MUC1-C oncoprotein suppresses reactive oxygen species-induced terminal differentiation of acute myelogenous leukemia cells.

Acute myeloid leukemia (AML) cells are characterized by unlimited self-renewal and an impaired capacity to undergo terminal differentiation. The MUC1 oncoprotein is aberrantly expressed in AML cells; however, there has been no evidence for involvement of MUC1 in myeloid leukemogenesis. Cell-penetrating peptide inhibitors of the MUC1-C subunit block its oligomerization and thereby oncogenic function. The present results demonstrate that treatment of human MOLM-14 and MV4-11 AML cells with these inhibitors is associated with arrest of growth, induction of late apoptosis/necrosis, and loss of self-renewal capacity. Similar results were obtained with primary blasts from patients with AML. Inhibition of MUC1-C was associated with increases in reactive oxygen species (ROS) and depletion of glutathione. Increases in ROS have been linked to induction of hematopoietic cell differentiation along the myeloid lineage. In this regard, inhibition of MUC1-C was associated with induction of a terminally differentiated myeloid phenotype in AML cell lines and primary blasts by an ROS-dependent mechanism. These findings indicate that MUC1-C function is of importance to AML cell self-renewal and that inhibition of MUC1-C represents a potential therapeutic approach to induce terminal differentiation of AML cells.

Vaccination with dendritic cell/tumor fusion cells results in cellular and humoral antitumor immune responses in patients with multiple myeloma.

We have developed a tumor vaccine in which patient-derived myeloma cells are chemically fused with autologous dendritic cells (DCs) such that a broad spectrum of myeloma-associated antigens are presented in the context of DC-mediated costimulation. We have completed a phase 1 study in which patients with multiple myeloma underwent serial vaccination with the DC/multiple myeloma fusions in conjunction with granulocyte-macrophage colony-stimulating factor. DCs were generated from adherent mononuclear cells cultured with granulocyte-macrophage colony-stimulating factor, interleukin-4, and tumor necrosis factor-α and fused with myeloma cells obtained from marrow aspirates. Vaccine generation was successful in 17 of 18 patients. Successive cohorts were treated with 1 × 10(6), 2 × 10(6), and 4 × 10(6) fusion cells, respectively, with 10 patients treated at the highest dose level. Vaccination was well tolerated, without evidence of dose-limiting toxicity. Vaccination resulted in the expansion of circulating CD4 and CD8 lymphocytes reactive with autologous myeloma cells in 11 of 15 evaluable patients. Humoral responses were documented by SEREX (Serologic Analysis of Recombinant cDNA Expression Libraries) analysis. A majority of patients with advanced disease demonstrated disease stabilization, with 3 patients showing ongoing stable disease at 12, 25, and 41 months, respectively. Vaccination with DC/multiple myeloma fusions was feasible and well tolerated and resulted in antitumor immune responses and disease stabilization in a majority of patients.

Fusion cell vaccination of patients with metastatic breast and renal cancer induces immunological and clinical responses.

PURPOSE: Dendritic cells (DCs) are potent antigen-presenting cells that are uniquely capable of inducing tumor-specific immune responses. We have conducted a Phase I trial in which patients with metastatic breast and renal cancer were treated with a vaccine prepared by fusing autologous tumor and DCs. EXPERIMENTAL DESIGN: Accessible tumor tissue was disrupted into single cell suspensions. Autologous DCs were prepared from adherent peripheral blood mononuclear cells that were obtained by leukapheresis and cultured in granulocyte macrophage colony-stimulating factor, interleukin 4, and autologous plasma. Tumor cells and DCs were cocultured in the presence of polyethylene glycol to generate the fusions. Fusion cells were quantified by determining the percentage of cells that coexpress tumor and DC markers. Patients were vaccinated with fusion cells at 3-week intervals and assessed weekly for toxicity, and tumor response was assessed at 1, 3, and 6 months after completion of vaccination. RESULTS: The vaccine was generated for 32 patients. Twenty-three patients were vaccinated with 1 x 10(5) to 4 x 10(6) fusion cells. Fusion cells coexpressed tumor and DC antigens and stimulated allogeneic T-cell proliferation. There was no significant treatment-related toxicity and no clinical evidence of autoimmunity. In a subset of patients, vaccination resulted in an increased percentage of CD4 and CD8+ T cells expressing intracellular IFN-gamma in response to in vitro exposure to tumor lysate. Two patients with breast cancer exhibited disease regressions, including a near complete response of a large chest wall mass. Five patients with renal carcinoma and one patient with breast cancer had disease stabilization. CONCLUSIONS: Our findings demonstrate that fusion cell vaccination of patients with metastatic breast and renal cancer is a feasible, nontoxic approach associated with the induction of immunological and clinical antitumor responses.

Induction of antitumor immunity ex vivo using dendritic cells transduced with fowl pox vector expressing MUC1, CEA, and a triad of costimulatory molecules (rF-PANVAC).

The fowl pox vector expressing the tumor-associated antigens, mucin-1 and carcinoembryonic antigen in the context of costimulatory molecules (rF-PANVAC) has shown promise as a tumor vaccine. However, vaccine-mediated expansion of suppressor T-cell populations may blunt clinical efficacy. We characterized the cellular immune response induced by ex vivo dendritic cells (DCs) transduced with (rF)-PANVAC. Consistent with the functional characteristics of potent antigen-presenting cells, rF-PANVAC-DCs demonstrated strong expression of mucin-1 and carcinoembryonic antigen and costimulatory molecules, CD80, CD86, and CD83; decreased levels of phosphorylated STAT3, and increased levels of Tyk2, Janus kinase 2, and STAT1. rF-PANVAC-DCs stimulated expansion of tumor antigen-specific T cells with potent cytolytic capacity. However, rF-PANVAC-transduced DCs also induced the concurrent expansion of FOXP3 expressing CD4CD25 regulatory T cells (Tregs) that inhibited T-cell activation. Moreover, Tregs expressed high levels of Th2 cytokines [interleukin (IL)-10, IL-4, IL-5, and IL-13] together with phosphorylated STAT3 and STAT6. In contrast, the vaccine-expanded Treg population expressed high levels of Th1 cytokines IL-2 and interferon-γ and the proinflammatory receptor-related orphan receptor γt (RORγt) and IL-17A suggesting that these cells may share effector functions with conventional TH17 T cells. These data suggest that Tregs expanded by rF-PANVAC-DCs, exhibit immunosuppressive properties potentially mediated by Th2 cytokines, but simultaneous expression of Th1 and Th17-associated factors suggests a high degree of plasticity.

Generation of tumor-specific T lymphocytes using dendritic cell/tumor fusions and anti-CD3/CD28.

Adoptive immunotherapy with tumor-specific T cells represents a promising treatment strategy for patients with malignancy. However, the efficacy of T-cell therapy has been limited by the ability to expand tumor-reactive cells with an activated phenotype that effectively target malignant cells. We have developed an anticancer vaccine in which patient-derived tumor cells are fused with autologous dendritic cells (DCs), such that a wide array of tumor antigens are presented in the context of DC-mediated costimulation. In this study, we demonstrate that DC/tumor fusions induce T cells that react with tumor and are dramatically expanded by subsequent ligation of the CD3/CD28 costimulatory complex. These T cells exhibit a predominantly activated phenotype as manifested by an increase in the percentage of cells expressing CD69 and interferon gamma. In addition, the T cells upregulate granzyme B expression and are highly effective in lysing autologous tumor targets. Targeting of tumor-specific antigen was demonstrated by the expansion of T cells with specificity for the MUC1 tetramer. Stimulation with anti-CD3/CD28 followed by DC/tumor fusions or either agent alone failed to result in a similar expansion of tumor-reactive T cells. Consistent with these findings, spectratyping analysis demonstrates selective expansion of T-cell clones as manifested by considerable skewing of the Vbeta repertoire following sequential stimulation with DC/tumor fusions and anti-CD3/CD28. Gene expression analysis was notable for the upregulation of inflammatory pathways. These findings indicate that stimulation with DC/tumor fusions provides a unique platform for subsequent expansion with anti-CD3/CD28 in adoptive T-cell therapy of cancer.

Direct targeting of the mucin 1 oncoprotein blocks survival and tumorigenicity of human breast carcinoma cells.

The mucin 1 (MUC1) oncoprotein is aberrantly overexpressed by approximately 90% of human breast cancers. However, there are no effective agents that directly inhibit MUC1 and induce death of breast cancer cells. We have synthesized a MUC1 inhibitor (called GO-201) that binds to the MUC1 cytoplasmic domain and blocks the formation of MUC1 oligomers in cells. GO-201, and not an altered version, attenuates targeting of MUC1 to the nucleus of human breast cancer cells, disrupts redox balance, and activates the DNA damage response. GO-201 also arrests growth and induces necrotic death. By contrast, the MUC1 inhibitor has no effect on cells null for MUC1 expression or nonmalignant mammary epithelial cells. Administration of GO-201 to nude mice bearing human breast tumor xenografts was associated with loss of tumorigenicity and extensive necrosis, which results in prolonged regression of tumor growth. These findings show that targeting the MUC1 oncoprotein is effective in inducing death of human breast cancer cells in vitro and in tumor models.

Fusions of dendritic cells with breast carcinoma stimulate the expansion of regulatory T cells while concomitant exposure to IL-12, CpG oligodeoxynucleotides, and anti-CD3/CD28 promotes the expansion of activated tumor reactive cells.

Vaccination of patients with dendritic cell (DC)/breast carcinoma fusions stimulated antitumor immune responses in a majority of patients with metastatic disease but only a subset demonstrate evidence of tumor regression. To define the factors that limit vaccine efficacy, we examined the biological characteristics of DC/breast carcinoma fusions as APCs and the nature of the vaccine-mediated T cell response. We demonstrate that fusion of DCs with breast carcinoma cells up-regulates expression of costimulatory and maturation markers and results in high levels of expression of IL-12 consistent with their role as activated APCs. Fusion cells also express the chemokine receptor CCR7, consistent with their ability to migrate to the draining lymph node. However, DC/breast cancer fusions stimulate a mixed T cell response characterized by the expansion of both activated and regulatory T cell populations, the latter of which is characterized by expression of CTLA-4, FOXP3, IL-10, and the suppression of T cell responses. Our results demonstrate that IL-12, IL-18, and TLR 9 agonist CpG oligodeoxynucleotides reduce the level of fusion-mediated regulatory T cell expansion. Our results also demonstrate that sequential stimulation with DC/breast carcinoma fusions and anti-CD3/CD28 results in the marked expansion of activated tumor-specific T cells. These findings suggest that DC/breast carcinoma fusions are effective APCs, but stimulate inhibitory T cells that limit vaccine efficacy. In contrast, exposure to TLR agonists, stimulatory cytokines, and anti-CD3/CD28 enhances vaccine efficacy by limiting the regulatory T cell response and promoting expansion of activated effector cells.

MUC1 oncoprotein regulates Bcr-Abl stability and pathogenesis in chronic myelogenous leukemia cells.

Chronic myelogenous leukemia (CML) results from expression of the Bcr-Abl fusion protein in hematopoietic stem cells. The MUC1 heterodimeric protein is aberrantly overexpressed in diverse human carcinomas. The present studies show that MUC1 is expressed in the human K562 and KU812 CML cell lines. The results show that MUC1 associates with Bcr-Abl through a direct interaction between the Bcr N-terminal region and the MUC1 cytoplasmic domain. Stable silencing of MUC1 decreased cytoplasmic Bcr-Abl levels by promoting Bcr-Abl degradation. Silencing MUC1 was also associated with decreases in K562 and KU812 cell self-renewal capacity and with a more differentiated erythroid phenotype. The results further show that silencing MUC1 increases sensitivity of CML cells to imatinib-induced apoptosis. Analysis of primary CML blasts confirmed that, as found with the CML cell lines, MUC1 blocks differentiation and the apoptotic response to imatinib treatment. These findings indicate that MUC1 stabilizes Bcr-Abl and contributes to the pathogenesis of CML cells by promoting self renewal and inhibiting differentiation and apoptosis.

Dendritic cells induce MUC1 expression and polarization on human T cells by an IL-7-dependent mechanism.

The MUC1 transmembrane mucin is expressed on the surface of activated human T cells; however, the physiologic signals responsible for the regulation of MUC1 in T cells are not known. The present studies demonstrate that IL-7, but not IL-2 or IL-4, markedly induces MUC1 expression on CD3+ T cells. MUC1 was also up-regulated by IL-15, but to a lesser extent than that found with IL-7. The results show that IL-7 up-regulates MUC1 on CD4+, CD8+, CD25+, CD69+, naive CD45RA+, and memory CD45RO+ T cells. In concert with induction of MUC1 expression by IL-7, activated dendritic cells (DC) that produce IL-7 up-regulate MUC1 on allogeneic CD3+ T cells. DC also induce MUC1 expression on autologous CD3+ T cells in the presence of recall Ag. Moreover, DC-induced MUC1 expression on T cells is blocked by a neutralizing anti-IL-7 Ab. The results also demonstrate that DC induce polarization of MUC1 on T cells at sites opposing the DC-T cell synapse. These findings indicate that DC-mediated activation of Ag-specific T cells is associated with induction and polarization of MUC1 expression by an IL-7-dependent mechanism.

Fusion of dendritic cells with multiple myeloma cells results in maturation and enhanced antigen presentation.

Dendritic cells (DCs) are potent antigen-presenting cells that are uniquely capable of inducing primary immune responses. Although tumour cells may directly inhibit DC maturation, exposure to tumour products may also result in their activation. Fusions of cancer cells and DCs are being explored as cancer vaccines. The effect of tumour cell fusion on DC maturation and their functional characteristics has not been defined. In the present study, immature and mature DC generated from human CD34+ and peripheral blood precursors were fused to multiple myeloma cells in the presence of polyethylene glycol. Fusion of both immature and mature DCs with tumour cells resulted in an activated phenotype. In this regard, fusion cells expressed interleukin-12, a cytokine essential for the induction of T-helper cell type 1 immunity. In contrast to immature DCs, fusion cells also strongly expressed CC-chemokine receptor R7, which is responsible for DC migration to draining lymph nodes. Fusions generated with both immature and mature DCs also potently stimulated T-cell expression of gamma-interferon and cytotoxic T lymphocyte killing of tumour targets. These findings demonstrate that tumour cell fusion induces DC maturation and the development of an activated phenotype necessary for their effectiveness as cancer vaccines.