Fig 2: Ipafricept blocks selection of WNT active cell types by paclitaxel in ovarian cancer.(A) Ipafricept is active when dosed either 1 or 2 days before paclitaxel. Ipafricept dosed 2 days before paclitaxel promotes mitotic catastrophe. Ipafricept (25 mg/kg) was dosed in 2-week cycles on the first day of each cycle. Paclitaxel (20 mg/kg) was dosed every 2 weeks, either on day 2, 3, or 5 of the cycle. Tumors at the end of study were paraffin-embedded and imaged for pHH3 and β-catenin (magnification, ×20). *P < 0.01; **P < 0.001 combination versus chemotherapy. Data are means + SEM; n = 8 to 10 per group. Q2W, once every 2 weeks. (B) Ipafricept dosed 2 days before paclitaxel, in 2-week cycles, prevents the accumulation of drug-resistant, WNT pathway–active ovarian tumor cells. Ipafricept (25 mg/kg) was dosed on days 1, 15, and 29 (blue arrows), and paclitaxel (20 mg/kg) was dosed on days 3, 17, and 31 (red arrows). Time course study: four to five tumors per treatment group per time point; time points were from days 2 to 7, 14, 21, 28, and 35, relative to first ipafricept dose on day 1. IHC performed from FFPE whole-tumor sections for pHH3, Aurora B, and LEF1. Digital scans were performed on an Aperio AT scanner and analyzed with Definiens Tissue Studio. Positive tumor nuclei from the control group of each time point were normalized to “1,” where a fold change of 1 implies no change, and the treatment groups are displayed as fold change as compared to the control from each corresponding time point and are mean + SEM; *P < 0.05 versus control. Representative images are from study day 35 (magnification, ×20).

Fig 3: Lef1 deletion increases tumor initiation and growth in Apc-mutant adenomas.(A) Schematic of the experiment. IHC, immunohistochemistry. (B) Kaplan-Meier survival curves of the LApc (n = 20) and LApcL (n = 19) mice. (C) Staining and quantification (means ± SEM) of nuclear β-catenin+ area in the small intestine (SI) 11 days after gene deletion. Scale bars, 100 μm. n = 12 per group, *P < 0.05 and **P < 0.01. (D to G) Immunostaining and quantification (means ± SD) of the percentage of (D and G) Lef1+, (E and G) EdU+, and (F and G) Casp3+ adenoma cells 18 days after gene deletion. Scale bars, 50 μm. n = 6 per group, *P < 0.05, **P < 0.01, and ****P < 0.001. (H) UMAP visualization of scRNA-seq results from epithelial cellular adhesion molecule (EpCAM)+ intestinal cells of the WT, LApc, and LApcL mice at days 29 and 21, respectively. (I) Cell distribution percentages in the indicated scRNA-seq clusters of WT, LApc, and LApcL cells. (J) Mki67 RNA expression in WT, LApc, and LApcL epithelial cells. Adjusted P value (p.adj) indicates the significance of the Mki67 expression in the LApcL versus LApc adenoma cells. (K) Representative images of LApc-TdTomatoLSL and LApcL-TdtomatoLSL crypts. Scale bars, 50 μm. (L to N) Quantification of the (L) crypt area, (M) crypt diameter, and (N) crypt cell number of LApc-TdtomatoLSL and LApcL-TdTomatoLSL intestines. n = 6 per group, *P < 0.05, **P < 0.01, and ***P < 0.005. The dashed lines in (C) to (F) indicate the nuclear β-catenin+ adenoma cell area. ns, not significant.

Fig 4: Tcf7l1 and Esrrb regulate density-sensitive genes during differentiation(A) Heatmap of RT-qPCR of Class I-IV density-sensitive genes after 72 h differentiation in -LIF at high or low density upon KD of ß-catenin, Tcf3 (Tcf7l1), Tcf7, or Lef1. All samples were normalized using the 2–??Ct method relative to Tcp1 expression and HD shCon (log2). LD shCon is an average of n = 3 biological replicates.(B) RT-qPCR of Class I and III density-sensitive genes after 72 h differentiation in -LIF at high density, low density, or high density upon dox-induced OE of Tcf7l1 (250 ng/mL dox) with or without 48 h treatment with 2.5 µM IWR-1, a ß-catenin inhibitor. All samples were normalized to no induction at HD (log2). Error bars indicate standard deviations (n = 2 biological replicates).(C) RT-qPCR of Classes I-IV density-sensitive genes after 72 h differentiation in -LIF at high or low density upon dox-induced OE of Esrrb (500 ng/mL dox for 48 h). All samples were normalized to no induction at HD (log2). Error bars indicate standard deviations (n = 2 biological replicates). Red text indicates presence of the Esrrb motif ±10 kb of the TSS, determined using TFmotifView (http://bardet.u-strasbg.fr/tfmotifview/).(D) RT-qPCR of Classes I-IV density-sensitive genes after 72 h differentiation in -LIF at high or low density upon KD of Esrrb or control KD. All samples were normalized to HD shCon (log2). Error bars indicate standard deviations (n = 2 biological replicates).(E) Representative confocal images (63× oil objective) of Tcf/Lef reporter mESCs after differentiation (-LIF, 72 h) upon either KD of Esrrb or control KD. Green indicates H2B-GFP. Nuclei were counterstained with NucBlue (Hoechst 33,342). Scale bar: 40 µm.(F) Quantification of fluorescent intensity of H2B-GFP in (E). For HD: shCon, n = 179 nuclei; KD1, n = 108 nuclei; KD2, n = 118 nuclei. For LD: shCon, n = 58 nuclei; KD1, n = 41 nuclei; KD2, n = 26 nuclei. Significance stars indicate p values from the ANOVA test for difference among all samples as well as the t test with Bonferroni-corrected p values between samples. * is 0.05 > p value > 0.01. ** is 0.01 > p value > 0.001. *** is 0.001 = p value.

Fig 5: Model of calcineurin regulation of Kcnk5b-mediated activation of developmental gene transcription.Kcnk5b activation results in reduced cytoplasmic K+, which is sufficient to induce the transcription of the shh ligand and lef1 transcription factor as well as components of other developmental pathways to induce coordinated allometric growth of the tissues of the fish fin appendage. Scaling information from the body or local tissues in the fin activate calcineurin so that it dephosphorylates Kcnk5b on S345 to reduce its K+-channel activity, which results in isometric growth of the fin.