TERRA G-quadruplex stabilization as a new therapeutic strategy for multiple myeloma

doi:10.21203/rs.3.rs-1627112/v1

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Research Article

TERRA G-quadruplex stabilization as a new therapeutic strategy for multiple myeloma

https://doi.org/10.21203/rs.3.rs-1627112/v1

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Journal Publication

published 27 Mar, 2023

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Background

Multiple Myeloma (MM) is a hematologic malignancy characterized by high genomic instability and telomere dysfunction is an important cause of acquired genomic alterations. Telomeric repeat-containing RNAs (TERRAs) transcripts are long non-coding RNAs (lncRNAs) involved in telomere stability through the interaction with shelterin complex. Dysregulation of TERRAs has been reported across several cancer types. We recently identified a small molecule, hit 17, which stabilizes the secondary structure of TERRA. In this study, we investigated in vitro and in vivo anti-MM activities of hit 17.

Methods

Anti-proliferative activity of hit 17 was evaluated in different MM cell lines by cell proliferation assay and apoptotic process was analyzed by flow cytometry. Gene and protein expression were detected by qRT-PCR and western blot, respectively. Microarray analysis was used to analyze transcriptome profile. The effect of hit 17 on telomeric structure was evaluated by chromatin immunoprecipitation (ChIP). Further evaluation in vivo was proceeded upon NCI-H929 and AMO-1 xenograft models.

Results

TERRA G4 stabilization induced in vitro dissociation of telomeric repeat-binding factor (TRF2) from telomeres leading to the activation of ATM-dependent DNA damage response, cell cycle arrest, proliferation block, and apoptotic death in MM cell lines. In addition, up-regulation of TERRA transcription was observed upon DNA damage and TRF2 loss. Transcriptome analysis followed by GSEA confirmed the involvement of the above-mentioned processes and other pathways such as E2F, MYC, oxidative phosphorylation, and DNA repair genes as early events following hit 17-induced TERRA stabilization. Moreover, hit 17 exerted anti-tumor activity against MM xenograft models.

Conclusion

Our findings provide evidence that targeting TERRA by hit 17 could represent a promising strategy for a novel therapeutic approach to MM.

multiple myeloma

TERRA

telomere dysfunction

TRF2

small molecule

DNA damage

genomic instability

long non-coding RNA

lncRNAs

Multiple myeloma (MM) is a plasma cell malignancy characterized by high genomic instability [1] that leads to progressive accumulation of genomic alterations which occur starting from pre-malignant stages of monoclonal gammopathy of undetermined significance (MGUS) and smoldering MM (SMM) to clinical overt MM [2]. Telomere dysfunction is an important source of genomic instability [3]. Telomeres are loop structures localized at chromosome ends and consisting of six nucleotide repetitive sequences (5′-TTAGGG-3′) associated with a group of shelterin proteins [4]. The maintenance of telomere architecture is crucial to prevent its targeting by DNA damage repair machinery and chromosome end-to-end fusion or structural rearrangements at chromosome ends. Telomeric repeat-containing RNAs (TERRA) transcripts are a class of long non-coding RNAs (lncRNAs) transcribed by RNA polymerase II (RNAPII) from the sub-telomeric to the partial telomeric regions of different chromosomes [5]. These lncRNAs are known to form G-quadruplex secondary structures (G4) which contain square planar alignment of four guanines connected by Hoogsteen hydrogen bonds and stabilized by a monovalent cation such as K⁺ and Na⁺ [6]. TERRA G4 actively participates in genome stability by interacting with the glycine-arginine rich (GAR) domain of the shelterin protein TRF2 [7, 8] that is required for t-loop formation and maintenance [9]. TERRAs are also negative regulators of human telomerase [10] and contribute to heterochromatin formation by their interaction with the histone methyltransferase Polycomb Repressive Complex 2 (PRC2) [11]. Transcriptional dysregulation of TERRA has been reported across several cancer types although with different trends. Notably, deregulation of TERRA has been observed in some studies, thus indicating that TERRA transcripts may act as tumor suppressors [12–14]. However, the role of TERRA in MM is still unclear. In the last years, one approach used for the investigation of lncRNAs in human cancer was based on the design of small molecules that are directed toward its secondary structures [15]. In particular, the targeting of telomeric components with G4 ligands has emerged as a therapeutic approach in human malignancies [16–19]. We recently reported that a benzofuran derivative named hit 17, identified by a virtual screening of small molecule libraries, can physically interact and stabilize TERRAs G4 [20]. To investigate the functional role of TERRAs in MM, we investigated in vitro and in vivo activity of the small molecule hit 17.

Hit 17 suppresses proliferation and induces apoptosis in MM cells

We first explored the role of TERRA in MM at mRNA level. Due to the absence of TERRA expression in public data repositories, we performed quantitative reverse transcription PCR (qRT-PCR) in a pilot cohort of patients including four MM patients and four healthy donors (HD). We used six different couple of primers, each specifically designed to detect TERRA transcripts from six different chromosome regions (2q, 7p, 10q, 15q, XpYp and XqYq). We observed a decreased expression of TERRA in plasma cells from MM as compared to peripheral blood mononuclear cells (PBMCs) from normal donors (Fig. 1A). We further examined the biological effect of TERRA G4 stabilization mediated by hit 17 on growth of MM cell lines. A panel of four MM cells, including bortezomib-resistant ABZB cells, were cultured for 48 hours at different concentrations of hit 17 (1µM, 2.5 µM, 5 µM). We observed that hit 17 exerted an anti-proliferative effect in all MM cell lines, showing IC₅₀ (half-maximal inhibitory concentration) values ranging from 2.5 to 5 µM at 48 hours (Fig. 1B). Moreover, to investigate the sensitivity of normal cells to hit 17, we evaluated cell viability in PBMCs from ten HD, and we did not reveal any cytotoxic effect in the same range of IC₅₀, suggesting a MM-specific vulnerability by this compound (Fig. 1C). To delineate the mechanism of cell death induced by hit 17 in MM cells, NCI-H929 and AMO-1 cells were treated with different concentrations of hit 17 and apoptosis was evaluated. As shown in Fig. 1D and 1E, hit 17 induced apoptosis in both cell lines with a dose-dependent increase of around 4-fold at 5 µM treatment respect to the control. This alteration was confirmed by western blot (WB) analysis showing an increase of cleaved caspase-3 and cleaved PARP1 in NCI-H929 cells (Fig. 1F).

TERRA G4 stabilization by hit 17 inhibits TERT and increases histone trimethylation marks at telomeric heterochromatin

TERRA transcripts repress telomerase activity and maintain a heterochromatic state at the ends of chromosomes through the interaction with PRC2. TERRA-PRC2 complex promotes deposition of H3K9me3, H4K20me3, H3K27me3 and HP1 protein to chromosome ends. To assess whether hit 17 modulates downstream targets of TERRA G4, by WB we evaluated the expression of the human telomerase subunit TERT, and the trimethylation of histone H3 (lysine 9 and 27) and histone H4 (lysine 20). We observed a significant dose-dependent decrease in human TERT (hTERT) protein level (Fig. 2A), while all three histone trimethylation marks resulted increased in both NCI-H929 and AMO-1 cell lines (Fig. 2B). These results demonstrate that hit 17 is a potent inhibitor of telomerase in vitro and a promoter of PRC2 activity.

Hit 17 induces dissociation of TRF2 from telomeres and enhances TERRA level

TERRA is known to interact with TRF2 increasing its specific binding to telomeric DNA [8]. To understand the effect of hit 17 on TRF2 recruitment on telomeres, we performed chromatin immunoprecipitation (ChIP) on the NCI-H929 cell line after small molecule treatment at 48 hours of 2.5 and 5 µM. As shown in Fig. 2C, we observed a dose-dependent TRF2 dissociation of 2-fold change from telomeres, consistent with previously reported observations [21], while no modulation of TRF2 expression levels was observed (Fig.S1). In addition, to analyze differences in TERRA expression following treatment, we performed qRT-PCR in MM cell lines and observed that TERRA levels increased after hit 17 treatments with remarkable fold change at the dose of 5 µM ranging from 2-fold to 10-fold (Fig. 3A-D). These results are in line with previous data reporting that TRF2 directly suppresses TERRA transcription through its homodimerization domain. When TRF2 is removed, the higher-order telomere structures are disrupted and the telomeric chromatin becomes accessible to RNAPII for TERRA transcription [22].

ATM activation by hit 17 induces DNA damage and cell cycle inhibitors

Activation of ATM also takes place in response to telomere damage. When telomeres become uncapped due to inhibition of TRF2 binding, phosphorylated ATM associates with telomeres [23, 24] and actives DNA damage response. To assess the possible changes induced by hit 17 treatments in this signaling pathway, we analyzed the expression levels of ATM by WB along with its downstream targets, including the cell cycle inhibitors cyclin-dependent kinase inhibitor 1A (CDKN1A, also known as p21) and cyclin-dependent kinase inhibitor 1B (CDKN1B, p27). As reported in Fig. 4A-C, hit 17 treatments in MM cell lines induced a dose-dependent increase of phosphorylated ATM (p-ATM), CHEK1 (p-Chk1), CHEK2 (p-Chk2) and H2AX (γH2AX) suggesting the activation of DNA damage response pathway. In addition, we observed by qRT-PCR a stronger up-regulation of p21 and p27 in the NCI-H929 cell line at 5 µM of hit 17 (Fig. 4D-E). Overall, our data are consistent with previous findings of the TRF2-dependent and ATM-mediated DNA damage response at telomeric ends [25, 26].

TERRA G4 downstream pathways modulated by hit 17

To explore molecular mechanisms induced by hit 17 in MM, we performed whole gene expression profiling (GEP) of the NCI-H929 cell line at two different doses (2.5 µM and 5 µM) and times (24 and 48 hours) of treatment. Microarray profiles were analyzed by gene set enrichment analysis (GSEA). This analysis revealed a significant enrichment of 31 gene sets (Table S1) after 48 hours of hit 17 treatment at 5 µM. In particular, TNFα, p53, and hypoxia pathways were significantly up-regulated in hit 17-treated cells with a normalized enrichment score (NES) ≥ 2 and p < 0.001. Conversely, targets of E2F and MYC, cell cycle, oxidative phosphorylation, mitotic spindle and DNA repair were significantly inhibited in NCI-H929-treated cells (NES ≤ -3, p < 0.001), and its modulation was already observed after 24 hours of exposure and 2.5 µM concentration, suggesting that these perturbations are early events in hit 17 treatment (Fig. 5). Focusing on the DNA repair pathway, and in particular on key regulators such as PARP1, RAD51, and DNA ligase I, we validated the down-regulation of this gene signature by qRT-PCR, confirming the inhibition of the DNA repair process (Fig. S2). Accordingly, the down-regulation of this DNA repair gene signature was also validated in three MM cell lines after hit 17 treatments (Fig. 6).

Diagnostic And Prognostic Evaluation Of Dna Repair Gene Signature

The gene expression patterns of DNA repair gene signature were further validated using GSE5900 and GSE19784 cohorts. LIG1, PARP1, and RAD51 expression are remarkably higher in MM samples when compared with HD samples (all P < 0.0001; Fig. 7A-C and Fig. S3). Survival analyses were performed in the GSE4581 dataset. As revealed in the Fig. 7D-F, MM patients with high expression of these three genes demonstrated a significantly unfavorable overall survival (OS) than patients with low expression (LIG1: probe-set 20226_at, HR = 1.7, P = 0.024; PARP1: probeset 208644_at, HR = 7.6x10^− 5; RAD51: probe-set 205024_s_at, HR = 2.6, P = 2.6x10^− 5). In summary, the DNA repair gene signature identified in this study show a correlation with MM outcome.

In vivo activity of hit 17

To assess the translational relevance of hit 17, we evaluated the anti-tumor activity against human MM xenografts in NOD-SCID mice. When NCI-H929 MM xenografts became palpable, mice were randomized into 2 groups and treated with hit 17 or saline. Intraperitoneal injection of 4 mg/kg was performed everyday treatment, 5 per week, for three weeks. We observed significant inhibition of tumor growth in hit 17 treated animals as compared to the control group (p < 0.05) in both experimental settings (Fig. 8A-B). Hematoxylin & Eosin (H&E) staining showed strong tumor shrinkage in the treated xenografts (Fig. 8C-D). Ki-67 immunohistochemistry (IHC) revealed a reduction of the proliferative index from 95% in the control group to 10% in treated group (Fig. 8E-F). Further, WB analysis of retrieved xenografts showed activation of DNA damage markers, the induction of apoptotic process by caspase-3, the down-regulation of TERT, and DNA repair gene signature in treated mice compared to the control group, consistent with hit 17 mechanism of action (Fig. 8G-H).

Genomic instability, an increased tendency to acquire genomic alterations, is a cancer hallmark and its frequency varies across different tumors [27]. Given its prognostic value, understanding mechanisms that lead to genomic instability is a crucial issue for the application of precision medicine. MM represents a suitable disease model to investigate genomic instability as it evolves through premalignant stages characterized by progressive accumulation of genomic alterations [28]. Several mechanisms have been proposed as causes of genomic instability in MM [1], including the dysfunction of the DNA repair machinery [29, 30]. Another important source of chromosomal instability is telomere uncapping and recently it has been demonstrated that telomere structure correlates with aggressiveness in MM at diagnosis [31, 32]. On the other hand, in the last years, several studies have highlighted how ncRNAs, among all miRNAs and lncRNAs, are driver regulators in biological processes and molecular functions and their dysregulation is a frequent event in numerous cancers [33, 34], including MM [35–37]. In this scenario, we explored in MM the functional role of TERRAs, a class of lncRNAs known to be involved in telomere stability. Transcriptional dysregulation of TERRA has been previously reported in several cancers although at different trends of expression. For instance, some authors reported that TERRA levels were lower in the tumor of patients with squamous cell carcinoma of the head and neck and correlated with a worse clinical outcome than patients in which TERRA in the tumor was higher or similar compared to the normal tissue [13]. However, to our knowledge, the role of TERRA in the pathogenesis of MM has not been studies. Therefore, we underwent TERRA investigation by targeting with hit 17, a small molecule that can bind and stabilize the G4 conformation. It has been reported that TERRAs G4 interact with the shelterin complex, including TRF2, at telomere ends. We demonstrated that hit 17 has anti-MM activity in vitro, decreasing proliferation and activating apoptosis. In our experimental model, by ChIP assay, we also observed that TERRA G4 stabilization by hit 17 reduces TRF2 DNA binding to telomeres, in a dose-dependent manner, with subsequent activation of ATM and its downstream targets. We confirmed a dose-dependent increase of phosphorylated ATM, CHEK1, CHEK2, and H2AX and cell cycle inhibitors CDKN1A/1B in MM cell lines. In addition, we demonstrated that hit 17 treatments increase TERRA levels from 2-fold to 10-fold at the dose of 5 µM, verifying that telomere disruption leads to an altered chromatin structure that enhances TERRA transcription. Next, we observed that TERRA G4 directly inhibits TERT expression and contributes to heterochromatin formation increasing H3K9me3, H3K27me3, and H4K20me3 through the activation of PCR2. After three weeks of treatment with hit 17, xenograft tumor growth was significantly inhibited as compared to control mice. However, we found that systemically administered hit 17 exerted a significant anti-MM activity in vivo as demonstrated by tumor growth inhibition associated with a strong reduction of proliferative Ki67 index. Whole transcriptome analysis revealed a signature of three differentially expressed genes, LIG1, PARP1, and RAD51. Following treatment with hit 17, the down-regulation of these genes was observed in both in vitro and in vivo experiments. In silico analysis showed that LIG1, PARP1 and RAD51 are over-expressed in MM patient samples compared to normal samples and have prognostic value. Different studies have reported overexpression of these genes in cancer including MM. For instance, in previous work, we observed that the expression level of PARP1 increased during disease progression and in high-risk MM subgroups harboring t(4;14) and t(14;16) translocations [38]. LIG1, PARP1, and RAD51 are involved in DNA damage response (DDR) through different DNA repair pathways: non-homologous end joining (LIG1 and PARP1) and homologous recombination (RAD51). The DDR has emerged as a hallmark in cancer resulting in the development of DDR inhibitors such as PARP inhibitors. A potential role for PARP inhibitors in the treatment of MM has already been proposed for MM patients with acquired homologous recombination deficiency [39]. In addition, several small molecules and peptides inhibiting RAD51 have been developed to increase MM cancer cell sensitivity to doxorubicin or melphalan [40, 41]. In this context, our results provide the rationale to consider TERRA as a new drug target that could improve the treatment of MM in combination with DNA-damaging agents.

The relationship between lncRNAs and cancer development and progression is well-recognized, thus the modulation of oncogenic or tumor suppressor lncRNAs remains an unchallenged resource. Our results provide the relevant role of TERRA in a genomic instability model such as MM. On the other hand, the stabilization of TERRA G4 by hit 17 leads to the activation of DDR pathways and in vitro and in vivo modulation of key target genes with diagnostic and prognostic values in MM. Taken together, our findings support the targeting of TERRA by hit 17 as a new promising therapeutic strategy against MM to enhance sensitivity to DDR inhibitors treatments.

Chemical compound

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.

Cell cultures

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 Attune^TMNxT 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® Turbo^TM 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 SYBR^TM Green PCR Master Mix (Thermo Fisher Scientific Inc.) and primers reported in Table S2.

Chromatin ImmunoPrecipitation (ChIP)

ChIP was performed using the Pierce^TM 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 SYBR^TM Green PCR Master Mix (Thermo Fisher Scientific Inc.) and telomere primers (Table S2) as previously described [42]. 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 GeneChip^TM Fluidics Station 450 and scanned using the GeneChip^TM 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 GeneChip^TM 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 [43]. 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 CONFIRM^TManti-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).

Statistical analysis

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.

Ethics approval and consent to participate

All in vivo experiments were approved by the Institutional Ethical Committee of the Magna Graecia University and all the procedures were performed according to the Institutional Animal Care-approved protocols and guidelines.

Consent for publication

Not applicable.

Availability of data and materials

The datasets used and analyzed in this study are available from the corresponding author upon reasonable request.

Competing interests

The authors declare that they have no competing interests.

Funding

This work has been supported by the Italian Association for Cancer Research AIRC IG No. 21588, 2019/24 PI: PT.

Author Contributions

F.S., G.J. and M.T.D.M. designed the experimental work and wrote the manuscript; F.S, G.J., R.R., D.C., C.R. N.P. and K.G., performed experiments and/or analyzed the data; B.C. contributed to histological analysis; M.A., S.A. provided critical evaluation; P.T. (Pierosandro Tagliaferri), P.T. (Pierfrancesco Tassone) and M.T.D.M. supervised the study and revised the manuscript. All authors have read and agreed to the published version of the manuscript.

Acknowledgments

We thank Maria Cerra for the valuable technical assistance in histological analysis.

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FigureS1.pdf
Figure S1. qRT-PCR of TERF2 encoding TRF2 protein. Hit 17 treatments on the NCI-H929 cell line showed no modulation of the expression level of TRF2.
FigureS2.pdf
Figure S2. Validation of DNA damage gene signature. (B-C) qRT-PCR of selected genes (LIG1, PARP1, RAD51) involved in DNA damage response in the NCI-H929 cell line after hit 17 treatments.
FigureS3.pdf
Figure S3. DNA repair gene signature validation. Gene expression level of LIG1, PARP1 and RAD51 in MM₂, MM₃, and MM₄ validation cohorts from GSE19784 each compared to HD samples from GSE5900. FC= fold change. ****P<0.0001
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TERRA G-quadruplex stabilization as a new therapeutic strategy for multiple myeloma

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