Combinatorial strategies targeting NEAT1 and AURKA as new potential therapeutic options for multiple myeloma

Abstract

Multiple myeloma (MM) is a dreadful disease, marked by the uncontrolled proliferation of clonal plasma cells within the bone marrow. It is characterized by a highly heterogeneous clinical and molecular background, supported by severe genomic alterations. Important de-regulation of long non-coding RNA (lncRNA) expression, which can influence progression and therapy resistance, has been reported in MM patients. NEAT1 is a lncRNA essential for nuclear paraspeckles and is involved in the regulation of gene expression. We showed that NEAT1 supports MM proliferation, making this lncRNA an attractive therapeutic candidate. Here, we used a combinatorial strategy integrating transcriptomic and computational approaches with functional high-throughput drug screening to identify compounds that synergize with NEAT1 inhibition in restraining MM cell growth. AURKA inhibitors were identified as top-scoring drugs in these analyses. We showed that the combination of NEAT1 silencing and AURKA inhibitors in MM profoundly impairs microtubule organization and mitotic spindle assembly, finally leading to cell death. Analysis of the large publicly available CoMMpass dataset showed that, in MM patients, AURKA expression is strongly associated with reduced progression-free survival (P<0.0001) and overall survival (P<0.0001) probabilities and patients with high levels of expression of both NEAT1 and AURKA have a worse clinical outcome. Finally, using RNA-sequencing data from NEAT1 knockdown MM cells, we identified the AURKA allosteric regulator TPX2 as a new NEAT1 target in MM and as a mediator of the interplay between AURKA and NEAT1, therefore providing a possible explanation for the synergistic activity observed upon their combinatorial inhibition.

Figure 1.

AURKA inhibition mimics the NEAT1 knockdown transcriptomic signature. (A) Framework overview: computational and functional approaches adopted to identify compounds exerting a synergistic activity with total NEAT1 silencing in multiple myeloma cells. (B) Volcano plot showing significantly down- (green) and up- (red) regulated genes in AMO-1 NEAT1 knockdown cells compared to the scramble condition (lnFC ≤ |0.7| Padj ≤0.05). (C) Pie chart showing the number of significantly down- and up-regulated genes in AMO-1 NEAT1 knockdown cells compared to the scramble condition lnFC ≤ |0.7| Padj ≤0.05 (D) Alluvial plot depicting top scoring molecules derived from the connectivity map query, in particular: inhibitors (left), the class of perturbation to which they belong (middle), and their target genes (right). (E) Heatmap showing the relative cMap score of the 33 top-scoring candidate compounds with a score >90. KD: knockdown; MM: multiple myeloma; HT: high throughput; cMAP: connectivity map; DEG: differentially expressed genes; FDR: false discovery rate; FC: fold change; gSCR: scramble guide RNA; NOT DE: not differentially expressed; NS: not significantly different.

Figure 2.

High throughput drug screening identifies AURKA inhibitors as promising synergistic agents when combined with NEAT1 inhibition. (A) Experimental overview of the drug screening. AMO-1 cells were seeded and silenced for NEAT1 expression (day -1) through gymnotic delivery of LNA-GapmeR (gNEAT1). Cell viability was assessed using an ATP assay after 24 hours (day 0) followed by treatment with three different concentrations of compounds. At 96 hours of NEAT1 silencing and 72 hours of treatment with the compounds (day 3), cell viability was assessed by ATP assay and NEAT1 expression was quantified through quantitative real-time polymerase chain reaction. The effect of the combined drugs was determined by Excess over Bliss (EOB) analysis. (B) Diagram illustrating the top 35 candidates (EOB >0.2) exhibiting a synergistic effect when combined with NEAT1 silencing. (C) Sunburst diagram depicting the categories of the compounds and the names of the drugs that exert a synergistic activity with NEAT1 knockdown. (D) The viability of AMO-1 cells was evaluated by ATP assay (upper diagram) in duplicate and flow-cytometry (lower diagram) in the presence or absence of gNEAT1. Statistical significance was measured with the Student t test. **P<0.01, ***P<0.001. TMRM: tetramethylrhodamine methyl ester; DMSO: dimethylsulfoxide.

Figure 3.

Human myeloma cell lines showed robust sensitivity to AURKA inhibitors. (A, B) Fluorescence activated cell sorting analysis of the distribution of cell cycle phases after treatment with alisertib (A) and an Aurora kinase A inhibitor I (AURKAi-I) (B) for 24 hours in AMO-1, NCI-H929 and MM1.S cells. The histograms show the percentages of cell cycle distribution; the standard deviation of three replicates is reported. ^*P<0.05, ^**P<0.01, ^***P<0.001, Student t test. (C) Western blot analyses showing pAURKA and AURKA protein expression after treatment with alisertib (24 hours) and AURKAi-I (6 hours) in AMO-1, NCI-H929 and MM1.S cells. (D) Western blot analyses showing CyCB1 and PLK1 cell cycle checkpoint proteins after treatment with alisertib and AURKAi-I (24 hours).

Figure 4.

Synergy assessment through the calculation of combination indexes. (A, B) Histograms depicting raw data of combination indexes, i.e., the fraction of viable AMO-1, NCI-H929, and MM1.S cells after 4 days of NEAT1 silencing and 3 days of treatment with the indicated concentrations of alisertib (A) or Aurora kinase A inhibitor I (AURKAi-I) (B).

Figure 5.

AURKA inhibition increases the cytostatic effect of NEAT1 inhibition in multiple myeloma over time. (A-C) By live cell imaging analysis, the proliferation rate was measured relative to T=0 h, in AMO-1 (A), NCI-H929 (B), and MM1.S (C) multiple myeloma cell lines with Incucyte S3 Live Cell Analysis (Sartorius). NEAT1 expression in the AMO-1, NCI-H929 and MM1.S cells was silenced with different concentrations of GapmeR and then the cells were treated with an inhibitory concentration (IC₂₀) of alisertib or Aurora kinase A inhibitor I (AURKAi-I). Values are represented as the ratio between the treated sample over the vehicle. The graph shows the mean ± standard error of the mean of two independent experiments. Statistical significance was determined with the Student t test, ^*P<0.05, ^**P<0.01, ^***P<0.001.

Figure 6.

NEAT1 transactivation determines increased resistance to AURKA inhibitors. (A, B) Half maximal inhibitory concentration (IC₅₀) curves of aisertib and Aurora kinase A inhibitor I (AURKAi-I) in AMO-1 SAM cell lines. The IC₅₀ value was calculated at 72 hours of treatment using Compusyn software. The fraction of alive cells (%) is provided on the vertical axis and the log (concentration) [µM] of alisertib (A) and AURKAi-I (B) on the horizontal axis. (C) Histograms showing the biological effects obtained in AMO-1 SCR and AMO-1 N#8 SAM cells treated with alisertib and AURKAi-I. Values are represented as the ratio between the treated samples over the vehicle. The graph shows the mean ± standard error of the mean of two independent biological replicates. Statistical significance was determined with the Student t test, ^*P<0.05, ^**P<0.01, ^***P<0.001.

Figure 7.

NEAT1 is involved in mitotic spindle dynamics by controlling AURKA activity through TPX2 transcriptional modulation. (A) Representative images of metaphase spindles. AMO-1 and NCI-H929 cells were treated with vehicle, gNEAT1, alisertib, or alisertib + gNEAT1. α-Tubulin was stained in green and DAPI was used to stain cell nuclei. Scale bar 20 µm. (B) Bar plots representing the percentages of mitotic cells with defective spindles in AMO-1 and NCI-H929 cells treated with vehicle, gNEAT1, alisertib, or alisertib + gNEAT1. (C) Dot plot of the top ten significantly down-regulated biological processes (false discovery rate <0.05) obtained in AMO-1 NEAT1 knockdown cells. (D) STRING node depicting functional and physical protein-protein interactions among the down-regulated genes related to mitotic spindle and microtubule organization in AMO-1 NEAT1 knockdown cells. (E) Quantitative real-time polymerase chain reaction of TPX2 in AMO-1 and NCI-H929 cells silenced for NEAT1 expression (gNEAT1) and in the relative control condition (gSCR), after GapmeR delivery and western blot analysis of TPX2 protein in AMO-1 and NCI-H929 cells silenced for NEAT1 expression (gNEAT1) and in the relative control condition (gSCR), after GapmeR delivery (N=3). (F) Quantitative real-time polymerase chain reaction of TPX2 in AMO-1 SAM gSCR and AMO-1 SAM gN#8 cells and western blot analysis of TPX2 protein in AMO-1 SAM gSCR and AMO-1 SAM gN#8 cells (N=2).