Dasatinib-Induced Apoptosis in Chronic Lymphocytic Leukaemia Is Abrogated by Selective Micro-Environmental Signals.

Recent advances in chronic lymphocytic leukaemia (CLL) biology indicate that a plethora of micro-environmental factors are important in promoting the pathogenesis of the disease. As B cell antigen receptor (BCR) signalling plays a critical role in CLL progression, we chose to study the effect of dasatinib, a dual src/abl tyrosine kinase inhibitor, on CLL cells, thus targeting the key, BCR-proximal tyrosine kinases Lyn and c-Abl that are over-expressed in CLL. We were interested to determine the impact of BCR-signalling directed therapy on CLL cell viability in the context of the leukaemic micro-environment.

Clinically relevant concentrations of dasatinib (100 nM) both inhibited ERK-MAPK and Akt phosphorylation, and prevented calcium mobilisation following BCR crosslinking. Furthermore, dasatinib prevented up-regulation of Mcl-1 observed on prolonged BCR stimulation, abrogating the BCR-driven increase in CLL cell survival. Dasatinib induced apoptosis of CLL cells, with a mean reduction in viability of 35.2% ± 3.8% following 48 hr exposure (n=26). Neither ZAP-70 expression nor cytogenetic abnormalities were predictive of response. In addition, dasatinib exhibited synergy with both fludarabine and chlorambucil, with mean ED50 combination indices of 0.29 and 0.62 respectively. Additional micro-environmental elements responsible for the maintenance and progression of CLL include stromal or ‘nurse-like’ cells and activated T lymphocytes, the latter of which express CD40 ligand and secrete IL-4. CLL cell co-culture with the murine bone marrow stromal cell line NT-L significantly inhibited both spontaneous and dasatinib induced apoptosis. As we observed that stromal co-culture significantly increased phosphorylation of both ERK-MAPK and the Akt/mTOR target p70 S6 kinase in CLL cells, we investigated whether pharmacological inhibition these signalling pathways may re-sensitise CLL cells to dasatinib in co-culture. Both the MEK inhibitor PD98059 and PI3K inhibitor LY294002 significantly increased the level of apoptosis observed on dasatinib treatment of CLL cells in stromal co-culture. Dasatinib also retained the ability to potentiate the effects of fludarabine and chlorambucil in stromal co-culture, however, CLL cells co-cultured with NT-L cells stably transfected with CD40 ligand (CD154L cells) were resistant to all drug combinations. Furthermore, on CD154L/IL-4 co-culture, dasatinib failed to prevent up-regulation of Bcl-xL and Mcl-1, or inhibit CLL cell proliferation.

While dasatinib was capable of reversing BCR-mediated survival signals, it was unable to overcome stromal-derived survival signals, indicating that combination of dasatinib with agents targeting antigen-independent micro-environmental pathways are required to effectively target CLL cells in tissue niches.

No relevant conflicts of interest to declare.