A screen of ReFRAME identifies pharmacological mechanisms for activating YAP.

(A) Schematic depicting screening workflow, which resulted in the identification of 9 kinase inhibitor-based YAP activating small molecules. (B) Scatter plots of TEAD-LUC activity from each of the screens conducted at the final compound concentrations of 200 nM, 2 μM, and 10 μM. Each data point represents a single compound. Hits were classified as having robust Z scores over five. (C) Pie charts indicating the classes of small molecules characterized as hits in each screen. (D) Structure and dose-response plots of TEAD-LUC activity of the nine identified kinase inhibitors (n=3, mean and s.d.).

SM04690 is a pharmacological activator of YAP.

(A) Relative luminance values after 24h treatment of 293A-TEAD-LUC cells with the indicated concentrations of SM04690 (n=3 biological replicates, mean and s.e.m.). (B) Relative transcript levels of YAP-dependent genes (ANKRD1 and CTGF) from HEK293A cells treated for 24h with SM04690, measured by RT-qPCR (n=3, mean and s.d.). (C) Western blot analysis of anti-phospho-YAP and total YAP levels from HEK293A cells treated with the indicated concentrations of SM04690 for 24h. (D) Relative transcript levels of ANKRD1 and CTGF from HEK293A cells expressing the indicated YAP targeting shRNAs and then treated with 1 μM SM04690, measured by RT-qPCR (n=3, mean and s.d.). Asterisks above samples treated with shRNAs to YAP and SM04690 refer to statistical significance as compared to scramble treated with SM04690. (E) Representative images of anti-YAP immunofluorescent staining of MDCK cells following treatment with 1 μM SM04690. Anti-YAP antibody in teal and Hoechst 33342 in pink to visualize nuclei (scale bar = 10 μm). (F) Quantification of anti-YAP and Hoechst 33342 (DNA) correlative immunofluorescent staining (n=4, mean and s.d.). (B, D, F) Statistical analyses are univariate two-sided t-tests (* P <0.0332, ** P <0.0021, *** P < 0.0002, **** P <0.0001).

Inhibition of CLK2 by SM04690 induces alternative splicing of Hippo pathway component AMOTL2.

(A) Relative transcript levels of CLK2 following knockdown by the indicated shRNA, measured by RT-qPCR (n=3, mean and s.d.). (B) Relative transcript levels of YAP-dependent genes (ANKRD1, CTGF, CYR61) expressing the indicated shRNA, measured by RT-qPCR (n=3, mean and s.d.). (C) Relative transcript levels of exon-included and exon-skipped spliceoforms following treatment with SM04690 for 24h in HEK293A cells, measured by RT-qPCR (n=3, mean and s.d.). (D) Anti-AMOTL2 immunoblotting from HEK293A cells treated with SM04690 for 24h. (E) Immunoblotting analysis of HA-YAP from anti-FLAG immunoprecipitated content from HEK293T cells. (F) Relative transcript levels of YAP-dependent genes (ANKRD1, CTGF, CYR61) following overexpression of 1 μg of AMOTL2 WT, Δ5, Δ9, Δ5Δ9, measured by RT-qPCR (n=3, mean and s.d.). (G) Relative transcript levels of YAP-dependent genes (ANKRD1, CTGF, CYR61) following overexpression of 2 μg WT AMOTL2 and treatment with 20 nM SM04690 for 24h (n=3, mean and s.d.). (A, B, F, G) Statistical analyses are univariate two-sided t-tests (* P <0.0332, ** P <0.0021, *** P <0.0002, **** P <0.0001).

Alternative splicing of AMOTL2 promotes delocalization from the membrane, essential to the activity of SM04690.

(A) Representative images of anti-AMOTL2 (yellow) and anti-E-cadherin (teal) immunofluorescent staining (with Hoechst 33342 in pink to visualize nuclei) of MDCK cells treated with control or 1 μM SM04690 for 24h (scale bar = 10 μm). (B) Quantification of anti-AMOTL2 and anti-E-cadherin correlative immunofluorescent staining (n=4, mean and s.d.). (C) Number of puncta in HEK293A cells treated with 1μM SM04690 for 24h (n=4, mean and s.d.). (D) Number of puncta per image field in HEK293A cells from 12-well plates overexpressing AMOTL2 spliceoforms (n=3, mean and s.d.). (E) Representative images of anti-AMOTL2 (green) with anti-PALS1 (red) immunofluorescent staining of MDCK cells treated with control or 1 μM SM04690 for 24h. Hoechst 33342 (blue) was used to visualize nuclei (scale bar = 20 μm). (F) Quantification of anti-AMOTL2 and anti-PALS1 correlative immunofluorescent staining (n=3, mean and s.d.). (I) Immunoblotting analysis of HA-MAGI1 WW1,2 from anti-FLAG immunoprecipitated content from HEK293T cells. (B, C, D, F) Statistical analyses are univariate two-sided t-tests (* P <0.0332, ** P <0.0021, **** P <0.0001).

Alternative splicing of AMOTL2 modulates Hippo pathway activity.

(A) A mechanistic model depicting that in the unperturbed system, AMOTL2 localizes at the membranes with proteins associated at tight junctions, bringing YAP in contact with the core kinase cascade of the Hippo pathway resulting in its phosphorylation and retention in the cytoplasm. (B) Inhibition of CLK2 by SM04690 decreases phosphorylation of serine/arginine-rich splicing factors, leading to exon skipping of exons 5 and 9 of AMOTL2. AMOTL2 lacking exons 5 and 9 cannot interact with membrane and membrane-associated tight junction proteins. As such, AMOTL2 cannot localize YAP to tight junctions, and so YAP localizes to the nucleus, interacts with TEADs, and activates its transcripts.

SM04690 induces YAP dependent proliferation at non-toxic concentrations.

(A) Representative Rhodamine B staining, and (B) number of cells of human keratinocytes (HaCaT) in 0.5% FBS treated with SM04690 for 7 days (n=4, mean and s.d.). Statistical test is a univariate two-sided t-test (* P <0.0332, ** P < 0.0021). (C) Cell viability dose-response of SM04690 in 293A-TEAD-LUC cells treated for 72h with SM04690 evaluated using CellTiter-Glo luminescent cell viability assay (n=3 biological replicates, mean and s.d.).

CLK2 inhibition activates YAP.

(A) Family tree of CLK, DYRK, and HIPK kinases. (B) Transcripts per million (TPM) of the transcripts corresponding to reported targets of SM04690 in HEK293A cells, as determined by RNA-sequencing of unstimulated cells. (C) Relative TEAD-LUC activity of CLK2-targeting inhibitors CC671, CLK-in-T3, and T025 in cells treated for 24h with compound (n=3 biological replicates, mean and s.d.). (D) Relative TEAD-LUC activity of non-CLK2-targeting inhibitors Harmine, INDY, Leucettine L41, ML167, and TG003 in cells treated for 24h with compound (n=3 biological replicates, mean and s.d.).

Top 40 transcripts with skipped exons in response to CLK2 inhibition.

Number of skipped exons of the indicated transcripts in response to CLK2 inhibition. Figure derived from Araki, S. et al., PLOS One, 2015.

Inhibition of CLK2 by SM04690 causes alternative splicing of AMOTL2 exons 5 and 9.

(A) Schematic of primer design to determine the presence of exon skipping in AMOTL2 by RT-qPCR. Exon inclusion primers were designed with the forward primer within the exon and the reverse primer at the start of the next flanking exon. Exon skipping primers were designed with the forward primer spanning the junction between the two exons flanking the skipped exon and the reverse primer within the adjacent exon. (B) Schematic of primer design to determine exon skipping in AMOTL2 by semi-quantitative PCR. The forward primer resides in the exon preceding the skipped exon and the reverse primer resides in the exon following the skipped exon. (C) Agarose gel electrophoresis of PCR amplified products using exon-specific primer sets. The primers amplified differently sized PCR amplicons depending on whether the template included the variable exon. Exon-included samples appear in the gel at the size of the exon plus the size of the primers. Exon-skipped samples appear in the gel at the size of only the primers (∼40 base pairs). (D) Schematic of C-terminal FLAG-tagged AMOTL2 constructs.

AMOTL2 displays density dependent localization to the plasma membrane.

(A) Representative images of anti-AMOTL2 (teal) and anti-E-cadherin (yellow) immunofluorescent staining of MDCK cells grown in sparse (10,000 cells) and dense (400,000 cells) cell conditions with Hoechst 33342 (pink) to visualize nuclei (scale bar = 20 μm). (B) Quantification of anti-AMOTL2 and anti-E-cadherin correlative immunofluorescent staining (n=4, mean and s.d.). Statistical analysis is a univariate two-sided t-test (**** P < 0.0001).

AMOTL2 spliceoforms are aggregation prone and do not localize to membranes. (A) Representative images of anti-AMOTL2 (white) immunofluorescent staining of HEK293A cells treated with control or 1μM SM04690 for 24h with Hoechst 33342 (blue) to visualize nuclei (scale bar = 20 μm). (B) Representative images of anti-AMOTL2 (green) immunofluorescent staining of HEK293A cells overexpressing AMOTL2 spliceoforms with Hoechst 33342 (pink) to visualize nuclei (scale bar = 10 μm).

SM04690 treatment delocalizes AMOTL2 from MAGI1. (A) Representative images of anti-AMOTL2 (green) and anti-MAGI1 (red) immunofluorescent staining of MDCK cells treated with control or 1 μM SM04690 for 24h. Hoechst 33342 (blue) was used to visualize nuclei (scale bar = 30 μm). (B) Quantification of anti-AMOTL2 and anti-MAGI1 correlative immunofluorescent staining (n=3, mean and s.d.). Statistical analysis is a univariate two-sided t-test (*P <0.0332).