@phdthesis{Kruber2019,
  author    = {Kruber, Philip},
  title     = {Functional analysis of DROSHA and SIX1 mutations in kidney development and Wilms tumor},
  doi       = {10.25972/OPUS-16141},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-161418},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {Wilms tumor (WT) is the most common kidney cancer in childhood. It is a genetically heterogeneous tumor and several genetic alterations have been identified in WT patients. Recurrent mutations were found in the homeo-domain of SIX1 and SIX2 in high proliferative tumors (18.1\% of the blastemal-type tumors) as well as in the microprocessor genes DROSHA and DGCR8 (18.2\% of the blastemal-type tumors), indicating a critical role of the SIX-SALL pathway and aberrant miRNA processing in WT formation. Underlined by the fact that a significant overlap between mutations in DROSHA and SIX1 was found, indicating a synergistic effect. To characterize the in vivo role of DROSHA and SIX mutations during kidney development and their oncogenic potential, I analyzed mouse lines with either a targeted deletion of Drosha or an inducible expression of human DROSHA or SIX1 carrying a tumor-specific E1147K or Q177R mutation, respectively. The DROSHA mutation E1147K was predicted to act in a dominant negative manner. Six2-cre mediated deletion of Drosha in nephron progenitors led to a lethal phenotype with apoptotic loss of progenitor cells and early termination of nephrogenesis. Mosaic deletions via Wt1-creERT2 resulted in a milder phenotype with viable offspring that developed proteinuria after 2-4 weeks, but no evidence of tumor formation. Activation of the DROSHA-E1147K transgene via Six2-cre, on the other hand, induced a more severe phenotype with apoptosis of progenitor cells, proteinuria and glomerular sclerosis. The severely growth-retarded mice died within the first two months. This strong phenotype was consistent with the predicted dominant-negative effect of DROSHA-E1147K. Analysis of the SIX1-Q177R mutation suggested that the mutation leads to a shift in DNA binding specificity instead of a complete loss of DNA binding. This may end up in subtle changes of the gene regulatory capacity of SIX1. Six2-cre mediated activation of SIX1-Q177R lead to a viable phenotype with no alterations or shortened life span. Yet a global activation of SIX1-Q177R mediated by Zp3-cre resulted in bilateral hydronephrosis and juvenile death of the mice. To mimic the synergistic effect of DROSHA and SIX1 mutations, I generated compound mutants in two combinations: A homozygous deletion of Drosha combined with an activation of SIX1-Q177R and a compound mutant with activation of DROSHA-E1147K and SIX1-Q177R. Each mouse model variant displayed new phenotypical alterations. Mice with Six2-cre mediated homozygous deletion of Drosha and activation of SIX1-Q177R were not viable, yet heterozygous deletion of Drosha and activation of SIX1-Q177R led to hydronephrosis, proteinuria and an early death around stage P28. Combined activation of DROSHA-E1147K and SIX1-Q177R under Six2-cre resulted in proteinuria, glomerulosclerosis and lesions inside the kidney. These mice also suffered from juvenile death. Both mouse models could confirm the predicted synergistic effect. While these results underscore the importance of a viable self-renewing progenitor pool for kidney development, there was no evidence of tumor formation. This suggests that either additional alterations in mitogenic or antiapoptotic pathways are needed for malignant transformation, or premature loss of a susceptible target cell population and early lethality prevent WT formation.},
  subject      = {Nephroblastom},
  language  = {en}
}
@phdthesis{Vardapour2022,
  author    = {Vardapour, Romina},
  title     = {Mutations in the DROSHA/DGCR8 microprocessor complex in high-risk blastemal Wilms tumor},
  doi       = {10.25972/OPUS-23140},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-231404},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2022},
  abstract  = {Wilms tumor (WT) or nephroblastoma is the most common kidney tumor in childhood. Several genetic alterations have been identified in WT over the past years. However, a clear-cut underlying genetic defect has remained elusive. Growing evidence suggests that miRNA processing genes play a major role in the formation of pediatric tumors, including WT. We and others have identified the microprocessor genes DROSHA and DGCR8 as key players in Wilms tumorigenesis. Exome sequence analysis of a cohort of blastemal-type WTs revealed the recurrent hotspot mutations DROSHA E1147K and DGCR8 E518K mapping to regions important for catalyic activity and RNA-binding. These alterations were expected to affect processing of miRNA precursors, ultimately leading to altered miRNA expression. Indeed, mutated tumor samples were characterized by distinct miRNA patterns. Notably, these mutations have been observed almost exclusively in WT, suggesting that they play a specific role in WT formation. The aim of the present work was to first examine the mutation frequency of DROSHA E1147K and DGCR8 E518K in a larger cohort of WTs, and to further characterize these microprocessor gene mutations as potential oncogenic drivers for WT formation. Screening of additional 700 WT samples by allele-specific PCR revealed a high frequency of DROSHA E1147K and DGCR8 E518K mutations, with the highest incidence found in tumors of high-risk histology. DROSHA E1147K was heterozygously expressed in all cases, which strongly implies a dominant negative effect. In contrast, DGCR8 E518K exclusively exhibited homozygous expression, suggestive for the mutation to act recessive. To functionally assess the mutations of the microprocessor complex in vitro, I generated stable HEK293T cell lines with inducible overexpression of DROSHA E1147K, and stable mouse embryonic stem cell (mESC) lines with inducible overexpression of DGCR8 E518K. To mimic the homozygous expression observed in WT, DGCR8 mESC lines were generated on a DGCR8 knockout background. Inducible overexpression of wild-type or mutant DROSHA in HEK293T cells showed that DROSHA E1147K leads to a global downregulation of miRNA expression. It has previously been shown that the knockout of DGCR8 in mESCs also results in a significant downregulation of canonical miRNAs. Inducible overexpression of wild type DGCR8 rescued this processing defect. DGCR8 E518K on the other hand, only led to a partial rescue. Differentially expressed miRNAs comprised members of the ESC cell cycle (ESCC) and let-7 miRNA families whose antagonism is known to play a pivotal role in the regulation of stem cell properties. Along with altered miRNA expression, DGCR8-E518K mESCs exhibited alterations in target gene expression potentially affecting various biological processes. We could observe decreased proliferation rates, most likely due to reduced cell viability. DGCR8-E518K seemed to be able to overcome the block of G1-S transition and to rescue the cell cycle defect in DGCR8-KO mESCs, albeit not to the full extent like DGCR8-wild-type. Moreover, DGCR8-E518K appeared to be unable to completely block epithelial-to-mesenchymal transition (EMT). Embryoid bodies (EBs) with the E518K mutation, however, were still able to silence the self-renewal program rescuing the differentiation defect in DGCR8-KO mESCs. Taken together, I could show that DROSHA E1147K and DGCR8 E518K are frequent events in WT with the highest incidence in high-risk tumor entities. Either mutation led to altered miRNA expression in vitro confirming our previous findings in tumor samples. While the DROSHA E1147K mutation resulted in a global downregulation of canonical miRNAs, DGCR8 E518K was able to retain significant activity of the microprocessor complex, suggesting that partial reduction of activity or altered specificity may be critical in Wilms tumorigenesis. Despite the significant differences found in the miRNA and mRNA profiles of DGCR8 E518K and DGCR8-wild-type mESCs, functional analysis showed that DGCR8 E518K could mostly restore important cellular functions in the knockout and only slightly differed from the wild-type situation. Further studies in a rather physiological environment, such as in a WT blastemal model system, may additionally help to better assess the subtle differences between DGCR8 E518K and DGCR8 wild-type observed in our mESC lines. Together with our findings, these model systems may thus contribute to better understand the role of these microprocessor mutations in the formation of WT.},
  subject      = {Nephroblastom},
  language  = {en}
}