Hit 17was obtained by a Virtual Screening on TERRA and DNA G4 (Rocca R., et al. "Identification of G-quadruplex DNA/RNA binders: structure-based virtual screening and biophysical characterization." Biochimica et Biophysica Acta (BBA)-General Subjects 1861.5 (2017): 1329-1340) and purchased from InterBioScreen (InterBioScreen Ltd). Dry powered was dissolved in DMSO at concentration of 10 mM and stored at -20 °C in small aliquots. For in vivo experiments, hit 17 saline solutions were used in one week, stored at -20 °C and avoid more than two defrost cycles.
NCI-H929, RPMI-8226, AMO-1 and AMO-BZB MM cell lines were purchased from DSMZ, certified authentication performed by short tandem repeat DNA typing, were cultured in RPMI-1640 media containing 10% FBS (Corning, Tewksbury MA, USA), 2 μmol/L glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin (Sigma Aldrich, St Louis, MO, USA) at 37°C in a 5% CO2 atmosphere. PBMCs were isolated by Ficoll-hypaque (Lonza Group, Basel, Switzerland) following manufacturer’s protocol.
Analysis of cell viability and apoptosis
Cell viability was evaluated by Cell Titer-Glo assay (Promega, Madison, WI, USA) according to manufacturer’s protocol. Apoptosis was assessed by AttuneTMNxT Flow cytometer (Thermo Fisher Scientific, Inc.) following annexin V-7-aminoactinomycin D staining (BD Pharmingen).
Western blotting analysis and antibodies
Whole cell protein extracts were prepared using NP40 lysis buffer containing Halt Protease Inhibitor cocktail (Thermo Fisher Scientific, Inc.), separated using 4–12% or 3-8%Nupage Bis-Tris SDS-acrylamide gels (Thermo Fisher Scientific, Inc.), and electro-transferred on nitrocellulose membranes (Bio-Rad) by a Trans-Blot® TurboTM Transfer Starter System for 30 min. Then, nitrocellulose membranes were blocked with milk and probed over-night with primary antibodies at 4°C, then membranes were washed 3 times in PBS-Tween, incubated with a secondary antibody conjugated with horseradish peroxidase for 2 hours at room temperature. Chemiluminescence was detected using SuperSignal West Pico PLUS Substrate (Thermo Fisher Scientific, Inc.) and visualized with a C-DiGit® Blot Scanner (LI-COR, version 5.0). WB was performed using Cell Signaling antibodies: p-ATM (#5883S), ATM (#2873S), p-CHK1 (#2348S), p-CHK2 (#21976), CASPASE-3 (#9665S), p21 (#2946S), H3 (#4499S), H3K9me3 (#5327S), H3K27me3 (#9733S), H4K20me3 (#5737S), H4 (#13919S), p-γH2AX(#9718S), RAD51 (#8875S). GAPDH (sc-25778), Actin (sc-58673), PARP1 (sc-8007), TERT (sc-377511) and LIGASE-I (sc-390235) were from Santa Cruz Biotechnology (Dallas, TX, USA).
Quantitative reverse transcription PCR (qRT-PCR)
Total RNA was extracted from cells using TRIzol® reagent (Gibco, Life Technologies, Carlsbad, CA), following the manufacturer’s instructions. The RNA quantity and quality was assessed through NanoDrop® ND-1000 Spectrophotometer. Reverse transcription was performing on 500 ng of total RNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according manufacturer’s protocol. TaqMan assays (Applied Biosystems) were used to detect and quantify the following genes: CDKN1B (Hs01597588_m1), CDKN1A (Hs00355782_m1), LIG1 (Hs01553527_m1), PARP1 (Hs00911376_g1), RAD51 (Hs00153418_m1), GAPDH (Hs02786624 g1). TERRA transcript levels were evaluated from six chromosomes (2q, 7p, 10q, 15q, XpYp, XqYq) amplifying cDNA with Power SYBRTM Green PCR Master Mix (Thermo Fisher Scientific Inc.) and primers reported in Table S2.
Chromatin ImmunoPrecipitation (ChIP)
ChIP was performed using the PierceTM Agarose ChIP Kit (Thermo Fisher Scientific, Inc.). Briefly, 1.5 x107 cells were crosslinked in 1% formaldehyde, lysed and enzymatic digestion with Micrococcal nuclease (MNase). Chromatin was divided into equal amounts of immunoprecipitation with the TRF2 antibody (Abcam, ab13579), or rabbit IgG as negative control (Santa Cruz Biotechnology). Chromatin extracts were incubated on a rotator with 20 µl of ChIP Grade Protein A/G Plus Agarose for 3 h at 4°C. Bound agarose beads were harvested by centrifugation (12.000 rpm, 15 seconds) and washed; the precipitated protein-DNA complexes were eluted from washed beads and incubated twice at 65°C for 1.5 h with NaCl and Proteinase K to revert cross-links. Purified DNA was subjected to qPCR using Power SYBRTM Green PCR Master Mix (Thermo Fisher Scientific Inc.) and telomere primers (Table S2) as previously described . Results are expressed as fold change as compared to IgG set as 1.
Microarray Gene Expression Profiling (GEP)
GEP was performed on NCI-H929 cells after24 or 48hours of exposure with 2.5 µM or 5 µM hit 17, or DMSO as control. TRNA was extracted by RNeasy Mini kit (Qiagen, Hilden, Germany). A total of 100 ng RNA was processed using the GeneChip® WT PLUS Reagent Kit (Applied Biosystems) and hybridized to GeneChip® Clariom D human array (Applied Biosystems) according to manufacture standard procedures. Arrays were washed and stained using the GeneChipTM Fluidics Station 450 and scanned using the GeneChipTM Scanner 3000. CEL files were analyzed by Transcriptome Analysis Console v4.0 (Applied Biosystems). Differential expression analysis was assessed using the Gene Level-SST-RMA Summarization Method, a fold change (FC) ≤ -1.5 or ≥ 1.5 and a p-value < 0.05. Annotation were performed by the Clariom_D_Human.na36.hg38.transcript.csv. Functional characterization of TERRA was performed using the GSEA software version 4.0.2, a tool to enrich the target pathways with statistically significant differences between hit 17 treated versus untreated cells. The gene set files selected from Molecular Signatures Database (MSigDB) and used for this analysis was “hallmark gene sets” browse 50 gene sets that represent specific well-defined biological states or processes. Gene set results were considered significant if the false discovery rate was ≤ 0.25 in a number of 1000 permutations.
Validation of gene expression pattern
Three independent Affymetrix GeneChipTM Human Genome U133 Plus 2.0 datasets were used to validate the biological value of the DNA repair signature. The GSE5900 contains expression data from plasma cells of 22 HD. The GSE19784 includes 320 newly diagnosed MM patients. From GSE19784 we randomly selected a training and three validation cohorts, namely respectively MM1 and MM2, MM3, MM4. The ratio of MM samples included in the training and validation cohorts was approximately 1:1. CEL files from GSE5900 and GSE19784 were downloaded from NCBI Gene Expression Omnibus. All microarray data were normalized by the robust multichip average (RMA) algorithm with the default option using TAC software. We applied a FC ≤ -1.5 or ≥ 1.5 and a p-value < 0.05. GSE19784 sample groups, included and analyzed in this study, are listed in Table S3. The GSE4581 dataset contains 414 untreated MM patients and was used to validate the prognostic signature. Survival analyses were performed with the tool-web GenomicScape (http://www.genomicscape.com).
Animals and in vivo model of Human MM
Male CB-17 NOD.SCID mice (6- to 8-weeks old; Harlan Laboratories, Inc., Indianapolis) were housed and monitored at University Magna Graecia Animal Research Facility. The rational design, the use of mice and the experimental procedures were approved by the National Directorate of Veterinary Services, Italy (n. 1138/2020-PR). Two independent animal experiments were conducted. In the first mice were subcutaneously inoculated into the interscapular area with 5x106 NCI-H929 cells while the second was performed by the use of5x106 AMO-1 cells. When tumors became detectable by palpation (100–200 mm3, approximately 10 days after the injection of MM cells) mice were randomized into 2 groups (5 animals per group) and treated intraperitoneally with 4 mg/kg of hit 17 or saline solution in control group. Tumor sizes were measured as previously described . The investigator was blinded to group allocation of each animal.
Histology and immunohistochemistry
Retrieved tumors from animals were fixed in 4% buffered formaldehyde then, after least 24 hours, washed, dehydrated, and embedded in paraffin. Four-µm thick sections were mounted on poly-lysine slides and stained with hematoxylin-eosin. For light microscopy analysis by an optical microscope AXIO Scope.A1 (Zeiss Oberkochen, Germany). For immunohistochemistry, 3-µm-thick tumor slices were deparaffinized and treated with Epitope Retrieval Solution 2 (EDTA-buffer pH 8.8) at 98°C for 20 min. After washing, peroxidase was blocked by exposing samples to the Bond Polymer for 10 min. All procedures were performed with the Benchmark XT - Automated Immunohistochemistry instrument (Ventana Medical Systems, Oro Valley, AZ, USA). Tissues were washed again and then incubated with the rabbit monoclonal primary antibody CONFIRMTManti-Ki-67 (Ventana, clone 30-9; 1∶150). Tissues were then incubated with DAB-Chromogen (8 min) and slides were counterstained with hematoxylin (12 min).
Each experiment was performed at least three times and values are reported as means ± standard deviations. Comparisons between groups were made with the Student t-test, while statistical significance of differences among multiple groups was determined by GraphPad software (www.graphpad.com). Graphs were obtained using GraphPad Prism version 6.0. P-values less than 0.05 were accepted as statistically significant.