Pharmacological inhibition of CLK2 activates YAP by promoting alternative splicing of AMOTL2

Reviewer #1 (Public Review):

In order to find small molecules capable of enhancing regenerative repair, this study employed a high throughput YAP-activity screen method to query the ReFRAME library, identifying CLK2 inhibitor as one of the hits. Further studies showed that CLK2 inhibition leads to AMOTL2 exon skipping, rendering it unable to suppress YAP.

The novelty of the study is that it showed that inhibition of a kinase not previously associated with the HIPPO pathway can influence YAP activity through modification of mRNA splicing. The major arguments appear solid.

There are several noteworthy points when assessing the results. In Figure S1C, 100nM drug was toxic to cells at 72 hours and 1nM drug suppressed cell proliferation by 60%. Yet such concentrations were used in Figure 1B and C to argue CLK2 inhibition liberates YAP activity (which one would assume will increase cellular proliferation). In Figure 1C it appears that 1nM drug treatment led to some kind of cellular stress, as cells are visibly enlarged. In Figure 1D, 1nM drug, which would have suppressed cell growth by 60%, did not affect YAP phosphorylation. Taken together, it appears even though CLK2 inhibitor (at high concentrations) liberates YAP activity, its toxicity may override the potential use of this drug as a YAP-activator to salve tissue regenerative repair, which was one of the goals hinted in the background section.

In Figure 2D, at 100nM concentration, the drug did not appear to affect AMOTL2 splicing. Even though at higher concentrations it did, this potentially put into question whether YAP activity liberated by this drug at 1nM (Fig 2A), 10-50nM (Fig 2C) concentrations is caused by altered AMOTL2 splicing. Discussions should be provided on the difference in drug concentrations in these experiments. Does the drug decay very fast, and is that why later studies required higher dose?

Likely impact of the work on the field: this study presented a high throughput screen method for YAP activators and showed that such an approach works. The hit compound found from ReFRAME library, a CLK2 inhibitor, may not be actually useful as a YAP activator, given its clear toxicity. Applying this screen method on other large compound libraries may help find a YAP activator that helps regenerative repair. The finding that CLK2 inhibition could alter AMOTL2 splicing to affect HIPPO pathway could bring a new angle to understanding the regulation of HIPPO pathway.

Reviewer #2 (Public Review):

In this manuscript, the authors have screened the ReFRAME library and identified candidate small molecules that can activate YAP. The found that SM04690, an inhibitor of the WNT signaling pathway, could efficiently activate YAP through CLK2 kinase which has been shown to phosphorylate SR proteins to alter gene alternative splicing. They further demonstrated that SM04690 mediated alternative splicing of AMOTL2 and rendered it unlocalized on the membrane. Alternatively spliced AMOTL2 prevented YAP from anchoring to the cell membrane which results in decreased YAP phosphorylation and activated YAP. Previous findings showed that WNT signaling more or less activate YAP. The authors revealed that an inhibitor of WNT siganaling could activate YAP. Thus, these findings are potentially interesting and important. However, the present manuscript provided a lot of indirect data and lacked key experiments.

Major points:
1. In Figure S3, since inhibition of CLK2 resulted in extensive changes in alternative splicing, why did the authors choose AMOTL2? How to exclude other factors such as EEF1A1 and HSPA5, do they affect YAP activation? Angiomotin-related AMOTL1 and AMOTL2 were identified as negative regulators of YAP and TAZ by preventing their nuclear translocation. It has been reported that high cell density promoted assembly of the Crumbs complex, which recruited AMOTL2 to tight junctions. Ubiquitination of AMOTL2 K347 and K408 served as a docking site for LATS2, which phosphorylated YAP to promote its cytoplasmic retention and degradation. How to determine that alternative splicing rather than ubiquitination of AMOTL2 affects YAP activity? Does AMOTL2 Δ5 affect the ubiquitination of AMOTL2? Does overexpression of AMOTL2 Δ5Δ9 cause YAP and puncta to co-localize?
2. The author proposed that AMOTL2 splicing isoform formed biomolecular condensates,.However, there was no relevant experimental data to support this conclusion. AMOTL2 is located not only on the cell membrane but also on the circulating endosome of the cell, and the puncta formed after AMOTL2 dissociation from the membrane is likely to be the localization of the circulating endosome. The author should co-stain AMOTL2 with markers of circulating endosomes, or conduct experiments to prove the liquidity of puncta to verify the phase separation of AMOTL2 splicing isoform.
3. The localization of YAP in cells is regulated by cell density, and YAP usually translocates to the nucleus at low cell density. In Figure 2E, the cell densities of DMSO and SM04690-treated groups are inconsistent. In Figure 4A, the magnification of t DMSO and SM04690-treated groups is inconsistent, and the SM04690-treated group seems to have a higher magnification.
4. There have been many reports that the WNT signaling pathway and the Hippo signaling pathway can crosstalk with each other. The authors should exclude the influence of the WNT signaling pathway by using SM04690.

Reviewer #3 (Public Review):

This study on drug repurposing presents the identification of potent activators of the Hippo pathway. The authors successfully screen a drug library and identify two CLK kinase inhibitors as YAP activators, with SM04690 targeting specifically CLK2. They further investigate the molecular basis of SM04690-induced YAP activity and identify splicing events in AMOTL2 as strongly affected by CLK2 inhibition. Exon skipping within AMOTL2 decreases the interactions with membrane bound proteins and is sufficient to induce YAP target gene expression. Overall the study is well designed, the conclusions are supported by sufficient data and represent an exciting connection between alternative splicing and the HIPPO pathway. The specificity of the inhibitor towards CLK2 and the mode of action via AMOTL2 could be supported by further data:

1. The inconsistent inhibitor concentrations and varying results reported in the paper can be distracting. For instance, the response of endogenous targets to 100 nM concentration is described as a >5-fold increase in Figure 2B, whereas it is reported as a 1-1.5-fold response to 1000 nM in Figure 2D. This inconsistency should be addressed and clarified to provide a more accurate and reliable representation of the findings.
2. In the absence of a strong inhibitor induced YAP target gene expression (Figure 2D), it is difficult to conclude the dependency on YAP expression, as investigated by siRNA mediated knockdown. In a similar experiment, the dependency of the inhibitor on CLK2 expression could be confirmed
3. To further support the conclusion that CLK2 is the direct target of SM04690, it would be informative to investigate the effects of CLK1/4 inhibition on AMOTL2 exons (for example within RNA-seq data). If CLK1/4 inhibitors do not induce changes in AMOTL2 exons, it would strengthen the evidence for CLK2's role as the direct target. Including the results in the discussion would enhance the comprehensiveness of the study.
4. It would be important to determine the specific dose of SM04690 required to induce changes in AMOTL2 splicing. The authors observe that AMOTL2 protein levels appear unaffected at doses below 50 nM in Figure 3D, while YAP target genes are already affected at 20 nM in Figure 3G. Although Western blotting may not be the most sensitive method to detect minor changes in splicing, performing PCR experiments at lower doses could provide more insight into the splicing changes. Therefore, it is suggested that the authors include PCR experiments at lower doses to determine if changes in splicing are visible and to better establish the relationship between splicing and gene expression changes.