MUC1 Inhibition Reverses the Poor Immunogenicity of Leukemia Stem Cells Rendering Them Susceptible to Immunotherapy

Acute myeloid leukemia (AML) arises from a malignant stem cell (LSC) population that is resistant to cytotoxic therapy and represents a reservoir for disease recurrence. A focus of research interest lies in developing cellular immunotherapy for the treatment of AML, in order to immunologically eradicate disease without the toxicity associated with allogeneic transplantation. In this study, we evaluated whether LSCs are effective targets for leukemia specific immunotherapy. To investigate this issue, we have developed a novel class of human T Cell Receptor-Like (TCRL) recombinant antibodies, which target MHC-peptide complexes mimicking their recognition. TCRL can be utilized to quantify the density of antigen presentation by LSCs; however, being able to distinguish LCSs from normal stem cells immunologically has been challenging. The tumor associated complex glycoprotein MUC1 may be used to define such an immature cell population.

We have previously demonstrated that LSCs strongly express the tumor associated antigen, MUC1, in contrast to normal hematopoietic stem cells. TCRL specific for the HLA-A2/MUC1 complex detectable by flow cytometry were constructed in order to quantify MUC1 antigen presentation. CD34+ cells were isolated from HLA-A2.1 AML patients with active disease and were analyzed for expression of MUC1 protein with the DF3 anti-MUC1 antibody and for HLA presentation of MUC1 peptide with the HLA-A2.1/MUC1 TCRL. Remarkably, despite the strong cell surface expression of the MUC1 protein (mean expression 68%, n=8), minimal levels of HLA bound MUC1 peptide was detected (mean expression 7.6%, n=8). CD34+ leukemia cells strongly expressed HLA-A2, indicating that the lack of TCRL recognition of the HLA-MUC1 complex was due to the lack of processing and presentation of the MUC1 peptide. Similarly, we demonstrated that leukemia progenitors minimally express HLA-A2-WT1 peptide complexes (mean expression 5%, n=3). These findings suggest that leukemia progenitors exhibit an immuno-privileged phenotype, potentially shielding them from leukemia specific immunotherapy. We have developed a MUC1 inhibitor peptide, G0-203, that has been shown to induce differentiation and apoptosis of leukemia progenitors. We evaluated whether, in addition to inducing differentiation of leukemia progenitors, exposure to GO-203 would augment antigen presentation. Isolated CD34+ AML cells were treated with 5μM GO-203 for 24 hours. Expression of HLA-A2/MUC1 peptide complexes, as determined by binding of the MUC1 TCRL antibody, was assessed before and after treatment. Exposure to GO-203 markedly upregulated the mean expression of HLA-A2.1/MUC1 peptide complexes by CD34+ leukemia cells (10% to 62%; n=4, p<0.05). Similarly, an upregulation in the mean expression of HLA-A2.1/WT1 peptide complexes was observed on CD34+ progenitors following exposure to GO-203 (mean expression 5% to 44%, n=3, p<0.05). Consistent with these findings, GO-203-treated leukemia progenitors exhibited an increase in potency as antigen presenting cells, as manifested by a 2 fold increase in autologous T cell proliferation in response to stimulation with GO-203 treated compared to untreated progenitors. In addition, an increase in MUC1 pentamer positive T cells was observed following stimulation with GO-203 treated compared to untreated AML progenitors (6.5% versus 3% MUC1 pentamer positive cells respectively, n=2). We subsequently evaluated whether the upregulation of antigen presentation correlated with enhanced targeting by immune effector cells. We have previously demonstrated that DC/AML fusion cells potently stimulate the expansion of leukemia specific T cells. We now show that GO-203 exposure results in an increased susceptibility of leukemia progenitors to targeting by vaccine stimulated T cells, as manifested by a 3-fold increase in cytotoxic T lymphocyte-mediated lysis as measured by Granzyme B activity.

The results demonstrate that leukemia progenitors exhibit low levels of HLA presentation of leukemia specific antigens such as MUC1 and WT1, resulting in their poor immunogenicity and potential resistance to tumor specific immunotherapy. Exposure of leukemia progenitors to a MUC1 inhibitor peptide, GO-203, upregulates HLA-based presentation of leukemia associated antigens, rendering these cells more susceptible to immune based targeting.

Kufe:genus oncology: Consultancy, Equity Ownership.