Background
Multiple myeloma (MM) is still an incurable hematologic malignancy characterized by clonal proliferation of malignant plasma cells (PCs) within the bone marrow (BM). Current MM therapy includes triple- or double-drug combination, based on proteasome inhibitors (PIs) and/or immune-modulatory drugs (IMiDs)
plus dexamethasone, with or without chemotherapeutic agents [
1]. Autologous stem cell transplant is reserved to selected patients as consolidation following induction treatment. However, despite recent advancements that significantly improved clinical outcome, patients invariably progress to drug resistance.
DNA repair mechanisms have a crucial role for the maintenance of the genome integrity, and their activation is fine tuned to resolve specific DNA damages. Currently, at least seven DNA repair active systems have been described in MM as protection from different DNA lesions [
2]. Specifically, base excision repair (BER), nucleotide excision repair (NER), and mismatch repair (MMR) pathways are involved in the repair of single-strand DNA damages; homologous recombination (HR), classical non-homologous end joining (c-NHEJ), and alternative NHEJ (a-NHEJ) pathways are conversely involved in double-strand breaks (DSBs), while Fanconi anemia pathway (together with NER and HR) is involved in the repair of interstrand crosslinks [
2,
3]. Dysregulation of these systems has been found to promote tumor progression, cell survival, and development of drug resistance [
2‐
4]. Furthermore, activation of DNA damage response (DDR) has been involved in the upregulation of ligands for activating receptors of natural killer (NK) lymphocytes. Indeed, besides participating in cell cycle control and induction of apoptosis, DDR works as a sensor for cellular stress or transformation, inducing recognition by the immune system [
5,
6].
Genomic instability is a major hallmark of MM and most of the drugs currently used in the treatment of MM have direct genotoxic activity (i.e., melphalan, doxorubicin, cyclophosphamide) or interfere with the DNA repair machinery (PIs or IMiDs) [
2]. Accordingly, these drugs have been reported to trigger the expression of DNAM-1 and NKG2D ligands on MM cells and to induce NK cells activation [
7,
8].
Herein, the expression and prognostic relevance of genes of DNA repair pathways in MM has been investigated. Since overexpression of NER pathway has been found, evaluation of the direct and immune-mediated anti-MM activity of the NER-targeting agent trabectedin in 2D and 3D experimental models of MM has been performed.
Methods
Cell lines, MM primary cells, and drugs
Multiple myeloma cell lines were cultured at 37 °C with 5% CO2. AMO-1, U266, and NCI-H929, SKMM1 were purchased from DSMZ (Braunschweig, Germany). AMO-BZB and AMO-CFZ were kindly provided by Dr. Christoph Driessen (Eberhand Karls University, Tübingen Germany), MM1S and RPMI-8226 were purchased from ATCC (Manassas, VA, USA), and OPM2 and RPMI-8226 DOX40 were kindly provided by Dr. K.C. Anderson (Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA). All these cells were cultured in RPMI-1640 medium (Gibco, Life Technologies) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 100 U/mL penicillin, and 100 μg/mL streptomycin (GIBCO; Thermo Fischer, Carlsbad, CA). U266 and U266 LR7 (kindly provided by Dr. A. Pandiella, Instituto de Biología Molecular y Celular del Cáncer, CSIC-Universidad de Salamanca, Salamanca, Spain) were cultured in RPMI-1640 with 20% FBS and 100 U/mL penicillin, and 100 μg/mL streptomycin. JJN3 were purchased from DSMZ and were cultured in Dulbecco’s modified Eagle medium supplemented with 20% FBS and 100 U/mL penicillin, and 100 μg/mL streptomycin.
NK-92 CI were obtained from NantKwest (Dr. Kerry S. Campbell) and cultured in alpha-MEM medium with ribonucleosides and deoxyribonucleosides (Gibco, Thermo Scientific) supplemented with 10% horse serum, 10% FBS, 0.2 mM myo-inositol (Sigma), 2 mM l-glutamine, 0.1 mM β-mercaptoethanol (Sigma), 0.002 mM folic acid (Fisher Scientific), 1x NEAA (Gibco, Thermo Scientific), 1 mM Na pyruvate (Gibco, Thermo Scientific), 100 U/mL penicillin, and 100 μg/mL streptomycin (Life Technologies) and 100 IU/mL of recombinant human IL-2 (IL-2 improved sequence, Miltenyi).
Primary MM cells were immune-magnetically sorted by using CD138 MicroBeads (MACS, Miltenyi, according to producers’ guidelines) from leftover samples of three MM patients’ bone marrow aspirates after all diagnostic procedures. All patients had provided the informed consent according to institutional bioethical standards, and all the samples have been anonymized before use (institutional approval n. 120/2015, within the project Innovative immunotherapeutic treatments of human cancer, MultiUnit—Multi Unit Regional n.16695).
Human monocytes were obtained by immune magnetical separation from healthy donor peripheral blood mononuclear cells (PBMCs) after Ficoll-Paque density-gradient separation. Specifically, BD IMag™ anti-human CD14 magnetic particles were used for positive selection of CD14+ monocytes according to producers’ guidelines.
Ascorbic acid, as antioxidant agent, was purchased from Sigma-Aldrich (Saint Louis, USA).
Trabectedin (PharmaMar, Madrid, Spain) was reconstituted in DMSO.
Virus generation and transduction of MM cells
MM cells stably expressing miR-17-92 cluster have been gently provided by Dr. E. Morelli. These cells have been obtained by using a PMIRH17-92PA-1 lenti-vector (System Biosciences, Palo Alto, CA, USA) through a methodology already described elsewhere [
9,
10]. Briefly, packaging of the miR-17-92 cluster constructs in pseudoviral particles was performed in 293Ta cells using the Lenti-Pac FIV Expression Packaging Kit (FPKLvTR-20), according to the manufacturer’s instructions (Genecopoeia, Rockville, MD, USA). After transfection of 293 T cells, supernatants containing miR-17-92 lentivirus were collected at 8-h intervals, filtered, and used for two rounds of transduction of U266 cells (1 × 10
6) in the presence of 8 mg/mL of polybrene (Sigma-Aldrich). Two days after transduction, selection with 1 μg/ml puromycin for 3 days was performed to achieve almost 100% transduced cells. Empty lentivirus transduced cells were used as a control for the experiments.
Apoptosis evaluation
MM cells (2 × 105) were treated with dose escalation of trabectedin (0-0.1-0.25-0.5-1-2.5 nM) and analyzed for apoptosis after 24, 48, and 72 h through Annexin V/7-AAD flow cytometry assay (Becton Dickinson). All experiments have been performed at least three times. Primary MM cells were exposed to 2.5 nM of Trabectedin and apoptosis was evaluated after 24 h. Apoptosis was further investigated at molecular level, analyzing the cleaved/total levels of caspase 3 and PARP by Western blot.
Cell cycle analysis
U266 and MM1S (1X106) were cultured in the presence of trabectedin (1 nM and 0.1 nM, respectively) in 6-wells plate for 48 h. Cells were then collected and washed twice with PBS 1X. Subsequently, 1 mL of 70% ice cold ethanol for each sample was added. Cells were stored at − 20 °C until used and then centrifuged and washed twice with PBS 1X. Cells were then resuspended in 1 mL of PI staining solution (100 μg/mL of ribonuclease A, 50 μg/mL of propidium iodide, and 0.01% of NP-40) and incubated for 1 h at room temperature, in the dark. Analysis was performed with flow cytometer and repeated three times.
3D model
We established a 3D in vitro model of MM cell lines alone or in the presence of human monocytes using Matrigel® matrix (Corning). Briefly, 1 × 105 MM cells (U266, OPM2, MM1S) alone or in co-culture with 0.5 × 105 CD14+ monocytes (2:1 MM/monocytes ratio) were resuspended in ice-cold matrigel and a matrigel drop of 35 μL was placed in 24-wells plate coated with a sterile parafilm dish to form a Matrigel-spheroid. After 30 min of incubation at 37 °C, 500 μL of medium with different concentrations of trabectedin was added to each well and the spheroids were incubated for 72 h. Matrigel-spheroids were then resuspended in Dispase (Sigma-Aldrich) and the recovered cells were stained with annexin-V/ 7AAD for analyzing apoptosis induction by flow-cytometry. Supernatants were collected to analyze cytokines expression. Alternatively, Matrigel-spheroids were stored for immunohistochemistry evaluation. All experiments have been performed at least three times.
Immunohistochemistry
Matrigel-spheroids of either tumor cells alone or co-cultured with CD14+ monocytes isolated from healthy donors were fixed in 0.3% glutaraldehyde, then in 4.21% formaldehyde, and subsequently paraffin-embedded. Serial section of 4-μm-thick were cut and mounted on acid-cleaned glass slides, which were dewaxed with xylene, and processed for hematoxylin-eosin staining and immunohistochemistry.
Slides were incubated overnight at 4 °C with anti-g-H2ax monoclonal antibody (Cell Signaling) and anti-cleaved caspase 3 (Santa Cruz Technologies) primary antibodies, washed with PBS three times and incubated with appropriate chromogen-conjugated secondary antibody for 1 h at room temperature. After washings using PBS, samples were observed by an optical microscope and images were acquired.
Single-cell gel electrophoresis (Comet) assay
Comet assay (Trevigen) was performed according to manufacturer’s instructions. Briefly, cells were harvested (1 × 105 cells per pellet), mixed with 200 mL low-melting agarose, and layered onto agarose-coated glass slides. The slides were immersed in lysis solution, and then placed into a horizontal electrophoresis apparatus filled with fresh alkaline or neutral electrophoresis buffer. After electrophoresis (30 min at 1 V/cm tank length), air-dried and neutralized slides were stained with Dapi and kept in a moist chamber in the dark at 4 °C. Images were acquired at × 63 oil immersion with an SP2 Leica Zeiss confocal laser-scanning microscope.
Mitochondrial membrane potential and ROS/superoxide analysis
MM cells (5 × 105) were seeded in 12-wells plate and were incubated for 24 h, untreated or treated with sub-lethal doses of trabectedin (depending on cell line), in the presence or absence of ascorbic acid (25 μM), as antioxidant agent. Trabectedin-induced changes in the production of mitochondrial membrane potential (MMP) and radical oxygen species (ROS) were evaluated by MitoScreen assay (Becton Dickinson) and Total ROS/Superoxides Detection kit (ENZO Life Sciences) respectively, by flow cytometry according to producer’s guidelines.
Flow cytometry and degranulation assay
The expression of the NKG2D and DNAM-1 ligands on different MM cells was evaluated, after 48 h of culture in the presence of trabectedin, by using fluorochrome-conjugated antibodies against MIC-A/B (Becton Dickinson), ULBP 1 (R&D Systems), ULBP 2-5-6 (R&D Systems), PVR (R&D Systems), and NECTIN-2 (Becton Dickinson) according to producer’s guidelines.
NK cell degranulation was evaluated using the CD107a staining. Specifically, trabectedin-treated MM cell lines were washed twice in complete medium and incubated with NK-92 CI cell line at effector/target (E:T) ratio of 1:1, in a U-bottom 96-well plate in complete medium at 37 °C and 5% CO2 in the presence of anti-CD107a/PE (Becton Dickinson) for 2 h. Cells were then stained with anti-CD3/PcP and anti-CD56/APC to identify NK cell population. NK cells positive for CD107a were considered as degranulating/activated cells able to induce cytotoxicity.
All experiments were acquired by an ATTUNE Nxt (Thermo Scientific) flow cytometer. For each sample, at least 1 × 104 events in the gate of interest were acquired.
RNA extraction and quantitative real-time-PCR
Total RNA from MM cells was prepared with TRIzol® Reagent (Life Technologies) according to manufacturer’s instructions. The integrity and quantity of total RNA was assessed using the NanoDrop Spectrophotometer (Thermo Scientific). The single-tube TaqMan miRNA assay (Life Technologies) was used to detect and quantify mature miR-17, miR-18a, miR-19a, miR-19b, miR-20a, miR-92a, performing a real-time polymerase chain reaction (RT-PCR) using TaqMan®Fast Universal PCR Master Mix on a ViiA7 RT reader (Life Technologies). MiRNAs expression was normalized on the RNU44 snoRNA (Life Technologies). Comparative RT–PCR was performed in triplicate, including no-template controls. Relative expression was calculated by using the ∆∆-cycle threshold (CT) method [
11].
Gene-expression profiling
U226 MM cells (3 × 10
6), obtained from two different experiments, were treated with PBS or 2.5 μM of trabectedin for 24 h. Gene expression profiling was performed as described elsewhere [
12]. Briefly, total RNA (tRNA) was extracted through column purification with RNeasy kit (Qiagen, Hilden, Germany). A total of 300 ng RNA was used as starting material for preparing the hybridization target by using the Ambion® WT Expression Kit (Ambion, Life Technologies). The integrity, quality, and quantity of tRNA were assessed by the Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA) and NanoDrop 1000 Spectrophotometer (Thermo Scientific, Wilmington, DE). The amplification of cRNA, the cleanup, and the fragmentation were performed according to the Affymetrix’s procedures. Microarray data were generated by Human transcriptom array 2.0 ST (Affymetrix Inc., Santa Clara, Ca). Arrays were scanned with an Affymetrix GeneChip Scanner 3000. Raw data produced by the Affymetrix Platform (i.e., CEL files) were processed and RMA normalized using Affymetrix Expression Console (EC). Data set has been deposited under the GEO accession number GSE128020.
Gene set enrichment analysis and gene ontology
We used the gene set enrichment analysis (GSEA) [
13] tool to enrich the target pathways with statistically significant differences between trabectedin treated versus untreated cells. Indeed, given a specific gene expression profile sorted by the expression ratio between the two conditions, the target pathway is considered significantly enriched if members are enriched in the top (up-regulated) or bottom (down-regulated) region of the profile. GSEA software then calculates an enrichment score (ES) by using a Kolmogorov-Smirnov test, to measure the degree to which the pathway is enriched in the top-ranked or bottom-ranked region of the profile. Next, ES is normalized (NES) according to the number of genes belonging to the pathways, in order to make comparable pathways with different size. Of note, this process takes into account the contribution of all genes included into the analysis, including those with minimum fold change. GSEA analysis parameters have been set as follows: number of permutations: 1000; permutation type: gene_set; metric for ranking genes: log2 ratio of Classes; size of genesets: 25–500 genes; gene sets evaluated: Hallmark gene sets and C2 curated gene sets from MSigDB.
Additionally, genes upregulated or downregulated with a fold change of at least 1.5 where analyzed with ClueGO, a Cytoscape plug in app that visualizes non-redundant biological terms for large clusters of gene sets in a functionally grouped network [
14,
15].
Western blot
Proteins were extracted from MM cells after lysing in NP40 CellLysis Buffer (Novex) containing a cocktail of protease and phosphatase inhibitors (Thermo Scientific, Waltham, MA). Whole cells lysates (20–30 μg/well) were loaded and separated on 4–12% NovexBis–Tris SDS–acrylamide gels or 3–8% Tris-Acetate Protein Gels (Gibco, Life Technologies). Proteins were then transferred on nitrocellulose membranes by Trans-Blot Turbo Transfer Starter System (Bio-Rad, Berkeley, CA). Subsequently, membranes were blotted with the following primary antibodies: anti-P21, anti-BCL2, anti-RAD51, anti-MCL1, anti-PRO-CASP3, anti-C-CASP3, anti-CDK6, anti-cyclinD1, anti-E2F1, anti-IKZF1, anti-XRCC1, anti-RPA32 (Cell Signaling) and anti-cPARP, anti-PARP, anti-BCL2, anti-IRF4, anti-PTEN, anti-XPF, anti-DDB2, anti-ERCC1, anti-actin, anti-vinculin, and GAPDH (Santa Cruz). To study major signaling checkpoints in response to active DNA damage, a specific commercial kit from Cell Signaling (DNA Damage Antibody Sampler Kit #9947) has been used, as already described in previous work by us and others [
16‐
18]. The kit includes the following primary antibodies: anti-phospho (p)P53 (Ser15), anti-gammaH2AX (Ser139), anti-pATM (Ser1981), anti-pATR (Ser428), anti-pCHEK1 (Ser345), anti-pCHEK2 (Thr68), and anti-pBRCA1 (Ser1524). All these forms are virtually absent in normal conditions and are activated in response to DNA damage to induce an attempt to DNA repair and block cell cycle progression (through, for example, p21). GAPDH expression has been used as a protein loading control for this kit. Blots were then incubated with goat anti-mouse or goat anti-rabbit HRP-conjugated antibodies (Santa Cruz Biotechnology); immunoreactive bands were detected by use of enhanced chemiluminescence (ECL) method, acquired through the C-DIGIT scanner (LI-COR) and quantified by Image Studio Lite 5.0 (LI-COR).
Immunostaining for confocal microscopy
Trabectedin-treated and control MM cells were seeded onto glass coverslips and cytospin for 5 min at 800 rpm was performed. Cells were then washed in PBS, fixed in 4% paraformaldehyde for 12 min, washed three times with PBS, followed by permeabilization with 0.01% Triton-X for 15 min, and again washed in PBS containing 0.5% BSA. Cells were then incubated with anti-g-H2ax monoclonal antibody (cell signaling) overnight at 4 °C, washed with PBS three times, and incubated with Alexa-fluor 488-conjugated secondary antibody (Molecular Probes, Life Technologies, NY) for 1 h at room temperature. Glass coverslips were then washed three times with PBS and mounted with Vecta-Shield mounting media containing DAPI. Samples were visualized and images captured using a Leica microscope. Images were acquired at × 63 oil immersion with an SP2 Leica Zeiss confocal laser-scanning microscope.
Cytokines analysis
A panel of different cytokines including IL1b, IL4, IL6, IL8, IL23, TNF, IFNg, G-CSF, IP10, MCP1, IL10, and VEGF were detected in the supernatants of CD14+ cells alone obtained by PBMCs of healthy donors and co-cultured with MM cells in 3D Matrigel-spheroids, in the presence or absence of Trabectedin, using BD CBA Human Soluble Protein Flex Set system (Becton Dickinson, Heidelberg, Germany). Samples from three different experiments were analyzed with an Attune Nxt Thermo Scientific flow cytometer.
A drop of 50 μL of Matrigel (CORNING) were used to coat 96-wells plates and allowed to polymerize at 37 °C for 30 min. Then, 15 × 10
3 HUVECs were seeded in each well and then 50 μL of conditioned medium from trabectedin-treated cells were added. After 1-h incubation at 37 °C, at least pictures of three representative fields per well were taken using phase contrast microscopy. The tubulogenic potential was quantified by estimating the total tube length and the number of nodal branchpoints (a single pixel connected to three or more pixels), through the “Pipeline 1.4” tool [
19] (
https://sourceforge.net/projects/pipelinetfaanalysis/). All experiments have been performed at least three times.
Gene expression datasets analysis
Datasets of gene expression profiling of MM were retrieved from GEO database (Table
1) or from the MMRF researcher gateway portal (
https://research.themmrf.org). The GSE47552 dataset includes data from 5 healthy donors (HD), 20 patients with MGUS, 33 high-risk sMM, and 41 MM; the GSE39754 includes results from 6 HD and 170 MM; the GSE6477 includes gene expression profiles of 22 MGUS, 24 sMM, 69 newly diagnosed MM, 32 relapsed MM, and 15 healthy subjects; and the GSE13591 dataset contains the gene expression profiles of immunomagnetically purified CD138
+ plasma cells obtained from 5 HD, 11 MGUS, 133 MM, and 9 plasma cell leukemia at diagnosis.
Table 1
Datasets of gene expression profiling of MM retrieved from GEO database
1 | GSE47552 | Affymetrix Human Gene 1.0 ST Array (GPL6244) | Centro de Investigación del Cáncer de Salamanca |
Homo sapiens
| Analysis of plasma cells from patients with monoclonal gammopathy of undetermined significance (MGUS) (n = 20), smoldering multiple myeloma (sMM) (n = 33), symptomatic MM (n = 41), and healthy donors (n = 5). |
2 | GSE39754 | Affymetrix Human Exon 1.0ST Array(GPL5175) | Dana-Farber Cancer Institute |
Homo sapiens
| Gene expression microarray datasets from CD138 purified plasma cells isolated from 170 patients with newly diagnosed MM and 6 healthy subjects. All patients received triple drug regime—Vincristine, Adriamycin, and Dexamethasone (VAD)—as induction therapy followed by autologous stem cell transplant (ASCT) as a maintenance therapy. |
3 | GSE6477 | Affymetrix Human Genome U133A Array (GPL96) | Mayo Clinic |
Homo sapiens
| Gene expression profile of CD138 purified plasma cells from 22 MGUS, 24 sMM, 69 newly diagnosed MM, 32 relapsed MM, and 15 healthy subjects. Each sample has been further characterized by FISH for the identification of hyperdiploidy. |
4 | GSE13591 | Affymetrix Human Genome U133A Array (GPL96) | University of Milan—Fondazione IRCCS Ospedale Maggiore Policlinico |
Homo sapiens
| This series of microarray experiments contains the gene expression profiles of immunomagnetically purified CD138+ plasma cells obtained from 5 normal donors, 11 MGUS, 133 MM, and 9 plasma cell leukemia at diagnosis |
The CoMMpass (Relating Clinical Outcomes in MM to Personal Assessment of Genetic Profile) Trial (NCT0145429), a longitudinal study in MM, relating clinical outcomes to genomic and immune-phenotypic profiles of CD138+ selected plasma cells from the BM of newly diagnosed MM patients (in the release used in this work (interim analysis 8, IA8), RNA-seq, together with clinical data, was available for 549 MM patients).
Datasets including MM cell lines gene expression profiling were retrieved from GEO database with the accession code GSE68379 and GSE6205. These data were normalized in Transcription analysis console (TAC, Thermo Scientific) software and result table processed through R Studio (R version: 3.5).
Statistical analysis
Differences between means were analyzed by using GraphPad statistical package. The results were expressed as the mean ± SD of at least three different experiments, and the significance assessed by the two-tailed Student t test or Mann-Whitney test according to samples distribution. A p value of 0.05 or less was considered statistically significant. Overall survival (OS) and progression-free survival (PFS) analyses (Kaplan-Meier curves and log-rank test) have been performed by using SPSS statistical software on data retrieved by the CoMMpass database.
Discussion
In this work, through a gene expression dataset meta-analysis, we demonstrated that among DNA repair systems, NER is the most upregulated in MM and it is strongly associated with patients’ prognosis. On these bases, for the first time, we decided to evaluate the activity of NER-targeting agent trabectedin in this setting. Trabectedin has been granted approval for the treatment of advanced soft tissue sarcoma and for relapsed ovarian cancer, while several studies are ongoing to evaluate its activity in other malignancies [
28]. In our experimental models, it exerts strong anti-myeloma activity on cell lines and primary cells at nanomolar concentrations, both in conventional 2D and in advanced 3D models. Interestingly, a quick response to trabectedin was associated to a high expression of ERCC1 protein, thus confirming its already known role in trabectedin mechanism of action [
22,
23]. Additionally, trabectedin showed a pleiotropic activity in MM, which includes DNA DSBs generation, cell cycle arrest, exacerbation of cellular stress, reduction of angiogenesis, and immunomodulation (Fig.
5h). Regarding the latters, two different aspects have been of our interest. Several studies showed that trabectedin has significant effects on tumor microenvironment by impairing tumor-associated macrophages [
32,
33]. Accordingly, in 3D Matrigel-spheroids, monocytes promoted MM cells viability and proliferation, which are strongly reduced by trabectedin. In the same setting, trabectedin modulated the pro-inflammatory cytokine/chemokine network by reducing MCP1, VEGF-A, and IL-10 which functionally translated into a decrease of pro-angiogenic potential. These effects are of particular relevance taking into account the role of inflammation and angiogenesis in MM [
12,
45]. A relevant finding is, in our opinion, the triggering activity of trabectedin on the innate immune response against MM by increased expression of NKG2D ligands MICA/B and ULBP1. The relevance of NK response in MM pathogenesis has been deeply investigated [
37,
46,
47], and in patients, a downregulation of surface expression of MICA on malignant plasma cells or a decline in NK-dependent immune-surveillance has been observed when MGUS progresses to symptomatic MM [
48,
49]. Furthermore, the expression of killer cell inhibitory receptor (KIR) ligands, such as MHC class I, increases in advanced disease, impairing the balance between stimulatory and inhibitory signaling to promotion of NK inactivation [
48]. Moreover, most of the anti-MM activity of immune-modulatory agents (IMiDs) has been attributed to their capability to induce proliferation and activation of NK cells [
50]. Along this line, our study provides novel evidence that trabectedin-treated MM cells are more recognizable by innate immune effectors and that the drug strongly induces NK cell activation. These results are consistent with previous evidence reporting that low doses of doxorubicin or melphalan are able to increase NK activating receptors ligands with a mechanism dependent on DNA damage response [
8].
In our work, we further investigated the regulatory network underlying trabectedin-mediated NKG2D ligands upregulation in MM. To this aim, the expression levels of all known MICA/B regulatory factors were evaluated by GEP and validated at protein level. Our results showed that trabectedin increased the expression level of the MICA/B-positive regulator E2F1 and reduced the expression of the negative regulators IRF4 and IKZF1. These findings are in line with recent reports where the phosphorylation of the kinases ATM/ATR, together with the production of ROS, induced the activation of E2F1 that, in turn, could promote MICA, MICB, and PVR transcription [
51]. Additionally, the inhibition of IRF4 and IKZF1/3 by several drugs such as bromodomain and extra terminal domain inhibitors (BETi) and IMiDs has been found to induce MICA and PVR transcription [
7,
52].
The mechanism through which trabectedin could affects E2F1, IRF4, and IKZF1 expression still remains elusive. Several reports in this field suggest the existence of possible networks between all these molecules that could account for the fine and reciprocal regulation we observed in this study [
53‐
56]. Specifically, CUL4A, one of the four components (together with DDB1, CRBN, and DDB2) of a complex ubiquitination machinery responsible for lenalidomide-induced IKZF1 and IKZF3 degradation, has been demonstrated to be necessary for trabectedin activity [
56]. Additionally, we observed an upregulation of three of the four genes of the ubiquitination machinery after trabectedin treatment. However, the strong activation of caspase 3 quickly induced by trabectedin treatment may account for the degradation of IKZF1 [
57], with a mechanism that is independent from CRBN expression (differently from what observed with IMiDs). Both mechanisms could than contribute to IKZF1 downregulation that in turn inhibits IRF4 expression at transcriptional level [
58], reducing the repressive regulation on MICA/B promoter.
On the other side, E2F1 has been demonstrated to be induced in response to several DNA-damaging agents, including UV radiation and a number of chemotherapeutic drugs [
59]. This translates in an increase in protein stability and in some cases apoptosis [
59]. Furthermore, taking into account that reprogramming the immune response requires rapid changes at both transcriptional and post-transcriptional level, we hypothesized a role for miRNAs in finely tuning this regulatory network. By using miRNA target prediction tools, miR-17 family has been identified as the most relevant in MM biology predicted to target at the same time MICA, MICB, and E2F1. We then confirmed that trabectedin downregulates miR-17 and miR-20a in MM cells and that miR-17-92 stable overexpression produced by a lentiviral construct reduces trabectedin-dependent upregulation of NKG2D ligands, making MM cells resistant to drug-induced apoptosis. The latter result appears to be in line with recent findings where miR-17-92 upregulation was found to be associated with resistance to trabectedin [
60]. Additionally, the downregulation of miR-17 and miR-20a may further increase the upregulation of E2F1 [
10], contributing to cell cycle arrest, and MICA/B surface expression. Thus, trabectedin could induce E2F1 upregulation through both DNA damage and miR-17 and miR-20a downregulation. However, the mechanism by which trabectedin reduces miR-17-92 transcription and impairs the miR-17-92/E2F1 auto-regulatory loop [
61,
62] is currently under investigation.
Conclusion
Altogether, our results demonstrated a potent and pleiotropic preclinical activity of trabectedin in multiple myeloma. Specifically, we here demonstrated an overexpression of NER genes in malignant MM plasma cells that strongly correlates with patients’ prognosis. Accordingly, we found that trabectedin exerts cytotoxicity on both MM cell lines (including drug-resistant derivatives) and primary MM patients derived cells at nanomolar concentrations, by inducing apoptosis, as confirmed by upregulation of caspase3 and downregulation of BCL-2, and cell cycle arrest increasing S-phase. Trabectedin also induces ROS production, with activation of stress response pathways, and DNA damage, enhancing the cytotoxic effect on MM cells. Importantly, trabectedin overcomes microenvironment-induced resistance, impairs MM-macrophages-mediated neo-angiogenesis, and induces NKG2D ligands upregulation enhancing NK-mediated killing. On the basis of these findings, trabectedin emerges as a new potential agent for the treatment of MM that deserves further translational and clinical investigation.