Inhibition of Histone Deacetylase 6 Effects on Interaction of Tumor and Immune Cells In Multiple Myeloma

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2996-2996
Author(s):  
Gullu Gorgun ◽  
Teru Hideshima ◽  
Naoya Mimura ◽  
Diana Cirstea ◽  
Loredana Santo ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Gene Transcription ◽  
Immune Cells ◽  
Immune Cell ◽  
Flow Cytometric Analysis ◽  
Histone Deacetylase 6 ◽  
Advisory Committees ◽  
Effector Cells ◽  
Immune Cell Subpopulations ◽  
Cell Subpopulations

Abstract Abstract 2996 Histone deacetylase 6 (HDAC6), a cytoplasmic enzyme, is a member of Class IIb HDAC family and catalyses the deacetylation of histones. Acetylation of histones induces chromatin relaxation leading gene transcription; conversely, deacetylation of histones induces chromatin condensation and silencing of gene transcription. HDAC6 regulates important cellular functions including histone modifications, gene transcription, and viral infection, degradation of misfolded proteins, cell migration, and immune synapse formation. HDAC inhibitors have been developed over the last decade as anti-tumor therapeutic agents in solid tumors and hematologic malignancies including multiple myeloma (MM). Myeloma represents a model system for the development of novel drugs which impact tumor cells as well as accessory cells in the tumor microenvironment. In this study we evaluated the effects of selective HDAC6 inhibition on immune accessory cells using a novel HDAC6 small molecule inhibitor, WT-161 compared with a control HDAC inhibitor trichostatin-A (TsA). Peripheral blood (PBMC) or bone marrow mononuclear cells (BMNC) from healthy donors or MM patients were cultured in the absence or presence of WT-161 (0.25-2.5uM) for 6h to 7 days. We first determined whether WT-161 induces HDAC6 inhibition in normal PBMCs by western blot of acetylated (Lys40)-tubulin, a target for HDAC6 activity. WT-161 induced acetylation of tubulin in PBMCs as early as 6 hours of exposure. Immunophenotypic changes were determined by flow cytometric analysis. Immunophenotypic analysis of PBMCs cultured with WT-161 showed significantly increased expression of PD1 in all immune cell subpopulations including CD4T, CD8T, CD3/CD8/CD56+ NKT and NK cells as well as in CD14+ myeloid cells. Conversely, decreased expression of negative costimulatory molecules CTLA4 and PDL1 was also noted, with no significant change in the expression of positive costimulatory molecules CD28, ICOS and ICOSL. Additionally, culture of PBMCs with WT-161 significantly increased expression of effector/memory T cell marker CXCR3 and decreased expression of CXCR4 in CD4T, CD8T, NKT, NK effector cells as well as CD14+ myeloid cells. WT-161 also induced ICAM1 expression on all immune cell subpopulations. WT-161 effects on the proliferation of immune cells were next assessed by CFSE-flow cytometric analysis. CFSE stained PBMCs were stimulated with anti-CD3 ab and cultured with WT-161 for 7 days. While WT-161 triggered proliferation of CD4T and CD8T cells, proliferation of NK and NKT cells decreased and B cells were unaffected. Interestingly, CD4/CD25/Foxp3 coexpressing regulatory T cells were also significantly diminished in both PBMCs and BMNCs cultured with WT-161 for 4 days. These data suggest that HDAC6 may have an immune regulatory function, and that inhibition of HDAC6 induces changes in the immune effector cells in MM microenvironment. Ongoing analysis of effects of HDAC6 inhibition on immune cells in the tumor microenvironment will further define the role of HDAC6 in disease pathogenesis and suggest novel immune-based epigenetic-targeted therapies. Disclosures: Raje: AMGEN: Consultancy; Celgene: Consultancy; Novartis: Consultancy; astraZeneca: Research Funding. Munshi:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Richardson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Johnson&Johnson: Membership on an entity's Board of Directors or advisory committees. Anderson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees.

Immunomodulatory EFFECTS of Lenalidomide and Pomalidomide ON INTERACTION of TUMOR and BONE MARROW Accessory CELLS IN MULTIPLE MYELOMA.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 950-950
Author(s):  
Gullu Gorgun ◽  
Elisabetta Calabrese ◽  
Teru Hideshima ◽  
Giulia Perrone ◽  
Giada Bianchi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Board Of Directors ◽  
Immune Cells ◽  
Research Funding ◽  
Effector Cell ◽  
Cytokine Signaling ◽  
Advisory Committees ◽  
Effector Cells ◽  
Healthy Donors ◽  
Socs3 Expression

Abstract Abstract 950 The bone marrow (BM) microenvironment consists of extracellular matrix and the cellular compartment including bone marrow stromal cells (BMSCs) and immune cells. Interaction between multiple myeloma (MM) cells and BM cells induces growth, survival, migration, and drug resistance in MM, via both cell-cell contact and cytokines. Even though MM cell interaction with BMSCs has been extensively studied, the role of immune cells in the MM BM milieu is not yet defined. The IMiDs® immunomodulatory agents lenalidomide (len) and pomalidomide (pom) target not only MM cells, but also MM cell-immune cell interactions and cytokine signaling. For example, we and others have shown that len stimulates T cell proliferation, secretion of IL2 and IFNγ, as well as promotes CTL and NK cell activity against MM cells. Here we examined the in vitro immunomodulatory effects of len or pom on cytokine signaling triggered by interaction of effector immune cells with MM cells and BMSCs. PBMCs or BMMNCs obtained from patients with rel/ref MM or healthy donors after informed consent. PBMCs were cultured either alone or with BMSC, in the absence or presence of len (1μM) or pom (1μM) for 1-48h. To determine whether len or pom regulate cytokine signaling in effector cells, we used flow cytometry to analyze their effects on suppressor of cytokine signaling proteins (SOCS, including SOCS1, SOCS2, SOCS3, CIS) expression in effector cells from both healthy donors and patients with MM. Len or pom diminished IL2 and IFNγ regulators SOCS1 and SOCS3 expression in effector cells from both BM and PB of MM patients. Additionally, coculture of MM cell lines, MM1S, U266, OPM1, RPMI, LR5 and DOX40, with healthy PBMCs induced SOCS1 and SOCS3 expression in effector cells; conversely, treatment with len or pom downregulated the SOCS1 and SOCS3 expression in effector cells. To assess effects of immunomodulatory agents on immune cell proliferation in their milieu, healthy or MM-PBMCs and MM-BMMNCs were prelabeled with CFSE and stimulated with PHA (5μg/ml) or anti-CD3 (1μg/ml) in the absence or presence of len or pom for 7 days. The proliferation of CD4T and CD8T, NKT, and NK cells was assessed by CFSE flow cytometric analysis. Len or pom induced CD4T cell (%Divided: Cont:55, len or pom >72), CD8 T cell (%Div: Cont:34, len or pom>60) and NKT cell (%Div: Cont:3.5, len or pom >8) proliferation, as well as stimulated IL2 (2-4 fold) and IFNγ (2 fold) production in effector cells from MM. It has been demonstrated that SOCS1 gene negatively regulates IL6 signaling and is silenced by methylation in MM cells. To understand the mechanism of cytokine inhibitory signaling in both effector cells and MM cells, we next analysed the interaction of effector cell with MM cells that were epigenetically modified to express SOCS1. SOCS1 methylation in MM cells was confirmed by SOCS1 gene methylation-specific polymerase chain reaction (SOCS1-MSP). Genomic DNA was isolated from MM cell lines (MM1S, RPMI8226, OPM1, INA6 and U266), sodium bisulfite-modified, and then subjected to MSP using MSP primers that specifically recognize unmethylated or methylated SOCS1 gene. SOCS1 gene was methylated and resulted in silenced SOCS1 protein expression in all MM cell lines. To delineate the role of SOCS in effector cell response against MM cells, MM cell specific cytotoxic T lymphocytes (CTL) were generated. T cells from healthy donors were stimulated with dendritic cells pulsed with apoptotic bodies of MM1S or U266 cells for 4 weeks, and cytotoxicity was measured by standard 51Cr-release assay. To reverse SOCS1 methylation, target MM cells were cultured with 5'-Azacytidine (Aza) or trichostatin A (TsA), alone or in combination with len or pom. CTLs were pretreated with len or pom for 24h and cocultured with DNA-modified or unmodified 51Cr-labeled target cells. Len induced more potent CTL response against MM cells that were treated with len and Aza combination (83% specific killing) than len alone (%50 specific killing). Len also showed more potent anti-MM activity, assessed by 3[H]thymidine proliferation assay, in the presence of Aza than alone (p<0.05). These data demonstrate that modulation of SOCS genes by blocking BMSC derived inhibitory cytokine signaling may enhance effector cell response and promote efficacy of len or pom in MM. Ongoing analysis of effects of len or pom on immune cells in the BM environment will both define their role in disease pathogenesis and suggest novel immune-based targeted therapies. Disclosures: Munshi: Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees. Richardson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees. Anderson:Millennium: Research Funding; Novartis: Research Funding; Celgene: Research Funding.

scholarly journals Tracking distinct microglia subpopulations with photoconvertible Dendra2 in vivo

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Eric B. Miller ◽  
Sarah J. Karlen ◽  
Kaitryn E. Ronning ◽  
Marie E. Burns
Keyword(s):  
Immune Cells ◽  
Large Scale ◽  
Fluorescent Protein ◽  
Immune Cell ◽  
Neuronal Damage ◽  
Ex Vivo ◽  
Fluorescent Markers ◽  
Immune Cell Subpopulations ◽  
Cell Subpopulations

Abstract Background The ability to track individual immune cells within the central nervous system has revolutionized our understanding of the roles that microglia and monocytes play in synaptic maintenance, plasticity, and neurodegenerative diseases. However, distinguishing between similar subpopulations of mobile immune cells over time during episodes of neuronal death and tissue remodeling has proven to be challenging. Methods We recombineered a photoconvertible fluorescent protein (Dendra2; D2) downstream of the Cx3cr1 promoter commonly used to drive expression of fluorescent markers in microglia and monocytes. Like the popular Cx3cr1–GFP line (Cx3cr1+/GFP), naïve microglia in Cx3cr1–Dendra2 mice (Cx3cr1+/D2) fluoresce green and can be noninvasively imaged in vivo throughout the CNS. In addition, individual D2-expressing cells can be photoconverted, resulting in red fluorescence, and tracked unambiguously within a field of green non-photoconverted cells for several days in vivo. Results Dendra2-expressing retinal microglia were noninvasively photoconverted in both ex vivo and in vivo conditions. Local in vivo D2 photoconversion was sufficiently robust to quantify cell subpopulations by flow cytometry, and the protein was stable enough to survive tissue processing for immunohistochemistry. Simultaneous in vivo fluorescence imaging of Dendra2 and light scattering measurements (Optical Coherence Tomography, OCT) were used to assess responses of individual microglial cells to localized neuronal damage and to identify the infiltration of monocytes from the vasculature in response to large scale neurodegeneration. Conclusions The ability to noninvasively and unambiguously track D2-expressing microglia and monocytes in vivo through space and time makes the Cx3cr1–Dendra2 mouse model a powerful new tool for disentangling the roles of distinct immune cell subpopulations in neuroinflammation.

scholarly journals Pathogenesis of Extramedullary Multiple Myeloma: From Resistance to Identification of Novel Therapeutic Targets

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2680-2680
Author(s):  
Tomas Jelinek ◽  
David Zihala ◽  
Tereza Sevcikova ◽  
Veronika Kapustova ◽  
Hana Sahinbegovic ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Single Cell ◽  
Board Of Directors ◽  
Immune Cells ◽  
Research Funding ◽  
Plasma Cells ◽  
Immune Cell ◽  
Biological Pathways ◽  
Advisory Committees ◽  
Pet Ct

Abstract I ntroduction Extramedullary disease (EMD) is a less frequent manifestation of multiple myeloma (MM), where MM plasma cells become independent of the bone marrow (BM) microenvironment and infiltrate other tissues and organs. The incidence of EMD is increasing and is associated with worse prognosis and drug resistance. The specific and efficient treatment is lacking. Therefore, a better understanding of EMD pathogenesis is desperately needed. Aims To identify biological pathways leading to EMD development and to evaluate therapeutic targets in EMD plasma cells with further focus on EMD tumor microenvironment to reveal presence of effector immune cells that are crucial for immunotherapy. Methods To identify EMD specific genes, FACS/MACS sorted aberrant plasma cells were collected from: i) fresh 11 EMD relapse tumors for which we had ii) 7 corresponding cryopreserved paired BM samples from the time of MM diagnosis (NDMM), iii) 9 unpaired fresh NDMM without EMD confirmed by PET-CT and iv) 6 unpaired fresh relapsed MM (RRMM). For library preparation, we used total RNA with rRNA depletion protocol and Illumina sequencing. Residual rRNA was filtered out by SortMeRNA. Differential expression analysis was performed using Salmon for read mapping and quantification and Deseq2 package. For single-cell RNAseq we used 10x Genomics technology for sequencing and CellRanger and Seurat for data processing and analysis. Results To better understand the aggressive nature of EMD, we have analyzed bulk RNA samples (7 EMD samples plus 7 corresponding cryopreserved paired BM samples from the time of MM diagnosis). Our preliminary analysis revealed a unique EMD profile (Fig 1A) with 423 up-regulated and 421 down-regulated genes in EMD samples (adjusted p-value &lt; 0.1; absolute fold change &gt; 1.5), with G2M checkpoint proteins being the most enriched hallmark pathways pointing to higher proliferation of EMD cells. EMD down-regulated genes mainly belong to genes of the adaptive immune response which together with lower immunoglobulin production suggest loss of mature plasma cell function. Among the top genes uniquely overexpressed in EMD (versus RRMM or NDMM) were SCD and ELOVL6 that regulate crucial steps in unsaturated fatty acids synthesis. Also their transcription factor SREBF1 was significantly up-regulated. The importance of these genes in EMD pathogenesis can be supported by the involvement of SREBP1 in stem cell differentiation and mediation of bortezomib resistance by ELOVL6 (Yi et al. 2018, Lipchick et al. 2021). Our dataset also revealed several deregulated lncRNA in EMD compared to NDMM. MALAT1 was highly expressed, however, we did not confirm results by Handa et al. 2017 showing lncRNA MALAT1 as upregulated in EMD. Furthermore, we aimed to evaluate expression of known immunotherapy MM targets being currently in use or under investigation. We compared the information about expression level in EMD vs paired NDMM, with unpaired NDMM without EMD lesion confirmed by PET/CT, and with RRMM. The analysis revealed a decrease in the expression of several antigens commonly used in anti-MM immunotherapy (e.g. CD38, SLAMF7, BCMA or PDL1) on EMD PCs (Fig 1B). Intriguingly, our data show EMD specific elevated expression of EZH2 gene being promising target in preclinical MM investigation which can prove efficient especially for the aggressive MM stage - EMD. Effective immunotherapy depends on the presence of effector immune cells. Therefore, we have evaluated immune cell types and their proportion in EMD tumors. Using flow cytometry we identified T and NK cells as the only immune cell subsets present in EMD tumors (median 0.9% and 0.5%, respectively). Single-cell RNAseq analysis of two EMD samples supported these findings. Conclusions Here, we present up to our knowledge the worldwide largest cohort of 11 EMD samples (including 7 longitudinal pre-EMD/EMD samples) analysed using RNAseq with focus on biological pathways and dysregulation of particular genes leading to EMD development. Drop of expression of several known drug targets may suggest limited efficacy of the modern treatment in EMD as already presented by Jelinek et al., 2021. Importantly, we are also providing the initial insight into the microenvironment (including single-cell RNA analysis) of EMD tumors, where we detected presence of T cell and NK cells in very limited numbers. Figure 1 Figure 1. Disclosures Hajek: Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria; Novartis: Consultancy, Research Funding; Pharma MAR: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

scholarly journals Allergic diseases influence symptom severity and T lymphocyte subgroups of children with tic disorders

2021 ◽  
pp. jim-2021-001788
Author(s):  
Xiumei Liu ◽  
Xueming Wang ◽  
Xiaoling Zhang ◽  
Ai hua Cao
Keyword(s):  
T Lymphocyte ◽  
Immune Cell ◽  
Clinical Manifestations ◽  
Allergic Diseases ◽  
Tic Disorders ◽  
Control Group ◽  
T Lymphocyte Subsets ◽  
Tic Severity ◽  
Immune Cell Subpopulations ◽  
Cell Subpopulations

Tic disorders (TD) are childhood-onset neurological disorders. Immune system dysregulation has been postulated to play a role in TD, and its mechanisms likely involve dysfunctional neural-immune cross-talk, which ultimately leads to altered maturation of the brain pathways that control different TD clinical manifestations and behavioral and emotional damages. Clinical studies have demonstrated an association between TD and allergies and overactive immune responses at a systemic level. In this study, the Yale Global Tic Severity Scale was taken as a global measure of tic severity. Compared with the control group, the group of children with TD plus allergic diseases displayed significantly increased Yale total scores (p<0.05), which suggests that children with TD plus allergic diseases have heavier tic symptoms. Both motor and vocal tic scores are higher in the group of children with TD plus allergy compared with the control group. We counted immune cell subpopulations using FACS. T lymphocyte subset comparison of CD3, CD4, CD8, and CD4:CD8 expression ratios revealed that the level of CD3, CD4, and CD4:CD8 in children with TD plus allergic diseases was significantly lower than those of children with TD without allergic diseases. These differences were statistically significant (p<0.05) and suggest that children with TD plus allergic diseases have imbalanced T lymphocyte subsets. We concluded that allergy increased the severity of TD through an imbalance in cellular immunity. Studies need to be done to show whether treatment of allergic symptoms leads to a decrease in TD manifestations.

scholarly journals Electronic Sorting of Immune Cell Subpopulations Based on Highly Plastic Genes

2016 ◽  
Vol 197 (2) ◽  
pp. 665-673 ◽  
Author(s):  
Pingzhang Wang ◽  
Wenling Han ◽  
Dalong Ma
Keyword(s):  
Immune Cell ◽  
Immune Cell Subpopulations ◽  
Cell Subpopulations

scholarly journals Single‐Cell Immune Profiling in Coronary Artery Disease: The Role of State‐of‐the‐Art Immunophenotyping With Mass Cytometry in the Diagnosis of Atherosclerosis

2020 ◽  
Vol 9 (24) ◽  
Author(s):  
Katharine A. Kott ◽  
Stephen T. Vernon ◽  
Thomas Hansen ◽  
Macha de Dreu ◽  
Souvik K. Das ◽  
...  
Keyword(s):  
Coronary Artery Disease ◽  
Coronary Artery ◽  
Single Cell ◽  
Immune Cell ◽  
Mass Cytometry ◽  
Artery Disease ◽  
Immune Cell Subpopulations ◽  
Cell Subpopulations ◽  
Serological Biomarkers

Abstract Coronary artery disease remains the leading cause of death globally and is a major burden to every health system in the world. There have been significant improvements in risk modification, treatments, and mortality; however, our ability to detect asymptomatic disease for early intervention remains limited. Recent discoveries regarding the inflammatory nature of atherosclerosis have prompted investigation into new methods of diagnosis and treatment of coronary artery disease. This article reviews some of the highlights of the important developments in cardioimmunology and summarizes the clinical evidence linking the immune system and atherosclerosis. It provides an overview of the major serological biomarkers that have been associated with atherosclerosis, noting the limitations of these markers attributable to low specificity, and then contrasts these serological markers with the circulating immune cell subtypes that have been found to be altered in coronary artery disease. This review then outlines the technique of mass cytometry and its ability to provide high‐dimensional single‐cell data and explores how this high‐resolution quantification of specific immune cell subpopulations may assist in the diagnosis of early atherosclerosis in combination with other complimentary techniques such as single‐cell RNA sequencing. We propose that this improved specificity has the potential to transform the detection of coronary artery disease in its early phases, facilitating targeted preventative approaches in the precision medicine era.

Immunomodulatory Effects of Histone Deacetylase 6 Inhibition in Suppressor Immune Cells in Multiple Myeloma

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 128-128
Author(s):  
Gullu Gorgun ◽  
Teru Hideshima ◽  
Noopur S. Raje ◽  
Naoya Mimura ◽  
James E. Bradner ◽  
...  
Keyword(s):  
Immune Response ◽  
T Cell ◽  
Board Of Directors ◽  
Immune Suppression ◽  
Immune Cells ◽  
Th17 Cells ◽  
Treg Cells ◽  
Advisory Committees ◽  
Healthy Donors ◽  
Effector Immune Response

Abstract Abstract 128 The interaction of myeloma (MM) cells with bone marrow accessory cells and/or the extracellular matrix induces genomic, epigenomic and functional changes which promote tumor development, progression, cell adhesion mediated-drug resistance (CAM-DR), and immune suppression. To develop the most efficient anti-MM treatment strategy and prevent tumor escape from immune recognition, both enhancing anti-MM effector immune response and overcoming MM-induced immune suppression is essential. Suppressive immune cells including myeloid derived suppressor cells (MDSC), regulatory T cells (Treg) and IL-17 secreting Th (Th17) cells act as tumor promoters and suppressors of effector immune response, and therefore represent a significant barrier to current anti-tumor therapeutic strategies. Since, we and others have reported increased numbers of Treg and Th17 cells in MM, we here assessed MDSCs in both peripheral blood (PBMC) and bone marrow (BMMC) of patients with MM compared to healthy donors. Phenotypic analysis by flow cytometry showed a significant increase in CD14−CD11b+HLA-DRlowCD15+ MDSCs in both PBMC and BMMC from MM patients compared to healthy donors (p<0.01). Furthermore, coculture of MM cell lines with healthy PBMCs for 6 days demonstrated that MM cells significantly induce MDSC differentiation in healthy PBMCs (p<0.03). Recent studies have demonstrated that histone deacytlase 6 (HDAC6) is an important regulator of monocyte/macrophage-mediated immune response. We therefore next analysed the immunomodulatory effects of WT-161, a novel small molecule inhibitor of HDAC6, alone or in combination with lenalidomide (len) and bortezomib (bort), on suppressive immune cells in the MMBM microenvironment. To keep cell-cell interaction intact reflective of the MMBM microenvironment, PBMCs or BMMCs from MM patients were cultured in the absence or presence of WT-161 (0.5–5uM), len (1–10uM), and/or bort (2–5nM), and individual cell populations were analysed by flow cytometry. Phenotypic characterization of suppressive immune cells showed a significant decrease in both CD4+CD25+Foxp3+ Treg cells and MDSCs in MM-PBMCs and MM-BMMCs cultured with WT-161, alone or in combination with len or bort (p<0.01); however, there was no change in the expression of Th17 cells. To determine the functional mechanism of immune suppression, MDSC and Treg cells were isolated by magnetic-Ab sorting and cultured for 6 days with autologous T cells (TCR/IL-2 stimulated), with or without WT-161, len and bort, alone or in combination. T cell proliferation (by 3H-thymidine assay) was significantly inhibited in the presence of MDSCs, whereas WT-161 notably reversed MDSC-mediated T cell suppression. In contrast, len and bort did not show any significant effect. Intracellular reactive oxygen species (ROS, an MDSC-derived metabolic immune inhibitory molecule) expression was significantly decreased in MDSCs from MM cultured with WT-161, alone or together with len and bort (p<0.05). Additionally, WT-161 also reversed Treg-mediated T cell suppression as well as len. Cytokine profiling by intracellular flow cytometric analysis demonstrated that WT-161 significantly decreased IL-6 and GM-CSFR expression in MDSCs, whereas it induced IFNγ and IL-12 production in effector CD4T, CD8T and NKT cells. Finally, unstimulated or IL-2 prestimulated (36h) PBMCs or NK cells were cultured with MM cell lines (MM1.S, RPMI8226), in the absence or presence of WT-161 alone or with len and bort (4h), and anti-MM cytotoxic activity was determined by Cr51-release cytotoxicity assay. While len (48% killing) and WT-161 (39% killing) induced CTL-mediated cytotoxicity, WT-161 (53% killing) and len (56% killing) induced more potent NK cell-mediated anti-MM cytotoxicity. These data suggest that HDAC6 may have an immune regulatory function, and that inhibition of HDAC6 induces changes in suppressor immune cells leading to enhanced anti-MM immune response in MM microenvironment. Ongoing analysis of the effects of HDAC6 inhibition on immune cells in the tumor microenvironment will further define the role of HDAC6 in disease pathogenesis and suggest novel immune-based epigenetic-targeted therapies. Disclosures: Hideshima: Acetylon: Consultancy. Raje:Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Acetylon: Research Funding. Bradner:Acetylon: Scientific Founder. Richardson:Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees. Munshi:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Onyx: Consultancy, Membership on an entity's Board of Directors or advisory committees. Anderson:Novartis: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Merck: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Acetylon: Membership on an entity's Board of Directors or advisory committees.

A Novel Agent SL-401 Triggers Anti-Myeloma Activity By Targeting Plasmacytoid Dendritic Cells: Implications for a Novel Immune-Associated Mechanism

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3000-3000 ◽  
Author(s):  
Arghya Ray ◽  
Deepika Sharma DAS ◽  
Yan Song ◽  
Vincent Macri ◽  
Christopher L. Brooks ◽  
...  
Keyword(s):  
Cell Growth ◽  
Board Of Directors ◽  
Xenograft Model ◽  
Advisory Committees ◽  
Immune Effector ◽  
Effector Cells ◽  
Murine Xenograft Model ◽  
Osteolytic Bone Disease

Abstract Introduction Multiple myeloma (MM) remains incurable despite novel therapies, highlighting the need for further identification of factors mediating disease progression and resistance. Our studies have identified an integral role of bone marrow (BM) plasmacytoid dendritic cells (pDCs) in MM pathogenesis. The functional significance of increased numbers of pDCs in MM BM is evident from our observations that pDCs: are relatively resistant to novel and conventional therapies; protect tumor cells from therapy-induced cytotoxicity; promote tumor growth and survival; and suppress immune responses (Chauhan et al, Cancer Cell 2009, 16:309-323). Aberrant pDC function is evidenced in their interactions not only with MM cells, but also with other immune effector T cells and NK cells in the MM BM milieu (Ray et al, Leukemia 2015, 29:1441-1444). Directly targeting pDC interactions with MM and immune effector cells in the MM BM milieu will be required to enhance both anti-tumor immunity and cytotoxicity. However, therapies targeting pDCs are lacking. We found that IL-3R is highly expressed on pDCs, and that pDC-MM interactions trigger secretion of IL-3, which in turn, promotes both pDC survival and osteolytic bone disease. Recent efforts have led to the development of a novel therapeutic agent SL-401, which specifically targets IL-3R-expressing pDCs. Here we examined the effect of SL-401 on pDC-induced MM cell growth both in vitro and in vivo, as well as on IL-3R-expressing osteoclasts. Methods Patient MM cells, pDCs, and MNCs were obtained from normal donors or MM patients. Cell growth/viability was analyzed using MTT/WST assays. OCL function and bone resorption were measured using the OsteoAssays and TRAP staining. The RPMI-8226 cell line was used to isolate MM-SPs by flow-cytometry based Hoechst 33342 staining. SL-401 is a recombinant protein expressed in E. coli. The hybrid gene is comprised of human IL-3 fused to truncated diphtheria toxin (DT). The IL-3 domain of SL-401, which replaces the native binding domain of DT, targets SL-401 to cells that overexpress IL-3R. SL-401 was obtained from Stemline Therapeutics, NY; bortezomib, lenalidomide, pomalidomide, and melphalan were purchased from Selleck Chemicals. For animal model studies, SL-401 (16.5 μg/kg) was administered intravenously daily for 2 weeks. Results SL-401 triggered significant apoptosis in pDCs (>95%) at low picomolar concentrations that are well within clinically achievable doses.Higher concentrations of SL-401 trigger a modest apoptosis (30%± 1.3% apoptosis at 83 ng/ml or 1.3 nM) in MM cells due to lower IL-3R expression versus pDCs. Moreover, SL-401 did not significantly induce apoptosis of normal PBMCs (8% ± 0.5% apoptosis at 83 ng/ml), suggesting a favorable therapeutic index for SL-401. SL-401 inhibited pDC-induced growth of MM cell lines and patient MM cells in a dose-dependent manner. Moreover, 6 of 9 MM samples were obtained from patients whose disease was progressing while on bortezomib, dexamethasone, and lenalidomide therapies. Combinations of SL-401 with melphalan, bortezomib, lenalidomide, or pomalidomide induced synergistic anti-MM activity (Combination index < 1). SL-401 blocked monocyte-derived osteoclast formation in a dose-dependent fashion, as well as restored MM patient BM-derived osteoblast formation. Having defined the efficacy of SL-401 in targeting pDCs and pDC-triggered MM cell growth in vitro, we validated these findings in vivo using our murine xenograft model of human MM, under auspices of protocols approved by our institutional animal protection committee. SL-401 inhibited pDC-induced MM cell growth in vivo and prolonged survival in a murine xenograft model of human MM. We also evaluated the efficacy of SL-401 in vivo using our SCID-human (SCID-hu) mouse model, which reflects the human BM milieu with human cytokines and extracellular matrix proteins. SL-401 significantly abrogated pDC-triggered MM cell growth in vivo in SCID-hu model. Conclusions Our data provide the basis for using SL-401 to directly target pDCs and inhibit the pDC-MM interaction as well as target osteolytic bone disease in novel therapeutic strategies in order to enhance MM cytotoxicity, overcome drug resistance, and improve patient outcome. The interactions of immune effector cells in the MM tumor microenvironment also provide a rationale for combining SL-401 with checkpoint inhibitors. Correspondence: Dharminder Chauhan Disclosures Macri: Stemline Therapeutics, Inc., New York, NY USA: Employment. Brooks:Stemline Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties. Rowinsky:Stemline Therapeutics: Employment, Equity Ownership. Richardson:Millennium Takeda: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gentium S.p.A.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees. Chauhan:Stemline Therapeutics: Consultancy.

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