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Inhibitors of CLK protein kinases suppress cell growth and induce apoptosis by modulating pre-mRNA splicing.

Araki S, Dairiki R, Nakayama Y, Murai A, Miyashita R, Iwatani M, Nomura T, Nakanishi O - PLoS ONE (2015)

Bottom Line: Additionally, the compounds resulted in splicing alterations of RPS6KB1 (S6K), and subsequent depletion of S6K protein.Taken together, these results suggest that CLK inhibitors exhibit growth suppression and apoptosis induction through splicing alterations in genes involved in growth and survival.These small molecule inhibitors may be valuable tools for elucidating the molecular machinery of splicing and for the potential development of a novel class of antitumor agents.

View Article: PubMed Central - PubMed

Affiliation: Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, Kanagawa, Japan.

ABSTRACT
Accumulating evidence has demonstrated the importance of alternative splicing in various physiological processes, including the development of different diseases. CDC-like kinases (CLKs) and serine-arginine protein kinases (SRPKs) are components of the splicing machinery that are crucial for exon selection. The discovery of small molecule inhibitors against these kinases is of significant value, not only to delineate the molecular mechanisms of splicing, but also to identify potential therapeutic opportunities. Here we describe a series of small molecules that inhibit CLKs and SRPKs and thereby modulate pre-mRNA splicing. Treatment with these small molecules (Cpd-1, Cpd-2, or Cpd-3) significantly reduced the levels of endogenous phosphorylated SR proteins and caused enlargement of nuclear speckles in MDA-MB-468 cells. Additionally, the compounds resulted in splicing alterations of RPS6KB1 (S6K), and subsequent depletion of S6K protein. Interestingly, the activity of compounds selective for CLKs was well correlated with the activity for modulating S6K splicing as well as growth inhibition of cancer cells. A comprehensive mRNA sequencing approach revealed that the inhibitors induced splicing alterations and protein depletion for multiple genes, including those involved in growth and survival pathways such as S6K, EGFR, EIF3D, and PARP. Fluorescence pulse-chase labeling analyses demonstrated that isoforms with premature termination codons generated after treatment with the CLK inhibitors were degraded much faster than canonical mRNAs. Taken together, these results suggest that CLK inhibitors exhibit growth suppression and apoptosis induction through splicing alterations in genes involved in growth and survival. These small molecule inhibitors may be valuable tools for elucidating the molecular machinery of splicing and for the potential development of a novel class of antitumor agents.

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Correlation between splicing activity and cell growth by CLK1 and CLK2.(A) Scatter plots comparing in vitro kinase inhibition with cellular splicing alteration of S6K after treatment with Cpd-1, Cpd-2, Cpd-3, and a series of other compounds. The X-axis shows the pIC50 [−log10(IC50)] value for CLK1, CLK2, SRPK1, SRPK2, or SRPK3 inhibition in cell-free enzymatic assays. The Y-axis shows the splicing induction activity defined as the drug concentration (μM) that induced 10% of the copy number of the aberrantly spliced S6K mRNA without skipping exon 7 compared with the copy number of the canonical mRNA (Rc0.1). R2: coefficient of determination; blue arrow: Cpd-1; red arrow: Cpd-2; orange arrow: Cpd-3. (B) Scatter plot analysis comparing GI50 values (concentration required to inhibit growth by 50%) on the X-axis with Rc0.1 values on the Y-axis for the same compounds shown in (A). Blue arrow: Cpd-1; red arrow: Cpd-2; orange arrow: Cpd-3. (C, D) MDA-MB-468 cells were transfected with CLK1 siRNA, CLK2 antisense oligonucleotide (ASO), or control Non-Silencing siRNA (NS) at the indicated concentrations. Cells were harvested after 24 and 48 h. The data represent means ± SD from three independent experiments. (C) The expression levels of CLK1 and CLK2 were measured by quantitative RT-PCR. (D) ASO and siRNA transfection experiments were performed to identify the kinases that altered the splicing pattern of S6K pre-mRNA. RT-PCR analyses of the expression levels of S6K mRNA exons 6–7 (canonical mRNA) and exons 6–8 (aberrantly spliced mRNA lacking skipped exon 7).
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pone.0116929.g003: Correlation between splicing activity and cell growth by CLK1 and CLK2.(A) Scatter plots comparing in vitro kinase inhibition with cellular splicing alteration of S6K after treatment with Cpd-1, Cpd-2, Cpd-3, and a series of other compounds. The X-axis shows the pIC50 [−log10(IC50)] value for CLK1, CLK2, SRPK1, SRPK2, or SRPK3 inhibition in cell-free enzymatic assays. The Y-axis shows the splicing induction activity defined as the drug concentration (μM) that induced 10% of the copy number of the aberrantly spliced S6K mRNA without skipping exon 7 compared with the copy number of the canonical mRNA (Rc0.1). R2: coefficient of determination; blue arrow: Cpd-1; red arrow: Cpd-2; orange arrow: Cpd-3. (B) Scatter plot analysis comparing GI50 values (concentration required to inhibit growth by 50%) on the X-axis with Rc0.1 values on the Y-axis for the same compounds shown in (A). Blue arrow: Cpd-1; red arrow: Cpd-2; orange arrow: Cpd-3. (C, D) MDA-MB-468 cells were transfected with CLK1 siRNA, CLK2 antisense oligonucleotide (ASO), or control Non-Silencing siRNA (NS) at the indicated concentrations. Cells were harvested after 24 and 48 h. The data represent means ± SD from three independent experiments. (C) The expression levels of CLK1 and CLK2 were measured by quantitative RT-PCR. (D) ASO and siRNA transfection experiments were performed to identify the kinases that altered the splicing pattern of S6K pre-mRNA. RT-PCR analyses of the expression levels of S6K mRNA exons 6–7 (canonical mRNA) and exons 6–8 (aberrantly spliced mRNA lacking skipped exon 7).

Mentions: To examine whether inhibition of a particular CLK and/or SRPK contributed to the alternative splicing of S6K pre-mRNA, we used an additional series of compounds with the same scaffold as Cpd-1, Cpd-2, and Cpd-3, but different selectivity. First, we examined the correlation between the enzymatic activity and the level of splicing alterations of S6K pre-mRNA for the individual compounds. We used the exon 7-skipped type of S6K variant to calibrate Rc0.1 values, i.e., the concentrations that induced 10% of the copy number of an alternative isoform compared with that of the canonical isoform. The inhibition of CLK1 and CLK2 activities was correlated with the splicing alteration activities (Rc0.1) (R2 = 0.3353 and 0.4255, respectively). In contrast, there was no correlation between the activities of SRPKs and the Rc0.1 values (R2 < 0.0468) (Fig. 3A). Next, we compared the levels of splicing alteration and growth inhibition of the compounds. The Rc0.1 values were significantly correlated with the GI50 values in MDA-MB-468 cells (R2 = 0.69; Fig. 3B). Similar correlations between Rc0.1 and GI50 values were also obtained in seven different cancer cell lines after treatment with Cpd-2 or Cpd-3 (Table 2). These results clearly suggest that inhibition of CLKs is the major driver for inducing alternative splicing of S6K pre-mRNA and subsequent inhibition of cell growth in a variety of cancer cell types.


Inhibitors of CLK protein kinases suppress cell growth and induce apoptosis by modulating pre-mRNA splicing.

Araki S, Dairiki R, Nakayama Y, Murai A, Miyashita R, Iwatani M, Nomura T, Nakanishi O - PLoS ONE (2015)

Correlation between splicing activity and cell growth by CLK1 and CLK2.(A) Scatter plots comparing in vitro kinase inhibition with cellular splicing alteration of S6K after treatment with Cpd-1, Cpd-2, Cpd-3, and a series of other compounds. The X-axis shows the pIC50 [−log10(IC50)] value for CLK1, CLK2, SRPK1, SRPK2, or SRPK3 inhibition in cell-free enzymatic assays. The Y-axis shows the splicing induction activity defined as the drug concentration (μM) that induced 10% of the copy number of the aberrantly spliced S6K mRNA without skipping exon 7 compared with the copy number of the canonical mRNA (Rc0.1). R2: coefficient of determination; blue arrow: Cpd-1; red arrow: Cpd-2; orange arrow: Cpd-3. (B) Scatter plot analysis comparing GI50 values (concentration required to inhibit growth by 50%) on the X-axis with Rc0.1 values on the Y-axis for the same compounds shown in (A). Blue arrow: Cpd-1; red arrow: Cpd-2; orange arrow: Cpd-3. (C, D) MDA-MB-468 cells were transfected with CLK1 siRNA, CLK2 antisense oligonucleotide (ASO), or control Non-Silencing siRNA (NS) at the indicated concentrations. Cells were harvested after 24 and 48 h. The data represent means ± SD from three independent experiments. (C) The expression levels of CLK1 and CLK2 were measured by quantitative RT-PCR. (D) ASO and siRNA transfection experiments were performed to identify the kinases that altered the splicing pattern of S6K pre-mRNA. RT-PCR analyses of the expression levels of S6K mRNA exons 6–7 (canonical mRNA) and exons 6–8 (aberrantly spliced mRNA lacking skipped exon 7).
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pone.0116929.g003: Correlation between splicing activity and cell growth by CLK1 and CLK2.(A) Scatter plots comparing in vitro kinase inhibition with cellular splicing alteration of S6K after treatment with Cpd-1, Cpd-2, Cpd-3, and a series of other compounds. The X-axis shows the pIC50 [−log10(IC50)] value for CLK1, CLK2, SRPK1, SRPK2, or SRPK3 inhibition in cell-free enzymatic assays. The Y-axis shows the splicing induction activity defined as the drug concentration (μM) that induced 10% of the copy number of the aberrantly spliced S6K mRNA without skipping exon 7 compared with the copy number of the canonical mRNA (Rc0.1). R2: coefficient of determination; blue arrow: Cpd-1; red arrow: Cpd-2; orange arrow: Cpd-3. (B) Scatter plot analysis comparing GI50 values (concentration required to inhibit growth by 50%) on the X-axis with Rc0.1 values on the Y-axis for the same compounds shown in (A). Blue arrow: Cpd-1; red arrow: Cpd-2; orange arrow: Cpd-3. (C, D) MDA-MB-468 cells were transfected with CLK1 siRNA, CLK2 antisense oligonucleotide (ASO), or control Non-Silencing siRNA (NS) at the indicated concentrations. Cells were harvested after 24 and 48 h. The data represent means ± SD from three independent experiments. (C) The expression levels of CLK1 and CLK2 were measured by quantitative RT-PCR. (D) ASO and siRNA transfection experiments were performed to identify the kinases that altered the splicing pattern of S6K pre-mRNA. RT-PCR analyses of the expression levels of S6K mRNA exons 6–7 (canonical mRNA) and exons 6–8 (aberrantly spliced mRNA lacking skipped exon 7).
Mentions: To examine whether inhibition of a particular CLK and/or SRPK contributed to the alternative splicing of S6K pre-mRNA, we used an additional series of compounds with the same scaffold as Cpd-1, Cpd-2, and Cpd-3, but different selectivity. First, we examined the correlation between the enzymatic activity and the level of splicing alterations of S6K pre-mRNA for the individual compounds. We used the exon 7-skipped type of S6K variant to calibrate Rc0.1 values, i.e., the concentrations that induced 10% of the copy number of an alternative isoform compared with that of the canonical isoform. The inhibition of CLK1 and CLK2 activities was correlated with the splicing alteration activities (Rc0.1) (R2 = 0.3353 and 0.4255, respectively). In contrast, there was no correlation between the activities of SRPKs and the Rc0.1 values (R2 < 0.0468) (Fig. 3A). Next, we compared the levels of splicing alteration and growth inhibition of the compounds. The Rc0.1 values were significantly correlated with the GI50 values in MDA-MB-468 cells (R2 = 0.69; Fig. 3B). Similar correlations between Rc0.1 and GI50 values were also obtained in seven different cancer cell lines after treatment with Cpd-2 or Cpd-3 (Table 2). These results clearly suggest that inhibition of CLKs is the major driver for inducing alternative splicing of S6K pre-mRNA and subsequent inhibition of cell growth in a variety of cancer cell types.

Bottom Line: Additionally, the compounds resulted in splicing alterations of RPS6KB1 (S6K), and subsequent depletion of S6K protein.Taken together, these results suggest that CLK inhibitors exhibit growth suppression and apoptosis induction through splicing alterations in genes involved in growth and survival.These small molecule inhibitors may be valuable tools for elucidating the molecular machinery of splicing and for the potential development of a novel class of antitumor agents.

View Article: PubMed Central - PubMed

Affiliation: Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, Kanagawa, Japan.

ABSTRACT
Accumulating evidence has demonstrated the importance of alternative splicing in various physiological processes, including the development of different diseases. CDC-like kinases (CLKs) and serine-arginine protein kinases (SRPKs) are components of the splicing machinery that are crucial for exon selection. The discovery of small molecule inhibitors against these kinases is of significant value, not only to delineate the molecular mechanisms of splicing, but also to identify potential therapeutic opportunities. Here we describe a series of small molecules that inhibit CLKs and SRPKs and thereby modulate pre-mRNA splicing. Treatment with these small molecules (Cpd-1, Cpd-2, or Cpd-3) significantly reduced the levels of endogenous phosphorylated SR proteins and caused enlargement of nuclear speckles in MDA-MB-468 cells. Additionally, the compounds resulted in splicing alterations of RPS6KB1 (S6K), and subsequent depletion of S6K protein. Interestingly, the activity of compounds selective for CLKs was well correlated with the activity for modulating S6K splicing as well as growth inhibition of cancer cells. A comprehensive mRNA sequencing approach revealed that the inhibitors induced splicing alterations and protein depletion for multiple genes, including those involved in growth and survival pathways such as S6K, EGFR, EIF3D, and PARP. Fluorescence pulse-chase labeling analyses demonstrated that isoforms with premature termination codons generated after treatment with the CLK inhibitors were degraded much faster than canonical mRNAs. Taken together, these results suggest that CLK inhibitors exhibit growth suppression and apoptosis induction through splicing alterations in genes involved in growth and survival. These small molecule inhibitors may be valuable tools for elucidating the molecular machinery of splicing and for the potential development of a novel class of antitumor agents.

Show MeSH
Related in: MedlinePlus