SFK Inhibition with Dasatinib Results In Selective Targeting of Primitive Human Acute Myeloid Leukemia Stem and Progenitor Cells.

Abstract 1053

The Src family tyrosine kinases (SFKs) are abnormally activated in AML compared to normal CD34⁺ hematopoietic progenitors. Studies using pharmacological and siRNA approaches indicate an important role for the SFK Lyn in AML progenitor cell growth and survival (Dos Santos et al., 2008). However the role of SFKs in AML leukemic stem cell (LSC) growth and survival is not clear. SFK activity in Lin^- CD34⁺ CD38^- primitive stem/progenitor cells, Lin^- CD34⁺ CD38⁺ committed progenitors, and Lin-CD34^- cells from primary human AML (n=14) and cord blood (CB) (n=6) samples was measured by analyzing SFK phosphorylation using flow cytometry. We observed significant increase of SFK phosphorylation in AML compared to CB Lin^- CD34⁺CD38^- cells (3.8±1.9 versus 1.9±0.7, p=0.006), Lin^- CD34⁺ CD38⁺ (3.9±1.7 versus 1.9±0.6, p=0.013) and Lin^- CD34^- cells (3.4±1.8 versus 1.1±0.5, p=0.0005). Dasatinib, a potent SFK and ABL kinase inhibitor, is approved for clinical use in chronic myeloid leukemia. We evaluated the effect of SFK inhibition using Dasatinib on the growth and viability of AML stem and progenitor cells. Dasatinib exposure resulted in dose-dependent inhibition of SFK phosphorylation in each subpopulation (in CD34⁺ CD38^-, 3.0 for the control versus 1.9 with 100nM and 1.6 with 500nM, in CD34⁺ CD38⁺, 2.8 for the control versus 1.9 with 100nM and 1.6 with 500nM) after 30 minutes of drug treatment). The addition of Dasatinib (10-500nM) to methylcellulose progenitor assays resulted in dose-dependent inhibition of AML colony forming cell (CFC) growth (83.9±16.1% inhibition with 500nM, and 48.1±6.8% inhibition with 10nM Dasatinib, n=8), to a greater extent than CB CFC to (CFU-GM inhibition 62.6±1.5% with 500nM, and 1.1±16.3% with 10nM Dasatinib, n=4). Short-term exposure to Dasatinib (10-500nM) for 48 hours also resulted in significantly greater inhibition of AML CFC (73.6±13% with 500nM, and 41.7±10.8% with 10nM Dasatinib, n=8) compared to CB CFC (CFU-GM inhibition 23±9.1% with 500nM, and 1.3±11.3% with 10nM Dasatinib, n=4). Importantly Dasatinib treatment (200nM) also resulted in reduction of AML stem/primitive progenitor growth in long term culture-initiating cells (LTC-IC) assays (56±23, 8 % inhibition, p=0.003, n=4), suggesting that SFK inhibition may inhibit AML stem cell maintenance. The effect of Dasatinib on apoptosis was evaluated by labeling cells with Annexin V and DAPI. Treatment with Dasatinib resulted in significant increase in apoptosis of Lin^- AML cells (41.5% ±10.7 of apoptosis with 200nM Dasatinib versus 25%±10.8 for the control, p=0,004, n=5) We studied the effects of Dasatinib on differentiation of Lin^- CB (n=3) and AML cells (N=5) cultured with SCF, IL-3, GM-CSF, G-CSF and EPO. In normal CD34⁺, Dasatinib (100 nM) treatment resulted in increased CD33+ and CD14+ cells and reduced CD34⁺, CD11b⁺, CD15⁺, GPA⁺ and CD71⁺ cell numbers, indicating that SFK increased monocytic but reduced granulocytic and erythroid differentiation. Treatment of AML cells with Dasatinib resulted in markedly reduced numbers of CD34⁺, CD33⁺ and CD71⁺ cells, but increased numbers of CD11b cells, in 3 of 5 samples, indicating a trend towards increased granulocytic differentiation in contrast to normal progenitors. Our results indicate that SFK activity is increased in primary human AML stem and progenitor cells and suggest that SFK blockade with Dasatinib may reduce maintenance of AML LSC/ primitive progenitors, through inhibition of progenitor proliferation, induction of apoptosis and enhancement of differentiation. These results support further evaluation of SFK blockade with Dasatinib for targeting of AML stem and progenitor cells in preclinical and clinical studies.