Defining Extrinsic Survival Pathways in CML Progenitor Cells.

The stem cell niche is thought to provide a protective microenvironment that allows for the persistence of malignant progenitors in various types of leukemia treated with chemotherapy. Tyrosine kinase inhibitor (TKI) treatment of chronic myeloid leukemia (CML) does not completely eliminate residual BCR/ABL positive progenitors, despite complete cytogenetic responses in the majority of patients, a phenomenon referred to as disease persistence. We hypothesize that niche factors may contribute to CML progenitor survival in the presence of imatinib and that the identification of defined survival factors may lead to therapeutic approaches to eliminate residual disease.

In an initial series of experiments CD34+ CML progenitor cells were cultured in (a) serum free media supplemented with low concentrations of cytokines (=control), (b) in direct contact with M210B4 murine stroma or (c) separated from M210B4 murine stroma through a transwell membrane, in the presence or absence of TKIs (dasatinib, imatinib). After 96h, cells were plated in semisolid media and CFU-GM scored. Murine stroma consistently increased CFU-GM survival 4-5-fold compared to controls, with most of the effect attributable to soluble factors. Similar observations were made with human stroma (HS-5), although direct contact accounted for approximately 40% of the protective effect. Since integrin-mediated adhesion has been shown to protect CML cells lines from imatinib and chemotherapy, we hypothesized that direct contact protection of CML CD34+ cells may be related to integrin engagement. To test this hypothesis we cultured CML CD34+ cells on plates coated or not with fibronectin and in the presence or absence of integrin activating (B44) or blocking antibodies (6S6), with imatinib added at 1.5 and 5 uM or dasatinib added at 5 or 50 nM. However, in several experiments (N=3), we found that integrin-mediated adhesion to fibronectin failed to promote CFU-GM survival in the presence of TKIs. To identify soluble factors responsible for progenitor cell survival in the presence of TKIs we measured cytokine and chemokine concentrations in HS-5 stroma conditioned media (CM) and found measurable levels of several cytokines with JAK2-dependent signal transduction, including IL3, IL6, GM-CSF and G-CSF. Based on this we asked whether a JAK2 inhibitor (CYT387) would synergize with imatinib to inhibit CML CFU-GM survival. Thus, CD34+ cells were cultured for 96h with imatinib and CYT387 HS-5 stroma cells. Imatinib (5uM) reduced CFU-GM survival by 79.4%. Addition of 5 uM of CYT387, a JAK2 inhibitor (IC50 for growth inhibition of BAF3 cells expressing JAK2V617F: 1.5uM) reduced CFU-GM survival by 92.9%. Combination of imatinib with graded concentrations of CYT387 led to a further reduction of CFU-GM in a dose-dependent manner (Table 1).

(a) Contact to and soluble factors produced by stromal cells appear to protect CD34+ CML progenitor cells in the presence of TKIs. (b) We find no evidence that integrin-mediated adhesion to fibronectin is involved in the protection of primary cells (unlike BCR-ABL expressing cell lines). (c) Combination of a JAK2 inhibitor with imatinib synergistically reduces colony formation, suggesting that this combination may be effective in targeting residual leukemia cells in vivo.

Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; Calistoga: Research Funding; Genzyme: Research Funding.