Chronic lymphocytic leukemia (CLL) is a B-cell neoplasia characterized by protumor immune dysregulation involving nonmalignant cells of the microenvironment, including T lymphocytes and tumor-associated myeloid cells. Although therapeutic agents have improved treatment options for CLL, many patients still fail to respond. Some patients also show immunosuppression. We have investigated trabectedin, a marine-derived compound with cytotoxic activity on macrophages in solid tumors. Here, we demonstrate that trabectedin induces apoptosis of human primary leukemic cells and also selected myeloid and lymphoid immunosuppressive cells, mainly through the TRAIL/TNF pathway. Trabectedin modulates transcription and translation of IL6, CCL2, and IFNα in myeloid cells and FOXP3 in regulatory T cells. Human memory CD8+ T cells downregulate PD-1 and, along with monocytes, exert in vivo antitumor function. In xenograft and immunocompetent CLL mouse models, trabectedin has antileukemic effects and antitumor impact on the myeloid and lymphoid cells compartment. It depletes myeloid-derived suppressor cells and tumor-associated macrophages and increases memory T cells. Trabectedin also blocks the PD-1/PD-L1 axis by targeting PD-L1+ CLL cells, PD-L1+ monocytes/macrophages, and PD-1+ T cells. Thus, trabectedin behaves as an immunomodulatory drug with potentially attractive therapeutic value in the subversion of the protumor microenvironment and in overcoming chemoimmune resistance.
Introduction of therapeutic agents including kinase and BCL-2 inhibitors has improved treatment options for patients with chronic lymphocytic leukemia (CLL), although some patients fail to respond, become resistant or relapse during treatment (1–3). Patients with lymphoid malignancies receiving the Bruton tyrosine kinase inhibitor ibrutinib are at risk of infection, including lethal invasive fungal infections (4), likely due to the inhibition of innate immune surveillance (5). Fludarabine-based chemoimmune therapy remains the first-line therapy for many patients, although the cytotoxic activity that fludarabine has against leukemic cells is accompanied by damage to normal immune cells, which leads to immune dysfunction and opportunistic infections (6–10). Alternative strategies are needed to bypass mechanisms that contribute to CLL cell resistance and immune dysfunction and to improve CLL patients' quality of life.
Leukemic cells depend on survival signals provided by microenvironment nonneoplastic cells (11). As in solid tumors, these signals enhance the frequency of immunosuppressive cells, including regulatory T cells (Treg; ref. 12), tumor-associated macrophages (13), and myeloid derived suppressor cells (14).
In CLL, T lymphocytes exhibit exhaustion and functional defects (ref. 15; e.g., impaired immunologic synapse formation and reduced cytotoxic capacity).
Clinical trials of CD19 chimeric antigen receptor (CAR) T-cell therapy revealed resistance in CLL patients likely due to T-cell defects that are characteristic of CLL and worsen with disease progression (16).
The CLL microenvironment is characterized by an excess of CD4+CD25+Foxp3+ Tregs that are resistant to apoptosis likely due to high Bcl-2 expression (17). CD4+ T cells seem to provide a niche that favors development and maintenance of the disease (18). Another component of the immune microenvironment is represented by nurse-like cells (NLC) that in vitro promote CLL cell survival mainly through SDF1 (19). NLCs resemble macrophages and have been identified as CLL-specific tumor-associated macrophages (TAM; ref. 20).
We demonstrated in CLL mouse models that TAMs support survival and proliferation of CLL and can be therapeutically targeted by CSF1R signaling blockade to restore leukemic cells' apoptosis sensitivity. Macrophage targeting via CSF1R inhibition reprograms the tumor microenvironment toward an antitumor phenotype by activating effector memory T cells and reducing immunosuppressive cells of the lymphoid and myeloid lineage such as Tregs and monocytic-myeloid derived suppressor cells (M-MDSC; ref. 13). These findings indicate bidirectional cross-talk between CLL cells, MDSCs, and Tregs (14).
These observations led us to investigate ET-743/trabectedin, a DNA binding sea squirt–derived compound, known to target TAMs in solid tumors. Trabectedin is approved for the treatment of soft-tissue sarcoma (21) and ovarian cancer (22). Unlike conventional chemotherapeutic agents (23), trabectedin binds the minor groove of DNA, blocks the cell cycle, affects gene transcription, and DNA-repair pathways (24, 25), and is a transcription-coupled nucleotide excision repair (TC-NER) agent. The targeting of TC-NER induces selective CLL cell death, regardless of genotype or preceding therapies (26). Trabectedin affects transcription regulation of cancer cells and some normal cells of the tumor microenvironment, which produce cytokines and factors promoting tumor growth (25). In solid tumors, trabectedin shows activity against tumor-associated myeloid cells (23, 27) and other immune cells (25, 28). In sarcoma, trabectedin targets cancer cells, shows selective TRAIL-mediated cytotoxicity against human monocytes, and inhibits the production of cytokines such as CCL2 and IL6 (29). We therefore hypothesized that trabectedin synergistically targets both leukemic cells and nonmalignant microenvironment cells. This study uncovered the immunomodulatory function of trabectedin in CLL. We showed that at very low doses, this drug repairs the immune system and kills leukemic cells.
Materials and Methods
Cells and reagents
For cell viability studies, human primary samples were obtained from CLL patients (RAI stage 0–2), who provided informed consent as approved by the Institutional Ethical Committee (protocol VIVI-CLL) of San Raffaele Scientific Institute in accordance with the Declaration of Helsinki. For all the other studies with human blood samples, specimens were obtained from CLL patients (any RAI stages) referred to the Leukemia Department at MD Anderson Cancer Center with the approval of MD Anderson's Institutional Review Board (protocol 2014-0678), in accordance with the Declaration of Helsinki. Written informed consent was obtained from the donors. The clinical and biological features of CLL patients analyzed are described in Supplementary Tables S1 and S2.
MEC1 cells are a CD5low/− CLL cell line established from a CLL patient in prolymphocytoid transformation to B-PLL, obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen (DMSZ) and cultured in RPMI-1640 medium (Invitrogen) with 10% fetal bovine serum and gentamicin (15 μg/mL; Sigma-Aldrich). MEC1 cells have been obtained in 2014 from DMSZ; they were cultured for 1 to 2 weeks and frozen at low passages (5–10). For in vitro and in vivo experiments, MEC1 cells were thawed, cultured for 1 to 2 weeks, and used at 15 to 20 passages. MEC1 cell lines regularly tested negative for Mycoplasma contamination (PCR mycoplasma detection kit, Applied Biological materials Inc.) and have not been reauthenticated in the past year. Trabectedin (Yondelis) was provided by Pharma Mar, S.A., Sociedad Unipersonal. For in vitro studies, trabectedin as pure powder was dissolved in DMSO to 1 mmol/L and kept at −20°C. For in vivo studies, trabectedin was provided as sterile lyophilized powder (including sucrose, potassium dihydrogen phosphate, phosphoric acid, and potassium hydroxide) and dissolved in physiologic solution, following the preparation guide for patient infusion of Yondelis. A monoclonal antibody (mAb) to human CD20 (GA101; ref. 30) was provided by Roche Innovation Center Zurich, Switzerland.
Human primary CD19+ cells and MEC1 cells were seeded in 96-well plates at a concentration of 3 × 106 cells/mL in 0.2 mL of RPMI. Vehicle (DMSO) as control and increasing concentrations of trabectedin (0.001 μmol/L, 0.01 μmol/L, 0.1 μmol/L, 1 μmol/L, 10 μmol/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 mononuclear cells (PBMC) or CD19+ cells from untreated CLL patients were seeded, in triplicate, at 3 × 106 cells/mL in culture medium and treated with trabectedin (0.01 μmol/L) or DMSO vehicle for 24 hours, in the presence or absence of anti-TRAIL-R2 (human, 1 μg/mL) mAb (HS201) from Adipogen AG. Specific percentages of remaining cells in the treated samples were calculated as (the absolute number of cells in treated samples/the absolute number of cells in control samples) × 100. For each condition, the absolute number of remaining cells was calculated as the total number of viable cells (trypan blue exclusion determination) × the percentage of viable cells (flow-cytometry analysis determination). Then, specific cell depletion was calculated as (100 − the specific percentage of remaining cells), as described (13). The flow-cytometry analysis of human myeloid and lymphoid cell types is described below and in Supplementary Tables S3 to S7. For transcriptional studies, fluorescence-activated cell sorting was performed after 15 hours of trabectedin treatment (described below and in Supplementary Table S8).
Human cell purification, flow cytometry, and cell sorting
For cytotoxicity studies, leukemic cells were purified immediately after blood withdrawal, by negative depletion, using a B-lymphocyte enrichment kit (RosetteSep; STEMCELL Technologies). The purity of all preparations was more than 99%, and the cells coexpressed CD19 and CD5 on their cell surfaces as assessed by flow cytometry; preparations were virtually devoid of natural killer cells, T lymphocytes, and monocytes. Phenotype analysis of human MEC1 leukemic cells in xenotransplanted mice was performed with PE-Cy7 Mouse Anti-Human CD19 (J3-119) purchased by Beckman Coulter. Surface expression was analyzed using Cytomics FC500 (Beckman Coulter).
For in vitro cell-depletion assays, 8-color flow-cytometry phenotype analysis of human live myeloid cells and 11-color flow-cytometry phenotype analysis of human live lymphoid cells were 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 Supplementary Tables S3 and S4. Finally, cells were incubated with ammonium chloride solution (STEMCELL Technologies) to lyse red cells. For lymphoid cell Foxp3 detection, surface-stained cells were further fixed and permeabilized using a Treg detection kit (Miltenyi Biotec) and finally stained with an anti-Foxp3 antibody.
For transcriptional and patient-derived xenograft studies, live myeloid cells were isolated by four-way fluorescence-activated cell sorting on a BD FACSAria II (BD Biosciences), after surface staining with the following antibodies: Alexa Fluor 700 mouse anti-human CD66b (G10F5), APC mouse anti-human Lineage Cocktail (CD3/CD19/CD20/CD56; ref. UCHTI, HIB19, 2H7, and 5.1H11), Brilliant Violet 786 mouse anti-human CD14 (MϕP9) purchased by BD Biosciences, PE mouse anti-human CD16 (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 staining with the following antibodies:
eFluor 450 mouse anti-human CD8a (SK1) purchased by eBioscience, PerCP mouse anti-human CD45RO (UCHL1) purchased by BioLegend, PE-Cy7 mouse anti-human CD45RA (L48) purchased by BD Biosciences, Brilliant Violet 605 mouse anti-human CD62L (DREG-56), PE-Dazzle 594 mouse anti-human CD4 (SK3), PE mouse anti-human CD25 (4E3) purchased by Milteny Biotec, APC mouse anti-human CD19 (J3-119) purchased by Beckman Coulter, FITC mouse anti-human CD127 (HIL-7R-M21) purchased by BD Biosciences. Live/Dead Fixable Aqua (Thermo Fisher Scientific) staining was first performed to allow the discrimination of Live/Dead cells.
Samples were analyzed with FCS Express 6 Flow-Cytometry Software (De Novo Software). Monocyte subsets have been identified by a negative exclusion gating strategy (31, 32). We excluded CD66b+ neutrophils, then, using a lineage (Lin) cocktail including mAbs to CD3, CD19, CD20, and CD56, we excluded T cells, B cells, and NK cells, respectively. CD14 and CD16 expression was then used to identify CD14+CD16++ nonclassic (NC), CD14++CD16+ intermediate (I), and CD14++CD16− classic (C) monocytes. T cells have been identified as follows: naïve hCD8+ CD45RO−CD45RA+CD62L+ T cells, central memory hCD8+ CD45RA−CD45RO+CD62L+ TCM, and effector memory hCD8+ CD45RA−CD45RO+CD62L− TEM.
Intracellular cytokine detection on human myeloid cells
For the detection of IFNα, TNFα, IL12, IL6, and CCL2 on human myeloid cells, fresh PBMCs from CLL patients were seeded at 1 × 106 cells/mL in culture medium and treated with trabectedin (0.01 μmol/L) or DMSO vehicle for 15 or 38 hours. Then, they were stimulated in vitro with ionomycin and Brefeldin A, and stained with the following surface antibodies: Alexa Fluor 700 mouse anti-human CD66b (G10F5), APC mouse anti-human Lineage Cocktail (CD3/CD19/CD20/CD56; UCHTI, HIB19, 2H7, and 5.1H11), Brilliant Violet 786 mouse anti-human CD14 (MϕP9) purchased by BD Biosciences, BUV737 mouse anti-human CD16 (3G8) purchased by BD Biosciences, PE mouse anti-human TRAIL-R2 purchased by BioLegend. The IntraPrep Permeabilization Kit (Beckman Coulter) was used for the intracellular detection of the cytokines described in Supplementary Table S5. Live/Dead Fixable Aqua (Thermo Fisher Scientific) staining was first performed to allow the discrimination of Live/Dead cells. Samples were acquired using an LSRFortessa X-20 and analyzed with FCS Express 6 Flow-Cytometry Software.
Violet proliferation dye–based phagocytosis assay
MEC1 cells were labeled with Calcein-violet proliferation dye (VPD; BioLegend) and cocultured with monocytes at an effector–target ratio of 1:1 in the presence or absence of 0.01 μmol/L trabectedin for 16 hours. To distinguish between phagocytosed VPD-positive MEC1 cells and free MEC1 cells, samples were counterstained with the following surface antibodies: anti-human CD66b, anti-human Lineage Cocktail (CD3/CD19/CD20/CD56), anti-human CD14, and anti-human CD68 antibody. The IntraPrep Permeabilization Kit (Beckman Coulter) was used for the intracellular detection of the CD68 macrophage marker. After the exclusion of neutrophils (through CD66b molecule), and then the exclusion of NK/NKT cells, T cells, and B cells (through Lineage Cocktail including CD56, CD3, CD19, CD20 molecules), VPD+ CD14− cells and VPD+ CD68− cells were identified by flow-cytometric analysis. The absolute number of surviving VPD+ CD68− target cells and of VPD+ CD14− target cells were calculated. The percentage of trabectedin-mediated phagocytosis by CD68+ macrophages was calculated using the previously described (33) formula: % trabectedin-mediated phagocytosis = 100 – [(absolute number of surviving VPD+ CD68− MEC1 cells in the presence of trabectedin/absolute number of surviving VPD+ CD68− MEC1 cells in the absence of trabectedin) × 100%]. The percentage of trabectedin-mediated phagocytosis by CD14+ monocytes was calculated using the formula: % trabectedin-mediated phagocytosis = 100 − [(absolute number of surviving VPD+ CD14− MEC1 cells in the presence of trabectedin/absolute number of surviving VPD+ CD14− MEC1 cells in the absence of trabectedin) × 100%]. The antibodies used in this assay are described in Supplementary Table S6.
Human T-cell in vitro functional assay and cytokine detection
CD8+ T lymphocytes (including hCD8+ TEM and hCD8+ TCM) were separated by fluorescence-activated cell sorting, coincubated in vitro at an effector–target ratio of 2:1 with MEC1 cells heated at 47°C for 1 hour to induce tumor-antigen-specific T-cell response (Ag-experienced; ref. 34), in the absence or presence of 0.01 μmol/L trabectedin for 15 hours. The cells were then stimulated in vitro with Brefeldin A (4 hours, 4 μg/mL) and stained with the surface antibodies described in Supplementary Table S7, after LIVE/DEAD fixable Aqua dye incubation and Fc receptor blocking. Surface-stained cells were further fixed and permeabilized using a Staining Buffer kit (Miltenyi Biotec) and finally stained with an anti-IFNγ antibody.
RNA extraction, RT-PCR amplification, and quantitative PCR
RNA extraction from sorted myeloid and lymphoid cells was performed using Mini Kit (QIAGEN). RNA sample quality and quantity checks were performed using an Agilent RNA 6000 Pico Assay (Agilent Technologies) at MD Anderson's Sequencing and Non-Coding RNA Program.
Reverse-transcription reaction from 50 to 200 ng RNA template was done using the RevertAid H Minus First-Strand cDNA Synthesis Kit (Fermentas, Thermo Fisher Scientific) according to the manufacturer's instructions. The sequences of primer pairs specific for each gene sequence (Primm Srl and Sigma-Aldrich) were designed with Beacon Designer (Premier Biosoft International) and NCBI-Primer Designing Tools (Supplementary Table S8).
Real-time PCR was performed using the SYBR Green PCR Master Mix (Applied Biosystems) with forward and reverse primers at a final concentration of 10 μmol/L. Each cDNA (2 μL) was used as template; 12.5 μL of 2× SYBR Green PCR Master Mix were mixed with template and primers. The total reaction volume was 25 μL. Three replicates for each cDNA sample were tested. 7900HT Fast Real-Time PCR System (Applied Biosystems) was used, and cycling conditions were 10 minutes at 95°C (1 cycle); 10 minutes at 95°C (1 cycle); 15 seconds at 95°C plus 1 m at 60°C (40 cycles). Data were normalized to β-actin expression.
All mice were housed and bred in specific pathogen–free animal facilities at San Raffaele Scientific Institute and at The University of Texas MD Anderson Cancer Center. Depending on the animal experiment, mice were treated in accordance with the European Union guidelines and with the approval of the San Raffaele Scientific Institute Institutional Ethical Committee (protocols 601 and 726) or with the approval of the Institutional Animal Care and Use Committee of The University of Texas MD Anderson Cancer Center (protocol 00001627-RN00) and conducted in accordance with the Animal Welfare Act. Rag2−/−γc−/− mice on a BALB/c background were kindly provided by CIEA or purchased by Taconic, Eμ-TCL1 transgenic mice on a C57BL/6 background were kindly provided by Dr. Byrd (The Ohio State University, Columbus, OH), and wild-type C57BL/6 mice were supplied by Taconic.
Eμ-TCL1 transgenic mice were genotyped for the hTCL1 transgene by PCR-based screening assay as described previously (35).
Eight-week-old male Rag2−/−γc−/− mice were i.v. transplanted (day 0) with 10 × 106 MEC1 cells in 0.1 mL of saline through a 27-gauge needle. In some experiments, mice were i.v. injected with 0.15 mg/kg of trabectedin or with saline (control) weekly. Depending on the experiments mice were monitored once per week for weight and humanely killed at different stages of leukemia. Peritoneal exudate (PE), peripheral blood (PB), spleen (SP), and femoral bone marrow (BM) were collected and analyzed.
For preclinical purposes, Rag2−/−γc−/− mice received s.c. injections of 10 × 106 MEC1 cells in 0.1 mL of saline into the left flank on day 0. Once the mice developed palpable subcutaneous tumors, they were i.v. injected with 0.15 mg/kg trabectedin or with saline (control). Mice were monitored once per week for weight and tumor growth (by measuring three perpendicular diameters by a caliper) and killed when the mean subcutaneous tumor volume reached 1,000 mm3 (before reaching clinical signs and symptoms, to avoid unnecessary pain and discomfort according to the ethical guidelines). Measurements were stopped when 75% of the originally treated mice were still surviving. For survival experiments, xenotransplanted mice were intravenously injected with 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-transplanted Rag2−/−γc−/− mice were injected intravenously on day 18 with patient-derived CD8+ memory T cells (treated with 0.01 μmol/L trabectedin for 16 hours in vitro or left untreated) from patients 41 to 43 (Supplementary Table S2) or with patient-derived monocytes (treated with 0.01 μmol/L trabectedin for 16 hours in vitro or left untreated) from patients 41 to 44 (Supplementary Table S2). Mice were humanely killed 5 days after the adoptive transfer. PB, SP, and BM were collected and analyzed.
Eight-week-old male syngeneic immunocompetent C57BL/6 mice were injected i.p. (day 0) with 10 × 106 cells purified from the SP of leukemic male Eμ-TCL1 transgenic mice using the EasySep mouse B-cell enrichment kit (STEMCELL Technologies). The purity of the transplanted CD19+ CD5+ Igκ+ cells was assessed by flow cytometry. Mice were monitored weekly for weight and leukemia development by flow-cytometric analysis of PB samples. Mice were i.v. injected with 0.15 mg/kg trabectedin every week starting when the frequency of CD19+CD5+ leukemic cells in the PB was 15% to 20%, compared with C57BL/6 wild-type mice. Mice were monitored weekly for weight and leukemia development by flow-cytometric analysis of the PB samples and, depending on the last trabectedin injection, humanely killed at different time points. PE, PB, and organs [SP, lymph node (LN), femoral BM] were collected and analyzed.
Murine cell preparations and flow cytometry
PB, PE, SP, and femurs were collected from mice, and cells were isolated. Erythrocytes from BM, PE, SP, and PB samples were lysed by incubation in ammonium chloride solution (NH4Cl) lysis buffer (NH4Cl 0.15 M, KHCO3 10 mmol/L, Na2EDTA 0.1 mmol/L, pH 7.2–7.4) for 5 minutes at room temperature. After blocking of fragment crystallizable (Fc) receptors with Fc block (BD Biosciences) for 10 minutes at room temperature, cells from PB, BM, PE, SP, and LNs were stained with the antibodies (15 minutes at 4°C) listed in Supplementary Tables S9 and S10. Depending on the experiments, cells were analyzed with a Beckman Coulter FC500 or with a BD LSRFortessa X-20 flow cytometer. Ten-color flow-cytometry phenotype analysis of live myeloid cell singlets and 13-color flow-cytometry phenotype analysis of live lymphoid cell singlets were performed using an LSRFortessa X-20. To develop the multicolor flow-cytometry panels, we used antibody-capture beads (UltraComp eBeads Invitrogen) for single-color compensation controls. A Live/Dead Fixable Aqua Dead Cell Stain Kit (Thermo Fisher Scientific) was used first to gate out dead cells. Further gating adjustments were made based on fluorescence-minus-one controls. Absolute cell numbers were obtained by multiplying the percentage of the cells by the total number of splenocytes, mesenteric LNs, peritoneal cells, and BM cells flushed from one femur.
The 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 or differences between experimental groups were assessed with an unpaired two-tailed Student t test (95% confidence interval) and considered statistically significant for P values less than 0.05. Survival curves were compared with the use of the log-rank test.
In cell-depletion experiments that involved different human myeloid and lymphoid cell types, normality was tested with a Shapiro–Wilk normality test. We applied the t test to normally distributed data, otherwise the Mann–Whitney–Wilcoxon test. A box-and-whisker plot [box plot represents first (lower bound) and third (upper bound) quartiles, whiskers represent 1.5 times the interquartile range] was used to visualize the data. The y-axis was plotted 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 significance was defined as a P value less than 0.05.
The outliers at end-point were never excluded. The number of biological and technical replicates for each experiment is detailed in the figure legends. All the experiments were repeated independently and are described in the figures and figure legends. Investigators were not blinded when assessing the analysis of the experimental outcome.
Trabectedin targets both CLL cells and their microenvironment
We first evaluated whether trabectedin affects human primary leukemic cells. As demonstrated previously (26), cytotoxicity studies performed on the MEC1 CLL cell line (Supplementary Fig. S1A) and purified human primary leukemic cells (Fig. 1A; Supplementary Fig. S1B; n = 5) revealed that trabectedin has a toxic effect on leukemic B cells in vitro. We selected the lowest trabectedin dose that reduced leukemic B-cell viability but did not completely kill them (e.g., 0.01 μmol/L; dotted line, Fig. 1A). The direct cytotoxic activity of trabectedin on CLL cells was confirmed in a flow-cytometry cell-depletion assay (Fig. 1B, empty circles). When unfractionated PBMCs of CLL patients (which contained both leukemic and normal hematopoietic cells) were treated with 0.01 μmol/L trabectedin, we observed depletion of both leukemic B cells and CD14+ myeloid cells (Fig. 1B, black circles). This confirmed the drug's cytotoxicity to mononuclear phagocytes demonstrated in solid tumors (23). Leukemic cell depletion induced by trabectedin increased significantly in the PBMCs as compared with purified leukemic cells, suggesting a role for CD14+ cells and other mononuclear cells (Fig. 1B; Supplementary Table S1).
We next evaluated the effect of trabectedin on monocyte subsets, identified by flow cytometry with a negative exclusion gating strategy (31, 32). CD14 and CD16 expression was used to identify CD14+CD16++ nonclassic (NC), CD14++CD16+ intermediate (I), and CD14++CD16− classic (C) monocytes (Fig. 1C, PB; Supplementary Fig. S2A, PBMC). This gating strategy enabled us to better characterize myeloid cells and to avoid nonspecific cell inclusion (31, 32, 36). Monocyte subsets were also screened for the surface expression of HLA-DR protein, because of the demonstrated immunosuppressive activity of HLA-DRlow/− classic monocytes in non-Hodgkin lymphoma patients (ref. 37; Supplementary 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; Supplementary Table S2), including their fractions with increased HLA-DR expression, were not depleted (Supplementary Figs. S2B, and S3A and S3B). Trabectedin also increased the absolute number of CD14++CD16− HLA-DR+ classic monocytes (Supplementary Fig. S4), high frequency of which has been found to correlate with improved overall survival in metastatic melanoma patients (36). These results led us to conclude that trabectedin affects myeloid cells.
We asked whether trabectedin also affects T lymphocytes. Human naïve, effector (TEM), and central memory (TCM) CD8+ T cells were identified based on the differential surface expression of CD45RO, CD45RA, and CD62L. CD4+ regulatory T lymphocytes expressing CD25 and Foxp3 were also analyzed. In addition to depleting the CD19+CD5+ leukemic clone, trabectedin reduced CD4+ Tregs. However, trabectedin did not deplete CD8+ TEM and TCM cells (Fig. 1D and F; Supplementary Tables S11 and S12).
Trabectedin triggers apoptosis of selected immune cells
Based on our findings, we hypothesized that trabectedin directly kills leukemic cells and tumor-supporting immune cells including CD4+ Tregs and some myeloid cell subtypes (M-MDSCs and I monocytes), and favors the activation of cytotoxic T lymphocytes.
Trabectedin's effects on transcription regulation affect both cancer cells and normal cells of the malignant microenvironment (25), but the effect of the drug on immune cell types from leukemic patients is unexplored. Therefore, we analyzed RNA from CLL patient lymphoid and myeloid cell types after 15 hours of trabectedin treatment. By means of two independent four-way fluorescence-activated cell-sorting strategies, we separated myeloid subsets including C, NC, I, M-MDSCs, and some of B-T cell types described in Fig. 1D, including leukemic CD19+CD5+ B cells, CD8+ TEM and TCM cells and Tregs. For RNA studies, CD4+ CD25+ Tregs were identified and sorted based on low-negative expression of CD127.
We then evaluated by RT-PCR the expression of RNA encoding effector molecules involved in TRAIL-, TNF-, and FAS/FASL-regulated death pathways on cell types depleted by trabectedin (Fig. 1D), including leukemic B cells, regulatory CD4+ T cells, I monocytes, and M-MDSCs. As described in Fig. 2, we observed upregulation of molecules involved in TRAIL and TNF pathways, including TRAIL-R2, TNFR1, BAX, BID, CASP3 transcripts in all the trabectedin-targeted cells types but not in memory CD8+ T cells (Supplementary Fig. S5). We confirmed the involvement of the TRAIL pathway by using a blocking anti-human TRAIL-R2 mAb. In 7 of 8 samples (Supplementary Fig. S6), the leukemic cell depletion induced by trabectedin was reduced when TRAIL signaling was blocked. Consistent with reported TRAIL-mediated activity of trabectedin in monocytes in solid tumors (23), we confirmed by flow cytometry that trabectedin modulated TRAIL-R2 receptor expression on monocytes and M-MDSCs (Supplementary Fig. S7A). We also observed the involvement of TRAIL-mediated cell death pathway in T cells treated with trabectedin (Fig. 2; Supplementary Fig. S6).
Trabectedin has immunomodulatory activity in vitro
We next evaluated the immunomodulatory activity of trabectedin on myeloid and lymphoid cell types. Trabectedin modified RNA expression of proinflammatory cytokines that promote the classic (M1) antitumor activation of macrophages (e.g., IFNα and TNFα), induce T-cell cytotoxic response (e.g., IFNα and IL12a), and control B lymphocytes (e.g., IL6; Fig. 3A). Expression of TNFα RNA was also upregulated in M-MDSCs (Fig. 3B).
Expression of RNA encoding CCL2, a monocyte chemoattractant, was reduced in C, I, and NC monocytes (Fig. 3A, C, and D), as demonstrated in solid tumors (23, 27). In a different set of CLL patient samples, we confirmed altered production of IL6, CCL2, and IFNα protein upon 38 hours of trabectedin treatment (Supplementary Fig. S7A–S7C). Patients 39 and 40 carried a 17p deletion and patient 40 (in red) was in Richter transformation, resistant to ibrutinib and venetoclax (Supplementary Table S2). In some patient PBMC samples, M-MDSCs disappeared after trabectedin treatment.
In Tregs, trabectedin downregulated FOXP3, the transcription factor that modulates the functionality of these cells and abolishes their suppressor activity (Fig. 3E). Unlike leukemic B lymphocytes (26), we did not observe TC-NER and homologous recombination (HR) pathways involvement in the selective targeting of Tregs by trabectedin (Supplementary Fig. S8).
The reduced immunosuppression was accompanied by upregulation of RNA encoding cytolytic factors in CD8+ TEM and TCM, including IFNy and GRANZYME B (GZMB; Fig. 3F and G).
Thus, trabectedin enhances the antitumor phenotype of monocytes and reprograms the tumor microenvironment toward immune effector T-cell cytotoxicity.
Trabectedin has therapeutic efficacy in CLL mouse models
We 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 MEC1 tumor growth (Fig. 4B) and induced a significant reduction of hCD19+ leukemic cells in lymphoid tissues, including BM and SP (SP; Fig. 4C).
We next performed a time-course experiment in which mice, transplanted intravenously with MEC1 cells, were sacrificed 1 or 10 days after the last of three trabectedin injections (days 27 and 36, respectively; Fig. 4D). The treatment reduced the leukemic expansion and stabilized the disease in the PB and lymphoid tissues, including SP and BM (Fig. 4E–G). Trabectedin also conveyed a survival benefit as both a single agent (Fig. 4H and I; P = 0.0009, trabectedin vs. untreated) and in combination with GA101, a glycoengineered type II CD20 mAb, GA101 (Fig. 4J and K).
Trabectedin affects immune cells of the tumor microenvironment in vivo
We tested whether trabectedin affected myeloid cells in CLL mouse models, including the xeno- and the TCL1 transgenic-transplantation systems (13). The antileukemic effect observed in xenotransplanted mice sacrificed at day 27, the day after the last injection of the drug (Fig. 4D–G), was associated with a significant reduction of CD11b+ F4/80+ TAMs in the SP and PE, with selective depletion of MRC1+ M2-like protumor TAMs in the PE (Supplementary Fig. S9A–S9E).
We next used a CLL immunocompetent mouse model, based on the transplantation of leukemic cells purified from the SP of Eμ-TCL1 transgenic (tg) mice into syngeneic C57/BL6 recipients (13). To evaluate the immune cell dynamics induced by trabectedin and their persistence over time, we performed three sets of experiments in which mice transplanted with leukemic cells from Eμ-TCL1 tg donors #1, #2, #3 were sacrificed 6, 9, or 13 days after the last trabectedin injection. When donor #1-transplanted mice were treated with trabectedin (starting at day 40) and sacrificed 6 days after the last injection of the drug (day 53), a significant reduction of the leukemic clone in the SP, BM, PE but not in the PB was observed (Supplementary Fig. S10A–S10D). This reduction was associated with decreased frequencies of CD11b+ CSF1R+ monocytes and F4/80+ macrophages and with selective depletion of PD-L1+ monocytes and MRC1+ and PD-L1+ M2-like TAMs in the PE (Supplementary Fig. S10E–S10I). Among monocytes, we observed a reduction of the Ly6Clow cell component expressing PD-L1 in the SP (Supplementary Fig. S10J): such a reduction suppresses T-cell immunity in solid tumors (41). Overall, these data are in line with findings on the PD-L1–mediated functional remodeling of monocytes and macrophages in colon and mammary carcinoma (42). We observed a decrease in the whole pool of Gr1+ MDSCs in the PB, BM, and SP (Supplementary Fig. S10K–S10M), an increase of CD8+ T lymphocytes in the BM and SP and an increase of CD8+ memory T cells in the PB and BM (Supplementary Fig. S10N–S10Q).
To clarify the long-term cell dynamics induced by trabectedin in vivo, we transplanted mice with splenic leukemic cells from TCL1 tg donor mouse #2 or donor mouse #3 and sacrificed them 9 (Fig. 5A, donor #2 recipients) and 13 days (Fig. 5B, donor #3 recipients) after the last trabectedin injection, respectively. In donor #2 recipient mice treated with trabectedin starting on day 25 (Fig. 5A), a significantly lower percentage of leukemic B cells in the PB was observed over time (Fig. 5C). Upon necropsy, comparative analysis of the treated and untreated mice confirmed 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 selectively depleted CD19+CD5+ leukemic cells expressing PD-L1 in the lymphoid tissues and PE (Supplementary Fig. S11A–S11C) of donor #3 recipient mice killed 13 days after the last trabectedin injection.
As for the myeloid cells, in mice sacrificed 9 days after the last trabectedin injection (Fig. 5A, donor #2 recipients), numbers of CSF1R+ monocytes (Fig. 5H) and CD11b+ PD-L1+ (Fig. 5I) monocytes were reduced in the BM, and the Ly6Clow subset was the most affected and significantly reduced in the BM and PB (Fig. 5J and K). Conversely, Ly6Chigh monocytes increased in the SP of treated mice (Supplementary Fig. S12A and S12B). In mice sacrificed 13 days after the last trabectedin injection (Fig. 5B, donor #3 recipients), the whole pool of monocytes (Fig. 5L and M), CD11b+ PD-L1+ monocytes (Fig. 5N and O), and Ly6Clow subset (Fig. 5P and Q) was reduced in the BM and PE of treated mice.
As for M-MDSCs and TAMs, we confirmed (Fig. 6A, donor #2 recipients) the reduced frequency of M-MDSCs in the PB, BM, SP, LN, and PE (Fig. 6B–F) and that of MRC1+, PD-L1+, and IAblow protumor TAMs in the BM (Fig. 6G–J). M-MDSCs remained reduced in the SP 13 days after the last trabectedin injection (Fig. 6K and L, donor #3 recipients), as did CD11b+ F4/80+ TAMs and MRC1+, PD-L1+, and IAblow protumor TAMs in the BM (Fig. 6M–P), SP (Supplementary Fig. S13A–S13E), and PE (Supplementary Fig. S13F and S13G).
In treated mice, myeloid cell targeting by trabectedin was associated with increased frequencies of CD8+, CD4+, CD8+ TCM, CD4+ TEM, and TCM in the PB (Supplementary Fig. S14A–S14E) and decreased frequency of potentially regulatory CD4+ CD25+ Tregs in the mesenteric LNs (Supplementary Fig. S14F). Thirteen days after last trabectedin treatment (Supplementary Fig. S14G, donor #3 recipients), CD8+ and CD4+ TEM cells were maintained at higher frequencies in circulation (Supplementary Fig. S14H–S14J), and these frequencies were associated with a significant reduction of CD4+ CD25+ T cells in the PE, SP, and LN (Supplementary Fig. S14K–S14M). These results are in line with the reduction of PD-1+ CD8+ memory T cells we observed in the BM and LN of trabectedin-treated mice sacrificed 9 (Supplementary Fig. S15A, donor #2 recipients) or 13 (Supplementary Fig. 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 on nonleukemic immune cells, we transplanted patient-derived monocytes or CD8+ memory T cells into leukemic MEC1-transplanted Rag2−/−γc−/− mice. Using fluorescence-activated cell sorting, we separated monocytes (including NC, I, and C) from patients 41 to 44 (Supplementary Table S2) and T cells (including CD8+ TEM and TCM) from patients 41 to 43 (Supplementary Table S2). After 16 hours of in vitro trabectedin treatment (0.01 μmol/L), monocytes or CD8+ T cells were adoptively transferred into xenotransplanted mice (Fig. 7A and B). Compared with mice injected with untreated T cells and monocytes, mice injected with trabectedin-treated T cells and monocytes benefited from significant antileukemic effects in the SP (Fig. 7C).
The functionality of CLL patient-derived CD14+ monocytes and CD68+ macrophages upon trabectedin was confirmed in vitro in a different set of patient samples by means of a VPD-based assay. The number of not-phagocytosed VPD-labeled CD14−/VPD+ MEC1 target cells or CD68−/VPD+ MEC1 target cells treated or not with trabectedin was quantified by flow cytometry. In most patient samples tested, trabectedin did not impair the phagocytic ability of monocytes and macrophages (Fig. 7D).
To confirm T-cell functionality upon trabectedin treatment in vitro, CD8+ memory T cells, in the absence or presence of antigen (Ag) stimulation with heated-MEC1 cells, were treated with 0.01 μmol/L trabectedin and then assessed for their expression of PD-1 checkpoint markers and ability to produce cytolytic proteins. Upon trabectedin treatment, absolute numbers of total CD8+ and CD8+ TEM cells were not affected (Supplementary Fig. S16A and S16B), whereas PD-1+ CD8+ and PD-1+ TEM cells were reduced significantly (Fig. 7E–H). CD8+ TEM cells retained their capacity for IFNγ and GRANZYME B production in both the presence and absence of trabectedin (Supplementary Fig. S16C and S16D). Indeed, T cells from CLL patients have increased expression of the PD-1 exhaustion marker but retain their ability to produce cytokines (43). We demonstrated that trabectedin restores T-cell functionality, likely interfering with PD-1 checkpoint molecule.
Overall, these findings indicate that trabectedin inhibits leukemia progression with a cascade of immunomodulatory effects in immune cell types of the malignant microenvironment (Supplementary Fig. S17).
In this study, we demonstrated that in CLL trabectedin interfered with leukemic cells and nonleukemic myeloid and lymphoid cells of the microenvironment.
In vitro, trabectedin was directly cytotoxic to human primary leukemic cells. It upregulated mRNAs for several cell death TRAIL and TNF pathway elements such as CASP3 (supporting the caspase-3 activity seen in CLL, ref. 26), while downregulating the mRNA for antiapoptotic MCL1.
Trabectedin depleted selected human myeloid cell subtypes by rapidly engaging CASPASES and other cell death pathway elements, corroborating previous findings with CASP8 (23). The murine counterpart of human CD16+ monocyte subsets is represented by Ly6Clow monocytes (44), which were depleted by trabectedin in our CLL mouse studies, in a T cell–independent manner. In mice, trabectedin depleted Ly6Clow monocytes expressing PD-L1, which inhibits T-cell proliferation and cytotoxic potential in solid tumors (41). In contrast, trabectedin has a selective activity on Ly6Chigh monocytes in murine fibrosarcoma (23), implicating a different leukemia-dependent cell mechanism (45). Patient-derived xenograft and in vitro phagocytosis studies showed that trabectedin enhanced antitumor functionality of monocytes and macrophages. These trabectedin-dependent effector cell mechanisms are reminiscent of the immunomodulatory activity of lenalidomide, a regulator of T-cell and macrophage function in CLL (33, 46).
Trabectedin had selective activity against human and mouse M-MDSCs, which inhibit T-cell activity (39) and limit antitumor activity through immunosuppression in CLL (14). In human in vitro studies and in vivo mouse models, targeting M-MDSCs was associated with reduced expression of transcription factor FOXP3 mRNA and the depletion of immunosuppressive Tregs, which often accumulate in CLL patients and are linked to worse prognosis and advanced disease (47). Trabectedin can break the immunosuppressive biological cross-talk among CLL cells, MDSCs, and Tregs.
Consistent with data in fibrosarcoma (23), long-term targeting of murine M2-like protumor TAMs was another determinant of in vivo trabectedin efficacy in CLL mouse models.
Effects other than tumor-associated myeloid cell depletion were involved in the antitumor activity of trabectedin, including downregulation of chemokine CCL2 RNA and protein expression in all monocyte subtypes. The CCL2/CCR2 axis is involved in the recruitment of myeloid cells at the tumor sites and several antitumor strategies interfere with this axis (48). Another effect of trabectedin was the rapid increase in mRNA expression of proinflammatory cytokines that promote the classic M1 antitumor activation of macrophages, inducing T-cell responses and interfering with B-cell activity (49). TNFα, IFNα, IL12a, and IL6 RNA and protein were differentially expressed by human classic monocytes and M-MDSCs. Cytokines, including IFNα, might be induced as a byproduct of the DNA-repair mechanisms (50). These transcriptional changes correlated with the induction of mRNA transcripts for IFNy and GZMB cytolytic factors in human memory T lymphocytes. Supporting our in vitro findings in human PBMCs, we observed induction of circulating memory CD4+ and CD8+ T cells in the TCL1 tg immunocompetent mouse system. Trabectedin effects on T cells were consistent with osteosarcoma showing that recruitment and expansion of adaptive T cells were induced by trabectedin (28). However, in our immunocompetent mouse system, trabectedin interfered with the PD-1/PD-L1 axis in the lymphoid tissues by reducing CLL cells as well as monocytes/macrophages expressing PD-L1, and also memory CD8+ T cells expressing PD-1. Unlike for osteosarcoma and pancreatic adenocarcinoma (28, 51), in primary CLL patient cells, trabectedin reduced PD-1 and restored CD8+ T-cell antitumor function. Alterations in DNA damage response and repair genes are associated with response to PD-1/PD-L1 blockade in patients with metastatic solid tumors (52). In addition, the downmodulation of PD-L1 we observed on myeloid cells is in line with evidence on the role of PD-1/PD-L1 checkpoint in monocyte/macrophage remodeling (42) and indicates that trabectedin has immunomodulatory activity.
Trabectedin, besides having a cytotoxic effect in leukemic cells, has immunomodulatory activity on several cell types of the microenvironment. Our findings suggest a therapeutic strategy in which one drug hits both the leukemic cell compartment and the protumor microenvironment, repairing CLL immune dysfunction. Future studies should aim to clarify the molecular mechanisms by which trabectedin functions in the distinct immune cell types.
Disclosure of Potential Conflicts of Interest
M. 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 ownership interest (including patents) in Roche. No potential conflicts of interest were disclosed by the other authors.
Conception and design: P. Banerjee, M.J. Keating, M.T.S. Bertilaccio
Development of methodology: P. Banerjee, R. Zhang, K. Clise-Dwyer, M.T.S. Bertilaccio
Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): P. Banerjee, R. Zhang, K. Clise-Dwyer, L. Scarfò, M.T.S. Bertilaccio
Analysis 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. Bertilaccio
Writing, 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. Bertilaccio
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): F. Barbaglio, C. Klein, M.T.S. Bertilaccio
Study supervision: F. Caligaris-Cappio, M.T.S. Bertilaccio
Other (performed in vitro and in vivo experiments and analyzed the data): G. Galletti
This study was supported by the CLL Global Research Foundation (to M.T.S. Bertilaccio and V. Gandhi), The University of Texas MD Anderson Cancer Center Moon Shots Program (to M.T.S. Bertilaccio, V. Gandhi, and M.J. Keating), Roche (to M.T.S. Bertilaccio), NCI P30CA016672 (to the South Campus Flow Cytometry and Cell Sorting Core MD Anderson Cancer Center), and Program Molecular Clinical Oncology-5 per mille number 9965 (to F. Caligaris-Cappio), Associazione Italiana per la Ricerca sul Cancro (AIRC, Italy). Eμ-TCL1 transgenic mice were from J. Byrd and C.M. Croce (Columbus, OH). Rag2−/−γc−/− mice on BALB/c background were provided by CIEA (Kawasaki, Japan) and Taconic. The authors thank PharmaMar, S.A., for providing trabectedin, and the Department of Scientific Publications, MD Anderson Cancer Center, for reviewing the manuscript and providing constructive comments. Figures were produced using Servier Medical Art: www.servier.com.
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