Jardine S¹, Anderson S², Babcock S², Leung G¹, Pan J¹, Dhingani N¹, Warner N¹, Guo C¹, Siddiqui I³, Kotlarz D⁴, Dowling JJ⁵, Melnyk R⁶, Snapper SB⁷, Klein C⁴, Thiagarajah JR², Muise AM⁸. Gastroenterology. 2019 Nov 16. pii: S0016-5085(19)41574-6. doi: 10.1053/j.gastro.2019.11.019. [Epub ahead of print]

Author information

1 SickKids Inflammatory Bowel Disease Center, The Hospital for Sick Children, Toronto, ON, Canada.

2 Division of Gastroenterology, Hepatology and Nutrition, Boston Children's Hospital, Boston, MA, USA, Harvard Medical School, Boston, MA, USA.

3 Division of Pathology, The Hospital for Sick Children, Toronto, ON, Canada.

4 Dr. von Hauner Children's Hospital, Department of Pediatrics, University Hospital, LMU Munich, Munich, Germany.

5 Division of Neurology, and Genetics and Genome Biology Program, Research Institute, The Hospital for Sick Children; Molecular Medicine Program, Research Institute, The Hospital for Sick Children, Toronto, ON, Canada.

6 Molecular Medicine Program, Research Institute, The Hospital for Sick Children, Toronto, ON, Canada.

7 Division of Gastroenterology, Hepatology and Nutrition, Boston Children's Hospital, Boston, MA, USA, Harvard Medical School, Boston, MA, USA; Division of Gastroenterology, Brigham and Women's Hospital, Boston, MA, USA.

8 SickKids Inflammatory Bowel Disease Center, The Hospital for Sick Children, Toronto, ON, Canada; Cell Biology Program, Research Institute, The Hospital for Sick Children, Toronto, ON, Canada; Pediatrics, Institute of Medical Science and Biochemistry, University of Toronto, The Hospital for Sick Children, Toronto, ON, Canada. Electronic address: aleixo.muise@utoronto.ca.

Abstract

BACKGROUND & AIMS: 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.

METHODS: 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.

RESULTS: 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.

CONCLUSIONS: 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.