Inhibition Of Lyn and Syk By Treatment With Dasatinib, Fludarabine, and Rituximab Correlates With Apoptosis and Clinical Response In Patients With Relapsed CLL

Survival, proliferation, and resistance to chemotherapy in CLL cells have been shown to be consistently associated with the activity of the B-cell receptor (BCR) and the associated downstream pathways activated by the BCR. Key molecules in this pathway are LYN and SYK (Spleen tyrosine kinase), as well as PI3K, BTK (Bruton’s tyrosine kinase), and others. Dasatinib, given at standard doses, allows for serum levels well above 11 nM, the IC50 for suppression of LYN kinase. We have previously shown that dasatinib used as a single agent in patients with relapsed CLL results in lymph node responses in 60% of patients and partial responses in 20% of patients as defined by NCI-WG criteria. In the current study, patients with relapsed CLL were treated with a regimen combining dasatinib at 140 mg/day, days 1-14, with fludarabine (F) 25 mg/m2/day, days 1-3, and rituximab (R) 375 mg/m2 per cycle repeated every 28 days, while effective up to 6 cycles. Patients were followed closely for response with CT scans every 2 months initially. Among the first 10 patients treated, all had responses according to IWCLL criteria as follows:

The median time to progression was 21 months.

In the first week multiple blood samples were taken for analysis of target inhibition and subsequent apoptosis. The schedule of administered agents was altered in the first week to determine which components were associated with which downstream effects. Hence, dasatinib was given on Day 1, no treatment was administered on Day 2, F and R without dasatinib on Day 3, dasatinib with FR on Day 4, and dasatinib with F on Day 5. Initial in vitro studies revealed inhibition of phosphorylation of Lyn at 6 hours after patients were given dasatinib on Days 1 and 4, with recovery by 24 hours. Day 3 treatment with FR but without dasatinib showed no such inhibition at 6 hours. Assessment of global tyrosine phosphorylation in CLL cells showed this same pattern, including that of Syk phosphorylation, specifically. Flow cytometry for annexin-V demonstrated that apoptosis was greatest on Day 4 after 6 hours of exposure to all 3 drugs. We conclude the following: 1) the combination of dasatinib with FR, as seen in the first 10 patients of this study, was associated with excellent responses in blood and lymph nodes as assessed by physical exam, 2) the combination was well tolerated with mainly hematologic toxicity, 3) the inhibition of phosphorylation of Lyn and Syk was associated with apoptosis and clinical response. This combination may have therapeutic promise in advanced CLL and is worthy of further investigation.