Targeting CLK2 and serine/arginine-rich splicing factors inhibits multiple myeloma through downregulating RAE1 by nonsense-mediated mRNA decay mechanism

Abstract

Multiple myeloma (MM) is closely related to abnormal RNA splicing in its pathogenesis. CDC2-like kinase-2 (CLK2) regulates RNA splicing by phosphorylating serine/arginine-rich splicing factors (SRSFs), but the role of CLK2 in MM remains undefined. This study was to explore the role and mechanism of CLK2 in MM. Analyzing GEO datasets of MM patients found that high CLK2 expression predicted poor prognosis. Overexpression of CLK2 promoted the cell proliferation and cell cycle progression of MM cell ARP1 and H929. Knockdown or inhibition of CLK2 suppressed cell proliferation and induced cell apoptosis and cell cycle arrest in ARP1 and H929 cells in vitro. An MM xenograft tumor experiment showed that CLK2 overexpression promoted tumor growth, while CLK2 inhibition suppressed tumor growth in vivo. Mechanistic studies revealed that interfering CLK2 inhibited SRSF phosphorylation, and induced exon 9 skipping of RAE1, resulting in nonsense-mediated mRNA decay (NMD) of RAE1. In addition, RAE1 knockdown inhibited cell proliferation in ARP1 and H929 cells. Moreover, RAE1 overexpression promoted cell proliferation and cell cycle progression of ARP1 and H929 cells, and partially reversed the antitumor effect of CLK2 knockdown. Targeting CLK2 shows antitumor effects on MM partially through inhibiting SRSF phosphorylation and inducing NMD of RAE1. Therefore, targeting the CLK2/SRSFs/RAE1 axis could be a potential therapeutic strategy for MM.

Keywords: CLK2; RAE1; SRSF; alternative splicing; nonsense‐mediated mRNA decay.

FIGURE 1

High CLK2 expression predicts poor prognosis in multiple myeloma (MM) patients, and promotes cell viability, proliferation, and cell cycle progression in MM cells. (A) Survival plots of MM patients stratified by the CLK2 (203229_s_at) mRNA levels using the GSE2658 database (n = 542). (B–D) In Kaplan–Meier Plotter, four datasets (GSE24080, GSE4204, GSE57317, and GSE9782) were used to assess the association of CLK2 mRNA level with (B) overall survival, (C) event‐free survival, and (D) postprogression survival in MM patients. (E) RNA levels of CLK2 in SKY92 high‐risk MM patients and standard‐risk ones (GSE87900). (F) CLK2 overexpression (CLK2‐OE) in ARP1 and H929 cells was evaluated by quantitative real‐time PCR. GAPDH was used as a loading control. (G, H) Western blotting was used to assess CLK2‐OE in ARP1 and H929 cells. (I, J) Effects of CLK2‐OE on cell viability in (I) ARP1 and (J) H929 cells were detected by cell viability assays for indicated times. (K, L) Effects of CLK2 overexpression on cell proliferation in ARP1 and H929 cells were measured by flow cytometry using EdU proliferation assay. (M, N) Effects of CLK2‐OE on the cell cycle in ARP1 and H929 cells were measured by flow cytometry using propidium iodide (PI) staining. *p < 0.05; **p < 0.01; ***p < 0.001. EV, empty vector.

FIGURE 2

CLK2 knockdown inhibits cell viability and proliferation, and induces cell apoptosis and cell cycle arrest in multiple myeloma cells. (A) CLK2 knockdown using three shRNAs in ARP1 and H929 cells was evaluated by quantitative real‐time PCR. (B, C) Western blotting was used to detect the knockdown efficiency of CLK2 shRNAs in ARP1 and H929 cells. (D) Effects of shCLK2#1 and shCLK2#2 on cell viability in ARP1 (top panel) and H929 (bottom panel) were detected by cell viability assay for indicated times. (E, F) Effects of shCLK2#1 and shCLK2#2 on cell apoptosis in ARP1 and H929 cells were measured by flow cytometry using annexin V‐APC/7‐AAD staining assay. (G) Effects of shCLK2#1 on cell proliferation in ARP1 and H929 cells were measured by flow cytometry using EdU assay. (H, I) Effects of shCLK2#1 on cell cycle in ARP1 and H929 cells were measured by propidium iodide (PI) staining assay. *, p < 0.05; **p < 0.01; ***p < 0.001. NC, negative control; ns, not significant.

FIGURE 3

CLK2 inhibitor CLK‐IN‐T3 shows antitumor effects on multiple myeloma in vitro and in vivo. (A) Cell viability assay was used to assess the effects of CLK2 inhibitor CLK‐IN‐T3 on cell viability of ARP1 and H929 cells. IC₅₀ values for ARP1 (273 nM) and H929 cells (484 nM) were calculated. (B, C) Effects of CLK‐IN‐T3 on cell apoptosis in ARP1 and H929 cells were measured by flow cytometry using annexin V‐FITC/propidium iodide (PI) staining kit. (D) Effects of CLK‐IN‐T3 (IC₅₀) on cell proliferation in ARP1 and H929 cells were measured by EdU assay. (E, F) Effects of CLK‐IN‐T3 (IC₅₀) on cell cycle in ARP1 and H929 cells were measured by flow cytometry using PI staining. (G) ARP1 and H929 cells were treated with CLK‐IN‐T3 (IC₅₀) for indicated times, western blotting was used to detect the phosphorylation of serine/arginine‐rich splicing factors (SRSFs). (H) Changes of tumor volume across time. NOD/SCID mice were injected s.c. with 2 × 10⁶ ARP1 cells expressing empty vector (EV) or CLK2 (CLK2‐OE), and were randomly assigned to receive i.p. injection of CLK2 inhibitor CLK‐IN‐T3 (20 mg/kg) or DMSO every 2 days (n = 6 in each group). (I) Photographic images of harvested tumor grafts at day 26. (J) Weight of harvested tumor grafts at day 26 in three groups. (K) Effect of CLK‐IN‐T3 on bodyweight in NOD/SCID mice was calculated. **p < 0.01; ***p < 0.001. ns, not significant.

FIGURE 4

CLK2 inhibition induces exon skipping (ES) events in nucleocytoplasmic transport‐related genes, and RAE1 predicts poor outcomes in multiple myeloma (MM) patients. (A) RNA sequencing was performed after H929 cells were treated with CLK2 inhibitor for 12 h. Differentially expressed genes (DEGs) included 5361 upregulated genes and 4624 downregulated genes. (B) Bubble chart shows the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of 4624 downregulated DEGs. (C–E) Alternative splicing (AS) events are classified into five types: exon skipping (ES), retention of intron (RI), mutually exclusive exons (MXE), alternative 5′ splice site (A5SS), and alternative 3′ splice site (A3SS). (C) Histogram shows the proportions of five AS types in each sample from CLK2 inhibitor and control group. (D) Differential AS events were counted according to five AS types. (E) KEGG pathway enrichment analysis of differential ES events. (F) Flowchart shows the conditions for screening downstream genes, and RAE1 was selected as the potential downstream gene. (G) Survival plots of MM patients stratified by the RAE1 (211318_s_at) levels using the GSE2658 database (n = 542). (H–J) In Kaplan–Meier Plotter, four datasets (GSE24080, GSE4204, GSE57317, and GSE9782) were used to assess the associations of RAE1 RNA level with (H) overall survival, (I) event‐free survival, and (J) postprogression survival in MM patients. (K) RNA levels of RAE1 in high‐risk MM patients and standard‐risk ones assessed by the SKY92 classifier (GSE87900). (L) Pearson correlation analysis of RAE1 (211318_s_at) with CLK2 (203229_s_at) based on the GSE2658 dataset. **p < 0.01.

FIGURE 5

CLK2 inhibition and knockdown induce exon 9 skipping of RAE1, and lead to nonsense‐mediated mRNA decay (NMD) of RAE1. (A) Histogram shows the RNA levels of RAE1 detected by RNA sequencing (RNA‐seq) in H929 cells treated with or without CLK‐IN‐T3. (B, C) Western blotting (WB) was used to measure the protein levels of RAE1 in ARP1 and H929 cells treated with or without CLK‐IN‐T3. (D) Effect of CLK‐IN‐T3 on RAE1 NMD transcript (ENST00000492498) in H929 cells was detected by RNA‐seq. (E) Schematic diagram shows exon 9 skipping of RAE1 in NMD transcript compared with protein coding transcripts, and the position of primers used for semiquantitative PCR. (F) Semiquantitative PCR was carried out to detect exon 9 skipping of RAE1 induced by CLK2 inhibitor in ARP1 and H929 cells. (G) The ratio of NMD transcripts to total transcripts was calculated. (H) Quantitative real‐time PCR was used to confirm the downregulating effects of CLK2 inhibition on RAE1, XPO1, and XPO5 mRNA levels. Cycloheximide (CHX, 10 μg/mL), an NMD pathway inhibitor, was used to assess whether their downregulation was through an NMD pathway. (I, J) The effect of CLK2 knockdown on RAE1 protein level in ARP1 and H929 cells was evaluated by WB. (K) Semiquantitative PCR was undertaken to detect exon 9 skipping of RAE1 induced by CLK2 knockdown in ARP1 and H929 cells. (L) The ratio of NMD transcripts to total transcripts was calculated. *p < 0.05; **p < 0.01; ***p < 0.001. FPKM, fragments per kilobase of transcript per million mapped reads; NC, negative control; ns, not significant.

FIGURE 6

RAE1 overexpression partially reverses the inhibiting effect of CLK2 knockdown on multiple myeloma (MM) cell proliferation. (A) RAE1 knockdown by three siRNAs in ARP1 and H929 cells was evaluated by quantitative real‐time PCR (qRT‐PCR). (B, C) Western blotting (WB) was used to assess RAE1 knockdown by siRNAs in ARP1 and H929 cells. (D) Cell viability assay was used to detect the cell viability of ARP1 and H929 cells treated with siRAE1 (#1 and #2) or negative control (siNC). (E) RAE1 overexpression (RAE1‐OE) in ARP1 and H929 cells was evaluated by qRT‐PCR. (F, G) WB was used to assess RAE1‐OE in ARP1 and H929 cells. (H, I) Effects of RAE1‐OE on cell viability in ARP1 (H) and H929 cells (I) were detected by cell viability assay for indicated times. (J) Effects of RAE1‐OE on cell proliferation in ARP1 and H929 cells were measured by flow cytometry using EdU proliferation assay. (K) Effects of RAE1‐OE on the cell cycle in ARP1 and H929 cells were measured by propidium iodide (PI) staining. (L) Cell viability assay was undertaken to assess the reversing effect of RAE1‐OE on cell viability inhibition induced by CLK2 knockdown in ARP1 (left) and H929 cells (right). (M) Schematic diagram shows the mechanism of targeting CLK2 in MM. *p < 0.05; **p < 0.01; ***p < 0.001. EV, empty vector; NC, negative control; ns, not significant; SRSF, serine/arginine‐rich splicing factor.