Myeloid-derived Suppressor Cells in Acute Myeloid Leukaemia

PhDThe tumour microenvironment consists of an immunosuppressive niche created by the complex interactions between cancer cells and surrounding stromal cells. A critical component of this environment are myeloid-derived suppressor cells (MDSCs), a heterogeneous group of immature myeloid cells arrested at different stages of differentiation and expanded in response to a variety of tumour factors. MDSCs exert diverse effects in modulating the interactions between immune effector cells and malignant cells. An increased presence of MDSCs is associated with tumour progression, poorer outcomes, and decreased effectiveness of immunotherapeutic strategies. In this project, we sought to quantify and characterise MDSC populations in patients with Acute Myeloid Leukaemia (AML) and delineate the mechanisms underlying their expansion. We have demonstrated that immune suppressive MDSCs are expanded in the peripheral blood and bone marrow of patients with AML. Furthermore, AML cells secrete extra-cellular vesicles (EVs) that skew the tumour microenvironment from antigen-presentation to a tumour tolerogenic environment, through the expansion of MDSCs. We then demonstrated that MDSC expansion is dependent on tumour and EV expression of the oncoproteins MUC1 and c-Myc. Furthermore, we determined that MUC1 signalling promotes c-MYC expression in a microRNA (miRNA) dependent mechanism. This observation lead us to elucidate the critical role of MUC1 in suppressing microRNA-genesis in AML, via the down-regulation of the DICER protein, a key component of miRNA processing machinery. Finally, exploiting this critical pathway, we showed that MDSCs can be targeted by MUC1 inhibition or by the use of a novel hypomethylating agent SGI-110.British Society for Haematology Royal College of Physicians, UK Professor David Avigan and the department of Bone Marrow Transplantation

Mucin-1 (MUC1) Oncoprotein in Multiple Myeloma Cells Inhibits the Th1 Responses By Down Regulating the Expression of Mir-200c and up-Regulating the PDL1 Expression

Abstract Introduction: The PDL1/PD-1 pathway is a critical mediator of immune escape in patients with multiple myeloma (MM). Regulation of this pathway has not been well characterized. MicroRNAs (miRNAs) are a conserved class of small (~22 nucleotides) RNAs that post-transcriptionally regulate gene expression by interacting with the 3′ untranslated region (3′ UTR) and, in some settings, coding regions of target mRNAs. MiRNAs suppress gene expression by promoting mRNA degradation or inhibiting translation. Of note, the 3’UTR of the PDL1 gene contains putative binding sites for miR-200 family of micro-RNA’s, suggesting a possible role of miR-200’s in regulation of PDL1 expression. We have previously demonstrated that miR-200c is suppressed by the MUC1 oncoprotein, and hypothesized that MUC1 expression on myeloma cells upregulates the expression of PDL1, via suppressing miR-200c. In the present study, we investigated the relationship between MUC1, miR-200c and PDL1 in multiple myeloma. Methods and Results: Lentivirus vectors expressing miR-200c or a control vector with green fluorescence protein (GFP) were transduced in two different MM cell lines (MM-RPMI, MM-U266). Cells were harvested sorted by Fluorescence-Activated Cell Sorting (FACS) after 72 hours of transduction, using a dual fluorescence for GFP and anti-PDL1 antibody to analyze the changes in PDL1 expression. MiR-200c transduction of U266 cells resulted in a decrease in mean expression of PDL1 from 69.55% to 1.4% (n=2). Similarly, RPMI cells demonstrated a reduction in mean expression of PDL1 from 62.5% to 1.9% (n=2) following miR-200c transduction. The abrogation of PDL1 expression in MM cells by ectopic expression of miR-200c was confirmed using western immunoblot analysis. Having previously demonstrated that miR-200c is suppressed by MUC1 in a solid tumor model, we evaluated the effect of silencing MUC1 in U266 and RPMI cell lines on miR-200c and PDL1 expression. MUC1 silenced stable cell lines of RPMI and U266 cells were generated using lentivirus shRNA vectors against MUC1 or a scrambled vector control. MUC1 silenced cells demonstrated an increase in miR-200c expression (&gt; 2 fold, p value &lt;0.05). Notably, PDL1 expression decreased from 52% to 3.7% and from 62.5% to 6.1% following silencing of MUC1 on U266 and RPMI cells respectively. Conclusions: Ectopic expression of micro-RNA miR-200c in RPMI-MM and U266-MM cell lines results in down regulation of PDL1 expression. Silencing MUC1 in RPMI-MM and U266-MM cell lines results in both increased expression of miR-200c and downregulation of PDL1 expression. These results support the hypothesis that MUC1 expression on myeloma cells contributes to tumor mediated immunosuppression, by suppressing miR-200c thereby enhancing PDL1 expression. Interfering with MUC1 mediated signaling represents a novel approach towards augmenting immune mediated targeting of myeloma. Disclosures No relevant conflicts of interest to declare. </jats:sec

A Novel Dendritic Cell-Derived Vaccine in Multiple Myeloma

Abstract Introduction: Despite recent advances in the treatment of multiple myeloma (MM), curative outcomes remain elusive with standard therapies. MM is characterized by immune dysregulation and a loss of tumor-associated T cell surveillance contributing to disease evolution. Cancer vaccines have emerged as a promising immunotherapeutic strategy that seeks to reestablish anti-tumor immunity through the effective presentation of tumor associated antigens in the context of positive costimulation and activation signals. DCOne is a cell based tumor vaccine derived from an acute myelogenous leukemia cell line, differentiated toward a dendritic cell (DC) phenotype (DCPrime, Netherlands). DCOne strongly expresses WT-1, PRAME, RHAMM, survivin, and MUC-1. Since MM cells commonly express these tumor-associated antigens as well, we explored the efficacy of DCOne in inducing myeloma-specific immunity. Methods/Results: We first investigated the capacity of DCOne to polarize T cells toward an activated phenotype. Peripheral blood mononuclear cells (PBMCs) derived from MM patients were cultured in the presence or absence of DCOne and pulsed with whole cell DCOne lysate 24 hours prior to analysis. Intracellular levels of IFN-γ and perforin in CD8+ T cells and intracellular levels of IL-10 in CD4+ T cells were assessed via FACS analysis. Following 10 days of co-culture, we observed an increased percentage of CD8+ IFN-γ+ T cells and an increased percentage of CD8+ perforin+ T cells in PBMCs co-cultured with DCOne versus those not co-cultured with DCOne (10.8% versus 3.3%, p=0.02, n=11 and 6.6% versus 1.7%, p=0.045, n=10, respectively). In contrast, exposure of PBMCs to DCOne did not alter the percentage of CD4+ IL-10+ T cells (p=0.53, n=8). Given the observed increase in IFN-γ- and perforin-positive CD8+ T cells after co-culture of MM PBMCs with DCOne, we investigated whether CD8+ T cells co-cultured with DCOne exhibited enhanced killing of autologous tumor cells in a standard cytotoxic T lymphocyte (CTL) assay measuring Granzyme B activity by FACS analysis. DCOne potently induced CTL-mediated killing of autologous MM cells as determined by an increased percentage of CD8+ Granzyme B+ cells in stimulated versus control cells (27.4% versus 12.5%, p=0.05, n=3). We next investigated the mechanism of action by which DCOne elicited a MM-specific response. We postulated that the allogeneic cell line would induce immune activation in part through the transfer of antigen to PBMC-derived antigen presenting cells; these antigen presenting cells would in turn be capable of eliciting response via MHC restricted presentation of antigen to autologous T cell populations. Consistent with this hypothesis, we demonstrated that 32% of patient-derived PBMCs exhibited uptake of DCOne RNA following co-culture (n=3). Uptake of DCOne RNA by patient-derived PBMCs was completely abrogated with Transwell separation. Next, we examined how patient-derived PBMCs take up DCOne antigens. Using Western Blot analysis, we found that MUC-1 and survivin are expressed in extracellular vesicles (EVs) derived from DCOne lysate. Thus, we conclude that DCOne antigens are trafficked via EVs secreted by the DCOne parent cell, whereupon they are taken up by MM patient-derived PBMCs. Conclusion: In conclusion, when cultured in vitro with MM patient-derived PBMCs, DCOne results in increased IFN-γ- and perforin-positive CD8+ T cells, as well as induction of autologous tumor killing by these cells. DCOne RNA is taken up by MM patient-derived PBMCs and trafficked to the extracellular environment via EVs. DCOne demonstrates in vitro efficacy as an "off-the-shelf" strategy for stimulating MM-specific immunity. A clinical trial is being planned. Disclosures Kruisbeek: DCPrime: Employment, Other: Founder, CSO, and CEO of DCPrime. Van Wetering:DCPrime: Employment. Arnason:Gilead: Consultancy. Rosenblatt:DCPrime: Research Funding; BMS: Research Funding; Astex: Research Funding. Avigan:Astex: Research Funding; DCPrime: Research Funding. </jats:sec

Myeloid-Derived Suppressor Cells Are Expanded in Patients with AML and Are Dependent on MUC1 Expression

Abstract Introduction: Myeloid-derived suppressor cells (MDSCs) are a critical component of the immunosuppressive milieu of the tumor microenvironment and play an important role in promoting immune tolerance and disease growth. They are comprised of granulocytic and monocytic compartments defined by a unique immunophenotypic signature. Importantly, the mechanism by which tumor cells evoke the expansion of MDSCs has not been well elucidated. In the present study, we examined the interaction of MDSCs with AML cells, a setting in which the presence and function of MDSC has not been well described. Methods and Results: Peripheral blood mononuclear cells (PBMCs) were isolated from patients with active AML and granulocytic (CD33+/CD11b+/HLADR-/CD15+) and monocytic (CD33+/CD11b+/HLADR-/CD15-) MDSCs were quantified by multichannel flow cytometry. AML patients had a significantly higher mean granulocytic MDSC population of 17.2% (n=3) compared to healthy controls 1.9%, (n=10) p=0.0083 and a mean monocytic MDSC population of 6.5% (n=3), which was similar to healthy controls (monocytic MDSCs 4.1%, n=10). MDSCs isolated from an AML patient exhibited immunosuppressive effects as measured by the suppression of dendritic cell mediated stimulation of T cells. The addition of AML derived MDSCs resulted in a 40% reduction in CD4+T cell production of IFNϒ and an 11 fold increase IL-10 secretion by CD4 and CD8 T cells following coculture with allogenic DC stimulation. The ability of AML blasts to directly induce the expansion of MDSC was assessed in vitro. Healthy donor PBMCs were co-cultured for 6 days with or without the AML cell lines MOLM-14 and THP-1 at a ratio of 100:1. MDSCs were quantified after 6 days. Coculture with MOLM-14 and THP-1 induced a 2.35 and 8.2 fold increase in MDSCs respectively (n=4). MUC1 is a critical oncogene expressed on leukemic blasts and leukemia initiating cells and plays an important role in the tumor microenvironment promoting tumor growth and immune escape. In the present study, we demonstrated that silencing of MUC1 via shRNA significantly diminishes AML recruitment and expansion of MDSCs in vitro. MOLM-14 cells underwent lentiviral transfection to silence MUC1-C expression which was confirmed by Western Blot. MOLM-14 wild type, MUC1 silenced, and control vector treated cells were co-cultured with healthy PBMCs for 6 days in a ratio of 100:1. Of note, MUC1-C silenced MOLM-14 and THP-1 cells exhibited decreased capacity to expand MDSCs upon co-culture with healthy donor PBMCs, as compared to the control vector (2.4 fold higher expansion of MDSCs with control vector MOLM-14 compared to MUC1-C silenced MOLM-14, n=4, 1.92 fold higher expansion of MDSCs with control vector THP-1 compared to MUC-1C silenced THP-1, n=4). In an in vivo model, NSG mice were irradiated and inoculated with THP-1 control and THP-1 MUC1 silenced cells. Following establishment of disease, mice were sacrificed and spleens were FACS analysed for MDSC quantification. Mice inoculated with THP-1 MUC1 silenced cells had mean MDSCs of 7.5%, compared to 16.25% in mice innoculated with THP-1 Wildtype cells (n=4). In conclusion, the data demonstrates that MDSCs are increased in the circulation of patients with AML, and that leukemic blasts directly induce the expansion of MDSCs. MUC1 expression on AML blasts contributes to the immunosuppressive milieu, and notably, silencing of MUC1 in AML cells blunts their capacity to induce the expansion of MDSCs. Incorporating strategies to inhibit the expansion of MDSC in AML, and reverse their immunosuppressive phenotype has the potential to improve response to therapy in AML. Disclosures No relevant conflicts of interest to declare. </jats:sec

Decitabine Priming Enhances Mucin 1 Inhibition Mediated Disruption of Redox Homeostasis in Cutaneous T-Cell Lymphoma

Abstract Background: Cutaneous T-cell lymphoma (CTCL) is a heterogeneous neoplasm and patients with relapsed/refractory disease exhibit resistance to standard cytotoxic therapies. We have previously demonstrated that the MUC1-C oncoprotein plays a critical role in regulating apoptosis and protection from oxidative stress in CTCL. Targeting of MUC1-C with a pharmacologic inhibitor, GO-203, was associated with late apoptosis and early necrosis and slowing of tumor growth in in vitro and in vivo models of CTCL, respectively. However, disease responses following MUC1-C inhibition were incomplete underscoring the need for combinatorial strategies that could exploit the vulnerability of CTCL cells to oxidative signals. Decitabine is a DNA methyltransferase inhibitor, previously shown to modulate oxidative stress in acute myeloid leukemia models. We hypothesized that treatment with decitabine and GO-203 would present a potent novel therapeutic combination for patients with CTCL. Method/Results: Our studies demonstrate that exposure of CTCL cells to decitabine in combination with GO-203, increased the generation of reactive oxygen species (ROS) and decreased levels of scavenger molecules, NADPH, glutathione (GSH), and TIGAR, a p53-inducible regulator of glycolysis and apoptosis, critical to intracellular redox homeostasis. This in turn led to marked increase in the ratio of stress activated proteins; phospho-p38 and phospho-JNK to Total-p38 and Total-JNK repectively. Further dual exposure to GO-203 and decitabine resulted in near abrogation of DNA methyl transferase 1 and 3b crucial for maintaining DNA methylation, demonstrating significant synergy of these agents for inducing hypomethylation. In concert with these findings, treatment with decitabine and GO-203 upregulated the ROS generating enzyme, NADPH oxidase 4 (Nox4) potentially due to their effect on epigenomic regulation of this protein. In concert with these findings, exposure to decitabine and GO-203 resulted in heightened apoptotic death of CTCL cell lines, primary patient derived samples and in a aggressive murine xenograft model. Conclusion: These findings indicate that decitabine augments MUC1-C inhibition induced redox imbalance and provides a novel combination of targeted and epigenetic agents for treatment of patients with CTCL. Accordingly an early phase multi-institution clinical trial to to investigate this combinatorial strategy based on epigenetic modulation of redox homeostasis in patients with relapsed/refractory T cell lymphomas is being planned. Disclosures Rosenblatt: Astex: Research Funding; BMS: Research Funding; DCPrime: Research Funding. Arnason:Gilead: Consultancy. Kufe:Genus Oncology LLC: Consultancy, Equity Ownership. </jats:sec