Development of an improved inhibitor of Lats kinases to promote regeneration of mammalian organs

Significance Many organs regenerate through the proliferation of cells that replace those that have succumbed to aging or injury. However, proliferation is largely absent in certain critical organs, including the heart, central nervous system, and sensory organs such as the inner ear and retina. The Hippo-Yap biochemical signaling pathway, a cascade of proteins that—when active—inhibits cell division, constitutes one impediment to proliferation. We earlier identified a small molecule that interrupts Hippo-Yap signaling and thus relieves this block for some non-proliferating mammalian cells. We have chemically modified the original substance to yield a more potent analog that is effective for several hours and initiates the proliferation of lesioned heart-muscle cells. Compounds of this family might prove useful in regenerative therapies.


Assays of Lats kinase inhibition
The activity of compounds was tested against a functional component of Lats kinases in an enzymological assay (1). The substances' ability to reduce the phosphorylation of Yap was also tested in HEK 293 cells, again as described previously. For the latter assay, variations in the control responses sometimes led to negative values or values exceeding 100 % for the percentage of inhibition, but the dependence of the results on the concentrations of the test compounds was highly reproducible.

Systemic administration of TDI-011536
procedures involved Swiss Webster mice (Charles River Laboratories) of both sexes and were conducted with the approval of Rockefeller University's Institutional Animal Care and Use Committee. Within each experiment, animals were age-and sex-matched and were randomly assigned to treated and control groups. When appropriate, 5-ethynyl-2′-deoxyuridine (EdU; E10187, Thermo Fisher) was administered intraperitoneally at a dosage of 5 mg/kg on each day of treatment with TDI-011536.

Measurement of in vivo Lats inhibition
Fifty milligrams of heart, liver, or skin tissue was lysed on ice in radioimmunoprecipitation-assay (RIPA) buffer solution (BP-115, Boston BioProducts) with protease inhibitors (Halt Protease Inhibitor Cocktail 87786, Thermo Fisher). After remaining on ice for 30 min, the samples were sonicated at low power for 2 min with 10 s exposures separated by 10 s interruptions, then centrifuged at 21,130 X g for 10 min at 4 °C. The supernatants were filtered and the lysates were immediately subjected to electrophoresis or stored at -80 °C.
A standard immunoblotting protocol was used. After lysates had been prepared, 10 mg of protein from the heart or liver or 20 mg of protein from the skin was resolved on a 4 %-12 % gradient bis-tris gel (NP0322, Thermo Fisher). The proteins were transferred to a PVDF membrane (1704156, BioRad) and blocked for one hour at room temperature (MB-070, Rockland). We used primary antibodies directed against Yap (101199, Santa Cruz Biotechnology), phosphorylated Yap S127 (4911, Cell Signaling Technology) and Gapdh (ab8245, Abcam), which were reconstituted at a dilution of 1:1000 in blocking solution. After a membrane had been exposed to primary antibodies overnight at 4 °C, it was subjected to three 5 min washes in tris-buffered saline solution (28358, Thermo Fisher) with 0.05 % Tween-20.
Secondary antibodies conjugated to horseradish peroxidase (W401B and W402B, Promega) were applied at a dilution of 1:10,000 in the same solution for one hour at room temperature before activity was detected (SuperSignal West Pico PLUS 34580, Thermo Fisher). Images were acquired with an iBright FL1000 system (Invitrogen).

Culture of human pluripotent stem cells and retinal organoids
After induced pluripotent stem cells of the WTC-11 line (Coriell Institute for Medical Research, Camden, NJ) had been grown by standard methods, human retinal organoids were produced (2) . The effects of Lats kinase inhibition were analyzed as described previously (1). For the preparation of protein lysates after a 24-hour treatment in two independent assays, five organoids 225-280 days of age were sampled per experimental group. In four independent proliferation assays, the organoids were incubated in long-term culture medium supplemented with 10 µM EdU and 10 µM TRULI or 3 µM TDI-011536. The extent of proliferation was assessed by quantifying the percentage cells doubly positive for EdU and Sox9 after five days in culture.

RNA sequencing
Sequence and transcript coordinates for the murine mm10 UCSC genome and gene models  With the animal supine on a thermostatted heating pad at 40 ˚C, an oblique, 15 mmlong incision was made from the xiphoid process parallel to the left lower costal margin and about 3 mm caudal to it. After the abdominal musculature and peritoneum had been transected with scissors in the same pattern, a miniature retractor was introduced to separate the two sides of the incision. This approach exposed the upper margin of the liver and provided a view of the beating heart and the base of the left lung through the transparent diaphragm.

Cryolesioning of the murine right ventricle
A cryolesion of the right ventricle was created with a round aluminum rod of mass 1.44 g secured to a plastic handle. A total of 76 mm in length, the rod was 3.2 mm in diameter over a distance of 59 mm at the base and 2.15 mm in diameter over the 17 mm at the tip. The rod was cooled for 20 s by complete immersion in liquid nitrogen, after which the slightly rounded, polished tip was immediately pressed against the diaphragm near the middle of the roughly rectangular area of exposure of the heart. A constant force, estimated as 0.8 N by simulation with an electronic balance, was exerted for 20 s, after which the probe was withdrawn. A successful lesion was marked by a 2 mm disk of frozen diaphragm-which rapidly thawedand often by a dark patch on the subjacent heart.
It proved important to avoid two possible complications. First, it was necessary to insert the probe at a steep enough angle with respect to the horizontal to avoid touching and lesioning the liver. And second, it was essential not to exert excessive force: if the diaphragm wrapped around the end of the probe, it adhered so strongly that its withdrawal occasionally perforated the diaphragm and caused an immediately fatal pneumothorax. After preliminary experiments, however, all of the five to ten mice operated on a given day survived the procedure and became active in a warmed cage 10-30 min after surgery. To maintain analgesia, each animal was administered an additional 100 µg/kg of buprenorphine subcutaneously at 12-hour intervals for each of the days on which TDI-011536 was injected.

Histological preparation and immunolabeling of cardiac muscle
After an animal had been anesthetized and sacrificed by cervical dislocation, its heart was rapidly removed. The site of the lesion was apparent as a 2 mm purple spot in the middle portion of the right ventricle (6). To facilitate the access of fixative, the base and apex of the heart were amputated and the lesioned segment was immersed for 16 hours in 4 % formaldehyde in phosphate-buffered saline solution (28906, Thermo Fisher). The specimen was then immersed overnight at 4 ˚C in 30 % sucrose solution, then infiltrated with cryoprotectant (OCT), frozen, and sectioned at a thickness of 5-8 µm.
Mounted on a glass slide, each section was blocked in a humidified chamber for one hour at room temperature with 3 % bovine serum albumin (AB 2336846, Jackson), 5 % normal Nuclei were stained with DAPI and sections were mounted with glass coverslips in a mounting medium (Prolong Gold Anti-fade P36934, Thermo Fisher).
Low-power images were obtained with a confocal microscope (LSM 780, Zeiss) equipped with a 10X plan-apochromatic objective lens of numerical aperture 0.45. The images were processed with Fiji (7) to estimate the density of EdU-labeled cells (SI Appendix, Fig. S4).
High-power images were acquired on an inverted microscope (IX81, Olympus) equipped with a super-resolution fluorescence illumination system (VT-iSIM, VisiTech International) and a      Tables   Table S1. Significantly upregulated Yap target genes

Liver Heart
Sgk1 Ccn2 The table lists Yap target genes (8)(9) significantly upregulated at the level Padj < 0.05 in the liver and heart four hours after TDI-011536 treatment. Bolded genes are highlighted in Table 1, and data relating to gene expression in response to TDI-011536 treatment are available at GEO (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE196322). The lists portray the top twenty-five gene-ontology terms for the liver (A) and heart (B), ranked by significance, enriched in RNA-sequencing data four hours after intraperitoneal administration of TDI-011536 at 200 mg/kg. Each image captured a field of 1417 µm X 1417 µm and therefore a total area of 2.008 mm 2 .
The density of labeled cells in 14 images from Specimen 1 was 15.2 ± 3.0 mm -2 and that in 19 images from Specimen 2 was 18.3 ± 3.4 mm -2 (means ± SDs). The two results were Each image captured a field of 1417 µm X 1417 µm and therefore a total area of 2.008 mm 2 .
The density of labeled cells in ten images from Specimen 3 was 5.4 ± 1.9 mm -2 and that in 18 images from Specimen 4 was 5.7 ± 2.3 mm -2 (means ± SDs). The two results were not significantly different at the level P = 0.80 by a double-tailed t-test with unequal variances.