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Survival of chronic lymphocytic leukemia (CLL) cells in vivo is supported by the tissue microenvironment, which includes components of the extracellular matrix. Interactions between tumor cells and the extracellular matrix are in part mediated by CD44, whose principle ligand in this respect is hyaluronic acid. Purpose: to evaluate the effect of CD44 engagement on the survival of CLL cells. Experimental Design: CD44 in CLL cells was engaged by anti-CD44 monoclonal antibody, or hyaluronic acid, and the effects of CD44 activation on CLL cell viability and pro-survival pathways were evaluated. Results: engagement of CD44 activated the PI3K/AKT and MAPK/ERK pathways and increased MCL-1 protein expression. Consistent with the induction of these anti-apoptotic mechanisms, CD44 protected CLL cells from spontaneous and fludarabine-induced apoptosis. Leukemic cells of the more aggressive CLL subtype that express unmutated IgVH genes (U-CLL) showed higher CD44 expression than IgVH-mutated CLL (M-CLL) cells, and acquired a greater survival advantage via CD44 activation. Thus, CD44 activation in the tissue microenvironment may contribute to increased MCL-1 protein levels, resistance to apoptosis, and could contribute to the more progressive nature of U-CLL. Furthermore, PI3K or MEK inhibitors as well as obatoclax, an antagonist of MCL-1, blocked the pro-survival effect of CD44. In addition, obatoclax synergized with fludarabine to induce apoptosis of CLL cells. Conclusions: components of the extracellular matrix may provide survival signals to CLL cells through engagement of CD44. Inhibition of MCL-1, PI3K, and MAPK/ERK pathways are promising strategies to reduce the anti-apoptotic effect of the microenvironment on CLL cells.

The online version of this article contains a supplementary appendix. BackgroundIn chronic lymphocytic leukemia lenalidomide causes striking immune activation, possibly leading to clearance of tumor cells. We conducted this study to investigate the mechanism of action of lenalidomide and the basis for its unique toxicities in chronic lymphocytic leukemia. Design and MethodsPatients with relapsed chronic lymphocytic leukemia were treated with lenalidomide 20 mg (n=10) or 10 mg (n=8) daily for 3 weeks on a 6-week cycle. Correlative studies assessed expression of co-stimulatory molecules on tumor cells, T-cell activation, cytokine levels, and changes in lymphocyte subsets. ResultsLenalidomide upregulated the co-stimulatory molecule CD80 on chronic lymphocytic leukemia and mantle cell lymphoma cells but not on normal peripheral blood B cells in vitro. T-cell activation was apparent in chronic lymphocytic leukemia, weak in mantle cell lymphoma, but absent in normal peripheral blood mononuclear cells and correlated with the upregulation of CD80 on B cells. Strong CD80 upregulation and T-cell activation predicted more severe side effects, manifesting in 83% of patients as a cytokine release syndrome within 8-72 h after the first dose of lenalidomide. Serum levels of various cytokines, including tumor necrosis factor-α, increased during treatment. CD80 upregulation on tumor cells correlated with rapid clearance of leukemic cells from the peripheral blood. In contrast, neither the severity of the cytokine release syndrome nor the degree of T-cell activation in vitro correlated with clinical response. ConclusionsUpregulation of CD80 on tumor cells and T-cell activation correlate with unique toxicities of lenalidomide in chronic lymphocytic leukemia. However, T-cell activation appears to be dispensable for the drug's anti-tumor effects. This provides a rationale for combinations of lenalidomide with fludarabine or alemtuzumab.