MUC1-C drives myeloid leukaemogenesis and resistance to treatment by a survivin-mediated mechanism

Acute myeloid leukaemia (AML) is an aggressive haematological malignancy with an unmet need for improved therapies. Responses to standard cytotoxic therapy in AML are often transient because of the emergence of chemotherapy-resistant disease. The MUC1-C oncoprotein governs critical pathways of tumorigenesis, including self-renewal and survival, and is aberrantly expressed in AML blasts and leukaemia stem cells (LSCs). However, a role for MUC1-C in linking leukaemogenesis and resistance to treatment has not been described. In this study, we demonstrate that MUC1-C overexpression is associated with increased leukaemia initiating capacity in an NSG mouse model. In concert with those results, MUC1-C silencing in multiple AML cell lines significantly reduced the establishment of AML in vivo. In addition, targeting MUC1-C with silencing or pharmacologic inhibition with GO-203 led to a decrease in active β-catenin levels and, in-turn, down-regulation of survivin, a critical mediator of leukaemia cell survival. Targeting MUC1-C was also associated with increased sensitivity of AML cells to Cytarabine (Ara-C) treatment by a survivin-dependent mechanism. Notably, low MUC1 and survivin gene expression were associated with better clinical outcomes in patients with AML. These findings emphasize the importance of MUC1-C to myeloid leukaemogenesis and resistance to treatment by driving survivin expression. Our findings also highlight the potential translational relevance of combining GO-203 with Ara-C for the treatment of patients with AML

STAT3 Inhibition Promotes Potent Th1 Responses By Down Regulating Pdl-1 Expression On Tumor Cells

Abstract STAT3 signaling is upregulated in cancer cells and is thought to be a critical mediator of tumor associated immune suppression. We examined the relationship of STAT3 and PDL-1, an important negative regulator of T cell immune responses via interactions with PD-1. The 5'upstream, putative promoter region of PDL-1 gene contains four potential STAT3 binding sites close to transcription start site, suggesting a possible role of STAT3 in transcriptional regulation of PDL-1. We investigated for a possible relationship between STAT3 and PDL-1 in multiple myeloma (MM-RPMI) cell line. Using chromatin immunoprecipitation assay (ChIP assay), we observed direct binding of STAT3 to the PDL-1 promoter region in MM-RPMI cells. Moreover, an enhanced occupancy of STAT3 on the PDL-1 promoter region was also observed when the cells were treated with IL-6 (20 ng/ml). Concurrent with the above observation, IL-6 treated MM-RPMI cells showed an enhanced expression of PDL-1 protein. Treatment of RPMI-MM cells with STAT3 inhibitors AG490 and CDDO-Me for 72 hrs resulted in PDL-1 expression levels of 24.5% and 15.1% respectively, compared to 97.5% for untreated control cells as determined by FACS analysis. Based on these observations, we evaluated the effect of STAT3 inhibition by exposure to small molecule STAT3 inhibitor on response to a tumor vaccine consisting of myeloma cells fused with dendritic cells (DC). DC/myeloma fusion cells were co-cultured with autologous T cells in the presence or absence of small molecule STAT3 inhibitor (CDDO-Me). Treatment of the cocultures with CDDO-Me resulted in an increased in the percentage of CD4/IFNγ (11.57±SEM 1.16; n=3) and CD8/IFNγ (23.37±SEM 2.25; n=3) expressing cells as compared to untreated cocultures (3.1± SEM 0.72; n=3) and (4.43±SEM 0.81; n=3) respectively. Moreover, we also observed a concomitant decrease in both CD4+ and CD8+ T cells expressing IL-10 or IL-4 compared to untreated cocultures. We also noted a decrease in the percentage of immunosuppressive regulatory T cells (CD4+/CD25+/FOXP3+) in treated cocultures (8.33±SEM 0.59; n=3) as compared to untreated cocultures (28.50±SEM 2.0; n=3). These findings indicate a pivotal role of inflammatory cytokine IL-6 and its downstream activator STAT3 in regulating PDL-1 expression. Thus, strategic inhibition of STAT3/IL-6 pathway and therefore down regulation of PDL-1 could enhance the potency of tumor/DC fusion cell based cancer vaccines for immunotherapeutic treatments. Furthermore, the data demonstrate that the availability of novel adjuvants offers the potential for combination therapy that may have profound effect on the potency and durability of responses to cellular immunotherapy. Disclosures: No relevant conflicts of interest to declare. </jats:sec

Transduction of Malignant Plasma Cells with Three Costimulatory Molecules (TRICOM) Elicits Myeloma-Specific Immune Response in Vitro – a Promising Strategy for Immunotherapy

Abstract Abstract 1908 Introduction: Tumor vaccines hold promise as a means of eliciting anti-myeloma immunity and controlling disease that may be resistant to chemotherapy and biologic therapy. We have developed a whole cell tumor vaccine, whereby patient derived plasma cells are transduced with an attenuated vaccinia vector that contains transgenes for the costimulatory molecules B7.1 (CD80), ICAM-1 (CD54), and LFA-3 (CD58), designated TRIad of COstimulatory Molecules (TRICOM). In this manner, a broad array of tumor antigens, including those which may be specific to a given patient, are presented in the context of costimulatory molecules that have been shown to be synergistic in the stimulation of the effector T-cells. In the present study, we evaluated the phenotype and functional characteristics of TRICOM transduced primary myeloma cells. Methods and results: Plasma cells were isolated from bone marrow aspirates obtained from patients with multiple myeloma following Ficoll density centrifugation. Bone marrow derived mononuclear cells were infected with a replication-defective poxviral vector, the modified vaccinia Ankara strain (MVA), encoding TRICOM, or a control empty MVA vector. The expression of costimulatory molecules was assessed using flow cytometric analysis 3 hrs following viral infection. Viral transduction using the TRICOM vector at the dose of 20 MOI (multiplicity of infection) increased the mean percentage of CD38+ cells expressing CD80, CD54 and CD58 from a minimal baseline level (below 5%) to 70%, 56% and 47%, respectively (n=4). Transduction with control MVA vector did not augment expression of costimulatory molecules on plasma cells (mean percent expression of CD80, CD54 and CD58 of 2.6%, 2.7% and 3.8%, respectively, n=4). Of note, compared to CD38+ plasma cells, the CD38 negative fraction of bone marrow derived mononuclear cells demonstrated a significantly lower TRICOM transduction efficiency (mean percent expression of CD80, CD54 and CD58 of 16%, 17% and 16%, respectively, n=4, p&lt;0.05 compared to CD38+ plasma cells). The ability of MVA-TRICOM transduced plasma cells to stimulate autologous T cell populations in vitro was assessed. Patient derived T-cells were purified from the non-adherent portion of PBMC by magnetic bead separation. MVA-TRICOM or empty MVA vector infected plasma cells were irradiated with 20Gy and co-cultured with autologous T cells at a 10:1 ratio of effector cells to vaccine for 7 days. MVA-TRICOM transduced plasma cells potently stimulated activated T cell responses, as assessed by the percentage of CD4+/CD25+/CD69+ T-cells (mean 7.8% of activated T-cells with TRICOM vaccine vs. 2.7% with control vaccine, n=3, p&lt;0.05). In contrast, vaccine stimulation did not result in regulatory T-cell expansion, assessed as the percentage of cells co-expressing CD4,CD25 and FoxP3 (2.4% vs. 2.3%, for TRICOM and control vaccine, respectively, n=3). In concert with these findings, vaccine stimulation resulted in a polarization towards Th1 cytokine secretion, with 7.9% of CD4+ T-cells expressing intracellular IFN-γ after stimulation with TRICOM vaccine as compared to 5.4% after stimulation with the control vaccine (n=3, p&lt;0.05). To further assess the expansion of tumor specific T cell populations, the ability of vaccine stimulated T cells to kill autologous tumor was assessed in a cell-based fluorogenic cytotoxicity assay. MVA-TRICOM transduced plasma cells potently stimulate the expansion of myeloma specific CTLs with the capacity to lyse autologous tumor targets. Mean CTL lysis was 20% and 8% for vaccine stimulated and unstimulated T cells respectively (n=2). Conclusions: Malignant plasma cells transduced with MVA-TRICOM strongly express costimulatory molecules, and potently stimulate activated, tumor reactive T cell populations. This preclinical data serves as a platform for developing a phase 1 clinical trial evaluating the use of MVA-TRICOM transduced autologous plasma cells in patients with multiple myeloma. Disclosures: No relevant conflicts of interest to declare. </jats:sec

Immunomodulatory Effect of SGI-110, a Novel Hypomethylating Agent in Acute Myeloid Leukemia (AML)

Abstract Introduction: We have developed a promising leukemia vaccine in which patient derived AML cells are fused with autologous dendritic cells (DCs) effectively presenting a broad array of antigens that capture the heterogeneity of the leukemia cell population. A major challenge of developing effective immunotherapy is overcoming the immunosuppressive milieu that characterizes patients with AML. AML cells evade immune targeting, in part, through the limited presentation of antigen by primitive leukemia progenitors. In contrast, induction of differentiation through upregulation of reactive oxygen species may enhance antigen presentation and the capacity to target AML cell by a tumor vaccine. Hypomethylating agents have been shown to be effective therapeutic agents for patients with AML and MDS, in part due to immunomodulatory effects. In the current study, we have examined the immunomodulatory properties of the novel DNA hypomethylating agent SGI-110, a dinucleotide of decitabine (DAC) and deoxyguanosine, which is resistant to in-vivo inactivation by cytidine deaminase. We have explored the in-vitro effect of treatment with SGI-110 on ROS, AML immunogenicity and efficacy of the AML/DC fusion vaccine. Methods and results: AML blasts were isolated by ficoll density centrifugation of bone marrow mononuclear cells (BMMCs) from patients with AML at presentation. AML blasts were cultured for 4 days in the presence or absence of 1 uM SGI-110, added twice daily on days 1 and 2 of culture. ROS as detected by levels of H2O2 expression demonstrated a 40% increase after treatment with SG1-110 (n=2). We subsequently evaluated whether the increase in ROS levels correlated with enhanced targeting by immune effector cells. SGI-110 or control treated AML cells were assessed for their susceptibility to T cell mediated targeting, using a standardized flourochrome CTL assay. Remarkably, a 72% increase in autologous cytotoxic T lymphocyte-mediated lysis as measured by Granzyme B activity, was demonstrated following SGI-110 treatment of AML blasts (n=3). The expression of co-stimulatory molecules CD80 and CD86, were unchanged following treatment with SGI-110 (n=3). In contrast, exposure to SGI-110 resulted in increased expression of the antigen processing TAP proteins by immunocytochemical analysis. We have previously demonstrated that DC/AML fusion cells potently stimulate the expansion of leukemia specific T cells. The effect of SGI-110 on T cell response to DC/AML fusion vaccine stimulation in vitro was assessed. Autologous Dendritic cells (DCs) were generated by culture of adherent peripheral mononuclear cells obtained from AML patients following remission in the presence of GM-CSF, IL-4 and TNF-α. DCs were fused with AML blasts by co-culture at a 1:1 ratio in the presence of polyethylene glycol (PEG). DC/AML fusions were cultured at a 1:10 ratio with autologous T cells for 5-6 days in the presence and absence of 1uM of SGI-110. Exposure of fusion stimulated autologous T cells to SGI-110 resulted in an increase in T cell expression of IFN-γ, with mean fold increase of 2.2 and 2.3 for CD4 and CD8 T cells respectively (n=4). The percentage of CD4+CD25+FOXP3+ regulatory T cells (TRegs) and T cell expression of PD1 was not significantly changed in the presence of SGI-110. The effect of SGI-110 on CTL mediated killing by vaccine stimulated T cells was assessed. Following stimulation with the DC/AML fusion vaccine, T cells mediated killing of autologous AML blasts increased to 11% from 4% following co-culture with unstimulated T cells. Interestingly, exposure of the AML cells to SGI-110 led to a further increase in T cells mediated killing with mean levels of 20% tumor lysis (n=2). Conclusions: The results demonstrate that treatment with SGI-110 results in increased ROS levels and an associated enhanced susceptibility of AML blasts to immune mediated targeting. The addition of SGI-110 to T cell stimulation by an autologous DC/AML fusion vaccine results in an increase in interferon gamma and increased susceptibility of AML cells to T cell mediated killing. The immunomodulatory properties of SGI-110, combined with its favorable pharmacologic and pharmacokinetic features, identify SGI-110 as a useful agent to implement novel combined epigenetic–immunotherapeutic strategies in AML. A clinical trial evaluating SGI-110 in combination with DC/AML fusion cell vaccination is planned. Disclosures Taverna: Astex Pharmaceuticals, Inc.: Employment. Avigan:Astex Pharmaceuticals: Research Funding. </jats:sec

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

Abstract Introduction 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. Methods and Results 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. Conclusions 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. Disclosures: Kufe: Genus Oncology: Consultancy, Equity Ownership. </jats:sec