CD44 Signaling Via PI3K/AKT and MAPK/ERK Pathways Protects CLL Cells from Spontaneous and Drug Induced Apoptosis

Chronic lymphocytic leukemia (CLL) is an incurable malignancy of mature B-cells. CLL cell proliferation and survival depends on host factors that are present in the tissue microenvironment. In–vitro these cells rapidly undergo apoptosis but can be supported by culture on stroma cells. The extracellular matrix is an important component of the microenvironment. Interactions between tumor cells and the extracellular matrix are, in part, mediated by CD44, whose principle ligand is hyaluronic acid. To explore a possible role of this adhesion receptor in CLL, we evaluated the effect of CD44 engagement on the survival of CLL cells and the induction of drug resistance. Dimerization of CD44 activated the PI3K/AKT and MAPK/ERK pathways and resulted in increased levels of MCL-1. There was no increase in MCL mRNA in CD44 stimulated cells, consistent with an effect on protein stabilization. Consistent with the induction of these anti-apoptotic mechanisms, CD44 protected CLL cells from spontaneous apoptosis: CD44 stimulated CLL cells had a 46% increase (range 7% – 181%) in viability over the corresponding unstimulated control cells (n=20, p<0.0001). Furthermore, CD44 activation also protected from fludarabine-induced apoptosis: cell viability for isotype treated cells 76 ±6%, for isotype and fludarabine treated cells 47 ±18% and for CD44 stimulated and fludarabine treated cells 69 ±16% (p=0.005). CLL cells of the IgVH unmutated subtype expressed CD44 more highly than IgVH mutated CLL cells (MFI ratio 224 ±43 to 122 ±44, respectively, p<0.0001) and derived a more pronounced survival advantage from CD44 activation: % live cells (CD44 stimulated - control) 21 ±9% vs. 13 ±6%, respectively (p=0.04), which could contribute to the more progressive nature of this subtype. PI3K or MEK inhibitors as well as obatoclax, an antagonist of MCL-1, blocked the pro-survival effect of CD44 activation. Furthermore, obatoclax was able to sensitize CLL cells to fludarabine. In conclusion, CD44 engagement protects CLL cells from spontaneous and fludarabine induced apoptosis. This prosurvival effect is mediated through PI3K/Akt and MAPK/ERK pathways and increased MCL-1 levels. Targeting either signaling pathway or MCL-1 with specific inhibitors could overcome the protective effect of the microenvironment in CLL and provides a rationale for combination therapy.