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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 de?ning the functional signi?cance 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 classi?ed 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-in?ltrating 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 de?ned the role of pDCs in regulating growth and survival of malignant plasma (MM)cells.28Speci?cally,we found increased numbers of pDCs in the MM BM microenvironment,which both mediate immune de?ciency 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-identi?ed 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.28Brie?y,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@https://www.wendangku.net/doc/d21589707.html, or Kenneth_Anderson@https://www.wendangku.net/doc/d21589707.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
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anti-BDCA-4antibody(Miltenyi Biotec)coupled to colloidal paramagnetic microbeads and passed through a magnetic separation column twice.Cells lacking lineage markers and CD11c were FACS sorted.The purity of pDCs was con?rmed by staining of cells with CD123PE-Cy5,HLA-DR Paci?c Blue and BDCA-2FITC(X99%purity).30The CD304-positive pDCs obtained by this method are lineage negative Lin-1(CD3,CD14,CD19,CD20,CD56and CD11c negative),MHC II positive and CD123/BDCA-2positive.pDCs were also puri?ed by negative depletion using LD columns(Miltenyi Biotec;99% BDCA2tCD123tcells).Cells were sorted using FACS Aria II cell sorter, and all?ow cytometric experiments were performed using BD Canto II or BD LSRFortessa machine(BD Biosciences,San Jose,CA,USA).Data were analyzed using a FACS DIVA(BD Biosciences)and FlowJO software(ver 7.6.5,Tree Star Inc.,Ashland,OR,USA).
Cytokines,antibodies and reagents
Human recombinant interleukin(IL)-3and IL-6were obtained from Peprotech Inc.(Rocky Hill,NJ,USA).Recombinant IFN-a,IFN-l and DR5-Alexa700were purchased from R&D Systems(Minneapolis,MN, USA).CD3-PE;CD4-FITC or APC-Cy7;CD40-FITC;CD80-FITC;CD83-FITC; CD86-FITC;CD123-PE/PE-Cy5;as well as CD138-FITC,PE were obtained from BD Biosciences.HLA-DR Violet Blue,BDCA-2FITC,CD14-PE and CD11c-APC were purchased from Miltenyi Biotec.TLR-9-FITC,TRAIL-PE and DR-4-FITC were obtained from Abcam.The CpG-C oligodeoxynucleotide C792was synthesized and puri?ed by standard techniques as previously described;29bortezomib,lenalidomide,and SAHA were purchased from Selleck Chemicals LLC(Houston,TX,USA);melphalan was purchased from Sigma Chemical Company(St Louis,MO,USA);and MyD88inhibitor was purchased from InVivoGen(San Diego,CA,USA).For assessing C792effect on the viability of freshly isolated pDCs,we cultured cells in DCP-MM medium(MatTek Corp,Ashland,MA,USA).
Cytokine assays
IFN-a,IFN-l and soluble TNF-related apoptosis-inducing ligand(sTRAIL) were measured by ELISA using commercially available kits,according to the manufacturer’s instructions(PBL Interferon Source,Piscataway,NJ,USA and R&D Systems).Brie?y,MM.1S cells(5?104cells/200m l per well)and pDCs(1?104cells/200m l per well)were cultured either alone or together at1:5(pDC:MM)ratio in96-well plates,in the presence or absence of C792; supernatants from these cocultures were collected and analyzed for cytokine secretion using ELISA.MM.1S cells were also treated with various concentrations of recombinant hu-IFN-a(R&D Systems)and analyzed for viability by MTT assay.
Flow cytometry and intracellular staining
For?ow cytometric analysis,single-cell suspensions were stained with different?uorophore-coupled monoclonal antibodies(BD Biosciences).All ?ow cytometric analysis was performed on gated populations:CD138tfraction for MM cells and CD123t/CD304t/CD303t/HLA-DRt/ CD11Càfraction for pDCs.Activation and maturation of pDCs were assessed by FACS analysis using CD40,CD80,CD83and CD86surface markers.Intracellular staining and cell/tissue?xation was assessed using the Cyto?x/Cytoperm buffer kit(BD Biosciences)as per manufacturer’s instructions.
Cell culture and assessment of cell viability,growth and apoptosis Cells were cultured in RPMI-1640medium supplemented with10%FBS, 100U/ml penicillin,100mg/ml streptomycin and2m M L-glutamine.MM.1S (Dexamethasone sensitive),MM.1R(Dexamethasone resistant),RPMI-8226, Doxorubicin(Dox)-resistant(Dox-40),Melphalan-resistant LR5and NCI-H929human MM cell lines were cultured in complete medium.ANBL-6 MM cell line was cultured in complete medium with IL-6(5ng/ml).Cell viability was assessed using colorimetric assay with3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyltetrazolium bromide(MTT;Calbiochem,EMD Millipore, Billerica,MA,USA).28MM cell proliferation in coculture experiments with pDCs or BM stromal cells was performed using WST(Clontech Laboratories, Mountain View,CA,USA)assays,as described previously.28Brie?y,pDCs were cocultured with MM.1S cells at1:5pDC/MM.1S ratio in the presence or absence of C792,followed by cell growth analysis using WST assay.T cell proliferation assay:MM pDCs were cultured with or without T cells,in the presence or absence of C792,and proliferation was assessed using WST assays.23,28Cell cycle was analyzed using propidium iodide staining. Apoptosis was quanti?ed using Annexin V-Fitc/Propidium iodide apoptosis detection kit as per manufacturer’s instructions(BD Biosciences),followed
by analysis on BD FACSCanto II or BD LSRFortessa(BD Biosciences).
Human plasmacytoma xenograft model
All animal experiments were approved by and conform to the relevant regulatory standards of the Institutional Animal Care and Use Committee at
the Dana-Farber Cancer Institute.In the human plasmacytoma murine xenograft model,CB-17SCID mice were randomized into three groups(four
mice each group):the?rst group was subcutaneously injected with MM.1S
cells alone(1.25?106cells in100m l of serum free RPMI-1640medium);the second group received resting pDC(0.25?106cells)plus MM.1S cells (1.25?106cells);and the third group was injected with ex vivo C792-activated
pDC plus MM.1S cells.Tumor growth was measured as previously described,28and animals were euthanized when their tumors reached2cm3. Statistical analysis
Statistical signi?cance of differences observed in drug-treated versus control cultures was determined using the Student’s t-test.The minimal
level of signi?cance was P o0.05.Tumor volume and survival in mice were measured using the GraphPad PRISM(GraphPad Software/version5, SanDiego,CA,USA).Isobologram analysis31was performed using
‘CalcuSyn’software program(Biosoft;Ferguson,MO,USA and Cambridge,UK).Combination index(CI)values of o1.0indicate synergism,and values41.0
antagonism.
Figure1.C792restores the ability of MM patient pDCs to trigger
both allogeneic and autologus T-cell proliferation(a)Normal CD4T
cells(1?105)were cultured with normal healthy donor(n-pDC)or
MM patient BM pDCs at1:10(pDC/T cell)in the presence or absence
of C792for5days,and then analyzed for T-cell proliferation with WST assay(BM-pDCs from5MM patients were utilized;mean±s.d.;
P o0.005for all patients).(b)MM patient BM pDCs(1?104)were cultured with autologous T cells(1?105)at1:10(pDC/T cell)ratio in
the presence or absence of C792(0.1m g/ml)for5days,and then analyzed for T-cell proliferation using WST assay(mean±s.d.;
P o0.01for all patients).
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RESULTS
C792stimulates MM patient pDCs to trigger T-cell proliferation Prior studies have reported immune dysfunction in MM,associated with decreased ability of MM patient BM-DCs to stimulate T-cell proliferation.21–23
In our study,we ?rst examined the in vitro function of pDCs 30(de?ned as HLA-DR t,CD123tand CD303/BDCA-2tcells)derived from normal healthy donors versus MM patients,assessed by their ability to stimulate allogeneic T-cell responses.As shown in Figure 1a,MM pDCs induce signi?cantly decreased T-cell proliferation versus normal pDCs (P ?0.02).We next examined whether treatment of MM patient BM-pDCs with TLR9agonist (CpG-ODNs)C792affects their T-cell stimulatory activity.Impor-tantly,C792restores the ability of MM patient BM-pDCs to induce both allogeneic (Figure 1a)and autologus T-cell proliferation (Figure 1b).Our data are consistent with other studies showing that targeting TLR9using agonists can activate pDC,and enhance their immune functions.17,18,32–35
C792blocks pDC-induced MM cell growth
Recent reports linked increased in?ltration of DCs in human tumors with unfavorable prognosis.6,10,36,37Our earlier study in MM showed that increased numbers of pDCs in MM patient’s BM
trigger growth of MM cells.28Having shown that C792restores MM patient BM-pDCs immune function,we next examined whether C792affects pDC-induced MM cell growth using WST proliferation assays.As shown in Figure 2a,C792inhibits patient-BM pDC-triggered MM.1S cell proliferation in a dose-dependent manner.Non-CpG ODN 1040served as negative control.C792also blocked pDC-induced growth of various other MM cell lines (MM.1R,ANBL-6,RPMI-8226,Dox-40or LR-5)(Supplementary Figure 1).These results were also con?rmed from an additional cell growth assay,using the CellTrace CFSE Proliferation Kit (data not shown;Life Technologies,Grand Island,NY,USA).No signi?cant growth of pDCs was observed in cocultures of pDCs and MM cells (data not shown).Importantly,C792treatment of pDCs or MM cell lines alone did not decrease viability of either cell type (Figure 2b and c,respectively).
Similar to our observation using MM cell lines,we next examined whether C792affects pDC-induced proliferation of patient MM cells using WST proliferation assays.As in our prior study,pDCs triggered proliferation of patient MM cells;and importantly,C792blocked pDC-induced patient MM cell growth (Figure 2d).Furthermore,C792inhibited MM patient BM pDC-induced growth of both autologous tumor cells (Figure 2e)and allogeneic MM.1S cell line (Figure 2f).Taken together,our ?ndings suggest that activation of pDCs with TLR9agonist C792
both
Figure 2.C792inhibits pDC-induced MM cell growth (a )MM.1S cells (5?104/200m l)and MM BM-pDCs (1?104/200m l)were cultured either alone or together at 1:5(pDC/MM)ratio in the presence or absence of C792for 72h,and then analyzed for growth using WST proliferation assays.Non-CpG-ODN 1040served as a negative control (mean ±s.d.;P o 0.05;n ?6).(b )Freshly isolated pDCs from six normal healthy donors were cultured in DCP-MM medium,in the presence of C792(5–10m g/ml)for 72h,and analyzed for viability by MTT assays (mean ±s.d.;P o 0.05).(c )MM.1S and IL-6-dependent ANBL-6cells were cultured with indicated concentrations of C792for 72h,and analyzed for viability by MTT assays (mean ±s.d.;P o 0.05,n ?3).Non-CpG-ODN 1040was also utilized as a control.(d )Patient MM cells were cultured with or without pDCs at 1:5(pDC/MM)ratio for 72h,in the presence or absence of C792,and cell growth was analyzed using WST assay (mean ±s.d.of triplicate cultures;P o 0.05for all samples).(e )Patient MM cells were cultured with or without autologous BM pDCs at 1:5(pDC/MM.1S)ratio in the presence of indicated concentrations of C792for 72h,and cell growth was analyzed using WST assay (mean ±s.d.;P o 0.04,n ?5).(f )Patient BM pDCs were cultured with or without MM.1S cells at 1:5(pDC/MM)ratio in the presence or absence of C792for 72h,and cell growth was analyzed by WST assay (mean ±s.d.;P o 0.01,n ?5).Data are fold change in MM cell growth normalized to growth in the absence of pDCs (mean ±s.d.;P o 0.05,n ?3).
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restores their ability to induce T-cell proliferation and reduces their MM cell growth-promoting activity.
C792inhibits pDC-induced MM cell growth in vivo in a MM xenograft model
Having shown the anti-MM activity of C792in vitro,we next utilized a subcutaneous model of human MM in SCID mice to examine whether C792similarly affects pDC-triggered MM cell growth in vivo.Mice were randomized into three groups:the?rst group was subcutaneously injected with MM.1S cells alone;the second group received pDC plus MM.1S cells;and the third group was injected with MM.1S cells plus pDCs that were ex vivo treated with C792.More robust tumor growth occurred in mice receiving MM.1S cells plus pDCs versus those injected with MM.1S cells alone(Figure3a),as in our prior study.28Importantly,signi?cantly reduced tumor growth was observed in mice receiving C792-treated pDCs plus MM.1S cells versus the mice injected with either MM.1S cells alone(P?0.0069)or untreated pDCs plus MM cells (P?0.0044)(Figure3a).Moreover,a marked prolongation of survival(P?0.002)was noted in mice who received C792-treated pDCs compared with in mice injected with either untreated pDCs plus MM.1S cells or MM.1S cells alone(Figure3b).Together,our in vitro and in vivo human MM xenograft studies provide evidence for the anti-MM activity of C792by virtue of its ability to block pDC-mediated MM cell growth.
Mechanism(s)mediating anti-MM activity of C792
Previous studies reported that CpG-ODNs can activate pDCs.38–41 To determine whether C792similarly activates freshly isolated MM patient pDCs,we treated pDCs with C792and examined expression of surface markers characteristic of activation and maturation.Treatment of pDCs with C792increases CD40,CD83, CD80,HLA-DR and CD86expression(Figure4a).Speci?cally,?ow cytometric data showed that C792triggered a signi?cant
increase
Figure3.C792inhibits pDC-induced MM cell growth in vivo in a murine xenograft model of human MM(a)Average and s.d.of tumor volume (mm3)in mice(n?4per group)versus time(days)when tumor was measured.CB-17SCID mice were randomized into three groups(four mice each group):the?rst group was subcutaneously injected with MM.1S cells alone(1.25?106cells in100m l of serum free RPMI-1640 medium);the second group received resting pDC(0.25?106cells)plus MM.1S cells(1.25?106cells);and the third group was injected with
ex vivo C792-activated pDCs plus MM.1S cells.Tumor growth was measured by calculating tumor volume using the formula:volume?(width)2?length/2.Error bars indicate s.d.(b)Kaplan–Meier survival plot shows signi?cantly increased survival of mice receiving C792-treated pDCs plus MM.1S cells versus mice injected with untreated pDCs plus MM.1S cells(P?0.0002,Log-rank(Mantel–Cox)test):median survival
was24days in MM.1S-injected mice;18days in mice injected with MM.1S plus untreated-pDCs;and49days in mice receiving MM.1S plus
C792-treated pDCs(CI95%).Tumor-bearing mice were killed with a tumor volume42cm2.
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in median ?uorescence intensity of CD40,CD83,CD80,HLA-DR and CD86expression (20,37,10,6and 13%increase,respectively).Non-CpG 1040served as a negative control (Figure 4a).C792is a TLR9agonist,and we found a signi?cant increase in TLR9expression in C792-treated pDCs (Figure 4b,left and right panels).To further con?rm that C792activates MM patient pDCs,we examined additional mechanism(s)associated with activation of pDCs.For example,prior studies have shown that CpG-ODN-activated pDCs secrete Type I and Type III IFNs;41–43this is exempli?ed in systemic lupus erythematous,which is linked to TLR-9-activated pDCs and Type I IFN-a release.18,33–35In view of these studies,we next examined (1)whether treatment of MM patient BM-pDCs with C792triggers IFN-a secretion;and (2)if IFN-a secretion is attenuated or augmented during pDC-MM cocultures.Treatment of pDCs with C792,but not non-CpG ODN control,triggered an increase in IFN-a secretion (Figure 4c).Importantly,C792induced a more robust dose-dependent IFN-a secretion in pDC-MM cocultures versus pDCs alone (Figure 4d).This phenomenon was also observed in C792-treated normal healthy donor pDC-MM cocultures versus normal pDC alone (data not shown).
IFN-a was undetectable in the supernatant from C792-treated MM cells.Our ?ndings are also consistent with earlier studies 40,41demonstrating a non-linear relationship between CpG-ODNs,like C792,and secretion of IFN-a .In addition to Type I IFN-a release,C792also triggered Type III IFN-l secretion in pDC-MM cocultures,albeit to a lesser extent than IFN-a (Supplementary Figure 2).We next examined the signaling mechanism mediating C792-induced IFN-a secretion in MM patient BM pDCs.As stated above,C792is a TLR9agonist and upregulates TLR9expression in pDCs (Figure 4b).To validate the functionality of TLR9signaling axis in pDCs,we examined downstream adaptor protein MyD88(Myeloid differentiation primary response gene).44,45In our study,biochemical inhibition of MyD88homodimer formation signi?cantly blocked C792-induced IFN-a secretion (Figure 4e).Together,our ?ndings suggest that the inhibitory effect of C792on pDC-induced MM cell growth is associated with activation of pDCs via TLR9-MyD88signaling and release of IFN-a .
Although C792-triggered activation of pDCs,explains the restoration of MM pDC ability to stimulate T-cell responses,it does not elucidate the mechanism whereby C792inhibits pDC-induced MM cell growth.One possibility is that C792
triggers
Figure 4.C792triggers maturation of pDCs,IFN release and activation of TLR9-MyD88signaling axis (a )MM BM-pDCs (CD123t/BDCA-2t/HLA-DR t/CD11c à)were cultured in the presence or absence of C792(1.0m g/ml)for 12h;cells were stained with ?uorophore-conjugated antibodies against CD40,CD80,CD83,CD86or HLA-DR,followed by ?ow cytometry analysis.Non-CpG 1040served as a negative control.Bar graph shows the percentage change in median ?uorescence intensity for indicated molecules in untreated versus C792-treated pDCs (mean ±s.d.;P o 0.05,n ?3).(b ,left panel)pDCs were treated with indicated concentrations of C792for 12h,followed by intracellular staining using FITC-conjugated TLR-9antibody.Isotype-matched antibody served as control.(b ,right panel)C792-triggered changes in TLR9,as shown in left panel,were quanti?ed.Intracellular TLR-9expression is presented as percentage change in relative ?uorescence intensity in C792-treated pDCs versus the untreated-pDCs (mean ±s.d.;P o 0.05,n ?3).(c )MM BM-pDCs (5?103cells)were cultured in the presence or absence of indicated concentrations of C792(Type C CpG ODN),Type B CpG ODN or non-CpG for 24h;supernatants from these cultures were analyzed for IFN-a using ELISA (mean ±s.d.;P o 0.05,n ?3).(d )pDCs (1?104),MM.1S cells (5?104)or pDCs plus MM.1S cells were cultured in the presence or absence of indicated concentrations of C792for 24h,and supernatant from these cultures were analyzed for IFN-a using ELISA (mean ±s.d.;P o 0.05,n ?4).(e )MM BM-pDCs (5?103)were pretreated with 50m M of MyD-88inhibitor peptide for 4h,followed by addition of C792for 24h;supernatants from these cultures were analyzed for IFN-a using ELISA (mean ±s.d.;P o 0.05,n ?3).
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secretion of soluble factors,which inhibit MM cell growth and/or trigger apoptosis.Interestingly,we found large amounts of IFN-a in C792-treated pDC-MM cocultures;and importantly,IFN-a has been utilized in the treatment of cancers,including MM.46–48We therefore examined whether IFN-a blocks pDC-induced MM cell growth.For these studies,we utilized human recombinant IFN-a at various concentrations,including those that were obtained in supernatants from C792-treated pDC-MM cocultures (Figure 4d).pDCs were cocultured with MM cell lines (MM.1S,MM.1R,LR-5and NCI-H929)in the presence or absence of rIFN-a ,followed by analysis of cell growth.A dose-dependent decrease in the pDC-induced MM cell growth was noted in response to treatment with rIFN-a (Figure 5a,and data not shown).These ?ndings suggest that IFN-a ,at least in part,mediates the anti-MM activity of C792.Previous studies have linked TLR9-stimulated pDCs to TRAIL,which binds to cells expressing TRAIL receptors TRAIL-R1(DR4)and TRAIL-R2(DR5)and results in apoptosis.49,50We next examined whether C792induces secretion of sTRAIL from pDCs.A signi?cant increase in sTRAIL was present in supernatants from C792-treated-versus untreated-pDC-MM cocultures (Figure 5b;
P o 0.005).As MM cells express TRAIL receptors DR4and DR5,51
and our ?ndings show that C792induce sTRAIL (Figure 5b),it is likely that TRAIL–TRAIL-R interactions mediate anti-MM activity of C792.We do not exclude the possibilty that other factors besides IFN-a or sTRAIL may also mediate MM cell cytotoxicity of C792in pDC-MM cultures,as we found that supernatants from C792-treated pDC-MM cocultures triggered a marked decrease in MM cell viability (Figure 5c).
We next examined whether MM cell growth inhibition in C792-treated pDC-MM cocultures is due to cell cycle alterations and/or apoptosis in MM cells.Treatment of pDC-MM cocultures with C792is associated with growth arrest in MM.1S cells:speci?cally,cell cycle analysis of CD138tMM cells obtained from pDC-MM.1S cocultures showed increased G2phase in C792-treated-versus untreated-pDC-MM cocultures (Figure 5d).Of note,treatment of MM cells without pDCs does not affect the cell cycle pro?le of MM cells;consistent with our ?nding that C792is not cytotoxic against MM cells alone (Figure 2c).Finally,we show that treatment of pDC-MM cocultures with C792triggers MM cell apoptosis (Figure
5e).
Figure 5.Mechanism(s)mediating C792activity (a )MM.1S cells were cultured with MM BM-pDCs in the presence or absence of recombinant human-IFN-a for 48h,and cell growth was analyzed using WST assay.Data are derived after normalizing cell growth in IFN-a -treated versus -untreated cultures (mean ±s.d.;P o 0.05,n ?3).(b )pDCs from two MM patients were treated with C792for 96h,and supernatants were analyzed for sTRAIL using ELISA (mean ±s.d.;P o 0.05,n ?3).(c )MM cell lines were treated for 72h with supernatants derived from untreated or C792-treated MM BM-pDC cultures,and cell viability was analyzed using MTT assay.Data are derived after normalizing MM cell viability in supernatants from untreated versus C792-treated pDC cultures (mean ±s.d.;P o 0.05,n ?3).(d )MM.1S and MM BM-pDCs cells were cocultured at 1:5(pDC/MM)ratio in the presence or absence of C792for 12h.Cells were then stained with propidium iodide.MM (CD138-positive)cell population was selectively gated by ?ow cytometry for cell cycle analysis.Bar graph shows the percentage of MM cells in cell cycle phases.A signi?cant accumulation of MM cells in G2M phase was noted in the C792-treated versus -untreated cocultures (P o 0.005,n ?3).(e )MM.1S and MM BM-pDCs cells were cocultured at 1:5(pDC/MM)ratio in the presence or absence of C792for 12h;the MM (CD138-positive)population was selectively gated by ?ow cytometry and analyzed for apoptosis using Annexin V/PI staining assays.A signi?cant increase in apoptotic MM cell population was noted in the C792-treated versus -untreated cocultures (25–30%increase in Annexin V t/PI àcells)(P o 0.005,n ?3).Bar graph shows the percentage of apoptotic MM cells.
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C792triggers synergistic anti-MM activity with bortezomib,SAHA,melphalan,and lenalidomide.
Our prior preclinical studies showing synergistic cytotoxicity have provided the basis for clinical trials of proteasome inhibitor bortezomib in combination with lenalidomide,Dex or HDAC inhibitors.1In our current study,we tested whether CpG-ODN C792triggers additive or synergistic anti-MM activity in combination
with proteasome inhibitor bortezomib,HDAC inhibitor SAHA,melphalan,or lenalidomide (Figure 6).Isobologram analysis 31demonstrated that the combination of low concentrations of C792with melphalan,SAHA,lenalidomide or bortezomib triggers synergistic anti-MM activity against various MM cell lines (MM.1S,MM.1R,RPMI-8226and LR5),albeit with differential extents of synergy (Figures 6a–d,and data not shown;CI o 1.0).
Although
Figure https://www.wendangku.net/doc/d21589707.html,bination of C792and bortezomib,SAHA,melphalan or lenalidomide triggers synergistic anti-MM activity (a )Cocultures of MM.1S plus BM-pDCs were treated with indicated concentrations of C792,bortezomib or C792plus bortezomib for 48h,and then analyzed for viability.Isobologram analysis shows the synergistic cytotoxic effect of C792and bortezomib.The graph (left)is derived from the values given in the table (right).The numbers 1–6in graph represent combinations shown in the table.CI o 1indicates synergy.(b )Cocultures of MM.1S plus BM-pDCs were treated with indicated concentrations of C792,SAHA or C792plus SAHA for 48h,and then analyzed for viability.Isobologram analysis shows the synergistic cytotoxic effect of C792and SAHA.The graph (left)is derived from the values given in the table (right).The numbers 1–6in graph represent combinations shown in the table.CI o 1indicates synergy.(c )Cocultures of MM.1S plus BM-pDCs were treated with indicated concentrations of C792,melphalan or C792plus melphalan for 48h,and then analyzed for viability.Isobologram analysis shows the synergistic cytotoxic effect of C792and melphalan.The graph (left)is derived from the values given in the table (right).The numbers 1–6in graph represent combinations shown in the table.CI o 1indicates synergy.(d )Cocultures of MM.1S plus BM-pDCs were treated with indicated concentrations of C792,lenalidomide or C792plus lenalidomide for 48h,and then analyzed for viability.Isobologram analysis shows the synergistic cytotoxic effect of C792and lenalidomide.The graph (left)is derived from the values given in the table (right).The numbers 1–6in graph represent combinations shown in the table.CI o 1indicates synergy.
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de?nitive evidence of decreased toxicity of combination therapy awaits results of clinical trials,the synergy observed in vitro may allow for use of lower doses and reduced toxicity. DISCUSSION
Our previous study identi?ed an integral role of pDCs in MM pathogenesis.28Speci?cally,using our in vitro and in vivo models of human MM in the BM milieu,we showed that increased numbers of pDCs in the MM BM confer growth,survival, chemotaxis and drug resistance.In addition,we found a decreased ability of MM BM pDCs versus normal pDCs to trigger T-cell proliferation.28A similar immune dysfunction in MM-pDCs has been noted in other reports.20,21Earlier studies have shown promising activity of immunostimulatory CpG-based formulations as immune adjuvants in vaccines or in the treatment of viral, bacterial and parasitic infections,as well as in cancers.29Indeed, both Type A and Type B CpG ODNs have been evaluated in clinical trials.52In the current study we examined whether C792,a novel next generation Type-C CpG-ODN targeting TLR9with a potent immune-enhancing activity,affects pDC-induced MM cell growth and/or stimulates pDC immune function.Our data show that treatment of MM BM pDCs with C792restores their ability to stimulate both autologous and allogeneic T-cell proliferation.A previous study using the B16mouse melanoma tumor model also showed that CpG-activated pDCs induce a systemic antitumor immunity through activation of NK cells and T cells.35Further studies are required to determine whether C792-activated MM pDCs generate antitumor immunity,which is clinically signi?cant in MM.
Importantly,we found that C792inhibits pDC-induced growth of various MM cell lines,including those sensitive and resistant to various conventional and novel therapies,as well as representing distinct cytogenetic subtypes.53,54For example,we studied isogenic cell lines Dex-sensitive MM.1S and Dex-resistant MM.1R with t(14;16)translocation,as well as RPMI-8266parental and Doxorubicin-resistant(8226/Dox-6)or melphalan-resistant(LR5) derivative cell lines.Similar?ndings were observed using MM cells from patients with relapsed MM refractory to various therapies. Importantly,C792blocked MM patient BM pDC-induced growth of both autologous tumor cells and allogeneic MM.1S cell line.Our data therefore highlight the ability of C792to overcome genetic heterogeneity and broadly inhibit pDC-induced growth in MM. Interestingly,treatment of MM cells or pDCs alone with C792does not affect their viability;the MM cell growth inhibitory activity of C792is only observed in the pDC-MM coculture setting.This ?nding con?rms the role of factors and/or MM BM accessory cells within the MM BM milieu,in this case pDCs,to impact the sensitivity versus resistance of MM cells.There is an urgent need to clinically evaluate novel agents targeting the tumor cell in the BM microenvironment,and clinical trials of C792will provide proof of principle.
In agreement with our in vitro data,C792also inhibited pDC-triggered MM cell growth in vivo.More rapid tumor growth occurred in mice receiving both pDCs and MM cells versus MM cells alone;and importantly,mice receiving C792-treated pDCs plus MM cells showed signi?cantly reduced tumor growth.The prolonged survival of pDCs in our xenograft model may be explained by our earlier study28showing:(1)that pDC–MM cell interactions trigger secretion of IL-3,a survival factor for pDCs;14and(2)increased IL-3 and pDCs(BDCA-2-positive cells)in tumor sections from mice receiving pDCs and MM cells versus MM cells alone.Our ongoing studies using C792are now evaluating the effects of C792on IL-3 and other cytokines/chemokines secreted during pDC–MM inter-actions.Together,our?ndings from both in vitro and human MM xenograft in vivo models suggest that C792inhibits pDC-induced MM cell growth,and provide the basis for targeting pDC–MM interactions as a therapeutic strategy in MM.
Treatment with C792is associated with activation/maturation of pDCs,evidenced by upregulation of CD83,CD40,CD80,CD86and
HLA-DR,as well as IFN-a release.Indeed,C792targets TLR-9to trigger both pDCs activation and release of IFN-a.Moreover,the enagagement of TLR9by C792activates downstream signaling via adaptor protein MyD88;conversely,blockade of MyD88inhibited
C792activity,evidenced by a marked decrease in IFN-a secretion from pDCs.Enhanced IFN-a secretion in response to C792may explain,at least in part,cytotoxicity of C792against MM cells in pDC–MM cocultures.Indeed,previous studies have shown cytotoxic activity of IFN-a against MM cells,46–48and clinical trials have shown anti-MM activity of IFN-a,either as a single agent or in combination.In addition to IFN-a,we also found sTRAIL in the supernatants from C792-treated pDC–MM cocul-tures,which may interact with TRAIL-R expressed on MM cells and trigger MM cell death.
Finally,we show that C792triggers synergistic anti-MM activity
with current anti-MM agents including proteasome inhibitor bortezomib,immunomodulatory agent lenalidomide,HDAC inhi-
bitor SAHA or alkylating agent melphalan.Most of these agents trigger a direct cytotoxic effect on MM cells,and combination trials with C792will allow not only targeting MM cells,but also bolster the immune response in MM patients.
Collectively,we show that targeting TLR9with CpG ODN C792 improves immune function of pDCs on the one hand and blocks pDC-induced MM cell growth on the other.Our preclinical study provides the framework for clinical trials of C792,either alone or in combination,to improve immune function,enhance cytotoxicity, overcome drug resistance and improve patient outcome in MM.
CONFLICT OF INTEREST
RLC is an employee of Dynavax Technologies.The remaining authors declare no
con?ict of interest.
ACKNOWLEDGEMENTS
This investigation was supported by National Institutes of Health Specialized Programs of Research Excellence(SPORE)grants P5*******,PO1-CA078378,and
RO1CA050947.KCA is an American Cancer Society Clinical Research Professor.
AUTHOR CONTRIBUTIONS
DC designed research,analyzed data and wrote the manuscript;AR performed
the experiments,analyzed data and wrote the manuscript;ZT and DSD helped
in?ow cytometry;RLC provided technical advice,analyzed data and reviewed
the manuscript;PR provided clinical samples;and KCA analyzed data and wrote
the manuscript.
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Supplementary Information accompanies this paper on the Leukemia website (https://www.wendangku.net/doc/d21589707.html,/leu)
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