Role of TRIM28 and BCOR-ITD in kidney development and the formation of pediatric kidney tumors

• Applicants: Privatdozent Dr. Matthias Becker; Professor Dr. Manfred Gessler
• Subject Area: Pediatric and Adolescent Medicine
• Term: since 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 518057412

Embryonal tumors like Wilms tumor are thought to result from failed or misguided differentiation and organ development. Several epigenetic regulators that control various developmental processes have been found mutated in pediatric kidney tumors. We identified loss of TRIM28 and internal tandem duplications of BCOR (BCOR-ITD), respectively, in epithelial Wilms tumors and in clear cell sarcoma of the kidney (CCSK). Both genes encode proteins that organize large repressor complexes involved in histone modification and gene silencing. Nevertheless, their mode of action and critical targets for tumorigenesis are still unknown. We aim to dissect the molecular mechanisms of how mutations in these epigenetic regulators disrupt normal kidney development and lead to embryonal tumors. We will use murine embryonic stem cells with cre-inducible mutant alleles to characterize the effects (1) on stem cell functions, (2) during in vitro kidney development from mES cells and (3) in conditional mutant mice. Our preliminary data show that Trim28 loss leads to severe renal developmental defects, underscoring its functional importance, but perinatal lethality precludes tumor formation. Therefore, we plan to use additional cell-type specific inducible cre-lines that allow controllable activation of the mutations in the developing kidney. We plan to study morphology and function of mutant cells and organs at multiple levels: proliferation and time course of differentiation via immunohistochemistry and mRNA in situ hybridization will inform on cellular states and adherence to differentiation programs. We will investigate the molecular functions of Trim28(-KO) and Bcor-ITD with RNA-seq analyses of controls vs. mutants. Global epigenetic changes will be visualized by ChIP-seq to define TRIM28 or BCOR-ITD binding patterns and the corresponding histone modifications underlying differences in gene activity. Depending on these results we may include single cell mRNA-seq or methylation profiling. We also plan to correct TRIM28 loss in appropriate organoid cultures by gene transfer to study dependency (addiction) and possible reversion of the tumor phenotype. The cellular and mouse models will be compared and validated against human tumors and cell cultures based on these molecular and functional data. Our analyses of the developmental defects in the Trim28-KO and Bcor-ITD models will help define the physiological functions of both genes in normal kidney development and pinpoint deviations leading to tumor formation. More generally, these models will demonstrate how epigenetic alterations lead to pediatric renal tumors and improve the concepts of cell-type specificity and future targeting options.

• DFG Programme: Research Grants