Network-based screen in iPSC-derived cells reveals therapeutic candidate for heart valve disease

Abstract

Mapping the gene-regulatory networks dysregulated in human disease would allow the design of network-correcting therapies that treat the core disease mechanism. However, small molecules are traditionally screened for their effects on one to several outputs at most, biasing discovery and limiting the likelihood of true disease-modifying drug candidates. Here, we developed a machine-learning approach to identify small molecules that broadly correct gene networks dysregulated in a human induced pluripotent stem cell (iPSC) disease model of a common form of heart disease involving the aortic valve (AV). Gene network correction by the most efficacious therapeutic candidate, XCT790, generalized to patient-derived primary AV cells and was sufficient to prevent and treat AV disease in vivo in a mouse model. This strategy, made feasible by human iPSC technology, network analysis, and machine learning, may represent an effective path for drug discovery.

Fig. 1.. Targeted RNA-seq and machine learning in human iPSC-derived cells revealed CAVD network-correcting small molecules

(A) Map of the gene network dysregulated by N1 haploinsufficiency in human iPSC-derived ECs (network inference based on DMSO-treated WT n=72 or N1^+/− n=79 and single replicates of N1^+/− treated with one of 1595 molecules). (B) Principal component analysis (PCA) plot of gene expression in WT (n=72) or N1^+/− (n=79) ECs. LOO=leave-one-out. (C) PCA plot of gene expression in N1^+/− ECs treated with one of 1595 molecules (each n=1) compared to DMSO-treated N1^+/− (n=79) or WT (n=72) ECs. (D) Example of hierarchical clustering of transcriptional profile of N1^+/− ECs treated with each of the small molecules (from LOPAC library plate 9 of 16) compared to DMSO-treated N1^+/− or WT ECs (each column n=1). (E) Percent of targeted RNA-seq validation replicates that classified as WT with true identities of DMSO-exposed WT (n=4), DMSO-exposed N1^+/− (n=4), or small molecule-treated N1^+/− (KNN hits n=3, hierarchical hits n=2). In (A-E): targeted RNA-seq.

Fig. 2.. Network-correcting small molecules broadly restored NOTCH1-dependent transcriptional dysregulation

(A) Correlation of gene expression in WT ECs with gene expression in N1^+/− ECs exposed to indicated molecules. (B) Extent of N1-dependent network restoration in N1^+/− ECs treated with indicated molecules or siRNA targeting SOX7 and TCF4 (*p<0.05, one-sided t-test vs. negative control Fmoc-leu, most to least significant: XCT790, TG003, GSK837149A, Biperiden, CB1954, R04929097). Positive values indicate correction towards or past WT expression level. Negative values indicate worsened dysregulation. (C) Gene expression of SOX7 and TCF4 regulatory nodes in response to indicated molecule treatment. In (A-C): whole transcriptome RNA-seq, n=3.

Fig. 3.. XCT790 alleviates dysregulation in primary ECs from calcified human aortic valves

(A) mRNA expression by whole transcriptome RNA-seq of genes dysregulated in primary valve ECs from patients with cTAV or cBAV as compared to ECs from patients with nTAV in response to DMSO or XCT790 treatment (p<0.05, negative binomial test, FDR correction). (B) Overlap of genes dysregulated in cTAV or cBAV with previously reported shear-responsive genes in iPSC-derived ECs (6) (p<0.05, X² test, Bonferroni correction). (C) Extent of dysregulated gene restoration towards nTAV in cTAV or cBAV ECs treated with XCT790. (Positive values indicate correction towards or past nTAV expression level. Negative values indicate worsened dysregulation.) (D) Overlap of genes dysregulated in cTAV or cBAV with genes dysregulated in N1-haploinsufficient iPSC-derived ECs as previously reported in Theodoris et al. 2015 (p<0.05, X² test, Bonferroni correction). (E) Expression of the key regulatory nodes SOX7, TCF4, and SMAD1 in nTAV, cTAV, or cBAV ECs by RNA-seq (*p<0.05, negative binomial test, FDR correction). In (A-E): nTAV n=5, cTAV n=9, cBAV n=12.

Fig. 4.. Network-correcting molecule XCT790 prevented and treated aortic valve disease in vivo

(A) AV or (B) PV peak velocity by echocardiography in N1^+/−/mTR^G2 mice treated with XCT790 (n=42) or control solvent (n=174) (AV *p=0.017, one-sided Mann-Whitney rank test). (C) AV peak velocity by echocardiography in N1^+/−/mTR^G2 mice with documented increased AV peak velocity at 4 weeks of age (AV peak velocity higher than any WT/mTR^G2 mice) treated with XCT790 or control solvent for 4 weeks (n=4, all male, *p=0.033, one-sided t-test). (D) AV and (E) PV thickness in N1^+/−/mTR^G2 mice treated with XCT790 (n=20) or control solvent (n=49) (AV *p=0.0018, PV *p=0.034, one-sided t-test). (F) Normalized number of mice with calcified AVs by Alizarin red staining in N1^+/−/mTR^G2 mice at the indicated number of days after completion of four weeks of treatment with XCT790 (n=37) or control solvent (n=106) to evaluate the sustainability of treatment effects after discontinuation. (G) Of the 106 N1^+/−/mTR^G2 mice treated with control solvent, 14 mice developed valve calcification (7 females, 7 males). Of the 37 N1^+/−/mTR^G2 mice treated with XCT790, only 2 developed any valve calcification (1 female, 1 male). Of the N1^+/−/mTR^G2 mice that developed valve calcification when treated by control solvent (n=14) compared to XCT790 (n=2), those treated with XCT790 had significantly less calcification by Alizarin red staining (*p=0.031, one-sided t-test). (H) Representative AV calcification by Alizarin red staining in mice treated with control solvent or XCT790. “AV”=valve lumen; arrows highlight Alizarin red staining. (I) Percentage of Runx2-positive cells by immunohistochemistry within the AV leaflets of N1^+/−/mTR^G2 mice treated with XCT790 (n=20) or control solvent (n=19) (*p=0.01, one-sided t-test). (J) Extent of N1-dependent network restoration by whole transcriptome RNA-seq in AVs from N1^+/−/mTR^G2 mice with increased AV peak velocity at 4 weeks of age treated for 1 month with XCT790 (n=3) compared to control solvent (n=3), where the WT network gene expression is defined by AVs from N1^WT/mTR^G2 mice with healthy valves at 4 weeks treated with control solvent for 1 month (n=4). All mice were male. Positive values indicate correction towards or past WT expression level. Negative values indicate worsened dysregulation. (*p<0.000001, one-sided Mann-Whitney rank test.) (K) Normalized qPCR gene expression of ERRα, SOX7, or TCF4 in WT ECs exposed to control (n=4) or one of three different ERRα (n=3) siRNAs (*p<0.05 by one-sided t-test). In (A-B): Boxes=interquartile range, whiskers=range, line=median, red circles=female, blue diamonds=male. In (C-E, G, I, K): Errors bars=standard error. NS=non-significant.