Src Kinase Inhibition by Dasatinib Enhances Targeting of Human AML Stem/Progenitor Cells by Chemotherapeutic Agents

Abstract 4270

Tyrosine kinases play a critical role in regulating cellular proliferation, differentiation and survival. We have previously shown that Src family tyrosine kinases (SFKs) including Lyn, Hck and Fgr are abnormally activated in Acute Myeloid Leukemia (AML) cells compared to normal CD34⁺ hematopoietic progenitors (Dos Santos et al., Blood 2008, 111:2269). Dasatinib is a potent small molecule inhibitor of SFKs and the ABL kinase that is approved for clinical use in Chronic Myeloid Leukemia. However the ability of Dasatinib to target AML is poorly documented. Here we evaluated the potential activity of Dasatanib on AML stem/progenitor cell maintenance and drug sensitivity. We assessed SFK phosphorylation in stem/progenitor populations from AML patients by flow cytometry (n=42). We observed significant increase of SFK phosphorylation in AML compared to normal Lin⁻ CD34⁺CD38⁻ cells (3.5±1.8 versus 1.7±0.48 fold increase compared to isotype control, p=0.008), Lin⁻ CD34⁺ CD38⁺ (3.6±1.6 versus 1.6±0.37, p=0.001) and Lin⁻ CD34⁻ cells (2.9±1.4 versus 1.1±0.4, p=0.001). Treatment with Dasatinib significantly reduced AML stem cell growth in LTCIC assays (56±23% inhibition with 200nM Dasatinib, p=0.003, n=5), and reduced growth of committed progenitors in CFC assays (73.6±13% inhibition with 500nM Dasatinib, and 41.7±10.8% inhibition with 10nM Dasatinib, n=9, p<0,0001), but did not reduce normal LTC-IC (n=4) or CFU-GM (23±9.1% inhibition with 500nM Dasatinib, and 1.3±11.3% with 10nM Dasatinib, n=6, p=0,007). Inhibition of growth was associated with increased apoptosis (41.5% ±10.7 with 200nM Dasatinib versus 26%±8.2 for the control, p=0,004, n=5), reduced proliferation (as evaluated with CFSE labeling, proliferation index 3.02±0.3 for the control and 2.27±0.6 with 200 nM Dasatinib, p=0.01 (n=5), and enhanced differentiation of AML stem/progenitor cells with significant increase in CD11b⁺ and CD15⁺ cells and reduction in CD34⁺, CD33⁺ and CD71⁺ cells, (Dasatinib 200 nM n=7). Exposure of AML cells (n=4) to Dasatinib (200nM) resulted in reduced phosphorylation of STAT3 (Y705), STAT5 (Y694) and Akt (Thr308) but did not affect MAPK (Thr202/Y204) phosphorylation. Interestingly, Dasatinib treatment strongly enhanced expression of p53 protein and its downstream targets, including p21 and Bax, in AML specimen (n=7), suggesting that Dasatinib treatment resulted in activation of p53 signaling in AML stem/progenitor cells. As compared to treatment with individual agents, the combination of Dasatinib with either daunorubicin (DNR) or Ara-C resulted in significantly increased apoptosis (14.9%±11 with Dasatinib, 15.8%±8.6 with 0.05μ M DNR and 25% with DNR+ Dasatinib=7, p=0.005), decreased proliferation (proliferation index 3.02±0.3 for control, 2.27±0.6 with 200 nM Dasatinib, 2.32±0.8 with 0.05μ M DNR and 1.29±0.4 for the combination n=5, p=0.01) and reduced CFC growth (n=6, 56.2±6.4% inhibition with 200nM Dasatinib, 35%±5.6 with 0.01μ M DNR, p=0.001, 82%±9.5 with DNR + Dasatinib, p=0.001, 51.8%±8.5 with 0.01μ M Ara-C and 83.2%±4.7 with Ara-C + Dasatinib, p=0.0002). The combination of Dasatinib with chemotherapeutic agents significantly enhanced expression of p53 and downstream targets compared with these agents alone. In conclusion, SFK activity is increased in primary human AML stem and progenitor cells. SFK blockade with Dasatinib reduces maintenance of AML stem and progenitor cells through inhibition of proliferation, induction of apoptosis and enhancement of differentiation. Dasatinib treatment enhances sensitivity of AML stem/progenitor cells to chemotherapeutic agents, potentially through inhibition of Akt and STAT signaling and activation of p53. These results support further preclinical and clinical evaluation of SFK blockade with Dasatinib to enhance AML stem and progenitor cells targeting by chemotherapeutic agents.