive statistical analysis of banked gene expression data from primary samples located at the Multiple Myeloma Research Consortium (MMRC) database revealed a significant correlation (p=0.01) between high expression levels of CD44 and a poor outcome prognosis, albeit irrespective of treatment regimens. Furthermore, current analysis of surface CD44 levels on primary MM bone marrow aspirates suggests that those patients who have received prior Len therapy have an overall increase in CD44 when compared to Len naïve patients.

Collectively, our results suggest that MM cell Len resistance is, at least in part, dependent on β-catenin and CD44 and that selective targeting of these cellular proteins in conjunction with Len treatment represents a rational approach for clinical treatment of Len-resistant MM.

Aukerman:Celgene Corporation: Employment. Lopez-Girona:Celgene Corp: Employment, Equity Ownership. Hussein:Celgene Corporation: Employment. Chopra:Celgene Corporation: Employment. Orlowski:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.