Targeting Lyn Kinase with Short Interfering RNA (siRNA)-A Novel Therapeutic Strategy for Drug Resistant Chronic Myelogenous Leukemia (CML).

Abstract

To examine the importance of Lyn kinase activity in CML cells, we employed Lyn targeted siRNA in Bcr/Abl-positive cell lines (K562, EM2, EM3), Bcr/Abl-negative cell lines (Mo7e, HL-60), and primary CML blast crisis cells isolated from patients with drug sensitive, and resistant disease. The siRNA were delivered by electroporation using an amaxa nucleoporator (Amaxa, Inc.) at a final working concentration between 1–5 μg/100 μl. By 48 hours after treatment, western blotting showed an 70–95% reduction in Lyn protein. Loss of Lyn protein was associated with an 3 fold inhibition of cell growth and viability at 72–96 hours in Bcr/Abl (+) cell lines. On the contrary, no inhibition in cell growth/viability was observed in Bcr/Abl (−) cell lines. Nucleoporation of control siRNA had no significant effect on cell growth in any of the lines examined. Based upon the differential sensitivity of Bcr/Abl (+) and (−) cell lines to Lyn ablation, we next asked if there were differential requirements for Lyn signaling in primary blast cells derived from Gleevec-resistant (n=3), and Gleevec-sensitive (n=2) patients, and whether normal hematopoietic progenitor cells also relied on Lyn signaling for growth. Anti-Lyn siRNA, delivered as described above, reduced Lyn protein, variably, in both malignant and normal primary cells. Lyn protein decrease ranged from 70 to 90% and was accompanied by a variable decrease in cell viability in CML blasts. Lymphoid blasts derived from Gleevec-resistant patients were particularly sensitive to Lyn ablation undergoing a massive induction of apoptosis by 48–96 hours after exposure to Lyn siRNA (90–100% reduction in cell viability). In contrast, CML cells from non-Gleevec resistant myeloid blast crisis patients appeared less sensitive to Lyn ablation since only 40–60% reduction in the cell viability was observed. Importantly, ablation of Lyn expression had no effect on the viability of normal CD34+ progenitors derived from healthy individuals (n=3). To ensure that growth arrest/apoptotic effects were not due to double stranded (ds) RNA triggering of the interferon (IFN) response pathway, we measured activation of dsRNA-dependent protein kinase, PKR, in K562 cells. While PKR kinase was robustly phosphorylated in long dsRNA treated cells (positive control), we did not detect any activation of PKR in anti-Lyn, or control siRNA-treated K562 cells. Finally, to ensure that “off target” silencing effects were not responsible for the observed decreases in cell viability and growth, we performed functional rescue experiments. To carry out these experiments, K562 cells were transfected with a pool of four individual SMARTselection-designed siRNA (Dharmacon, Inc.) designed to silence endogenous Lyn expression by specifically targeting the 3′UTR. Treated cells were then co-transfected with a Lyn expression construct that was lacking the 3′UTR. We observed that cells treated with siRNA targeting the 3′UTR underwent growth arrest and apoptosis. Cells co-transfected with the Lyn expression construct continued to grow and did not undergo apoptosis. In conclusion, these results demonstrate that Bcr/Abl (+) leukemias are dependent on Lyn kinase for survival and proliferation. Normal cells, and Bcr/Abl (−) leukemic cell lines, are not Lyn dependent and can tolerate its loss without apparent ill effect. Accordingly, Lyn is a legitimate therapeutic target in CML blast crisis and siRNAs targeting Lyn may find utility in the treatment of this disease, especially when Gleevec resistant.