Drug Screen Identifies Leflunomide for Treatment of Inflammatory Bowel Diseases Caused by TTC7A Deficiency

Jardine, S., Anderson, S., Babcock, S., Leung, G., Pan, J., Dhingani, N., Warner, N., Guo, C., Siddiqui, I., Kotlarz, D., Dowling, J.J., Melnyk, R., Snapper, S.B., Klein, C., Thiagarajah, J.R., Muise, A.M.
Gastroenterology   158(4): 1000-1015 (Journal)
Registered Authors
Dowling, Jim
animal model, cell death, genetic, monogenic IBD
MeSH Terms
  • Adaptor Proteins, Signal Transducing/metabolism
  • Animals
  • Apoptosis/drug effects*
  • Apoptosis Regulatory Proteins/metabolism
  • Colon/cytology
  • Enzyme Inhibitors/pharmacology*
  • Gene Knockout Techniques
  • Haploidy
  • Humans
  • Inflammatory Bowel Diseases/drug therapy*
  • Inflammatory Bowel Diseases/genetics
  • Leflunomide/pharmacology*
  • Phosphorylation/drug effects
  • Proteins/genetics*
  • X-Linked Inhibitor of Apoptosis Protein/metabolism
31743734 Full text @ Gastroenterology
Mutations in the tetratricopeptide repeat domain 7A gene (TTC7A) cause intestinal epithelial and immune defects. Patients can become immune deficient and develop apoptotic enterocolitis, multiple intestinal atresia, and recurrent intestinal stenosis. The intestinal disease in patients with TTC7A deficiency is severe, untreatable, and recurs despite resection or allogeneic hematopoietic stem cell transplant. We screened drugs for those that prevent apoptosis of in cells with TTC7A deficiency and tested their effects in an animal model of the disease.
We developed a high-throughput screen to identify compounds approved by the Food and Drug Administration that reduce activity of caspases 3 and 7 in TTC7A-knockout HAP1 (human haploid) cells and reduce the susceptibility to apoptosis. We validated the effects of identified agents in HeLa cells that stably express TTC7A with point mutations found in patients. Signaling pathways in cells were analyzed by immunoblots. We tested the effects of identified agents in zebrafish with disruption of ttc7a, which develop intestinal defects, and colonoids derived from biopsies of patients with and without mutations in TTC7A. We performed real-time imaging of intestinal peristalsis in zebrafish and histologic analyses of intestinal tissues from patients and zebrafish. Colonoids were analyzed by immunofluorescence and for ion transport.
TTC7A-knockout HAP1 cells have abnormal morphology and undergo apoptosis, due to increased levels of active caspases 3 and 7. We identified drugs that increased cell viability; leflunomide (used to treat patients with inflammatory conditions) reduced caspase 3 and 7 activity in cells by 96%. TTC7A-knockout cells contained cleaved caspase 3 and had reduced levels of phosphorylated AKT and XIAP; incubation of these cells with leflunomide increased levels of phosphorylated AKT and XIAP and reduced levels of cleaved caspase 3. Administration of leflunomide to ttc7a-/- zebrafish increased gut motility, reduced intestinal tract narrowing, increased intestinal cell survival, increased sizes of intestinal luminal spaces, and restored villi and goblet cell morphology. Exposure of patient-derived colonoids to leflunomide increased cell survival, polarity, and transport function.
In a drug screen, we identified leflunomide as an agent that reduces apoptosis and levels of caspase 3 and activates AKT signaling and in TTC7A-knockout cells. In zebrafish with disruption of ttc7a, leflunomide restores gut motility, reduces intestinal tract narrowing, and increases intestinal cell survival. This drug might be repurposed for treatment of TTC7A deficiency.
Genes / Markers
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Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Engineered Foreign Genes