Targeting MUC1 as a Marker for Myeloid Leukemia Stem Cells by DC/AML Fusions.

The epithelial mucin antigen (MUC1) is aberrantly expressed in many epithelial tumors and hematologic malignancies and promotes oncogenesis and tumor progression. MUC1 is recognized by the T cell repertoire and has served as a target for cellular immunotherapy. In the present study, we examined MUC1 as a marker for myeloid leukemia cells and their progenitors and its capacity to serve as a target for leukemia stem cells. Myeloid leukemia cells were isolated from bone marrow aspirates or peripheral blood in patients with high levels of circulating disease. MUC1 was not expressed on unselected leukemia samples (mean expression 3%, n=12). Similarly, low levels of MUC1 expression were seen in leukemic blasts with monocytoid differentiation (mean expression 2.7%, n=5). A subset of leukemia specimens underwent CD34 selection by magnetic bead separation. In contrast to unselected cells, 38% of CD34+ leukemia cells expressed MUC1 (n=5). The leukemia stem cell compartment was isolated by separating CD34+/CD38−/ lineage- fractions by flow cytometric sorting. Leukemia stem cells demonstrated strong expression of MUC-1 by immunohistochemical staining and FACS analysis. Similarly, we examined MUC1 expression on progenitor cells derived from chronic phase chronic myeloid leukemia and following blast transformation. MUC1 was seen in only 4% of CD34+ cells obtained from chronic phase CML samples (n=4) while uniform expression was observed in samples derived from patients with accelerated/blastic phase disease. These data suggest that MUC1 serves as a marker for early leukemia progenitors and is associated with blastic transformation. We assessed the capacity of a cancer vaccine consisting of dendritic cell (DC)/myeloid leukemia fusions to stimulate immune responses that target MUC1 and other antigens expressed by the stem cell compartment. DCs were generated from adherent mononuclear cells that were cultured with GM-CSF and IL-4 and matured with TNFa. DCs were fused with patient derived myeloid leukemia cells using polyethylene glycol as previously described. Fusion cells were quantified by determining the percentage of cells that expressed unique DC and leukemia antigens. DC/AML fusions induced the expansion of MUC1 specific T cells. Stimulation of autologous T cells with DC/AML fusions resulted in a mean 3 fold increase in CD8+ cells binding the MUC-1 tetramer (N=4). DC/AML fusions stimulated anti-tumor immune responses that targeted leukemia stem cells. Fusion stimulated T cells demonstrated increased expression of IFNγ following exposure to lysate generated from unselected leukemia cells (29 fold) and leukemia stem cells (28 fold). In contrast, exposure to renal carcinoma lysate generated only a 5 fold increase in IFNγ. In summary, these findings suggest that leukemic progenitors in AML and accelerated/blast phase CML express MUC-1. DC/tumor fusion vaccines target the MUC-1 protein and the stem cell compartment, and may be a potent immunotherapeutic strategy to eliminate the malignant stem cell clone in AML.