Mechanism and treatment of Wilms Tumors caused by CTR9 mutations

PROJECT SUMMARY/ABSTRACT Wilms tumor (WT) is the most common pediatric cancer of the urinary tract. Although the overall prognosis for WT patients is generally favorable after chemotherapy, relapse and deaths still occur in a significant proportion of patients. Therefore, there is a critical need to elucidate the molecular mechanisms underlying the disease causal mutations and develop better treatments. Around 30 gene mutations have been associated with WT. CTR9 germline mutations were identified in some WT families. The mutations caused alternative splicing and deletion of exon 9 of CTR9. However, how one missing exon contributes to the initiation of WT is unknown. CTR9 is the scaffold protein of the PAF complex, which has been well-characterized to regulate multiple steps in transcription. We have modeled the CTR9 exon 9 deletion in an epithelial WT cell line, WiT-49, and found that WiT-49 acquires stem cell features when exon 9 of CTR9 is deleted. When WiT-49 exon 9 deletion cells are implanted into the kidney capsules of mice, they grow more aggressively than parental cells. Gene expression and biochemical analyses uncovered that exon 9 deletion of CTR9 results in profound changes in gene expression and disruption of the integrity of the transcription elongation complex. Flag-CTR9, but not Flag-CTR9exon9, could pull down Elongin A (ELOA) and its associated splicing factors such as SF3B3 and SF3A1. We hypothesize that exon 9 deletion of CTR9 blocks kidney-specific differentiation by dislodging Elongin complex and disrupting transcriptional elongation, resulting in development of WT. We have engineered cell lines to delineate how exon 9 deletion of CTR9 results in the disturbance of transcriptional elongation and alternative mRNA splicing. Our recent studies have shown that CTR9 is essential for precluding the repressive chromatin from spreading by counteracting the activity of EZH2. Therefore, we will also examine if exon 9 deletion of CTR9 acquires stem cell properties to WiT-49 and renders cells sensitive to EZH2 inhibitors and inhibitors of splicing machinery. Successful completion of this project is expected to enhance the understanding of pathogenesis of WT caused by CTR9 mutations and establish the invaluable experimental platform for identifying effective therapies for WT.