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  • 1
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    Immunomodulatory Effect of SGI-110, a Novel Hypomethylating Agent in Acute Myeloid Leukemia (AML)
    American Society of Hematology ; 2014
    In:  Blood Vol. 124, No. 21 ( 2014-12-06), p. 2303-2303
    Details
    In: Blood, American Society of Hematology, Vol. 124, No. 21 ( 2014-12-06), p. 2303-2303
    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.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V124.21.2303.2303
    RVK:
    XA 33000
    RVK:
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    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2014
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  • 2
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    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
    American Society of Hematology ; 2014
    In:  Blood Vol. 124, No. 21 ( 2014-12-06), p. 2072-2072
    Details
    In: Blood, American Society of Hematology, Vol. 124, No. 21 ( 2014-12-06), p. 2072-2072
    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 ( 〉 2 fold, p value 〈 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.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V124.21.2072.2072
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2014
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  • 3
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    Phase I Clinical Trial Evaluating Abatacept in Patient with Steroid-Refractory Chronic Graft Versus Host Disease
    American Society of Hematology ; 2016
    In:  Blood Vol. 128, No. 22 ( 2016-12-02), p. 387-387
    Details
    In: Blood, American Society of Hematology, Vol. 128, No. 22 ( 2016-12-02), p. 387-387
    Abstract: Introduction: Chronic graft versus host disease (cGVHD) remains a major source of morbidity and mortality following allogeneic transplantation. While corticosteroids remain first line therapy for cGVHD, they are associated with significant toxicity, and a substantial proportion of patients fail to completely respond. Treatments for steroid-refractory cGVHD are limited. While the pathophysiology of chronic GVHD is complex, activated T cells play a critical role, driven by allo-antigen stimulation. As such, inhibition of T cell activation via blockade of co-stimulation has potential as a therapeutic target in cGVHD. Abatacept is a recombinant fusion protein consisting of the extracellular domain of human CTLA-4 and a fragment of the Fc domain of human IgG1 that has been modified to prevent complement fixation and antibody-dependent cellular cytotoxicity. Abatacept is the first drug in a class of agents termed "selective co-stimulation modulators." The CTLA-4 moiety of Abatacept binds specifically to CD80 and CD86 and down-modulates the CD28-mediated co-stimulation of T cells. We conducted a phase I clinical trial was conducted to evaluate the safety, clinical and immune effects of Abatacept in patients with steroid-refractory cGVHD. Methods: The study followed a 3+3 design with two escalating doses of Abatacept to determine the maximum tolerated dose (MTD): 3 mg/kg and 10 mg/kg. Dose-limiting toxicities (DLTs) were defined as Grade 3 or 4 toxicities judged to be probably or definitely related to Abatacept. Infection was not considered a DLT. Abatacept was administered for a total of 6 doses. Doses 1-3 were administered at two-week intervals. One month following Dose 3, Abatacept was given at four-week intervals for three doses (Doses 4-6). Inclusion criteria included recipients of allogeneic bone marrow or stem cell transplantation with myeloablative or reduced intensity conditioning, with cGVHD defined by NIH consensus criteria. Patients must have had treatment with ≥ 0.5 mg/kg/day of prednisone for at least 4 weeks. Patients with active malignant disease relapse or other active malignancy and patients with uncontrolled infection were excluded. Peripheral blood was drawn prior to each dose of Abatacept and following completion of therapy to assess the effect of treatment on circulating T cells. PD-1 expression on circulating T cells, and T cell expression of interferon gamma versus IL-10 was assessed by multichannel FACS analysis. Results: 17 subjects were treated. Three patients were treated at a dose of 3 mg/kg without DLT. Three evaluable patients completed treatment on cohort 2, at a dose of 10mg/kg without DLT. A forth participant withdrew consent following one dose of treatment and therefore is not evaluable. Ten patients were treated on an expansion cohort at a dose of 10mg/kg. We observed one grade 4 pulmonary infection, and three grade 3 pulmonary infections which resolved. Other Abatacept related adverse events included grade 2 gastritis (n=1), grade 2 pain (n=1), and grade 1 diarrhea (n=2), fatigue (n=2), rash (n=1), and skin pain (n=1). Of the 16 evaluable patients, 7 (44%) achieved a clinical partial response as defined by improvement of two disease systems based on the 2011 NIH consensus criteria. Abatacept resulted in a 51.3% reduction in prednisone usage in clinical responders with a mean baseline dose of 27mg compared to a mean dose of 14mg 1 month following the 6th dose of Abatacept (p = 0.01). PD-1 expression on circulating CD4+ and CD8+ T cells increased from a mean of 3.4% and 2.7% respectively at baseline to a mean of 8.9% and 7.6% respectively at one month following the 6thdose of Abatacept in clinical responders (n=3; p 〈 0.05). In contrast, no change in T cell expression of PD-1 was observed in non-responders. A shift from Th1 to Th2 cytokine secretion was observed in clinical responders, with a mean 2.8 fold decrease in interferon gamma and a mean 2.5 fold increase in IL-10 secretion by circulating T cell populations (n=4). Conclusion: Abatacept is well-tolerated in the treatment of steroid-refractory cGVHD. Abatacept resulted in the improvement in NIH cGVHD scores in 44% of patients with steroid-refractory GVHD with a significant decrease in prednisone dose. An increase in PD-1 expression and a skewing toward Th2 cytokines was observed in clinical responders. Based on this promising data, a phase II trial is being initiated. Disclosures Soiffer: GentiumSpA/Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Arnason:Gilead: Consultancy. Avigan:Astex: Research Funding; DCPrime: Research Funding. Rosenblatt:Astex: Research Funding; BMS: Research Funding; DCPrime: Research Funding.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V128.22.387.387
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2016
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  • 4
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    Bone Marrow Stroma Protects Myeloma Cells from Cytotoxic Damage Via Induction of the Oncoprotein MUC1
    American Society of Hematology ; 2014
    In:  Blood Vol. 124, No. 21 ( 2014-12-06), p. 3378-3378
    Details
    In: Blood, American Society of Hematology, Vol. 124, No. 21 ( 2014-12-06), p. 3378-3378
    Abstract: Introduction : Stromal cells in the bone marrow microenvironment of patients with multiple myeloma (MM) are thought to play a vital role in promoting cell growth and protection from cytotoxic injury. Targeting of stromal-myeloma cell interactions to enhance anti-myeloma treatment represents a promising therapeutic strategy. The MUC1 oncoprotein is a critical oncoprotein that is expressed in the majority of primary myeloma cells and regulates downstream pathways such as NFkB and β-catenin/wnt that modulate myeloma growth and survival. Inhibition of MUC1 via a cell penetrating peptide (GO-203) that blocks down stream signaling reverses resistance to bortezomib (BZT). Herein we studied the influence of bone marrow stromal cells (BMSC) on MUC1 expression on MM cells, and its link to drug resistance. Methods and Results : Coculture of MM human cell lines (RPMI and U266) with a stromal cell line (HS-5), resulted in an upregulation of MUC1 expression as determined by an approximately 2 fold increase in the mean fluorescent intensity (MFI) of MUC1 as measured by flow cytometry. Similar findings were observed following coculture of MM cells with stromal cells isolated from primary bone marrow mononuclear cells (BMSC) of MM patients. Stromal cell mediated upregulation of MUC1 expression was subsequently confirmed by Western blot analysis. Patient derived MM cells were also noted to increase their MUC1 expression 2.9 fold when co-cultured with stroma (HS-5 cell line). MUC1 expression was also increased following coculture of MM cells with stromal cells in transwell plates, suggesting the effect was mediated by soluble factors not requiring cell-cell contact. Consistent with these findings, we demonstrated that addition of recombinant IL-6, a stromal cell derived cytokine, to MM cells resulted in a 2 fold increase in MFI of MUC1 expression. Moreover, coculture of MM cells with IL-6 neutralizing antibodies abrogated the effect of BMSC on MUC1 expression. These results suggest that stromal cell secretion of IL-6 plays a role in upregulation of the oncoprotein MUC1 on MM cells. We subsequently evaluated the effect of stromal cell induction of MUC1 expression on resistance to anti-myeloma agents. Increased MUC1 expression following coculture of MM cells with BMSC was associated with a higher level of resistance to BTZ (20nM), resulting in 48% less cell death by CellTiter-Glo and annexin/propidium iodide (PI) staining. Conversely, we demonstrated that silencing of MUC1 expression using a lentiviral siRNA resulted in enhanced sensitivity to anti-myeloma agents. Cell viability in MUC1 silenced as compared to wild type RPMI cells decreased by 18%, 43%, and 50% when treated with 10mg/ml cyclophosphamide (Cy), 5nM BZT, and 0.1mM melphalan, respectively. MUC1 silenced U266 cells demonstrated a decrease in cell viability by 24%, 34%, and 45% when treated with 10mg/ml Cy, 5nM BZT, and 1mM lenalidomide respectively. Similarly, exposure of primary MM cells to the MUC1 inhibitor GO-203 resulted in enhanced MM cell sensitivity to bortezomib and cyclophosphamide evidenced by a 60% and 39% decrease in cell viability respectively, compared to each drug alone. Conclusions : Our results delineate one of the mechanisms by which the bone marrow microenvironment confers drug resistance in MM. MM cells co-cultured with BMSC have enhanced expression of MUC1, mediated by IL-6 secretion. Overexpression in turn confers MM cell resistance to standard anti-myeloma agents. Importantly inhibition of MUC1 via silencing of expression or exposure to a small molecule inhibitor can overcome drug resistance to known anti-myeloma drugs, providing the rationale for clinical evaluation of combination therapy. Disclosures Kufe: Genus Oncology: Consultancy, Equity Ownership.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V124.21.3378.3378
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2014
    detail.hit.zdb_id: 1468538-3
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  • 5
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    MUC1 Inhibition Overcomes Chemotherapy Resistance in Acute Myeloid Leukemia
    American Society of Hematology ; 2015
    In:  Blood Vol. 126, No. 23 ( 2015-12-03), p. 2473-2473
    Details
    In: Blood, American Society of Hematology, Vol. 126, No. 23 ( 2015-12-03), p. 2473-2473
    Abstract: Chemotherapy is not curative for the majority of patients with acute myeloid leukemia (AML) due to the presence of leukemia stem cells (LSCs) and the emergence of other clonal populations that exhibit resistance to cytotoxic therapy. Understanding the pathways responsible for the development of chemotherapy resistance is critical for developing novel strategies that more effectively target AML. We have previously demonstrated that the MUC1 oncogene is expressed on AML cells including LSCs. MUC1 is a heterodimeric glycoprotein where the MUC1-C subunit functions as an oncoprotein. Importantly in AML, MUC1-C facilitates the nuclear translocation of active β-catenin necessary for downstream effectors including survivin, a negative regulator of apoptosis. Recent data has demonstrated that survivin inhibition is crucial in conferring susceptibility to chemotherapeutic agents in a leukemia model. In the current study, we sought to examine the effect of MUC1-C inhibition on survivin levels and the sensitivity of leukemic cells to cytotoxic chemotherapy. To assess the effect of MUC1-mediated signaling on survivin expression, MUC1-C was silenced using a lentiviral shRNA hairpin against MUC1-C in two AML cell lines, MOLM-14 and THP-1. Silencing of MUC1-C was confirmed by flow cytometric and western blot analyses and resulted in the downregulation of β-catenin and its target, survivin, at both the protein and mRNA level. In contrast, MUC1-C overexpression led to increased survivin expression. The role of MUC1-C as a mediator of resistance to cytotoxic chemotherapy was assessed. A stable MUC1-C gene knockdown of the AML cell line, MOLM-14, was generated using CRISPR/Cas9 technology. The MOLM-14 CRISPR and MOLM-14 wild-type (WT) cell lines were independently treated with increasing doses of the cytotoxic chemotherapeutic agent, cytarabine (Ara-C 50-1000 nM). The MOLM-14 CRISPR cell line demonstrated reduced cell viability utilizing an ATP-based luminescence assay (CTG, Promega) as compared to the MOLM-14 WT cell line at 72 hours (14% vs. 32%) and 96 hours (6% vs. 28%) after treatment with Ara-C. The results demonstrate that MUC1-C confers resistance to chemotherapy, and that the loss of MUC1-C in leukemic blasts significantly increases AML susceptibility to cytotoxic chemotherapy. Next, we investigated if the functional inhibition of MUC1-C would increase the sensitivity of AML to Ara-C. A novel cell-penetrating peptide, GO-2O3, binds to the MUC1-C subunit and blocks MUC1-C homodimerization and function. Two AML cell lines, MOLM-14 and MV4-11, were treated with increasing doses of Ara-C (25-1000nM) and GO-2O3 (1-5uM) to establish dose-dependent cytotoxicity curves. Based on the cytotoxicity curves, doses of Ara-C (50, 100, 125 nM) and GO-2O3 (1.0, 1.5, 2.0 uM) were selected for combination therapy. Analysis at 48 hours utilizing CTG demonstrated statistically significant synergy validated by the combination index (CI) calculated through CompuSyn [MV4-11 (0.54) and MOLM-14 (0.86)] where CI values 〈 0.90 indicate drug synergy. These results were confirmed in both the MOLM-14 and MV4-11 cell lines by staining for Annexin V/PI with FACS analysis after 48 hours of treatment. In MOLM-14, apoptosis and necrosis were noted as follows: no treatment (1.5%, 0.6%), GO-2O3 (3.7%, 6%), Ara-C (12%, 9.8%), and the combination (17%, 34%). In MV4-11, apoptosis and necrosis were noted as follows: no treatment (2.1%, 0.9%), GO-2O3 (4%, 6%), Ara-C (17.3%, 11.2%), and the combination (14%, 40%). The functional inhibition of MUC1-C in combination with Ara-C resulted in both decreased cell viability and increased cell death as compared to either agent alone. In conclusion, the data demonstrates that MUC1 expression on AML cells plays a critical role in conferring resistance to chemotherapy. Via its effector, survivin, MUC1-C inhibition renders leukemia cells more susceptible to cytotoxic injury in synergy with Ara-C. A clinical trial evaluating the combination of Ara-C and GO-203 in patients with relapsed AML is planned. Disclosures Kufe: Genus Oncology: Consultancy, Equity Ownership.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V126.23.2473.2473
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2015
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  • 6
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    MUC-1 Regulates MiR34a Expression in Acute Myeloid Leukemia Cells Resulting in an Accumulation of Granulocytic Myeloid-Derived Suppressor Cells
    American Society of Hematology ; 2015
    In:  Blood Vol. 126, No. 23 ( 2015-12-03), p. 643-643
    Details
    In: Blood, American Society of Hematology, Vol. 126, No. 23 ( 2015-12-03), p. 643-643
    Abstract: Acute myeloid leukemia (AML) is characterized by an immunosuppressive milieu that blunts effector cell function and the generation of tumor specific immunity. Myeloid-derived suppressor cells (MDSCs) are a critical component of the immunosuppressive tumor microenvironment that fosters immune tolerance and disease growth. The role of MDSCs in AML and the mechanism by which tumor cells evoke the expansion of MDSCs has not been well elucidated. MUC1 is an oncoprotein that is aberrantly expressed on a majority of primary AML. The C-terminus of MUC1 (MUC1-C) forms dimers and translocates to the nucleus where it mediates downstream signaling. The effect of MUC1-C mediated signaling on immune modulation in AML has not been well characterized. In prior studies, we have demonstrated that in vitro expansion of MDSCs is correlated with MUC1 expression by the AML cells. In the present study, we sought to characterize the effect of MUC1 on in vivo recruitment of MDSCs and examine the mechanism by which this is accomplished. The murine AML cell line TIB-49 was transplanted in C57BL/6J mice. Following establishment of disease, the mice were euthanized, along with healthy controls, and bone marrow and spleen CD11b+ Gr1+ MDSCs were quantified by flow cytometry. Engrafted mice had an average MDSC burden of 47% in the marrow and 8.7% in the spleen, compared with 35% and 2% in control mice, respectively. The increase in MDSCs was granulocyte predominant, consistent with our findings in patients with AML. Gr1+CD11b+ cells derived from the marrow and spleens of engrafted mice showed higher levels of Arginase-1 compared to MDSCs from control mice, suggesting an increase in immune suppressive phenotype. To investigate the role of MUC1 on the expansion of MDSCs, expression of MUC-1C, the signaling C-terminus of MUC1, was silenced in TIB-49 cells by stable expression of a MUC1-C shRNA as determined by Western Blot analysis. MUC1 silenced or control vector transduced TIB-49 cells were transplanted into C57BL/6J mice. Following establishment of disease, the mice were euthanized, and bone marrow and spleens were quantified for MDSCs. Control AML engrafted mice had an average splenic MDSC burden 2-fold higher than MUC1 silenced AML engrafted mice (n=4). We have developed a cell-penetrating peptide (GO-203) that disrupts homodimerization of the MUC1-C subunit necessary for its downstream signaling. C57BL/6J mice were challenged with TIB-49 AML cells and after 24 hours were treated daily with GO-203. Mice treated with the MUC1 inhibitor had a 2-fold decrease in splenic MDSCs, compared to control mice (n=3) at time of analysis following disease establishment. Noncoding RNAs have emerged as a critical biologic effector of oncogenic pathways. MicroRNAs (miRNAs) post-transcriptionally regulate gene expression by interacting with the 3′ untranslated region (3′ UTR) of target mRNAs. miR-34a has been implicated in regulating the expansion of MDSCs. In the present study we demonstrated that silencing of MUC1 expression via lentiviral transduction with a MUC1 specific shRNA resulted in a significant increase in miR-34a expression, as quantified by q-PCR. Consistent with the MUC1 mediated regulation of MDSC expansion by modulation of miR-34a levels, MDSCs induced by co-culture of healthy donor PBMCs with MUC1 silenced AML cells contained 4-fold higher levels of miR-34a, as compared to controls. AML cells were pre-incubated with SYTO® RNASelect™ Green Fluorescent cell stain. After 4 hours, co-cultures of PBMCs and AML cells were analyzed via flow cytometry. AML cells were excluded and cells positive for MDSC markers and containing green fluorescing exosome dye were quantified. AML derived exosomes were found in 18% of MDSCs expanded from AML cells (n=3), demonstrating exosome trafficking from tumor to MDSCs. Finally, miR-34a was over-expressed in MOLM-14 using lentiviral transduction. Over-expression of miR-34a in MUC1 expressing MOLM-14 cells resulted in a 30% reduction in MDSC expansion in co-cultured PBMCs (n=3). In conclusion, MUC1 regulates MDSC expansion in AML, via its effects on miR34a, acting as a critical mediator of tumor mediated immune suppression. Incorporating strategies to reverse the expansion of MDSCs in AML, potentially by targeting MUC1 and increasing miR-34a expression offers a novel therapeutic approach for cancer immunotherapy. Disclosures Küfe: Genus Oncology, LLC: Equity Ownership.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V126.23.643.643
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2015
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  • 7
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    Co-Expression Of The MUC1 Oncoprotein and CD34 On Primary Myeloma Bone Marrow Cells Identifies a Population With Myeloma Initiating Potential
    American Society of Hematology ; 2013
    In:  Blood Vol. 122, No. 21 ( 2013-11-15), p. 127-127
    Details
    In: Blood, American Society of Hematology, Vol. 122, No. 21 ( 2013-11-15), p. 127-127
    Abstract: A major challenge in the development of effective myeloma (MM) therapy is addressing tumor heterogeneity, with the presence of sub-clones that exhibit resistance to standard therapy. An ongoing area of investigation focuses on identification of myeloma initiating cells that demonstrate greater capacity for self-renewal and serve as a potential reservoir for disease recurrence. It has been postulated that MM cells arise from a primitive B cell precursor population distinct from the more differentiated malignant plasma cell population. The critical feature of these myeloma-propagating cells is thought to be the ability to efficiently recapitulate MM in immunocompromised mice. MUC1 is an oncoprotein aberrantly expressed in malignant cells, including multiple myeloma, that interacts with multiple transcription factors, such as NF-κB and the β-catenin/TCF4 complex, that regulate cell survival and proliferation critical for malignant transformation. We have previously demonstrated that MUC1 is expressed by AML leukemic blasts, as compared to normal hematopoietic stem cells, and blockade of MUC1 signaling prevents establishment of leukemia in immunocompromised animals. In the present project, we identify a unique population of CD34+/MUC1+/CD138+/CD20+ cells in primary MM bone marrow samples that exhibit features of myeloma initiating cells, as manifested by high levels of enzymatic ALDH activity, the ability to efflux Hoechst dye represented as “side population” (SP), and the ability to establish disease in immunocompromised mice. Of note, MM engraftment of unselected primary myeloma cells in a xenograft model has a low success rate, and typically requires the introduction of an artificial stromal support network. Methods and results Bone marrow aspirates were obtained from newly diagnosed MM patients using an established protocol approved by the IRB. Expression of MUC1, myeloid and lymphoid markers was assessed using multicolor flow cytometric analysis. While MUC1 shows only a minimal expression ( 〈 5%, n=8) in normal CD34+ hematopoietic progenitors, we have demonstrated that on average 54% of CD34+ cells isolated from bone marrow samples of MM patients expressed MUC1 (n=7, p 〈 0.05), in addition to other MM and lymphoid markers. MM derived CD34+MUC1+ cells segregated with SP by the ability to efflux Hoechst dye and expressed high levels of ALDH as assessed by the Aldefluor assay (11% of CD34+MUC+ cells had high ALDH activity as opposed to less than 1% in bulk MM marrow cells, n=3). CD34+MUC+ cells co-expressed CD138+, CD20+, and were CD38 dim (n=7), consistent with the phenotypic markers that have been previously described in association with myeloma propagating cells. In order to study the capacity of CD34+MUC1+ cells to recapitulate MM in a murine model, a bone marrow sample was obtained from a patient with newly diagnosed MM with a cytogenetic abnormality characterized by the rearrangement of the CCND1/IGH loci. Primary bone marrow cells were fluorescently labeled and CD34-MUC+ (consistent with mature CD138+CD38hi plasma cells) and CD34+MUC+ populations of cells were isolated using FACS sorting. Cells from each population were injected into an irradiated NOD/SCID mouse (0.5x106cells/mouse). After 13 weeks, no human engraftment was detected in the 4/4 mice injected with CD34-MUC+ population of mature plasma cells. In contrast, 2/2 mice injected with CD34+MUC+ cells demonstrated human engraftment. Engrafted cells were isolated by FACS sorting and transferred onto glass slides for cytogenetic analysis by FISH. Notably, the engrafted cells harbored rearrangement at CCND1/IGH loci consistent with the originating MM clone. In another experiment, 2/2 NOD/SCID mice inoculated with CD34+MUC1+ primary MM cells demonstrated MM engraftment after 12 weeks, characterized by the presence of CD138+CD45- human plasma cells in the murine bone marrow. Conclusions We have identified a subpopulation of primitive myeloma cells that coexpress CD34 and the MUC1 oncoprotein. CD34+MUC1+ cells express CD20 and CD138, and express high levels of ALDH. CD34+MUC1+ cells demonstrate the capacity to engraft human MM cells in immunocompromised mice, even without an artificial stromal framework. Inhibition of MUC1 signaling thus may offer new avenues to target critical myeloma subclones. Disclosures: No relevant conflicts of interest to declare.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V122.21.127.127
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2013
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  • 8
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    Rituximab Directly Decreases T Cell Activation, Both In Vivo and In Vitro
    American Society of Hematology ; 2010
    In:  Blood Vol. 116, No. 21 ( 2010-11-19), p. 3935-3935
    Details
    In: Blood, American Society of Hematology, Vol. 116, No. 21 ( 2010-11-19), p. 3935-3935
    Abstract: Abstract 3935 Rituximab, a chimeric anti-C20 monoclonal antibody, has been extensively used over the last decade for the therapy of B cell malignancies. Recent clinical data suggest that rituximab may affect T cell function, increasing the risk of T cell dependent infections in heavily-treated patients. The current study was designed to investigate the effect of rituximab on T cell activation and assess T cell function following the addition of rituximab to purified T cells. The T cell activation profile, dependent on rituximab administration, was evaluated in vivo and in vitro. Peripheral blood mononuclear cells (PBMCs) generated from B-cell non-Hodgkin lymphoma (NHL) patients prior and immediately after the administration of 375 mg/m2 rituximab, were examined for the expression of inflammatory cytokines. The in vitro studies were performed by using CD25 depleted PBMCs or B cell depleted T cells (CD3+CD25-CD19-). The obtained cells were stimulated with allogeneic dendritic cells (DCs), in the absence or presence or 2 mg/ml rituximab. T cell activation was evaluated using immunophenotypic markers, cytokine profile and T cell proliferation assay. Eight NHL patients participated in the study. The level of T cells expressing inflammatory cytokines was significantly decreased following the administration of a single dose of rituximab. T cells expressing IL-2 declined from a mean level of 26.5% to 11.5% and the level of IFN- γ decreased from 22% to 4.2%. Further administration of rituximab, up to 4 weekly doses, resulted in an additional decline in the amount of inflammatory cytokine producing T cells to a level of 1.4% for IL-2 and 3.5% for IFN-g. However, repeated evaluation, performed at 4 months after completing rituximab, showed restoration of the inflammatory population. In accord with this inhibitory effect, in vitro stimulation of T cells with allogeneic DCs, in the presence of rituximab, resulted in a significant decrease in activation markers (CD25, GITR and CTLA-4) (Table 1). These changes were accompanied by a marked reduction in inflammatory cytokine production and proliferative capacity. Of interest, these inhibitory effects were also obtained whilst using B cell depleted T cells (CD3+CD25-CD19-). In conclusion, rituximab administration results in a transient T cell inactivation, demonstrated through the reduction in inflammatory cytokine production and T cell proliferation capacity. This effect appears to be non-B cell dependent, being obtained in the absence of B cell in the culture, and may account for clinical observations in ameliorating T-cell dependent disorders, such as graft-versus-host disease. Table 1. Activation profile depending on rituximab (in vitro) Without rituximab With rituximab *Activation marker (%) CD25 27 9 GITR 15.6 4.7 CTLA4 17.7 7 *Cytokines expression (%) IL-2 22 2 IL12 16 4 IFN-gamma 21 1.8 T cells proliferation (O.D.) DC stimulation 1.528 0.580 CMV stimulation 1.563 0.570 anti CD3/CD28 stimulation 0.705 0.407 * Gated out of lymphocytes Disclosures: No relevant conflicts of interest to declare.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V116.21.3935.3935
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2010
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  • 9
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    Clinical Trial Evaluating DC/AML Fusion Cell Vaccination In AML Patients
    American Society of Hematology ; 2013
    In:  Blood Vol. 122, No. 21 ( 2013-11-15), p. 3928-3928
    Details
    In: Blood, American Society of Hematology, Vol. 122, No. 21 ( 2013-11-15), p. 3928-3928
    Abstract: We have developed a promising leukemia vaccine in which patient derived AML cells are fused with autologous dendritic cells (DCs), presenting a broad array of antigens. We are conducting a clinical trial in which AML patients who are not candidates for allogeneic transplantation undergo vaccination with DC/AML fusion cells following chemotherapy induced remission. Twenty-six patients (14 males, 12 females) underwent collection of AML cells at disease presentation for vaccine generation and immune monitoring studies. Median age of the patients is 66 years. Tumor was collected from either a bone marrow aspirate (N=16), 20 cc of peripheral blood (N=7), or leukapheresis product (N=3) at the time of presentation with newly diagnosed AML (N=25) or first relapsed AML (N=1). The mean yield of AML cells was 109 x 106 cells with a mean viability of 91%. Eligible patients achieving CR following chemotherapy (N=16) underwent leukapheresis for DC generation and vaccine preparation. Adherent peripheral blood mononuclear cells were isolated, cultured in the presence of GM-CSF and IL-4 for 5-7 days, and exposed to TNFα for 48-72 hours to generate mature DCs. The mean yield of DCs was 177 x106 cells with a mean viability of 89%. Fusion cells were generated by co-culture of DCs with AML cells in the presence of 50% polyethylene glycol and identified as cells co-expressing antigens that were unique to the DC and tumor population. Mean fusion efficiency and viability was 38% and 85%, respectively. As a measure of their activity as antigen presenting cells, the capacity of fusion cells to stimulate allogeneic T cell proliferation ex vivo was quantified. In contrast to the leukemia preparation (mean stimulation index (SI) 3.81), the DC and fusion cell preparation were potent stimulators (mean SI 19.61 and 13.48, respectively). Vaccination with DC/leukemia fusion cells was initiated within 12 weeks from count recovery following the final cycle of chemotherapy. 13 patients received at least two monthly vaccinations at a dose of 5x106 fusion cells. 8 patients had intermediate risk cytogenetics, 3 patients had good risk cytogenetics, and 2 patients had a complex karyotype. Vaccination was well tolerated, and importantly, was not associated with clinically significant auto-immunity. Possibly related adverse events were transient and of grade 1-2 intensity, including vaccine site reactions, pruritis, arthalgias, myalgias, eosinophilia, leukopenia, thrombocytopenia. Biopsy of vaccine site reactions demonstrated a dense infiltrate of CD4 and CD8 T cells consistent with recruitment of reactive T cell populations to the vaccine bed. To date, 9 patients remain in remission (69%), with a mean follow up of 23 months. Peripheral blood samples were collected prior to each vaccination and at 1, 3, and 6 months following completion of vaccination. Vaccination resulted in the potent induction of leukemia specific immunity as measured by an increase in CD8 T cells expressing IFNγ in response to ex vivo exposure to autologous leukemia cell lysates (mean fold increase 8, n=6). Bone marrow derived T cells were isolated prior to and following vaccination in patients who are HLA2.1+. Vaccination resulted in the expansion of bone marrow infiltrating T cells recognizing MUC1 (9 fold increase), WT1 (5 fold increase), PRAME (12 fold increase) tumor antigens by tetramer analysis (n=2). In conclusion, DC/AML fusion cell vaccination results in the potent expansion of leukemia reactive T cells and durable remissions following chemotherapy. Enrollment to a second cohort is being initiated, in which patients with be treated with DC/AML fusion cell vaccination in conjunction with PD1 blockade. Disclosures: No relevant conflicts of interest to declare.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V122.21.3928.3928
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2013
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  • 10
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    Cobomarsen, an Oligonucleotide Inhibitor of miR-155, Slows DLBCL Tumor Cell Growth In Vitro and In Vivo
    American Association for Cancer Research (AACR) ; 2021
    In:  Clinical Cancer Research Vol. 27, No. 4 ( 2021-02-15), p. 1139-1149
    Details
    In: Clinical Cancer Research, American Association for Cancer Research (AACR), Vol. 27, No. 4 ( 2021-02-15), p. 1139-1149
    Abstract: miRNA-155 is an oncogenic miRNA highly expressed in B-cell malignancies, particularly in the non–germinal center B-cell or activated B-cell subtype of diffuse large B-cell lymphoma (ABC-DLBCL), where it is considered a potential diagnostic and prognostic biomarker. Thus, miR-155 inhibition represents an important therapeutic strategy for B-cell lymphomas. In this study, we tested the efficacy and pharmacodynamic activity of an oligonucleotide inhibitor of miR-155, cobomarsen, in ABC-DLBCL cell lines and in corresponding xenograft mouse models. In addition, we assessed the therapeutic efficacy and safety of cobomarsen in a patient diagnosed with aggressive ABC-DLBCL. Experimental Design: Preclinical studies included the delivery of cobomarsen to highly miR-155–expressing ABC-DLBCL cell lines to assess any phenotypic changes, as well as intravenous injections of cobomarsen in NSG mice carrying ABC-DLBCL xenografts, to study tumor growth and pharmacodynamics of the compound over time. To begin to test its safety and therapeutic efficacy, a patient was recruited who underwent five cycles of cobomarsen treatment. Results: Cobomarsen decreased cell proliferation and induced apoptosis in ABC-DLBCL cell lines. Intravenous administration of cobomarsen in a xenograft NSG mouse model of ABC-DLBCL reduced tumor volume, triggered apoptosis, and derepressed direct miR-155 target genes. Finally, the compound reduced and stabilized tumor growth without any toxic effects for the patient. Conclusions: Our findings support the potential therapeutic application of cobomarsen in ABC-DLBCL and other types of lymphoma with elevated miR-155 expression.
    Type of Medium: Online Resource
    ISSN: 1078-0432 , 1557-3265
    URL: Article
    DOI: 10.1158/1078-0432.CCR-20-3139
    RVK:
    XA 36791
    Language: English
    Publisher: American Association for Cancer Research (AACR)
    Publication Date: 2021
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