Dasatinib Enhances Targeting of Human AML Leukemia Stem Cells by Chemotherapeutic Agents Via Activation of p53

Abstract 3590

Tyrosine kinases play a critical role in regulating cellular proliferation, differentiation and survival in acute myeloid leukemia (AML). Tyrosine kinase inhibitors (TKI) can induce remissions of AML patients by reducing leukemia burden, but fail to eradicate primitive leukemia stem cells (LSC) that likely contribute to AML relapse after completion of treatment. Therefore, new strategies to enhance targeting of AML LSCs and increase probability of cure are required. We have previously reported that pharmacological inhibition of Src Family Kinases (SFKs) using Dasatinib selectively reduced AML progenitor growth in vitro (Dos Santos et al, Blood 2011, 118:4270A). Since Dasatinib monotherapy has had limited success in malignancies other than CML, we evaluated whether Dasatinib treatment could enhance targeting of AML LSCs by the commonly used chemotherapeutic agents Daunorubicin (DNR) and Cytarabine (Ara-C). Combined treatment with Dasatinib and DNR or Ara-C resulted in a significantly greater inhibition of AML CD34⁺ cell proliferation compared to Dasatinib, DNR or Ara-C alone (PI=3.1±0.3 for control, 2.3±0.6 for Dasatinib, 2.3±0.9 for DNR, 1.6±0.7 for Ara-C n=5; 1.3±0.4 for Dasatinib and DNR, and 1.1±0.1 for Dasatinib and Ara-C). The combination of Dasatinib with DNR or Ara-C also resulted in significantly increased AML CD34⁺ cell apoptosis, and significantly greater reduction of AML colony forming cells (CFC), compared to Dasatinib, Ara-C or DNR alone. Importantly, AML CD34⁺ cells treated with the combination of Dasatinib with DNR (p=0.0006) or Ara-C (p=0.008) and transplanted into NSG mice demonstrated significantly reduced engraftment in murine BM compared with cells treated with Dasatinib, DNR or Ara-C alone. The combination of Dasatinib with DNR or Ara-C did not inhibit engraftment of normal HSC compared to DNR and Ara-C alone, suggesting that Dasatinib specifically enhanced chemotherapy-induced targeting of AML LSC. To evaluate the efficacy of the Dasatinib and chemotherapy combination in vivo, we used a conditional knock-in mouse model (Cbfb^56M/+/Mx1-Cre) of inversion 16 AML (Kuo et al, Cancer Cell 2006, 9:57). Although leukemic cells were reduced in BM and spleen of mice treated with chemotherapy alone, compared to controls, they were further significantly reduced with the combination of Dasatinib and chemotherapy (p<0.0039 compared to chemotherapy alone). To determine treatment effects on LSC capable of regenerating leukemia, BM cells from treated mice were transplanted into secondary recipients. Treatment with the combination of Dasatinib and chemotherapy resulted in significantly enhanced survival in secondary transplant recipients (after 40 days, 0% survival for controls and 10% for Dasatinib treated mice, 45% survival for chemotherapy treated mice and 91% survival for the Dasatinib plus chemotherapy combination treated mice, p=0.0008), indicating enhanced elimination of cells with LSC capacity. AML CD34⁺ cells treated with the combination of Dasatinib with DNR showed enhanced expression of p53 mRNA (n=8, p=0.008) and protein, and several p53 target genes, including BAX (p=0.01), PUMA (=0.03), P21 (p=0.04), NOXA (p=0.03) and DR5 (p=0.01), compared to untreated cells or cells treated with Dasatinib or DNR alone. We show that p53 knockdown results in a significant reduction in apoptosis in cells treated with the combination of Dasatinib and chemotherapy (54.4±11.4% apoptosis with combination treatment and control siRNA 37.2±12.5% apoptosis with p53 siRNA, p=0.01). Our results suggest that the increased p53 response is related to inhibition of the important p53 regulatory gene MDM2 by the combination of Dasatinib and DNR, resulting from reduced AKT-mediated phosphorylation of MDM2 on the Ser166 regulatory site. In conclusion, we have found that co-treatment with the SFK inhibitor Dasatinib enhances p53 activation in response to chemotherapy in AML LSC, and lead to their increased elimination. These promising preclinical studies are being applied to a clinical trial of Dasatinib in combination with chemotherapy in high risk AML patients.