Stromal Cell-Derived Secreted Factors Contribute to the Innate Imatinib Resistance of Leukemia Stem Cells In a Genetically Defined Murine Model of Chronic Myeloid Leukemia Blast Crisis

Abstract 204

Treatment of chronic myeloid leukemia (CML) patients with tyrosine kinase inhibitors (TKIs), such as imatinib, nilotinib, and dasatinib, results in a dramatic reduction in proliferating BCR-ABL expressing leukemia cells. However, these agents do not eliminate the CML stem cell population, indicating that inhibiting BCR-ABL kinase activity alone is not sufficient to eradicate the disease. In vitro studies of human CML cell lines and CD34+ cells isolated from CML patients, have shown that bone marrow stromal cell factor (BMSF) conditioned media can maintain important pro-survival and self-renewal activities downstream of BCR-ABL in the presence of TKIs, suggesting a role for secreted BMSFs in innate resistance to BCR-ABL kinase inhibition. However, the ability of BMSFs to maintain the leukemic potential of CML stem cells upon exposure to TKIs has not been reported. We used a standard murine retroviral transduction system to model CML blast crisis (BC-CML) and obtain cells highly enriched for leukemia initiating potential. Purified LIN-, Sca-1+, CD117+ cells (LSKs) were isolated from the bone marrow of C57BL6/J mice and retrovirally-transduced with BCR-ABL-GFP and Nup98/HoxA9-YFP then injected intravenously into recipient C57BL6/J mice. All animals developed leukemia within 21 days characterized by leukocytosis and extensive infiltration of bone marrow and spleen with leukemic blasts. LSKs expressing both BCR-ABL-GFP and Nup98/HoxA9-YFP (GFP+/YFP+ LSKs) were purified from the spleens or bone marrows of leukemic mice and cultured for 72 hrs in BMSF conditioned media across a range of concentrations (0% - 50%) in the presence and absence of imatinib (0 - 1000 nM). BMSF conditioned media reduced the cytotoxic effects of imatinib on GFP+/YFP+ LSKs as assessed by cell counts, trypan blue viability assays, and Annexin V expression by flow cytometry. Furthermore, BMSF conditioned media reduced the inhibitory effects of imatinib on GFP+/YFP+ LSK colony formation in methylcellulose, and beta-catenin expression as assessed by flow cytometry. These observations strongly suggest that signaling by stromal cell-derived soluble factors protects BC-CML stem cells from imatinib therapy by re-activating pro-survival and self-renewal pathways. The ability of BMSFs to reduce the inhibitory effect of imatinib on BC-CML stem cell self-renewal in vivo was assessed by performing secondary transplantation assays. GFP+/YFP+ LSKs were purified from primary CML mice and transplanted into secondary recipients following in vitro exposure to BMSF conditioned media in the presence and absence of 1000 nM imatinib. Survival after transplantation was compared in cohorts of 5 mice per experimental condition: Group 1 (0% BMSF, 0 nM imatinib), Group 2 (50% BMSF, 0 nM imatinib), Group 3 (50% BMSF, 1000 nM imatinib) and Group 4 (0% BMSF, 1000 nM imatinib). Survival was significantly prolonged in Group 4 mice treated with 1000 nM imatinib and this effect was abrogated by treatment with 50% BMSF conditioned media, indicating that cell-derived soluble factors contribute to maintaining BC-CML stem cell function in the presence of imatinib. Our findings strongly suggest that signaling by soluble BMSFs plays an important role in the innate imatinib resistance of CML stem cells, implicating these factors in disease relapse. Genetically defined murine models of CML provide a powerful in vivo system to identify and target soluble factors that contribute to stromal-mediated cytoprotection of CML stem cells from TKIs.