A unique subset of low-risk Wilms tumors is characterized by loss of function of TRIM28 (KAP1), a gene critical in early renal development: A Children's Oncology Group study

Abstract

This study explores the genomic alterations that contribute to the formation of a unique subset of low-risk, epithelial differentiated, favorable histology Wilms tumors (WT), tumors that have been characterized by their expression of post-induction renal developmental genes (Subset 1 WT). We demonstrate copy neutral loss of heterozygosity involving 19q13.32-q13.43, unaccompanied by evidence for imprinting by DNA methylation. We further identified loss-of-function somatic mutations in TRIM28 (also known as KAP1), located at 19q13, in 8/9 Subset 1 tumors analyzed. An additional germline TRIM28 mutation was identified in one patient. Retrospective evaluation of previously analyzed WT outside of Subset 1 identified an additional tumor with anaplasia and both TRIM28 and TP53 mutations. A major function of TRIM28 is the repression of endogenous retroviruses early in development. We depleted TRIM28 in HEK293 cells, which resulted in increased expression of endogenous retroviruses, a finding also demonstrated in TRIM28-mutant WT. TRIM28 has been shown by others to be active during early renal development, and to interact with WTX, another gene recurrently mutated in WT. Our findings suggest that inactivation of TRIM28 early in renal development contributes to the formation of this unique subset of FHWTs, although the precise manner in which TRIM28 impacts both normal renal development and oncogenesis remains elusive.

Fig 1. Copy-neutral loss of heterozygosity on chromosome 19 in five S1 favorable histology Wilms tumors.

The beta allele frequency values from the Illumina Human 610-quad beadchip were filtered to include only regions on 19q for which the beta value is < 15% or > 85% for ≥ 10 consecutive probes. The filtered files were converted to .bed format and imported into IGV for visualization. The red arrow indicates the location of TRIM28. The regions were verified to have normal copy number using BioDiscovery Nexus 6.1 software (see S1 File).

Fig 2. TRIM28 mutations identified in Wilms tumors.

TRIM28 protein structure according to the UniProt database is illustrated using DOG 1.0 software (http://bioinformatics.lcd-ustc.org/dog). TRIM28 includes an N-terminal tripartite RBCC (Ring finger, two B-box zinc fingers, and a coiled coil) domain, which is necessary for interaction with the family of KRAB ZNF transcription factors, a central TIF1 signature sequence (TSS) domain, a HP1 (heterochromatin protein 1)-binding domain, a C terminal combination plant homeodomain (PHD), and a bromodomain. Illustrated are TRIM28 pathogenic mutations identified in 8 S1 tumors and in one anaplastic WT from this study, depicted in black font above the TRIM28 protein image. TRIM28 pathogenic mutations identified by Halliday et al [13] are depicted in gray font below the TRIM28 protein image. One mutation (a) was reported by Halliday et al. [13] in two siblings and their mother.

Fig 3. Human endogenous retrovirus expression in TRIM28 CRISPR clones.

The expression of four human endogenous retroviruses up-regulated in TRIM28-mutant WT was evaluated in HEK293 cells (HEK293 parent), two CRISPR clones with normal TRIM28 gene and protein levels (CRISPR Ctrl 1 and 2), and three TRIM28 knockdown CRISPR clones (CRISPR Clone 1, 2, and 3) by SYBR Green PCR. The endogenous retrovirus levels were normalized to GAPDH and are presented as the relative quantitative (RQ) value compared to HEK293 parent cells. Error bars represent the standard deviation of two PCR replicates.