IGF1 (median of probes 209540_at, 209541_at, 209542_x_at, and 211577_s_at), IGF1R (median of probes 203627_at and 225330_at), IRS1 (probe 204686_at), and IRS2 (median of probes 209184_s_at and 209185_s_at) mRNA expression data from samples from healthy donors (bone marrow plasma cells [n = 5]) or MM patients (n = 30) were derived from the publicly accessible data portal AmaZonia! database 2008 (http://amazonia.transcriptome.eu).28 The gene expression values were obtained from cDNA microarray experiments by the Affymetrix HUG133 plus 2.0 arrays system and the data were crossed using tumor-specific identification numbers. Gene dependency scores for IGF1, IGF1R, IRS1, and IRS2 derived from high throughput CRISPR-Cas9 screens using twenty MM cell lines (Supplementary Table 1) were obtained from the MyeloDB database (https://project.iith.ac.in/cgntlab/myelodb/).

MM.1S, MM.1R, and U266 cells were kindly provided by Prof. Sara Teresinha Olalla Saad (University of Campinas, Campinas, Brazil). RPMI 8226 cells were kindly provided by Prof. Gisele Wally Braga Colleoni (Federal University of São Paulo, São Paulo, Brazil). Cells were cultured in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) plus 1% penicillin/streptomycin and maintained at 5% CO2 and 37ºC. MM cells were used for up to 12 passages after thawing. NT157 was obtained from Sun-Shinechem (Wuhan, China) and prepared as 10 mM stock solutions in dimethyl sulfoxide (DMSO).

In total 2 × 104 cells per well were seeded in a 96-well plate in appropriate medium in the presence of vehicle or NT157 (0.8; 1.6; 3.2; 6.4; 12.5; 25 and 50 µM) for 24, 48, and/or 72 h. Next, 10 μl methylthiazoletetrazolium (MTT) solution (5 mg/ml) was added and incubated at 37°C, 5% CO2 for 4 h. The reaction was stopped using 150 μl 0.1N HCl in anhydrous isopropanol. Cell viability was evaluated by measuring the absorbance at 570 nm. IC50 values were calculated using nonlinear regression analysis in GraphPad Prism 8 (GraphPad Software, Inc., San Diego, CA, USA).

Autonomous colony formation assays were carried out in semi-solid methylcellulose medium (1 × 103/ml; MethoCult 4230; StemCell Technologies Inc., Vancouver, BC, Canada) in the presence of vehicle or NT157 (1.6, 3.2, 6.4, 12.5, and 25 μM). Colonies were detected after 10-14 days of culture by adding 100 μl (5 mg/ml) MTT reagent and scored using Image J quantification software (U.S. National Institutes of Health, Bethesda, MD, USA).

In total 1 × 105 cells per well were seeded in 6-well plates supplemented with vehicle or NT157 (0.8, 1.6, 3.2, 6.4, and 12.5 μM), harvested at 24 h, fixed with 70% ethanol and stored at 4°C for at least 4 h. Next, the fixed cells were stained with 20 μg/ml propidium iodide (PI) containing 10 μg/ml RNase A for 30 min at room temperature in a light-protected area. DNA content distribution was acquired using flow cytometry (FACSCalibur; Becton Dickinson, Franklin Lakes, NJ, USA) and analyzed using FlowJo software (Treestar, Inc., San Carlos, CA, USA).

In total 4 × 104 cells per well were seeded in a 24-well plate in RPMI-1640 medium with 10% FBS in the presence of vehicle or NT157 (0.8, 1.6, 3.2, 6.4, and 12.5 μM) for 48 h. Next, the cells were washed with ice-cold phosphate-buffered saline (PBS) and resuspended in a binding buffer containing 1 μg/ml propidium iodide (PI) and 1 μg/ml APC-labeled annexin V. All specimens were analyzed by flow cytometry (FACSCalibur; Becton Dickinson) after incubation for 15 min at room temperature in a light-protected area. Ten thousand events were acquired for each sample.

Total protein extraction was performed from MM.1S and MM.1R cells treated with vehicle or NT157 (3.2 and 6.4 µM) for 24h using a buffer containing 100 mM Tris (pH 7.6), 1% Triton X-100, 150 mM NaCl, 2 mM PMSF, 10 mM Na3VO4, 100 mM NaF, 10 mM Na4P2O7, and 4 mM EDTA. Equal amounts of protein were used from total extracts followed by SDS-PAGE and a Western blot analysis with the indicated antibodies, as previously described.16 Western blot analysis was performed using a SuperSignalTM West Dura Extended Duration substrate system (Thermo Fisher Scientific, San Jose, CA, USA) and a G: BOX Chemi XX6 gel document system (Syngene, Cambridge, UK). Antibodies against phospho(p)-IGF1RTyr1135 (#3918), IGF1R (#3027), IRS1 (#3407), IRS2 (#3089), p-STAT3Tyr705, (#9131) STAT3 (#4905), p-STAT5Tyr694 (#9359), STAT5 (#25656), p-RPS6Ser235/236 (#4858), RPS6 (#2217), p-ERK1/2Thr202/204 (#9101), ERK1/2 (#9102), PARP1 (#9542), γH2AX (#9718), and α-tubulin (#2144) were obtained from Cell Signaling Technology (Danvers, MA, USA).

Total RNA from MM.1S and MM.1R cells treated with vehicle or NT157 (6.4 µM) for 24h was obtained using the TRIzol reagent (Thermo Fisher Scientific). cDNA was synthesized from 1 µg of RNA using a High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). The quantitative PCR (qPCR) was performed using a QuantStudio 3 Real-Time PCR System in conjunction with a SybrGreen System using specific primers for NT157-related genes (Supplementary Table 2).15-18HPRT1 and ACTB were used as reference genes. Relative quantification values were calculated using the 2−ΔΔCT equation.29 A negative “No Template Control” was included for each primer pair. Data were visualized using the multiple experiment viewer (MeV) 4.9.0 software.30

Statistical analyses were performed using GraphPad Prism 8 (GraphPad Software, Inc.). For comparisons, Mann-Whitney, Student t-test or ANOVA test and Bonferroni post-test were used. Correlation analysis was performed using the Spearman test and RStudio software (version 1.4.1717, RStudio, PBC) and the Corrplot plugin. A p-value < 0.05 was considered statistically significant.

Firstly, the levels of genes related to IGF1R-mediated signaling were evaluated in healthy donors and patients with multiple myeloma. IGF1 and IRS1 mRNA expression were significantly increased, while IGF1R and IRS2 mRNA expression were significantly decreased in MM patients (all p < 0.05; Figure 1A). A positive correlation was observed between the expression of IGF1R and IRS2 (r = 0.38, p = 0.03; Figure 1B) in MM patients. MM cell lines showed a heterogeneous expression of elements related to IGF1R-mediated signaling. MM.1S and MM.1R cells showed the highest levels of p-IGF1R, IGFR, IRS1, and IRS2, while U266 cells showed low levels of IGF1R and IRS1 and did not show detectable expression of IRS2 and p-IGF1R. RPMI 8226 cells showed high levels of IGF1R, IRS1, and IRS2 but not p-IGF1R (Figure 1C).

Figure 1.

Differential expression of IGF1R signaling-related genes in multiple myeloma patients. (A) IGF1, IGF1R, IRS1, and IRS2 mRNA levels in samples from healthy donors and multiple myeloma (MM) patients. The y-axis represents gene expression data obtained from the AmaZonia! database 2008, which were measured using Affymetrix HGU133 plus 2.0 arrays. The data sets were cross-referenced using tumor-specific identification numbers. The numbers of subjects for each group are indicated. The p-values are also indicated; Mann–Whitney U test. (B) Correlogram of IGF1R signaling-related genes in MM patients. The correlation analysis was performed using the Spearman test and RStudio software and the Corrplot plugin. The sizes of the circle indicate the r values and the color indicates the direction of the correlation (blue: positive correlation, red: negative correlation). (C) Western blot analysis p-IGF1R, IGF1R, IRS1, and IRS2 in the total extract from MM cell lines. The membranes were re-probed for the detection of α-tubulin and developed using a SuperSignal™ West Dura Extended Duration Substrate system and a G:BOX Chemi XX6 gel doc system. (D) Gene dependency scores for IGF1, IGF1R, IRS1, and IRS2 of 20 MM cell lines were obtained from the MyeloDB database. For gene effect, a score less than -0.5 represents depletion in most cell lines, while less than -1 represents strong killing. (E) Representation of the NT157 chemical structure. CAS, Chemical Abstracts Service.

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The dependence score was 0.032 for IGF1, -0.519 for IGF1R, -0.503 for IRS1, and -0.455 for IRS2, indicating that the receptor and intracellular targets of the pathway are more relevant as targets than the autocrine production of IGF1, which could be obtained via paracrine pathways (Figure 1D). Therefore, NT157, a multitarget inhibitor, was selected for functional studies (Figure 1E). Among the targets of NT157 are the proteins IRS1/2 and STAT3/5.12,13,15

Next, we investigated the effects of NT157 on cell viability and autonomous clonal growth of MM cells. NT157 reduced the dose- and time-dependent manner cell viability of all MM cells evaluated, being more potent and efficient in MM.1S, MM.1R, and U266 cells (Figure 2A). IC50 values ranged from 2.9 to >50 µM for MM.1S cells, from 2.6 to >50 µM for MM.1R cells, from 2.7 to 21.9 µM for U266 cells, and from 18.5 to 36.1 µM for RMPI 8226 cells. NT157 also strongly reduced autonomous clonal growth in all MM cell lines (all p < 0.05; Figure 2B), which suggests that NT157 had a suppressive effect on the ability of MM cells to grow and multiply independently.

Figure 2.

NT157 reduces cell viability and autonomous clonal growth of multiple myeloma cells. (A) Dose- and time-response cytotoxicity was analyzed by methylthiazoletetrazolium (MTT) assay for multiple myeloma (MM) cells treated with graded concentrations of NT157 (ranged from 0.8 to 50 µM) for 24, 48, and 72 h. Values are expressed as the percentage of viable cells for each condition relative to vehicle-treated controls. Results are shown as the mean ± SD of at least three independent experiments. (B) Colonies containing viable cells were detected by adding an MTT reagent after 10-14 days of culturing the cells in the presence of vehicle or NT157 (ranged from 1.6 to 25 µM). Colony images are shown for one experiment and bar graphs show the mean ± SD of at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001; ANOVA test and Bonferroni post-test.

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In an effort to gain insight into the cellular mechanisms induced by NT157 that lead to the reduction in cell viability, we conducted additional experiments. The analysis of cell cycle progression revealed that NT157 had a significant effect on the increase of cell population in subG1 and reduced the number of cells in the proliferative phases (S and G2/M phases) in the MM cell models studied (all p < 0.05, Figure 3A). The increase in the subG1 population suggests that NT157 is inducing cell cycle arrest, particularly in cells that have damaged DNA or are undergoing programmed cell death. Complementing these results, the annexin V externalization analyses indicated that NT157 was a potent inducer of apoptosis at concentrations as low as 0.8 µM after 48 h of exposure (all p < 0.05, Figure 3B). This further confirms the compound's ability to trigger cell death pathways in MM cells.

Figure 3.

NT157 triggers cell cycle arrest and cell death in multiple myeloma cells. (A) Cell cycle progression was determined by flow cytometry in multiple myeloma (MM) cells treated with the indicated concentrations of NT157 for 24 h. A representative histogram for each condition is illustrated. (B) Bar graphs represent the mean ± SD of the percent of cells in subG1, G0/G1, S, and G2/M phase upon vehicle or NT157 exposure (ranging from 0.8 to 12.5 µM) for 24 h and represent at least three independent experiments. The p values and cell lines are indicated in the graphs. *p < 0.05, **p < 0.01, ***p < 0.001; ANOVA test and Bonferroni post-test. (C) Cell death was detected by flow cytometry in MM cells treated with vehicle or NT157 (ranging from 0.8 to 12.5 µM) for 48 h using an annexin V/PI staining method. Representative dot plots are shown for each condition; the upper and lower right quadrants (Q2 plus Q3) cumulatively contain the cell death population (annexin V+ cells). (D) Bar graphs represent the mean ± SD of at least three independent experiments. The p values and cell lines are indicated in the graphs. ***p < 0.001; ANOVA test and Bonferroni post-test.

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The effects of NT157 were investigated on molecular targets previously associated with the compound and which participate in cell proliferation and survival. NT157 reduced the expression of IRS2 and the activation of STAT3, STAT5, and RPS6. The compound increases the activation of ERK1/2, a mechanism previously associated with cellular stress and degradation of IRS proteins.12 Markers of apoptosis (cleaved PARP1) and DNA damage (γH2AX) were also strongly induced by the compound (Figure 4A). Finally, NT157 reduced the expression of important oncogenes such as MYB, MYC, and BCL2 and increased the expression of tumor suppressors associated with cellular stress (ERG1, FOS, and JUN), cell cycle arrest (CDKN1A and CDKN1B), and apoptosis in response to DNA damage (BBC3 and PMAIP1) (all p < 0.05, Figure 4B). Overall, these results suggest that NT157 exerts its anti-proliferative and pro-apoptotic effects on multiple myeloma cells by modulating a network of key molecular targets involved in cell survival and growth, and promoting DNA damage and apoptosis.

Figure 4.

NT157 favors a tumor-suppressive molecular profile in multiple myeloma cells. (A) Western blot analysis phospho(p)-IGF1R, IGF1R, IRS1, IRS2, p-STAT3, STAT3, p-STAT5, STAT5, p-RPS6, RPS6, p-ERK1/2, ERK1/2, PARP1 (total and cleaved), and γH2AX in the total extract from MM.1S and MM.1R cells treated with vehicle or NT157 (6.4 µM) for 24 h. The membranes were re-probed for the detection of α-tubulin and developed using a SuperSignal™ West Dura Extended Duration Substrate system and a G:BOX Chemi XX6 gel doc system. (B) Heatmap of the gene expression in MM.1S and MM.1R cells treated with vehicle or NT157 (3.2 and 6.4 μM). The data are represented as the fold-change of vehicle-treated cells, and downregulated and upregulated genes are shown by blue and red colors, respectively. Genes, fold-change (FC), standard deviation (SD) and p values (Student t test) are indicated.

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