MUC1 Expression Is Essential For Establishment Of Acute Myeloid Leukemia In Vivo

MUC1 is an oncoprotein aberrantly expressed on AML cells that interacts with multiple transcription factors, such as NFK-B and the β-catenin/TCF4 complex, that regulate cell survival and proliferation and are linked to malignant transformation. We have demonstrated that inhibition of MUC1 results in AML cell death and differentiation. We have also demonstrated that MUC1 is strongly expressed by leukemia initiating cells but not normal hematopoietic stem cells. In a xenogeneic murine model with primary AML cells, transplantation of the subset of cells expressing high levels of MUC1 results in a high level of efficiency of disease engraftment. Conversely, treatment with a MUC1 inhibitor prevents leukemia engraftment and is capable of eradicating the established disease. In the present study, we sought to better elucidate the effect of MUC1 on AML pathogenesis.

To study the significance of MUC1-C expression on engraftment of AML in vivo, MUC1-C was silenced in MOLM-14 AML cells using lentiviral shRNA hairpin against MUC1-C. Following the infection, these cells were shown to have significantly decreased MUC1-C expression at both mRNA and protein levels. As a control, wild type MOLM-14 (wt) cells and MOLM -14 cells infected with control shRNA (control) were analyzed.

MUC1-C silenced MOLM-14, wt and control cells were transplanted (10x10³ cells/ mouse) into sub-lethally irradiated NSG mice in cohort of 8 mice/group using retro orbital injections. The animals were sacrificed at day 14 following injection and analyzed for leukemia establishment. The mice inoculated with wt and control cells developed large tumors at the injection site. Furthermore, flow cytometric analysis of the mice bone marrow revealed mean involvement of 54% and 48% with human CD45+ leukemia cells for the wt and control MOLM-14 AML cells respectively. Infiltration with leukemia cells was observed in all recipient mice (8/8) in the two control groups. Remarkably, 8 mice that were inoculated with MUC1-C silenced MOLM-14 cells showed no symptoms of leukemia and had no tumors at the injection site. Bone marrow analysis of these mice revealed mean AML involvement of 6% of the bone marrow cells that was significantly lower than that observed in the wt and the control groups p=.003 and p=.01 respectively.

The MUC1 oncoprotein facilitates the nuclear translocation of active β-catenin necessary for downstream signaling including the regulation of cyclin D1, Myc and survivin expression. In the present model, we demonstrated that MUC1-C/β-catenin colocalized in the nucleus of MOLM-14 cells using immunoflourescence (IF) staining. A significant decrease in colocolization of MUC1-C/β-catenin complex in the nucleus of the MUC1-C silenced MOLM-14 cells as opposed to control MOLM-14 cells. Intriguingly the silencing of MUC1 resulted in the down-regulation of mRNA and protein expression of survivin, a factor shown to regulate leukemia stem cell activity. These observations suggest that the loss of AML engraftment of MUC1 silenced MOLM-14 cells in mouse bone marrow depends on survivin, a downstream target of β-catenin/TCF4 pathways.

The results demonstrate that MUC1 is essential for establishment of AML in vivo. Silencing of MUC1 markedly diminishes the engraftment capacity of AML cells. MUC1-C colocalizes with activated β-catenin in the nucleus. Silencing of MUC1 down modulates expression of survivin, which is critical for the support of leukemia initiating cells. MUC1 a novel therapeutic target for AML and a clinical trial of a MUC1 inhibitor is being planned for patients with recurrent AML.

Kufe:Genus Oncology: Consultancy, Equity Ownership.