trabectedin reveals a strategy of immunomodulation in chronic lymphocytic leukemia · research...

Research Article

Trabectedin Reveals a Strategy ofImmunomodulation in Chronic LymphocyticLeukemiaPriyanka Banerjee1, Ronghua Zhang1,Cristina Ivan1,Giovanni Galletti1, KarenClise-Dwyer2,Federica Barbaglio3, Lydia Scarf�o3,4, Miguel Aracil5, Christian Klein6,William Wierda7,William Plunkett1, Federico Caligaris-Cappio8, Varsha Gandhi1, Michael J. Keating7, andMaria Teresa S. Bertilaccio1

Abstract

Chronic lymphocytic leukemia (CLL) is a B-cell neoplasiacharacterized by protumor immune dysregulation involvingnonmalignant cells of the microenvironment, including Tlymphocytes and tumor-associated myeloid cells. Althoughtherapeutic agents have improved treatment options for CLL,many patients still fail to respond. Some patients also showimmunosuppression. We have investigated trabectedin, amarine-derived compound with cytotoxic activity on macro-phages in solid tumors. Here, we demonstrate that trabectedininduces apoptosis of human primary leukemic cells and alsoselected myeloid and lymphoid immunosuppressive cells,mainly through the TRAIL/TNF pathway. Trabectedin mod-ulates transcription and translation of IL6, CCL2, and IFNa in

myeloid cells and FOXP3 in regulatory T cells. Humanmemory CD8þ T cells downregulate PD-1 and, along withmonocytes, exert in vivo antitumor function. In xenograft andimmunocompetent CLL mouse models, trabectedin has anti-leukemic effects and antitumor impact on the myeloid andlymphoid cells compartment. It depletes myeloid-derivedsuppressor cells and tumor-associated macrophages andincreases memory T cells. Trabectedin also blocks the PD-1/PD-L1 axis by targeting PD-L1þCLL cells, PD-L1þmonocytes/macrophages, and PD-1þ T cells. Thus, trabectedin behaves asan immunomodulatory drug with potentially attractive ther-apeutic value in the subversion of the protumor microenvi-ronment and in overcoming chemoimmune resistance.

IntroductionIntroduction of therapeutic agents including kinase and BCL-2

inhibitors has improved treatment options for patients withchronic lymphocytic leukemia (CLL), although some patients

fail to respond, become resistant or relapse during treat-ment (1–3). Patients with lymphoid malignancies receiving theBruton tyrosine kinase inhibitor ibrutinib are at risk of infection,including lethal invasive fungal infections (4), likely due to theinhibition of innate immune surveillance (5). Fludarabine-based chemoimmune therapy remains the first-line therapyfor many patients, although the cytotoxic activity that fludar-abine has against leukemic cells is accompanied by damage tonormal immune cells, which leads to immune dysfunctionand opportunistic infections (6–10). Alternative strategies areneeded to bypass mechanisms that contribute to CLL cellresistance and immune dysfunction and to improve CLLpatients' quality of life.

Leukemic cells depend on survival signals provided by micro-environment nonneoplastic cells (11). As in solid tumors, thesesignals enhance the frequency of immunosuppressive cells,including regulatory T cells (Treg; ref. 12), tumor-associatedmacrophages (13), and myeloid derived suppressor cells (14).

In CLL, T lymphocytes exhibit exhaustion and functionaldefects (ref. 15; e.g., impaired immunologic synapse formationand reduced cytotoxic capacity).

Clinical trials of CD19 chimeric antigen receptor (CAR) T-celltherapy revealed resistance in CLL patients likely due to T-celldefects that are characteristic of CLL and worsen with diseaseprogression (16).

The CLL microenvironment is characterized by an excess ofCD4þCD25þFoxp3þ Tregs that are resistant to apoptosis likelydue to high Bcl-2 expression (17). CD4þ T cells seem to provide aniche that favors development and maintenance of the

1Department of Experimental Therapeutics, The University of Texas MDAnderson Cancer Center, Houston, Texas. 2Department of Stem Cell Transplan-tation and Cellular Therapy, The University of Texas MD Anderson CancerCenter, Houston, Texas. 3B-cell Neoplasia Unit, Division of Experimental Oncol-ogy, IRCCS San Raffaele Scientific Institute, Milan, Italy. 4Universit�a Vita-SaluteSan Raffaele, Milan, Italy. 5R&D, PharmaMar, Madrid, Spain. 6Roche PharmaResearch and Early Development, Oncology Discovery, Roche Innovation Cen-ter Zurich, Zurich, Switzerland. 7Department of Leukemia, The University ofTexas MD Anderson Cancer Center, Houston, Texas. 8Division of ExperimentalOncology, IRCCS San Raffaele Scientific Institute, Milan, Italy.

Note: Supplementary data for this article are available at Cancer ImmunologyResearch Online (http://cancerimmunolres.aacrjournals.org/).

Current address for P. Banerjee: HoustonMethodist Research Institute, Houston,Texas; current address for G. Galletti: Humanitas Clinical and Research Center,Rozzano, Milan, Italy; and current address for F. Caligaris-Cappio: AssociazioneItaliana per la Ricerca sul Cancro (AIRC), Milan, Italy.

P. Banerjee and R. Zhang contributed equally to this article.

CorrespondingAuthor:Maria Teresa S. Bertilaccio, Department of ExperimentalTherapeutics, The University of Texas MD Anderson Cancer Center, 1901 EastRoad, Houston, TX 77054. Phone: 713-563-6128; Fax: 713-745-9231; E-mail:[email protected]

Cancer Immunol Res 2019;7:2036–51

doi: 10.1158/2326-6066.CIR-19-0152

�2019 American Association for Cancer Research.

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disease (18). Another component of the immune microenviron-ment is represented by nurse-like cells (NLC) that in vitro promoteCLL cell survival mainly through SDF1 (19). NLCs resemblemacrophages and have been identified as CLL-specific tumor-associated macrophages (TAM; ref. 20).

We demonstrated in CLL mouse models that TAMs supportsurvival and proliferation of CLL and can be therapeuticallytargeted by CSF1R signaling blockade to restore leukemic cells'apoptosis sensitivity. Macrophage targeting via CSF1R inhibi-tion reprograms the tumor microenvironment toward an anti-tumor phenotype by activating effector memory T cellsand reducing immunosuppressive cells of the lymphoidand myeloid lineage such as Tregs and monocytic-myeloidderived suppressor cells (M-MDSC; ref. 13). These findingsindicate bidirectional cross-talk between CLL cells, MDSCs,and Tregs (14).

These observations led us to investigate ET-743/trabectedin, aDNA binding sea squirt–derived compound, known to targetTAMs in solid tumors. Trabectedin is approved for the treatmentof soft-tissue sarcoma (21) and ovarian cancer (22). Unlikeconventional chemotherapeutic agents (23), trabectedin bindsthe minor groove of DNA, blocks the cell cycle, affects genetranscription, and DNA-repair pathways (24, 25), and is a tran-scription-coupled nucleotide excision repair (TC-NER) agent. Thetargeting of TC-NER induces selective CLL cell death, regardless ofgenotype or preceding therapies (26). Trabectedin affects tran-scription regulation of cancer cells and some normal cells of thetumor microenvironment, which produce cytokines and factorspromoting tumor growth (25). In solid tumors, trabectedinshows activity against tumor-associated myeloid cells (23, 27)and other immune cells (25, 28). In sarcoma, trabectedin targetscancer cells, shows selective TRAIL-mediated cytotoxicity againsthumanmonocytes, and inhibits the production of cytokines suchas CCL2 and IL6 (29).We therefore hypothesized that trabectedinsynergistically targets both leukemic cells and nonmalignantmicroenvironment cells. This study uncovered the immunomod-ulatory functionof trabectedin inCLL.We showed that at very lowdoses, this drug repairs the immune system and kills leukemiccells.

Materials and MethodsCells and reagents

For cell viability studies, human primary samples wereobtained from CLL patients (RAI stage 0–2), who providedinformed consent as approved by the Institutional Ethical Com-mittee (protocol VIVI-CLL) of San Raffaele Scientific Institute inaccordance with the Declaration of Helsinki. For all the otherstudies with human blood samples, specimens were obtainedfrom CLL patients (any RAI stages) referred to the LeukemiaDepartment at MD Anderson Cancer Center with the approvalof MD Anderson's Institutional Review Board (protocol 2014-0678), in accordance with the Declaration of Helsinki. Writteninformed consent was obtained from the donors. The clinical andbiological features of CLL patients analyzed are described inSupplementary Tables S1 and S2.

MEC1 cells are a CD5low/� CLL cell line established from aCLL patient in prolymphocytoid transformation to B-PLL,obtained from Deutsche Sammlung von Mikroorganismen undZellkulturen (DMSZ) and cultured in RPMI-1640 medium(Invitrogen) with 10% fetal bovine serum and gentamicin (15

mg/mL; Sigma-Aldrich). MEC1 cells have been obtained in 2014fromDMSZ; they were cultured for 1 to 2 weeks and frozen at lowpassages (5–10). For in vitro and in vivo experiments, MEC1 cellswere thawed, cultured for 1 to 2 weeks, and used at 15 to 20passages. MEC1 cell lines regularly tested negative forMycoplasmacontamination (PCR mycoplasma detection kit, Applied Biolog-ical materials Inc.) and have not been reauthenticated in the pastyear. Trabectedin (Yondelis) was provided by Pharma Mar, S.A.,Sociedad Unipersonal. For in vitro studies, trabectedin as purepowder was dissolved in DMSO to 1mmol/L and kept at�20�C.For in vivo studies, trabectedin was provided as sterile lyophilizedpowder (including sucrose, potassium dihydrogen phosphate,phosphoric acid, and potassium hydroxide) and dissolved inphysiologic solution, following the preparation guide for patientinfusion of Yondelis. A monoclonal antibody (mAb) to humanCD20 (GA101; ref. 30)was provided by Roche Innovation CenterZurich, Switzerland.

Cytotoxicity assayHuman primary CD19þ cells and MEC1 cells were seeded in

96-well plates at a concentration of 3� 106 cells/mL in 0.2 mL ofRPMI. Vehicle (DMSO) as control and increasing concentrationsof trabectedin (0.001mmol/L, 0.01mmol/L, 0.1mmol/L, 1mmol/L,10 mmol/L) were added, and cell viability was assessed at 24, 48,and 72 hours using CellTiter-Glo chemoluminescence assay(Promega).

In vitro cultures and quantitative flow cytometry–based cell-depletion assay from CLL patient samples

Depending on the experiments, fresh peripheral blood mono-nuclear cells (PBMC) or CD19þ cells from untreated CLL patientswere seeded, in triplicate, at 3 � 106 cells/mL in culture mediumand treated with trabectedin (0.01 mmol/L) or DMSO vehicle for24 hours, in the presence or absence of anti-TRAIL-R2 (human, 1mg/mL)mAb (HS201) fromAdipogen AG. Specific percentages ofremaining cells in the treated samples were calculated as (theabsolute number of cells in treated samples/the absolute numberof cells in control samples) � 100. For each condition, theabsolute number of remaining cells was calculated as the totalnumber of viable cells (trypan blue exclusion determination) �the percentage of viable cells (flow-cytometry analysis determi-nation). Then, specific cell depletion was calculated as (100� thespecific percentage of remaining cells), as described (13). Theflow-cytometry analysis of human myeloid and lymphoid celltypes is described below and in Supplementary Tables S3 to S7.For transcriptional studies, fluorescence-activated cell sorting wasperformed after 15 hours of trabectedin treatment (describedbelow and in Supplementary Table S8).

Human cell purification, flow cytometry, and cell sortingFor cytotoxicity studies, leukemic cells were purified immedi-

ately after blood withdrawal, by negative depletion, using a B-lymphocyte enrichment kit (RosetteSep; STEMCELL Technolo-gies). The purity of all preparations was more than 99%, and thecells coexpressed CD19 and CD5 on their cell surfaces as assessedby flow cytometry; preparations were virtually devoid of naturalkiller cells, T lymphocytes, andmonocytes. Phenotype analysis ofhuman MEC1 leukemic cells in xenotransplanted mice was per-formed with PE-Cy7 Mouse Anti-Human CD19 (J3-119) pur-chased by Beckman Coulter. Surface expression was analyzedusing Cytomics FC500 (Beckman Coulter).

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For in vitro cell-depletion assays, 8-color flow-cytometryphenotype analysis of human live myeloid cells and 11-colorflow-cytometry phenotype analysis of human live lymphoid cellswere performed using LSRFortessa X-20 (BD Biosciences).

PBMCs were first incubated with LIVE/DEAD fixable Aqua dye(Thermo Fisher Scientific); then, after the blocking of Fc receptors,cells were stained with surface antibodies described in Supple-mentary Tables S3 and S4. Finally, cells were incubated withammonium chloride solution (STEMCELL Technologies) to lysered cells. For lymphoid cell Foxp3 detection, surface-stained cellswere further fixed and permeabilized using a Treg detection kit(Miltenyi Biotec) andfinally stainedwith an anti-Foxp3 antibody.

For transcriptional and patient-derived xenograft studies, livemyeloid cells were isolated by four-way fluorescence-activatedcell sorting on a BD FACSAria II (BD Biosciences), after surfacestaining with the following antibodies: Alexa Fluor 700 mouseanti-human CD66b (G10F5), APC mouse anti-human LineageCocktail (CD3/CD19/CD20/CD56; ref. UCHTI,HIB19, 2H7, and5.1H11), Brilliant Violet 786 mouse anti-human CD14 (MjP9)purchased byBDBiosciences, PEmouse anti-humanCD16 (3G8)purchased by BioLegend, APC-Cy7 mouse anti-human HLA-DR(L243).

Live lymphoid cells were isolated by four-way fluorescence-activated cell sorting on a BD FACSAria II after surface stainingwith the following antibodies:

eFluor 450 mouse anti-human CD8a (SK1) purchased byeBioscience, PerCP mouse anti-human CD45RO (UCHL1) pur-chased by BioLegend, PE-Cy7mouse anti-human CD45RA (L48)purchased by BD Biosciences, Brilliant Violet 605 mouse anti-human CD62L (DREG-56), PE-Dazzle 594 mouse anti-humanCD4 (SK3), PE mouse anti-human CD25 (4E3) purchased byMilteny Biotec, APC mouse anti-human CD19 (J3-119) pur-chased by Beckman Coulter, FITC mouse anti-human CD127(HIL-7R-M21) purchased by BD Biosciences. Live/Dead FixableAqua (Thermo Fisher Scientific) staining was first performed toallow the discrimination of Live/Dead cells.

Samples were analyzed with FCS Express 6 Flow-CytometrySoftware (De Novo Software). Monocyte subsets have beenidentified by a negative exclusion gating strategy (31, 32). WeexcludedCD66bþneutrophils, then, using a lineage (Lin) cocktailincluding mAbs to CD3, CD19, CD20, and CD56, we excluded Tcells, B cells, and NK cells, respectively. CD14 and CD16 expres-sion was then used to identify CD14þCD16þþ nonclassic (NC),CD14þþCD16þ intermediate (I), and CD14þþCD16� classic (C)monocytes. T cells have been identified as follows: na€�ve hCD8þ

CD45RO�CD45RAþCD62Lþ T cells, central memory hCD8þ

CD45RA�CD45ROþCD62Lþ TCM, and effector memory hCD8þ

CD45RA�CD45ROþCD62L� TEM.

Intracellular cytokine detection on human myeloid cellsFor the detection of IFNa, TNFa, IL12, IL6, and CCL2 on

humanmyeloid cells, fresh PBMCs fromCLLpatientswere seededat 1 � 106 cells/mL in culture medium and treated with trabecte-din (0.01mmol/L) orDMSOvehicle for 15or 38hours. Then, theywere stimulated in vitro with ionomycin and Brefeldin A, andstained with the following surface antibodies: Alexa Fluor 700mouse anti-human CD66b (G10F5), APC mouse anti-humanLineage Cocktail (CD3/CD19/CD20/CD56; UCHTI, HIB19,2H7, and 5.1H11), Brilliant Violet 786mouse anti-human CD14(MjP9) purchased by BD Biosciences, BUV737 mouse anti-human CD16 (3G8) purchased by BD Biosciences, PE mouse

anti-human TRAIL-R2 purchased by BioLegend. The IntraPrepPermeabilization Kit (Beckman Coulter) was used for the intra-cellular detection of the cytokines described in SupplementaryTable S5. Live/Dead Fixable Aqua (Thermo Fisher Scientific)staining was first performed to allow the discrimination ofLive/Dead cells. Samples were acquired using an LSRFortessaX-20 and analyzed with FCS Express 6 Flow-Cytometry Software.

Violet proliferation dye–based phagocytosis assayMEC1 cells were labeled with Calcein-violet proliferation dye

(VPD; BioLegend) and cocultured withmonocytes at an effector–target ratio of 1:1 in the presence or absence of 0.01 mmol/Ltrabectedin for 16 hours. To distinguish between phagocytosedVPD-positive MEC1 cells and free MEC1 cells, samples werecounterstained with the following surface antibodies: anti-human CD66b, anti-human Lineage Cocktail (CD3/CD19/CD20/CD56), anti-human CD14, and anti-human CD68 anti-body. The IntraPrep Permeabilization Kit (Beckman Coulter) wasused for the intracellular detection of the CD68 macrophagemarker. After the exclusion of neutrophils (through CD66bmolecule), and then the exclusion of NK/NKT cells, T cells, andB cells (through Lineage Cocktail including CD56, CD3, CD19,CD20 molecules), VPDþ CD14� cells and VPDþ CD68� cellswere identified by flow-cytometric analysis. The absolute numberof surviving VPDþ CD68� target cells and of VPDþ CD14� targetcells were calculated. The percentage of trabectedin-mediatedphagocytosis by CD68þ macrophages was calculated using thepreviously described (33) formula: % trabectedin-mediatedphagocytosis ¼ 100 – [(absolute number of surviving VPDþ

CD68� MEC1 cells in the presence of trabectedin/absolute num-ber of surviving VPDþ CD68� MEC1 cells in the absence oftrabectedin) � 100%]. The percentage of trabectedin-mediatedphagocytosis by CD14þ monocytes was calculated using theformula: % trabectedin-mediated phagocytosis ¼ 100 � [(abso-lute number of survivingVPDþCD14�MEC1 cells in the presenceof trabectedin/absolute number of surviving VPDþCD14�MEC1cells in the absence of trabectedin)� 100%]. The antibodies usedin this assay are described in Supplementary Table S6.

Human T-cell in vitro functional assay and cytokine detectionCD8þ T lymphocytes (including hCD8þ TEM and hCD8þ TCM)

were separated by fluorescence-activated cell sorting, coincubatedin vitro at an effector–target ratio of 2:1 with MEC1 cells heatedat 47�C for 1 hour to induce tumor-antigen-specific T-cellresponse (Ag-experienced; ref. 34), in the absence or presence of0.01 mmol/L trabectedin for 15 hours. The cells were then stim-ulated in vitro with Brefeldin A (4 hours, 4 mg/mL) and stainedwith the surface antibodies described in Supplementary Table S7,after LIVE/DEAD fixable Aqua dye incubation and Fc receptorblocking. Surface-stained cells were further fixed and permeabi-lized using a Staining Buffer kit (Miltenyi Biotec) and finallystained with an anti-IFNg antibody.

RNA extraction, RT-PCR amplification, and quantitative PCRRNA extraction from sorted myeloid and lymphoid cells was

performed using Mini Kit (QIAGEN). RNA sample quality andquantity checks were performed using an Agilent RNA 6000 PicoAssay (Agilent Technologies) at MD Anderson's Sequencing andNon-Coding RNA Program.

Reverse-transcription reaction from50 to 200 ng RNA templatewas done using the RevertAid H Minus First-Strand cDNA

Banerjee et al.

Cancer Immunol Res; 7(12) December 2019 Cancer Immunology Research2038




Synthesis Kit (Fermentas, Thermo Fisher Scientific) according tothe manufacturer's instructions. The sequences of primer pairsspecific for each gene sequence (Primm Srl and Sigma-Aldrich)were designed with Beacon Designer (Premier Biosoft Interna-tional) and NCBI-Primer Designing Tools (SupplementaryTable S8).

Real-time PCR was performed using the SYBR Green PCRMaster Mix (Applied Biosystems) with forward and reverse pri-mers at a final concentration of 10 mmol/L. Each cDNA (2 mL) wasused as template; 12.5 mL of 2� SYBRGreen PCRMasterMix weremixed with template and primers. The total reaction volume was25mL. Three replicates for each cDNA samplewere tested. 7900HTFast Real-Time PCR System (Applied Biosystems) was used, andcycling conditions were 10minutes at 95�C (1 cycle); 10 minutesat 95�C (1 cycle); 15 seconds at 95�Cplus 1m at 60�C (40 cycles).Data were normalized to b-actin expression.

MiceAll mice were housed and bred in specific pathogen–free

animal facilities at San Raffaele Scientific Institute and at TheUniversity of Texas MD Anderson Cancer Center. Depending onthe animal experiment, mice were treated in accordance with theEuropean Union guidelines and with the approval of the SanRaffaele Scientific Institute Institutional Ethical Committee (pro-tocols 601 and 726) or with the approval of the InstitutionalAnimal Care and Use Committee of The University of Texas MDAnderson Cancer Center (protocol 00001627-RN00) and con-ducted in accordance with the Animal Welfare Act. Rag2�/�gc�/�

mice on a BALB/c background were kindly provided by CIEA orpurchased by Taconic, Em-TCL1 transgenic mice on a C57BL/6background were kindly provided by Dr. Byrd (The Ohio StateUniversity, Columbus, OH), and wild-type C57BL/6 mice weresupplied by Taconic.

Mouse genotypingEm-TCL1 transgenic mice were genotyped for the hTCL1 trans-

gene by PCR-based screening assay as described previously (35).

Xenograft studiesEight-week-old male Rag2�/�gc�/�mice were i.v. transplanted

(day 0) with 10 � 106 MEC1 cells in 0.1 mL of saline through a27-gauge needle. In some experiments, mice were i.v. injectedwith 0.15 mg/kg of trabectedin or with saline (control) weekly.Depending on the experiments mice were monitored once perweek for weight and humanely killed at different stages ofleukemia. Peritoneal exudate (PE), peripheral blood (PB), spleen(SP), and femoral bone marrow (BM) were collected andanalyzed.

For preclinical purposes, Rag2�/�gc�/�mice received s.c. injec-tions of 10� 106MEC1 cells in 0.1mL of saline into the left flankon day 0. Once the mice developed palpable subcutaneoustumors, they were i.v. injected with 0.15 mg/kg trabectedin orwith saline (control). Mice were monitored once per week forweight and tumor growth (by measuring three perpendiculardiameters by a caliper) and killed when the mean subcutaneoustumor volume reached 1,000mm3 (before reaching clinical signsand symptoms, to avoid unnecessary pain anddiscomfort accord-ing to the ethical guidelines). Measurements were stopped when75%of the originally treatedmicewere still surviving. For survivalexperiments, xenotransplanted mice were intravenously injectedwith trabectedin on days 11 and 18 or with GA101 mAb on days

12 and 19 as single agents or in combination settings. For patient-derived xenograft studies,MEC1-transplantedRag2�/�gc�/�micewere injected intravenously on day 18with patient-derived CD8þ

memoryT cells (treatedwith0.01mmol/L trabectedin for 16hoursin vitro or left untreated) from patients 41 to 43 (SupplementaryTable S2) or with patient-derived monocytes (treated with 0.01mmol/L trabectedin for 16 hours in vitro or left untreated) frompatients 41 to 44 (Supplementary Table S2).Mice were humanelykilled 5 days after the adoptive transfer. PB, SP, and BM werecollected and analyzed.

Transgenic studiesEight-week-old male syngeneic immunocompetent C57BL/6

mice were injected i.p. (day 0) with 10 � 106 cells purified fromthe SP of leukemic male Em-TCL1 transgenic mice using theEasySep mouse B-cell enrichment kit (STEMCELL Technologies).The purity of the transplanted CD19þ CD5þ Igkþ cells wasassessed by flow cytometry. Mice were monitored weekly forweight and leukemia development by flow-cytometric analysisof PB samples.Micewere i.v. injectedwith 0.15mg/kg trabectedinevery week starting when the frequency of CD19þCD5þ leukemiccells in the PB was 15% to 20%, compared with C57BL/6 wild-type mice. Mice were monitored weekly for weight and leukemiadevelopment by flow-cytometric analysis of the PB samples and,depending on the last trabectedin injection, humanely killed atdifferent time points. PE, PB, and organs [SP, lymph node (LN),femoral BM] were collected and analyzed.

Murine cell preparations and flow cytometryPB, PE, SP, and femurs were collected frommice, and cells were

isolated. Erythrocytes fromBM, PE, SP, and PB sampleswere lysedby incubation in ammonium chloride solution (NH4Cl) lysisbuffer (NH4Cl 0.15M, KHCO3 10mmol/L, Na2EDTA 0.1 mmol/L, pH 7.2–7.4) for 5minutes at room temperature. After blockingof fragment crystallizable (Fc) receptors with Fc block (BD Bios-ciences) for 10 minutes at room temperature, cells from PB, BM,PE, SP, and LNs were stained with the antibodies (15 minutes at4�C) listed in Supplementary Tables S9 and S10. Depending onthe experiments, cells were analyzed with a Beckman CoulterFC500 or with a BD LSRFortessa X-20 flow cytometer. Ten-colorflow-cytometry phenotype analysis of live myeloid cell singletsand 13-color flow-cytometry phenotype analysis of live lymphoidcell singlets were performed using an LSRFortessa X-20. To devel-op the multicolor flow-cytometry panels, we used antibody-capture beads (UltraComp eBeads Invitrogen) for single-colorcompensation controls. A Live/Dead Fixable Aqua Dead CellStain Kit (Thermo Fisher Scientific) was used first to gate outdead cells. Further gating adjustments were made based onfluorescence-minus-one controls. Absolute cell numbers wereobtained by multiplying the percentage of the cells by the totalnumber of splenocytes, mesenteric LNs, peritoneal cells, and BMcells flushed from one femur.

Statistical analysisThe statistical analysis of the data was performed using the

GraphPad Prism 5.0 Software. Data were expressed as means �standard deviations (SD), and comparison of growth curves ordifferences between experimental groups were assessed with anunpaired two-tailed Student t test (95% confidence interval)and considered statistically significant for P values less than0.05. Survival curves were compared with the use of the log-rank test.






In cell-depletion experiments that involved different humanmyeloid and lymphoid cell types, normality was tested with aShapiro–Wilk normality test. We applied the t test to normallydistributed data, otherwise the Mann–Whitney–Wilcoxon test. Abox-and-whisker plot [boxplot representsfirst (lower bound) andthird (upper bound) quartiles, whiskers represent 1.5 times theinterquartile range] was used to visualize the data. The y-axis wasplotted on a logarithmic scale for convenience of representation.The analysis and graphical representation were performed in R(version 3.0.1; http://www.r-project.org/) and the statistical sig-nificance was defined as a P value less than 0.05.

The outliers at end-point were never excluded. The number ofbiological and technical replicates for each experiment is detailedin the figure legends. All the experiments were repeated indepen-dently and are described in the figures and figure legends. Inves-tigators were not blinded when assessing the analysis of theexperimental outcome.

ResultsTrabectedin targets both CLL cells and their microenvironment

We first evaluated whether trabectedin affects human primaryleukemic cells. As demonstrated previously (26), cytotoxicitystudies performed on the MEC1 CLL cell line (SupplementaryFig. S1A) and purified human primary leukemic cells (Fig. 1A;Supplementary Fig. S1B; n ¼ 5) revealed that trabectedin has atoxic effect on leukemic B cells in vitro. We selected the lowesttrabectedin dose that reduced leukemic B-cell viability but did notcompletely kill them (e.g., 0.01 mmol/L; dotted line, Fig. 1A). Thedirect cytotoxic activity of trabectedin on CLL cells was confirmedin a flow-cytometry cell-depletion assay (Fig. 1B, empty circles).When unfractionated PBMCs of CLL patients (which containedboth leukemic and normal hematopoietic cells) were treated with0.01mmol/L trabectedin, we observed depletion of both leukemicB cells and CD14þ myeloid cells (Fig. 1B, black circles). Thisconfirmed the drug's cytotoxicity to mononuclear phagocytesdemonstrated in solid tumors (23). Leukemic cell depletioninduced by trabectedin increased significantly in the PBMCs ascompared with purified leukemic cells, suggesting a role forCD14þ cells and other mononuclear cells (Fig. 1B; Supplemen-tary Table S1).

We next evaluated the effect of trabectedin on monocyte sub-sets, identified by flow cytometry with a negative exclusion gatingstrategy (31, 32). CD14 andCD16 expressionwas used to identifyCD14þCD16þþ nonclassic (NC), CD14þþCD16þ intermediate(I), and CD14þþCD16� classic (C) monocytes (Fig. 1C, PB;Supplementary Fig. S2A, PBMC). This gating strategy enabled usto better characterize myeloid cells and to avoid nonspecific cellinclusion (31, 32, 36). Monocyte subsets were also screened forthe surface expression of HLA-DR protein, because of the dem-onstrated immunosuppressive activity of HLA-DRlow/� classicmonocytes in non-Hodgkin lymphoma patients (ref. 37; Supple-mentary Fig. S2A). Moreover, M-MDSCs were analyzed (38, 39).PBMCs from 10 CLL patients were treated with trabectedin:after 24 hours, I monocytes and M-MDSCs were depleted,whereas NC and C monocytes (Fig. 1D and E; SupplementaryTable S2), including their fractions with increased HLA-DRexpression, were not depleted (Supplementary Figs. S2B,and S3A and S3B). Trabectedin also increased the absolute num-ber of CD14þþCD16�HLA-DRþ classic monocytes (Supplemen-tary Fig. S4), high frequency of which has been found to correlate

with improved overall survival in metastatic melanomapatients (36). These results led us to conclude that trabectedinaffects myeloid cells.

We asked whether trabectedin also affects T lymphocytes.Human na€�ve, effector (TEM), and central memory (TCM) CD8þ

T cells were identified based on the differential surface expressionof CD45RO, CD45RA, and CD62L. CD4þ regulatory T lympho-cytes expressingCD25andFoxp3were alsoanalyzed. In addition todepleting the CD19þCD5þ leukemic clone, trabectedin reducedCD4þ Tregs. However, trabectedin did not deplete CD8þ TEM andTCM cells (Fig. 1D and F; Supplementary Tables S11 and S12).

Trabectedin triggers apoptosis of selected immune cellsBased on our findings, we hypothesized that trabectedin direct-

ly kills leukemic cells and tumor-supporting immune cells includ-ing CD4þ Tregs and somemyeloid cell subtypes (M-MDSCs and Imonocytes), and favors the activation of cytotoxic T lymphocytes.

Trabectedin's effects on transcription regulation affect bothcancer cells and normal cells of the malignant microenviron-ment (25), but the effect of the drug on immune cell types fromleukemic patients is unexplored. Therefore, we analyzed RNAfrom CLL patient lymphoid andmyeloid cell types after 15 hoursof trabectedin treatment. By means of two independent four-wayfluorescence-activated cell-sorting strategies, we separated mye-loid subsets including C, NC, I, M-MDSCs, and some of B-T celltypes described in Fig. 1D, including leukemic CD19þCD5þ Bcells, CD8þ TEM and TCM cells and Tregs. For RNA studies, CD4þ

CD25þ Tregs were identified and sorted based on low-negativeexpression of CD127.

We then evaluated by RT-PCR the expression of RNA encodingeffector molecules involved in TRAIL-, TNF-, and FAS/FASL-regulated death pathways on cell types depleted by trabectedin(Fig. 1D), including leukemic B cells, regulatory CD4þ T cells, Imonocytes, and M-MDSCs. As described in Fig. 2, we observedupregulation of molecules involved in TRAIL and TNF pathways,including TRAIL-R2, TNFR1, BAX, BID, CASP3 transcripts in allthe trabectedin-targeted cells types but not in memory CD8þ Tcells (Supplementary Fig. S5). We confirmed the involvement ofthe TRAIL pathway by using a blocking anti-human TRAIL-R2mAb. In 7 of 8 samples (Supplementary Fig. S6), the leukemic celldepletion induced by trabectedin was reduced when TRAIL sig-naling was blocked. Consistent with reported TRAIL-mediatedactivity of trabectedin in monocytes in solid tumors (23),we confirmed by flow cytometry that trabectedin modulatedTRAIL-R2 receptor expression on monocytes and M-MDSCs(Supplementary Fig. S7A). We also observed the involvementof TRAIL-mediated cell death pathway in T cells treated withtrabectedin (Fig. 2; Supplementary Fig. S6).

Trabectedin has immunomodulatory activity in vitroWe next evaluated the immunomodulatory activity of trabec-

tedin on myeloid and lymphoid cell types. Trabectedin modifiedRNA expression of proinflammatory cytokines that promotethe classic (M1) antitumor activation of macrophages (e.g., IFNa

and TNFa), induce T-cell cytotoxic response (e.g., IFNa andIL12a), and control B lymphocytes (e.g., IL6; Fig. 3A). Expressionof TNFa RNA was also upregulated in M-MDSCs (Fig. 3B).

Expression of RNA encoding CCL2, a monocyte chemoattrac-tant,was reduced inC, I, andNCmonocytes (Fig. 3A,C, andD), asdemonstrated in solid tumors (23, 27). In a different set of CLLpatient samples, we confirmed altered production of IL6, CCL2,

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and IFNa protein upon 38 hours of trabectedin treatment (Sup-plementary Fig. S7A–S7C). Patients 39 and 40 carried a 17pdeletion and patient 40 (in red) was in Richter transformation,resistant to ibrutinib andvenetoclax (Supplementary Table S2). In

some patient PBMC samples, M-MDSCs disappeared after tra-bectedin treatment.

In Tregs, trabectedin downregulated FOXP3, the transcriptionfactor thatmodulates the functionality of these cells and abolishes

Figure 1.

Trabectedin depletes selected human primary lymphoid andmyeloid cells. A, Human primary CLL cells obtained from CLL patients (n¼ 5, patients 1–5;Supplementary Table S1) were incubated with increasing concentrations of trabectedin [or with vehicle (DMSO v1, v2)] and subjected to a luminescent assay at24 and 48 hours to assess the cells' sensitivity to the drug. Cell viability of each sample was normalized to its control. Dot lines indicate CLL cell viability at 0.01mmol/L trabectedin. B, The percentage of depletion of hCD19þ CD5þ cells (plated alone, empty circles; plated with total PBMCs, black circles) and hCD14þ cells(plated with total PBMCs, black circles) after 24 hours of treatment (n¼ 7, patients 6–12; Supplementary Table S1) with 0.01 mmol/L of trabectedin was analyzedby flow cytometry. Purple and orange dots identify 2 representative CLL patients. � , P < 0.05, Student t test. C,Monocytes were identified as CD14þ CD16þþ

nonclassic (green), CD14þþ CD16þ intermediate (red), and CD14þþ CD16� classic (blue) subsets. A representative PB sample from CLL patient 24(Supplementary Table S2) is described. D, Classic monocyte, nonclassic monocyte, intermediate monocyte, and hCD14þHLADRlow/�M-MDSC cell depletion andCD8þ T cell, na€�ve T cell, hCD8þ TCM, hCD8

þ TEM, regulatory hCD4þCD25þFoxp3þ Treg, and hCD19þ, hCD19þCD5þ, and hCD19þCD5þCD23þ lymphoid cell

depletion (n¼ 10; patients 20–26, 45, 48, and 49; Supplementary Table S2) after 24 hours of treatment with 0.01 mmol/L of trabectedin were analyzed bymulticolor flow cytometry and calculated by the formula described in Materials and Methods. The statistical analysis is described in Supplementary Tables S11and S12. E and F, An alternate presentation of the distribution of data from D showing selected paired-cell type comparisons is depicted by box-and-whiskerplots. P value is given by Mann–Whitney–Wilcoxon test; � , P < 0.05; �� , P < 0.01.






their suppressor activity (Fig. 3E). Unlike leukemic B lympho-cytes (26), we did not observe TC-NER and homologous recom-bination (HR) pathways involvement in the selective targeting ofTregs by trabectedin (Supplementary Fig. S8).

The reduced immunosuppression was accompanied by upre-gulation of RNA encoding cytolytic factors in CD8þ TEM and TCM,including IFNy and GRANZYME B (GZMB; Fig. 3F and G).

Thus, trabectedin enhances the antitumor phenotype ofmono-cytes and reprograms the tumor microenvironment towardimmune effector T-cell cytotoxicity.

Trabectedin has therapeutic efficacy in CLL mouse modelsWe verified the in vivo antitumor activity of trabectedin in the

subcutaneous MEC1-based xenograft model of CLL (Fig. 4A;ref. 40). The treatment significantly reduced subcutaneous MEC1tumor growth (Fig. 4B) and induced a significant reduction ofhCD19þ leukemic cells in lymphoid tissues, including BM andSP (SP; Fig. 4C).

We next performed a time-course experiment in which mice,transplanted intravenously with MEC1 cells, were sacrificed 1 or10 days after the last of three trabectedin injections (days 27 and36, respectively; Fig. 4D). The treatment reduced the leukemicexpansion and stabilized the disease in the PB and lymphoidtissues, including SP and BM (Fig. 4E–G). Trabectedin alsoconveyed a survival benefit as both a single agent (Fig. 4H andI; P ¼ 0.0009, trabectedin vs. untreated) and in combinationwithGA101, a glycoengineered type II CD20mAb,GA101 (Fig. 4Jand K).

Trabectedin affects immune cells of the tumormicroenvironment in vivo

We tested whether trabectedin affected myeloid cells in CLLmouse models, including the xeno- and the TCL1 transgenic-transplantation systems (13). The antileukemic effect observed inxenotransplanted mice sacrificed at day 27, the day after the lastinjectionof thedrug (Fig. 4D–G),was associatedwith a significant

Figure 2.

Trabectedin-induced cell death mechanisms in selected primary lymphoid andmyeloid cells from CLL patients. Relative mRNA expression of TRAILR2, TNFR1,BAX, BID, FADD, CASP3, CASP8, CASP9, andMCL1 in hCD19þCD5þ human primary CLL cells, CD14þHLADRlow/�M-MDSCs, intermediate monocytes, andCD4þCD25þCD127low/� Tregs separated by FACS from PBMCs (n¼ 6; patients 173, 538, 665, 915, 089, and 642; Supplementary Table S2) and plated in 6-wellplates alone or with 0.01 mmol/L of trabectedin for 15 hours. Three technical replicates were analyzed for each sample. Data were normalized to b-actinexpression. Gene expression was determined by calculating the difference (DCt) between the threshold cycle (Ct) of each gene and that of the reference gene,and was expressed as the mean of three replicates� SEM. Then the relative quantification values were calculated as the fold change expression of the gene ofinterest over its expression in the selected cell type reference sample, i.e., the untreated sample (considered as the calibrator sample), by the formula 2 � DDCt.Finally, the treated sample relative mRNA expression was normalized to the vehicle. Samples with undetermined Ct values are not included.

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Figure 3.

Immunomodulatory activity of trabectedin in humanmyeloid and lymphoid cell types in CLL in vitro.A, Relative mRNA expression of IFNa, TNFa, IL12a, IL6, andCCL2 in classic monocytes; B, TNFa in M-MDSCs; C, CCL2 in intermediate monocytes; D, CCL2 in nonclassic monocytes; E, FOXP3 on CD4þCD25þCD127low/�

Tregs; F, IFNy in hCD8þ TEM; and G, IFNy and GZMB in hCD8þ TCM. All cells were separated by FACS from CLL patient PBMCs (n¼ 6; patients 173, 538, 665, 915,089, and 642; Supplementary Table S2), and plated in 6-well plates alone or with 0.01 mmol/L of trabectedin for 15 hours. Three technical replicates wereanalyzed for each sample. Data were normalized to b-actin expression. The gene expression was determined by calculating the difference (DCt) between thethreshold cycle (Ct) of each gene and that of the reference gene, and was expressed as the mean of three replicates� SEM. Then the relative quantificationvalues were calculated as the fold change expression of the gene of interest over its expression in the selected cell type reference sample, i.e., the untreatedsample (considered as the calibrator sample), by the formula 2 �DDCt. Finally, the treated sample relative mRNA expression was normalized to the vehicle.Samples with undetermined Ct values are not included.






reduction of CD11bþ F4/80þ TAMs in the SP and PE, withselective depletion of MRC1þ M2-like protumor TAMs in the PE(Supplementary Fig. S9A–S9E).

We next used a CLL immunocompetent mouse model, basedon the transplantation of leukemic cells purified from the SP ofEm-TCL1 transgenic (tg)mice intosyngeneicC57/BL6recipients (13).

Figure 4.

Antileukemic effect and survival benefit of trabectedin in the CLL xenotransplantation systems.A–C, Rag2�/�gc�/�mice subcutaneously transplanted withMEC1 cells (day 0) received weekly i.v. injections of 0.15 mg/kg of trabectedin on days 22, 29, and 36 (n¼ 5, white circles) or were left untreated (n¼ 4, blackcircles). B, Tumor size was evaluated as described in Materials and Methods. Statistically significant differences were calculated using the Student t test; � , P <0.05; �� , P < 0.01; �� , P < 0.001. C, Themean value� SD of the relative contribution of CD19þ cells in SP, BM, and PE is shown in the graph. Statistical analysis: � , P< 0.05, Student t test.D–G, Rag2�/�gc�/�mice intravenously transplanted with MEC1 cells (day 0) were left uninjected (n¼ 10, black circles) or i.v. treated (daysþ13,þ19,þ26) with 0.15 mg/kg of trabectedin (n¼ 8, white circles) and killed at days 27 and 36. The mean value� SD of the percentage of hCD19þ in the PB (E),the mean value of the absolute number of hCD19þin the SP (F), and the mean value� SD of the absolute number of hCD19þin the BM (G) are shown in graphs. � , P< 0.05; �� , P < 0.01; ��P < 0.001, Student t test. H and I, Rag2�/�gc�/�mice intravenously transplanted with MEC1 cells (day 0) were left untreated (n¼ 6, blackcircles) or treated i.v. (daysþ11,þ18) with 0.15 mg/kg of trabectedin (n¼ 7, white circles) andmonitored for survival. I, Kaplan–Meier survival curve isrepresented; statistical analysis was performed using the log-rank test. P¼ 0.0009. J and K, Rag2�/�gc�/�mice intravenously transplanted with MEC1 cells wereleft untreated (n¼ 6, black circles) or treated with 0.15 mg/kg i.v. of trabectedin (n¼ 7, white circles; daysþ11,þ18), 30mg/kg i.p. of anti-CD20 GA101 (n¼ 5,black triangles; daysþ12,þ19), or 0.15 mg/kg i.v. of trabectedin (daysþ11,þ18)þ 30mg/kg i.p. of anti-CD20 GA101 (daysþ12,þ19; n¼ 5, white triangles) andmonitored for survival. K, Kaplan–Meier survival curve is represented; statistical analysis was performed using the log-rank test. Trabectedin versus control: P¼0.001; aCD20 versus control: P¼ 0.0014; trabectedinþ aCD20 versus control: P¼ 0.0014; trabectedinþ aCD20 versus aCD20: P¼ 0.0023; trabectedinþaCD20 versus trabectedin: P¼ 0.0011. abs. number, absolute number; Trab, trabectedin.

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To evaluate the immune cell dynamics induced by trabectedinand their persistence over time, we performed three sets ofexperiments in which mice transplanted with leukemic cellsfrom Em-TCL1 tg donors #1, #2, #3 were sacrificed 6, 9, or13 days after the last trabectedin injection. When donor #1-transplanted mice were treated with trabectedin (starting atday 40) and sacrificed 6 days after the last injection of the drug(day 53), a significant reduction of the leukemic clone in theSP, BM, PE but not in the PB was observed (SupplementaryFig. S10A–S10D). This reduction was associated withdecreased frequencies of CD11bþ CSF1Rþ monocytes andF4/80þ macrophages and with selective depletion of PD-L1þ

monocytes and MRC1þ and PD-L1þ M2-like TAMs in the PE(Supplementary Fig. S10E–S10I). Among monocytes, weobserved a reduction of the Ly6Clow cell component expressingPD-L1 in the SP (Supplementary Fig. S10J): such a reductionsuppresses T-cell immunity in solid tumors (41). Overall,these data are in line with findings on the PD-L1–mediatedfunctional remodeling of monocytes and macrophages incolon and mammary carcinoma (42). We observed a decreasein the whole pool of Gr1þMDSCs in the PB, BM, and SP (Supp-lementary Fig. S10K–S10M), an increase of CD8þ T lympho-cytes in the BM and SP and an increase of CD8þmemory T cellsin the PB and BM (Supplementary Fig. S10N–S10Q).

To clarify the long-term cell dynamics induced by trabectedinin vivo, we transplanted mice with splenic leukemic cells fromTCL1 tg donormouse #2or donormouse #3 and sacrificed them9(Fig. 5A, donor #2 recipients) and 13 days (Fig. 5B, donor #3recipients) after the last trabectedin injection, respectively. Indonor #2 recipient mice treated with trabectedin starting on day25 (Fig. 5A), a significantly lower percentage of leukemic B cells inthe PB was observed over time (Fig. 5C). Upon necropsy,comparative analysis of the treated and untreated mice con-firmed that in both donor #2 and donor #3 recipient mice(Fig. 5A and B) leukemic cell expansion was reduced in the PE,SP, BM, LN (Fig. 5D–G). Trabectedin treatment selectivelydepleted CD19þCD5þ leukemic cells expressing PD-L1 in thelymphoid tissues and PE (Supplementary Fig. S11A–S11C) ofdonor #3 recipient mice killed 13 days after the last trabectedininjection.

As for the myeloid cells, in mice sacrificed 9 days after the lasttrabectedin injection (Fig. 5A, donor #2 recipients), numbers ofCSF1Rþ monocytes (Fig. 5H) and CD11bþ PD-L1þ (Fig. 5I)monocytes were reduced in the BM, and the Ly6Clow subset wasthe most affected and significantly reduced in the BM and PB(Fig. 5J and K). Conversely, Ly6Chigh monocytes increased in theSP of treated mice (Supplementary Fig. S12A and S12B). In micesacrificed 13 days after the last trabectedin injection (Fig. 5B,donor #3 recipients), the whole pool of monocytes (Fig. 5L andM), CD11bþ PD-L1þ monocytes (Fig. 5N and O), and Ly6Clow

subset (Fig. 5P and Q) was reduced in the BM and PE of treatedmice.

As for M-MDSCs and TAMs, we confirmed (Fig. 6A, donor #2recipients) the reduced frequency of M-MDSCs in the PB, BM, SP,LN, and PE (Fig. 6B–F) and that of MRC1þ, PD-L1þ, and IAblow

protumor TAMs in the BM (Fig. 6G–J). M-MDSCs remainedreduced in the SP 13 days after the last trabectedin injection(Fig. 6K and L, donor #3 recipients), as did CD11bþ F4/80þ TAMsand MRC1þ, PD-L1þ, and IAblow protumor TAMs in the BM(Fig. 6M–P), SP (Supplementary Fig. S13A–S13E), and PE (Sup-plementary Fig. S13F and S13G).

In treated mice, myeloid cell targeting by trabectedin wasassociated with increased frequencies of CD8þ, CD4þ, CD8þ

TCM, CD4þ TEM, and TCM in the PB (Supplementary Fig. S14A–S14E) and decreased frequency of potentially regulatory CD4þ

CD25þ Tregs in the mesenteric LNs (Supplementary Fig. S14F).Thirteen days after last trabectedin treatment (SupplementaryFig. S14G, donor #3 recipients), CD8þ and CD4þ TEM cells weremaintained at higher frequencies in circulation (SupplementaryFig. S14H–S14J), and these frequencies were associated with asignificant reduction of CD4þCD25þ T cells in the PE, SP, and LN(Supplementary Fig. S14K–S14M). These results are in line withthe reduction of PD-1þ CD8þ memory T cells we observed in theBM and LN of trabectedin-treated mice sacrificed 9 (Supplemen-tary Fig. S15A, donor #2 recipients) or 13 (SupplementaryFig. S15B, donor #3 recipients) days after the last treatment(Supplementary Fig. S15C and S15D).

Trabectedin repairs immune dysfunction of CLL patient–derived cells

To conclusively delineate the direct effect of trabectedin onnonleukemic immune cells, we transplanted patient-derivedmonocytes or CD8þ memory T cells into leukemic MEC1-trans-planted Rag2�/�gc�/� mice. Using fluorescence-activated cellsorting, we separated monocytes (including NC, I, and C) frompatients 41 to 44 (Supplementary Table S2) and T cells (includingCD8þ TEM and TCM) from patients 41 to 43 (SupplementaryTable S2). After 16 hours of in vitro trabectedin treatment (0.01mmol/L), monocytes or CD8þ T cells were adoptively transferredinto xenotransplanted mice (Fig. 7A and B). Compared withmice injected with untreated T cells andmonocytes, mice injectedwith trabectedin-treated T cells and monocytes benefited fromsignificant antileukemic effects in the SP (Fig. 7C).

The functionality of CLL patient-derived CD14þ monocytesand CD68þ macrophages upon trabectedin was confirmedin vitro in a different set of patient samples by means of aVPD-based assay. The number of not-phagocytosed VPD-labeled CD14�/VPDþ MEC1 target cells or CD68�/VPDþ

MEC1 target cells treated or not with trabectedin was quanti-fied by flow cytometry. In most patient samples tested, tra-bectedin did not impair the phagocytic ability of monocytesand macrophages (Fig. 7D).

To confirm T-cell functionality upon trabectedin treatmentin vitro, CD8þ memory T cells, in the absence or presence ofantigen (Ag) stimulation with heated-MEC1 cells, were treatedwith 0.01 mmol/L trabectedin and then assessed for their expres-sion of PD-1 checkpoint markers and ability to produce cytolyticproteins. Upon trabectedin treatment, absolute numbers of totalCD8þ and CD8þ TEM cells were not affected (SupplementaryFig. S16A and S16B), whereas PD-1þ CD8þ and PD-1þ TEM cellswere reduced significantly (Fig. 7E–H). CD8þ TEM cells retainedtheir capacity for IFNg and GRANZYME B production in both thepresence and absence of trabectedin (Supplementary Fig. S16Cand S16D). Indeed, T cells from CLL patients have increasedexpression of the PD-1 exhaustion marker but retain their abilityto produce cytokines (43). We demonstrated that trabectedinrestores T-cell functionality, likely interfering with PD-1 check-point molecule.

Overall, these findings indicate that trabectedin inhibits leu-kemia progression with a cascade of immunomodulatory effectsin immune cell types of the malignant microenvironment (Sup-plementary Fig. S17).






Figure 5.

Long-term impact of trabectedin on leukemic cells and onmonocytes in the TCL1 tg transplantation system. A, C57BL/6mice intraperitoneally transplanted withleukemic B cells from Em-TCL1 transgenic mouse donor #2, left untreated (n¼ 6, black circles) or treated (daysþ25,þ32) with 0.15 mg/kg i.v. of trabectedin (n¼5, white circles), were killed on day 41 (9 days after the last injection of trabectedin) and analyzed by flow cytometry. B, C57BL/6 mice intraperitoneallytransplanted with leukemic B cells from Em-TCL1 transgenic mouse donor #3, left untreated (n¼ 4, black circles) or treated (daysþ19,þ26) with 0.15 mg/kg i.v.of trabectedin (n¼ 4, white circles), were killed on day 39 (13 days after the last injection of trabectedin) and analyzed by flow cytometry. C, Themean value�SD of the relative contribution of CD19þ CD5þ cells to the whole B-cell pool over time in PB of mice described in A is shown in the graph. The mean value� SD ofthe absolute number of CD19þ CD5þ cells gated on CD19þ in PE (D), SP (E), BM (F), and LN (G) of mice described inA and B is shown in graphs. H, The meanvalue� SD of the absolute number of CD11bþ CSF1Rþ cells gated on CD45þ in BM of mice described in A is shown in graph. I, Themean value� SD of theabsolute number of CD11bþ PDL1þ cells to the whole monocyte pool (CD11bþ CSF1Rþ) gated on CD45þ in BM of mice described in A is shown in the graph. J, Themean value� SD of the absolute number of the CD11bþ Ly6Clowmonocyte subset gated on CD45þ in the BM of mice described in A is shown in the graph. K, Themean value� SD of the relative contribution of the CD11bþ Ly6Clow monocyte subset gated on CD45þ in the PB of mice described in A is shown in the graph. Themean value� SD of the absolute number of CD11bþ CSF1Rþ cells gated on CD45þ in BM (L) and PE (M) of mice described in B is shown in graphs. The mean value� SD of the absolute number of CD11bþ PD-L1þ cells to the whole monocyte pool (CD11bþ CSF1Rþ) gated on CD45þ in BM (N) and PE (O) of mice described in B isshown in graphs. The mean value� SD of the absolute number of the CD11bþ Ly6Clow monocyte subset gated on CD45þin the BM (P) and PE (Q) of micedescribed in B is shown in graphs. � , P < 0.05; �� , P < 0.01; Student t test. abs. number, absolute number; Trab, trabectedin.

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Figure 6.

Long-term impact of trabectedin on leukemic cells, M-MDSCs, and TAMs in the TCL1 tg transplantation system. A, C57BL/6 mice intraperitoneally transplantedwith leukemic B cells from Em-TCL1 transgenic mouse donor #2, left untreated (n¼ 6, black circles) or i.v. injected (daysþ25,þ32) with 0.15 mg/kg oftrabectedin (n¼ 5, white circles), were killed on day 41 (9 days after the last trabectedin injection) and analyzed by flow cytometry. The mean value� SD of therelative contribution of monocytic Ly6Cþ Ly6G� gated on CD11bþ F4/80low cells in the PB (B) and of the absolute number� SD of monocytic Ly6Cþ Ly6G�

gated on CD11bþ F4/80low cells in BM (C), in SP (D), in LN (E), and in PE (F) of mice described inA is shown in graphs. G, The mean value� SD of the absolutenumber of CD11bþ F4/80þ cells gated on CD45þ in BM is shown in the graph. The mean value� SD of the absolute number of CD11bþMRC1þ cells (H), of CD11bþ

PD-L1þ cells (I), and of CD11bþ IAb low cells (J) to the whole macrophage pool (CD11bþ F4/80þ) gated on CD45þ in BM of mice described in A is shown in graphs.K, C57BL/6 mice intraperitoneally transplanted with leukemic B cells from Em-TCL1 transgenic mouse donor #3, left untreated (n¼ 4, black circles) or i.v. injected(daysþ19,þ26) with 0.15 mg/kg of trabectedin (n¼ 4, white circles), were killed on day 39 (13 days after the last trabectedin injection) and analyzed by flowcytometry. L, The mean value� SD of the absolute number of monocytic Ly6Cþ Ly6G� gated on CD11bþ F4/80low cells in SP of mice described in K is shown inthe graph.M, The mean value� SD of the absolute number of CD11bþ F4/80þ cells gated on CD45þ in BM is shown in the graph. The mean value� SD of theabsolute number of CD11bþMRC1þ cells (N), of CD11bþ PD-L1þ cells (O), and of CD11bþ IAb low cells (P) to the whole macrophage pool (CD11bþ F4/80þ) gated onCD45þ in BM of mice described in K is shown in graphs.� , P < 0.05; �� , P < 0.01; �� , P < 0.001, Student t test. abs. number, absolute number; Trab, trabectedin.






Figure 7.

In vivo and in vitro antitumor activity of patient-derived T cells and monocytes/macrophages. A and B, Rag2�/�gc�/�mice intravenously injected with MEC1 cells(day 0) were adoptively transferred on day 18 with monocytes (i.v.: 35.000–156.000 cells depending on the patient, including NC, C, and I subsets) or CD8þ

memory cells (i.v.: 36.000–226.000 cells depending on the patient, including TEM and TCM cells) separated from the PBMCs of patients 41–44 (SupplementaryTable S2) and in vitro pretreated with 0.01 mmol/L trabectedin for 15 hours. Mice injected with the same patient sample cells (either treated or untreated, n¼ 4)received the same number of cells and were killed at day 23. C, The mean value� SD of the absolute number of hCD19þ in the SP frommice transplanted withmonocytes (left) and T cells (right) is shown in the graph. � , P < 0.05, Student t test. Trabe, trabectedin; Unt, untreated.D,Monocytes (including NC, C, and I) frompatient 43 and patients 45–49 (n¼ 6) were separated by cell sorting and coincubated with VPD-labeled MEC1 cells at an effector–target ratio of 1:1 in thepresence or absence of 0.01 mmol/L trabectedin for 16 hours. The percentage of viable MEC1 target cells was measured by flow cytometry to determine absolutenumbers of surviving MEC1 cells. The percentage of trabectedin-mediated phagocytosis by CD68þmacrophages and CD14þmonocytes was calculated asdescribed in Materials and Methods. E–H, CD8þ T lymphocytes (including TEM and TCM) from PBMCs of patients 45–49 (n¼ 5) were separated by cell sorting andcoincubated in vitrowith heated-MEC1 cells (at an effector–target ratio of 2:1) to induce tumor antigen–specific T-cell response (Ag-experienced) in the absence(C) or presence (Trab) of 0.01 mmol/L trabectedin for 15 hours. E, Themean value� SD of the absolute number of PD-1þ T cells to the whole CD8þ T-cell poolgated on live cells is shown in the graph. � , P < 0.05; �� , P < 0.001, Student t test. F, Flow-cytometry detection of PD-1 by CD8þ T cells from representativepatient 46. The percentage of PD-1–expressing cells is indicated. G, The mean value� SD of the absolute number of PD-1þ cells to the TEM cell pool gated onCD8þ T cells is shown in the graph. H, Flow-cytometry detection of PD-1 on CD8þ TEM cells from representative patient 46. The percentage of PD-1 expressingcells is indicated. abs. number, absolute number; Trab, trabectedin.

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DiscussionIn this study, we demonstrated that in CLL trabectedin inter-

fered with leukemic cells and nonleukemic myeloid and lym-phoid cells of the microenvironment.

In vitro, trabectedin was directly cytotoxic to human primaryleukemic cells. It upregulated mRNAs for several cell death TRAILand TNF pathway elements such as CASP3 (supporting thecaspase-3 activity seen in CLL, ref. 26), while downregulating themRNA for antiapoptotic MCL1.

Trabectedin depleted selected humanmyeloid cell subtypes byrapidly engaging CASPASES and other cell death pathway ele-ments, corroborating previous findings with CASP8 (23). Themurine counterpart of human CD16þ monocyte subsets is repre-sented by Ly6Clow monocytes (44), which were depleted bytrabectedin in our CLL mouse studies, in a T cell–independentmanner. Inmice, trabectedin depleted Ly6Clowmonocytes expres-sing PD-L1, which inhibits T-cell proliferation and cytotoxicpotential in solid tumors (41). In contrast, trabectedin has aselective activity on Ly6Chigh monocytes in murine fibrosarco-ma (23), implicating a different leukemia-dependent cell mech-anism (45). Patient-derived xenograft and in vitro phagocytosisstudies showed that trabectedin enhanced antitumor functional-ity of monocytes andmacrophages. These trabectedin-dependenteffector cell mechanisms are reminiscent of the immunomodu-latory activity of lenalidomide, a regulator of T-cell and macro-phage function in CLL (33, 46).

Trabectedin had selective activity against human and mouseM-MDSCs, which inhibit T-cell activity (39) and limit antitumoractivity through immunosuppression in CLL (14). In humanin vitro studies and in vivo mouse models, targeting M-MDSCswas associated with reduced expression of transcription factorFOXP3 mRNA and the depletion of immunosuppressive Tregs,which often accumulate in CLL patients and are linked to worseprognosis and advanced disease (47). Trabectedin can break theimmunosuppressive biological cross-talk among CLL cells,MDSCs, and Tregs.

Consistent with data in fibrosarcoma (23), long-term targetingof murine M2-like protumor TAMs was another determinant ofin vivo trabectedin efficacy in CLL mouse models.

Effects other than tumor-associated myeloid cell depletionwere involved in the antitumor activity of trabectedin, includingdownregulation of chemokineCCL2 RNA and protein expressionin all monocyte subtypes. The CCL2/CCR2 axis is involved in therecruitment of myeloid cells at the tumor sites and several anti-tumor strategies interfere with this axis (48). Another effect oftrabectedin was the rapid increase in mRNA expression of proin-flammatory cytokines that promote the classic M1 antitumoractivation of macrophages, inducing T-cell responses and inter-fering with B-cell activity (49). TNFa, IFNa, IL12a, and IL6 RNAand protein were differentially expressed by human classicmono-cytes and M-MDSCs. Cytokines, including IFNa, might beinduced as a byproduct of the DNA-repair mechanisms (50).These transcriptional changes correlated with the induction ofmRNA transcripts for IFNy and GZMB cytolytic factors in humanmemory T lymphocytes. Supporting our in vitro findings inhuman PBMCs, we observed induction of circulating memoryCD4þ andCD8þ T cells in the TCL1 tg immunocompetentmousesystem. Trabectedin effects on T cells were consistent with oste-osarcoma showing that recruitment and expansion of adaptiveT cells were induced by trabectedin (28). However, in our immu-nocompetent mouse system, trabectedin interfered with the

PD-1/PD-L1 axis in the lymphoid tissues by reducing CLL cellsas well as monocytes/macrophages expressing PD-L1, and alsomemory CD8þ T cells expressing PD-1. Unlike for osteosarcomaand pancreatic adenocarcinoma (28, 51), in primary CLL patientcells, trabectedin reduced PD-1 and restored CD8þ T-cell antitu-mor function. Alterations in DNA damage response and repairgenes are associated with response to PD-1/PD-L1 blockade inpatients with metastatic solid tumors (52). In addition, thedownmodulation of PD-L1 we observed on myeloid cells is inline with evidence on the role of PD-1/PD-L1 checkpoint inmonocyte/macrophage remodeling (42) and indicates that tra-bectedin has immunomodulatory activity.

Trabectedin, besides having a cytotoxic effect in leukemic cells,has immunomodulatory activity on several cell types of themicroenvironment. Our findings suggest a therapeutic strategyin which one drug hits both the leukemic cell compartment andthe protumor microenvironment, repairing CLL immune dys-function. Future studies should aim to clarify the molecularmechanisms by which trabectedin functions in the distinctimmune cell types.

Disclosure of Potential Conflicts of InterestM. Aracil is a project leader, R&D, at PharmaMar. C. Klein is a department

head for, has a family member who is an employee of, and has ownershipinterest (including patents) in Roche. No potential conflicts of interest weredisclosed by the other authors.

Authors' ContributionsConception and design: P. Banerjee, M.J. Keating, M.T.S. BertilaccioDevelopment of methodology: P. Banerjee, R. Zhang, K. Clise-Dwyer,M.T.S. BertilaccioAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): P. Banerjee, R. Zhang, K. Clise-Dwyer, L. Scarf�o,M.T.S. BertilaccioAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): P. Banerjee, C. Ivan, G. Galletti, K. Clise-Dwyer,W. Wierda, V. Gandhi, M.T.S. BertilaccioWriting, review, and/or revision of the manuscript: P. Banerjee, R. Zhang,M. Aracil, C. Klein, W. Wierda, W. Plunkett, V. Gandhi, M.J. Keating,M.T.S. BertilaccioAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): F. Barbaglio, C. Klein, M.T.S. BertilaccioStudy supervision: F. Caligaris-Cappio, M.T.S. BertilaccioOther (performed in vitro and in vivo experiments and analyzed the data):G. Galletti

AcknowledgmentsThis study was supported by the CLL Global Research Foundation (toM.T.S.

Bertilaccio andV.Gandhi), TheUniversity of TexasMDAndersonCancer CenterMoon Shots Program (toM.T.S. Bertilaccio, V.Gandhi, andM.J. Keating), Roche(to M.T.S. Bertilaccio), NCI P30CA016672 (to the South Campus Flow Cyto-metry and Cell Sorting Core MD Anderson Cancer Center), and ProgramMolecular ClinicalOncology-5 permille number 9965 (to F. Caligaris-Cappio),Associazione Italiana per la Ricerca sulCancro (AIRC, Italy).Em-TCL1 transgenicmicewere from J. Byrd andC.M.Croce (Columbus,OH). Rag2�/�gc�/�mice onBALB/c backgroundwere provided by CIEA (Kawasaki, Japan) and Taconic. Theauthors thank PharmaMar, S.A., for providing trabectedin, and the Departmentof Scientific Publications, MD Anderson Cancer Center, for reviewing themanuscript and providing constructive comments. Figures were produced usingServier Medical Art: www.servier.com.

The costs of publicationof this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

ReceivedMarch 5, 2019; revised June 14, 2019; accepted September 11, 2019;published first September 17, 2019.





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2019;7:2036-2051. Published OnlineFirst September 17, 2019.Cancer Immunol Res Priyanka Banerjee, Ronghua Zhang, Cristina Ivan, et al. Lymphocytic LeukemiaTrabectedin Reveals a Strategy of Immunomodulation in Chronic

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