Modeling high-risk Wilms tumors enables the discovery of therapeutic vulnerability

Abstract

Wilms tumor (WT) is the most common pediatric kidney cancer treated with standard chemotherapy. However, less-differentiated blastemal type of WT often relapses. To model the high-risk WT for therapeutic intervention, we introduce pluripotency factors into WiT49, a mixed-type WT cell line, to generate partially reprogrammed cells, namely WiT49-PRCs. When implanted into the kidney capsule in mice, WiT49-PRCs form kidney tumors and develop both liver and lung metastases, whereas WiT49 tumors do not metastasize. Histological characterization and gene expression signatures demonstrate that WiT49-PRCs recapitulate blastemal-predominant WTs. Moreover, drug screening in isogeneic WiT49 and WiT49-PRCs leads to the identification of epithelial- or blastemal-predominant WT-sensitive drugs, whose selectivity is validated in patient-derived xenografts (PDXs). Histone deacetylase (HDAC) inhibitors (e.g., panobinostat and romidepsin) are found universally effective across different WT and more potent than doxorubicin in PDXs. Taken together, WiT49-PRCs serve as a blastemal-predominant WT model for therapeutic intervention to treat patients with high-risk WT.

Keywords: HDAC inhibitors; Panobinostat; Romedipsin; WIT49; Wilms tumor model; iPSC.

Graphical abstract

Figure 1

Generation and characterization of transgene-free WiT49-PRC (A) Schematic diagram illustrating the timeline for the establishment of the reprogrammed cell line. E8: Essential 8 Medium, which is a xeno-free and feeder-free medium specially formulated for the growth and expansion of human pluripotent stem cells (PSCs). (B) Represented pictures of morphologic changes observed in WiT49 cells during the cellular reprogramming process. The border or the edge of the formed iPSC clones was indicated by the red arrow. (C–H) Detection of pluripotency markers OCT4 (C), NANOG (D), SOX2 (E), C-MYC (F), KLF4 (G), and Lin28A (H) in both WiT49 and WiT49-PRC-1 by RT-qPCR, data are represented as the mean ± SEM. (I–N) Detection of relative mRNA levels of the germ layer-associated markers TBXT (I) and CXCR4 (J) (mesoderm), PAX6 (K) and NES (L) (ectoderm), and SOX17 (M) and FOXA2 (N) (endoderm) in WiT49 and WiT49-PRC-1 by RT-qPCR, data are represented as the mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Figure 2

Reprogramming of WiT49 cells to partial iPSCs enhances metastasis (A) Schematic diagram of the experiment for orthotopic tumor formation of WiT49 cells. (B) Representative tumor pictures (left) and Ki67 IHC staining (right) of WiT49-parental and WiT49-PRC-1 xenografts. (C) Tumor weight of WiT49-parental and WiT49-PRC-1 tumors grow in vivo. (D–F) Assessment of WiT49 parental and WiT49-PRC-1 metastasis by ex vivo bioluminescence imaging. Photographic image and bioluminescence imaging analysis of the liver and lung of mice with WiT49-parental (n = 12) and WiT49-PRC-1 (n = 9) transplants in the kidney (D). Quantification of the luciferase signal in the liver (E) and lung (F) is shown. Data are represented as the mean ± SEM. (G) Representative pictures of Snail2, Vimentin, and HLAB IHC staining of WiT49-parental and WiT49-PRC-1 tumors. (H–J) Detection of cancer metastasis markers Snail2 (H), Vimentin (I), and HLAB (J) in both WiT49 and WiT49-PRC-1 by RT-qPCR, data are represented as the mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Figure 3

Reprogrammed WiT49-PRCs recapitulate the histology of blastemal tumors and share gene expression signatures of blastemal tumors (A) Representative pictures of H&E staining of Wilms tumor patient samples, PDX samples, and orthotopic tumors formed by WiT49-parental and WiT49-PRC-1 cells. (B) Volcano plot of RNA-seq data of cultured WiT49 and WiT49-PRC-1 cells showed 2,195 genes increased while 3,296 genes decreased in WiT49-PRC-1 cells. Indicated genes are the upregulated and downregulated genes. (C and D) The gene expression heatmap shows 32 genes measured from Wilms tumors with high-blastemal and low-blastemal Wilms tumor patient (C) and PDX (D) histology, as well as the WiT49-parental and WiT49-PRC-1. It can be seen that the WiT49-PRC-1 cells overexpress gene sets associated with patient and PDX samples categorized into high blastemal content. Hierarchical clustering was done using Pearson correlation distance and average linkage. (E–G) Representative pictures of IHC staining of SIX2, NCAM1, EZH2, and KRT18 using Wilms tumor patient samples (E), PDX samples (F), WiT49, and WiT49-PRC-1 tumors (G). Red arrows in 3E indicated the representative epithelial structures in tumors with triphasic histology.

Figure 4

WiT49-parental and WiT49-PRCs exhibit different drug sensitivity profiles (A) Scatterplot of cell viabilities of WiT49-parental and WiT49-PRC-1 cells. Circle represents epigenetic drugs; triangle represents FDA approved chemotherapy drugs; green triangle represents FDA approved drugs for treating Wilms tumor; and red circle represents selected epigenetic drugs. (B–J) Cell viability of WiT49-parental and WiT49-PRC-1 cells treated with panobinostat (B), doxorubicin (C), alisertib (D), cytarabine (E), romidepsin (F), CY10683 (G), RGFP966 (H), BRD4354 (I), and CXD101 (J). IC₅₀ is shown in the figure. The WiT49-PRC-1 cells are 100-fold more sensitive to cytarabine and 20-fold more sensitive to alisertib than the parental WiT49 cells. (K and L) ZIP synergy matrices of combination of panobinostat and doxorubicin. WiT49-parental (K) and WiT49-PRC-1 (L) cells were treated for 72 h with the doses of panobinostat and doxorubicin shown.

Figure 5

Tumor growth and metastasis of WiT49-PRCs to the liver and lung are abrogated by HDAC inhibitors (A–D) Orthotopic xenograft was established by kidney capsule transplantation of WiT49-parental and WiT49-PRC-1 cells and treated with doxorubicin, panobinostat, and romidepsin at indicated doses for 16 days. The rough tumor size was estimated by injection of luciferin followed by measuring the radiance of tumors using bioluminescence imaging (BLI). The tumor volume was measured as radiance. Quantification of the bioluminescent signal of WiT49-parental and WiT49-PRC-1 tumor of each treatment group was shown in (A) and (B). The tumor weight of each treatment group was shown in (C) and (D). (E) Representative pictures of Ki67 IHC staining of WiT49-parental and WiT49-PRC-1 tumors treated with vehicle, doxorubicin, panobinostat, and romidepsin. (F–I) Assessment of the abrogation of liver and lung metastasis by HDAC inhibitors using ex vivo bioluminescence imaging. Orthotopic xenograft was established by kidney capsule transplantation of WiT49-parental and WiT49-PRC-1 cells and treated with doxorubicin, panobinostat, and romidepsin at indicated doses for 16 days. Representative ex vivo bioluminescent images of the liver and lung of WiT49-parental and WIT9-PRC-1 tumor-bearing mice were shown in (F) and (G). Quantification of the luciferase signal in the liver (H) and lung (I) of WiT49-PRC-1 tumor-bearing mice is shown. Data are represented as the mean ± SEM. WiT49-parental, control, n = 7, doxorubicin, n = 6, panobinostat, n = 7, romidepsin, n = 7; WiT49-PRC-1, control, n = 9, doxorubicin, n = 7, panobinostat, n = 7, romidepsin, n = 7, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. (J) Representative pictures of IHC staining of cancer metastasis markers Snail2, Vimentin, and HLAB in WiT49-parental and WiT49-PRC-1 tumors treated with vehicle, doxorubicin, panobinostat, and romidepsin.

Figure 6

Blastemal Wilms tumor PDXs exhibit a similar drug sensitivity profile as WiT49-PRCs The therapeutic response of doxorubicin, panobinostat, and alisertib in one epithelial-predominant WT PDX 226, and two blastemal-predominant WT PDXs 207 and 253. PDXs were established by intramuscular transplantation and treated with drugs at indicated doses. Tumor size was measured every 2–3 days. (A–C) Tumor volume was calculated. Tumors of PDX 226 and PDX 207 were collected and weighed. (D and E) The tumor weight of each treatment group. PDX226, control, n = 14, doxorubicin, n = 12, panobinostat, n = 14, alisertib, n = 16; PDX207, control, n = 10, doxorubicin, n = 6, panobinostat, n = 9, alisertib, n = 12; PDX253, control, n = 8, doxorubicin, n = 8, panobinostat, n = 11, alisertib, n = 9, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. (F) Representative pictures of Ki67 IHC staining of epithelial and blastemal PDXs treated with vehicle, doxorubicin, panobinostat, and alisertib.