Abstract LB-258: Smoothening the way for human leukemia stem cell inhibition in chronic myeloid leukemia

Chronic myeloid leukemia (CML) progresses from a chronic phase (CP) state that harbors the BCR-ABL fusion gene, to CML blast crisis (BC), characterized by activation of -catenin within granulocyte-macrophage progenitors (GMP) (Jamieson et al, NEJM 2004). BC CML GMP serially transplant leukemia in vivo and form myeloid sarcomas (Abrahamsson et al, PNAS 2009). Wnt, GSK3 and Shh family molecules are implicated in driving leukemia progression and because GSK3 is a negative regulator of both Wnt and Shh signaling pathways, we evaluated whether GSK3 deregulation activates -catenin and Gli simultaneously. We investigated whether treatment with a SMO antagonist in combination with dasatinib decreases human BC CML engraftment in vivo. BC CML tumor cells were treated for 0 and 7 days in vitro with vehicle (DMSO) or SMO antagonist (Pfizer; PF-04449913) and harvested for Q-PCR analysis. FACS-purified BC CML progenitors (CD34+CD38+Lin-PI−) were transplanted intrahepatically into newborn RAG2−/− c−/− mice and serially transplanted for propagation of tumors. Harvested tumors were then CD34-selected and transplanted to tertiary recipients (1×105 cells per mouse). Mice were treated for 14 days starting at 8 weeks of age. Mice were dosed once daily by oral gavage with 200µl of drug or vehicle (50% PEG in HBSS). The treatment included PF-04449913 (100mg/kg) with or without dasatinib (50mg/kg). After treatment the percentage of LSC (CD34+CD38+CD45RA+CD123+Lin-PI−) was determined by FACS analysis of the bone marrow and liver and number of tumors evaluated. BC CML progenitors harbors an up regulation of Gli mRNA and a downregulation of Ptch1 mRNA (n=3) compared to normal cord blood. Gli mRNA expression decreases following in vitro treatment with PF-04449913. Treatment with PF-04449913 alone in human BC CML LSC transplanted mice (n=3 exp., n=15 mice) does not significantly inhibit engraftment compared with vehicle (n=3 exp., n= 27 mice). Although dasatinib alone decreases tumor formation (P≤0.05), it does not eradicate the LSC in the liver or bone marrow (n=3 exp., n=20 mice). Combination treatment with PF-04449913 and dasatinib revealed a significant decrease in LSC engraftment in livers (P≤0.05) compared with the PF-04449913 or dasatinib alone. The combination treatment completely eradicated the LSC capability of tumor formation (0.0) in secondary recipients (n=5) compared with dasatinib that had an average of 1.8 tumors (n=6), PF-04449913 that had an average of 4.9 (n=7) and vehicle group that had 3.8 tumors (n=13). Colony formation assays were performed with cord blood CD34+ progenitors (n=3 samples) and no toxicity could be seen with PF-04449913 (1µM) compared with vehicle. There was no significant inhibition of normal CD34+ colony replating following PF-04449913 compared with vehicle treatment. A phase I clinical trial with PF-04449913 in combination with dasatinib is currently being executed for advanced phase CML. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-258.