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ray2014

ORIGINAL ARTICLE

A novel TLR-9agonist C792inhibits plasmacytoid dendritic cell-induced myeloma cell growth and enhance cytotoxicity of bortezomib

A Ray 1,Z Tian 1,DS Das 1,RL Coffman 2,P Richardson 1,D Chauhan 1,3and KC Anderson 1,3

Our prior study in multiple myeloma (MM)patients showed increased numbers of plasmacytoid dendritic cells (pDCs)in the bone marrow (BM),which both contribute to immune dysfunction as well as promote tumor cell growth,survival and drug resistance.Here we show that a novel Toll-like receptor (TLR-9)agonist C792restores the ability of MM patient-pDCs to stimulate T-cell proliferation.Coculture of pDCs with MM cells induces MM cell growth;and importantly,C792inhibits pDC-induced MM cell growth and triggers apoptosis.In contrast,treatment of either MM cells or pDCs alone with C792does not affect the viability of either cell type.In agreement with our in vitro data,C792inhibits pDC-induced MM cell growth in vivo in a murine xenograft model of human MM.Mechanistic studies show that C792triggers maturation of pDCs,enhances interferon-a and interferon-l secretion and activates TLR-9/MyD88signaling axis.Finally,C792enhances the anti-MM activity of bortezomib,lenalidomide,SAHA or melphalan.Collectively,our preclinical studies provide the basis for clinical trials of C792,either alone or in combination,to both improve immune function and overcome drug resistance in MM.Leukemia (2014)28,1716–1724;doi:10.1038/leu.2014.46

Keywords:myeloma;immunotherapy;dendritic cells;pDC;TLR-9;CpG-ODN-C792;interferons

INTRODUCTION

Multiple myeloma (MM)remains incurable due to the develop-ment of drug resistance mediated by mechanisms intrinsic to the tumor cells as well as interaction of MM cells with the accessory cells in the bone marrow (BM)microenvironment.1–3BM stromal cells,osteoclasts,osteoblasts,myeloid cells and immune effector cells,that is,myeloid-derived suppressor cells,can promote growth and drug resistance in MM cells.4–6Research efforts are now focused on defining the functional significance of tumor cell interaction with BM accessory cells in the MM niche to identify novel therapeutic strategies.

Dendritic cells (DCs)7,8mediate immune function and promote tumor growth.9–11Human DCs have been classified into two major subtypes 12–14based on the their origin,phenotype and function:myeloid DCs and plasmacytoid DCs (pDCs).myeloid DCs have been extensively characterized,12–16and recent studies have also begun to characterize pDCs and their functionality.pDCs express CD123,CD303,CD304and HLA-DR,and lack lineage cell markers for B,NK and T cells,as well as monocytes.The antigen presenting function of pDCs is,at least in part,mediated via Toll-like receptors (TLRs;TLR7and TLR9),which recognize viral RNA template or unmethylated bacterial DNA,thereby facilitating secretion of Type I and Type II interferons (IFN).17–19These pleiotropic cytokines in turn activate multiple components of the immune system including T cells,B cells and NK cells.Early reports 20,21showed that pDCs from MM patients are defective in their antigen-presenting function;indeed,the loss of immune function of tumor-infiltrating DCs has been linked to suppressive effects

of the tumor microenvironment in multiple cancers,including MM.22,23

Besides generating an antiviral immune response,pDCs also have a role in normal B-cell development into plasmablasts,differentiation into antibody-secreting plasma cells and survi-val.24–27In this context,our recent study defined the role of pDCs in regulating growth and survival of malignant plasma (MM)cells.28Specifically,we found increased numbers of pDCs in the MM BM microenvironment,which both mediate immune deficiency characteristic of MM,as well as promote tumor cell growth,survival and drug resistance.In the present study,we show that a novel TLR-9agonist C79229both restores pDC immune function and inhibits pDC-induced MM cell growth and drug resistance.Our study provides the basis for targeting pDC–MM interactions using TLR9agonist C792as a potential therapeutic strategy in MM.

MATERIALS AND METHODS

Isolation and phenotypic analysis of pDCs

Studies involving patient MM cells were performed following IRB-approved protocols at Dana-Farber Cancer Institute and Brigham and Women’s Hospital (Boston,MA,USA).Informed consent was obtained,and the samples were de-identified before experimental use.pDCs were isolated from both BM and peripheral blood mononuclear cells by magnetically activated cell sorting using CD304(BDCA-4/Neuropilin-1)microbeads kit (Miltenyi Biotec,Auburn,CA,USA),as previously described.28Briefly,mononuclear cells from healthy donors and MM patients were isolated by Ficoll Hypaque density gradient centrifugation,magnetically labeled with

1

Department of Medical Oncology,The LeBow Institute for Myeloma Therapeutics and Jerome Lipper Myeloma Center,Dana-Farber Cancer Institute,Harvard Medical School,Boston,MA,USA and 2Dynavax Technologies,Berkeley,CA,USA.Correspondence:Dr D Chauhan or Dr KC Anderson,Department of Medical Oncology,The LeBow Institute for Myeloma Therapeutics and Jerome Lipper Myeloma Center,Dana-Farber Cancer Institute,Harvard Medical School,M561,450Brookline Ave,Boston,MA 02215,USA.E-mail:Dharminder_Chauhan@http://www.wendangku.net/doc/d152196e227916888586d706.html or Kenneth_Anderson@http://www.wendangku.net/doc/d152196e227916888586d706.html 3

These authors contributed equally to this work.

Received 2January 2014;accepted 17January 2014;accepted article preview online 30January 2014;advance online publication,18February 2014

Leukemia (2014)28,1716–1724

&2014Macmillan Publishers Limited All rights reserved 0887-6924/14

http://www.wendangku.net/doc/d152196e227916888586d706.html/leu

ray2014

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