DNA methylation, chromatin-binding proteins, and DNA looping are common components regulating genomic imprinting which leads to parent-specific monoallelic gene expression. Loss of methylation (LOM) at the human imprinting center 2 (IC2) on chromosome 11p15 is the most common cause of the imprinting overgrowth disorder Beckwith-Wiedemann Syndrome (BWS). Here, we report a familial transmission of a 7.6 kB deletion that ablates the core promoter of KCNQ1. This structural alteration leads to IC2 LOM and causes recurrent BWS. We find that occupancy of the chromatin organizer CTCF is disrupted proximal to the deletion, which causes chromatin architecture changes both in cis and in trans. We also profile the chromatin architecture of IC2 in patients with sporadic BWS caused by isolated LOM to identify conserved features of IC2 regulatory disruption. A strong interaction between CTCF sites around KCNQ1 and CDKN1C likely drive their expression on the maternal allele, while a weaker interaction involving the imprinting control region element may impede this connection and mediate gene silencing on the paternal allele. We present an imprinting model in which KCNQ1 transcription is necessary for appropriate CTCF binding and a novel chromatin conformation to drive allele-specific gene expression.

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Beckwith‐Wiedemann Syndrome (BWS) is the most common human overgrowth disorder caused by structural and epigenetic changes to chromosome 11p15. Patients with BWS are predisposed to developing hepatoblastoma (HB). To better understand the mechanism of HB oncogenesis in this cancer predisposition background, we performed the first multi‐dimensional study of HB samples collected from patients diagnosed with BWS. This multi‐omic investigation of seven BWS HB and five matched nontumor BWS liver samples from 7 unique patients included examination of whole exome sequences, messenger RNA/microRNA expression, and methylation levels to elucidate the genomic, transcriptomic, and epigenomic landscape of BWS‐associated HB. We compared the transcriptional profiles of the BWS samples, both HB and nontumor, to that of control livers. Genes differentially expressed across BWS tissues were identified as BWS HB predisposition factors; this gene group included cell cycle regulators, chromatin organizers, and WNT, mitogen‐activated protein kinase (MAPK), and phosphoinositide 3‐kinase (PI3K)/AKT members. We also compared transcriptional changes associated with non‐syndromic HB carrying BWS‐like 11p15 alterations compared to those without, as well as to BWS HB. Through this analysis, we identified factors specific to 11p15‐altered HB oncogenesis, termed the BWS oncogenesis network. We propose that 11p15 alterations drive HB oncogenesis by initially dysregulating cell‐cycle regulators and chromatin organizers, including histone deacetylase 1 (HDAC1), ATP‐dependent helicase X, and F‐Box and WD repeat domain containing 7. Furthermore, we found oncogenic factors such as dickkopf WNT signaling pathway inhibitor 1 and 4, WNT16, forkhead box O3 (FOXO3), and MAPK10 are differentially expressed in 11p15‐altered HB in both the BWS and non‐syndromic backgrounds. These genes warrant further investigation as diagnostic or therapeutic targets.

In mammals, the X and Y chromosomes share only small regions of homology called pseudo-autosomal regions (PAR) where pairing and recombination in spermatocytes can occur. Consequently, the sex chromosomes remain largely unsynapsed during meiosis I and are sequestered in a nuclear compartment known as the XY body where they are transcriptionally silenced in a process called meiotic sex chromosome inactivation (MSCI). MSCI mirrors meiotic silencing of unpaired chromatin (MSUC), the sequestration and transcriptional repression of unpaired DNA observed widely in eukaryotes. MSCI is initiated by the assembly of the axial elements of the synaptonemal complex (SC) comprising the structural proteins SYCP2 and SYCP3 followed by the ordered recruitment of DNA Damage Response (DDR) factors to effect gene silencing. However, the precise mechanism of how unsynapsed chromatin is detected in meiocytes is poorly understood. The sex chromosomes in eutherian mammals harbor multiple clusters of SYCP3-like amplicons comprising the Xlr gene family, only a handful of which have been functionally studied. We used a shRNA-transgenic mouse model to create a deficiency in the testis-expressed multicopy Xlr3 genes to investigate their role in spermatogenesis. Here we show that knockdown of Xlr3 in mice leads to spermatogenic defects and a skewed sex ratio that can be traced to MSCI breakdown. Spermatocytes deficient in XLR3 form the XY body and the SC axial elements therein, but are compromised in their ability to recruit DDR components to the XY body.

Wilms Tumor (WT), or nephroblastoma, is the most common pediatric renal cancer. This cancer is thought to arise from the expansion of embryonal nephrogenic rest cell population. The most common (epi)genetic alterations in WT occur on chromosome 11p15, accounting for about 75% of WT. These same changes on chromosome 11p15 also cause one of the most common WT predisposition syndromes, Beckwith-Wiedemann Syndrome (BWS). BWS is a pediatric overgrowth disorder affecting numerous tissues including the kidney and up to 28% of patients with BWS develop cancer. As patients with BWS have the same 11p15 epigenetic and structural changes that arise sporadically in non-syndromic WT, BWS WT provides a unique opportunity to investigate the specific molecular effect and mechanism by which 11p15 causes oncogenesis. Using clinical data and patient samples from the International BWS Registry and Biorepository, we used whole exome sequencing (WES) and methylation array technologies to examine the genomic and epigenomic landscape of BWS non-tumor kidney (NT) and WT tumors compared to control kidney samples. We found several large-scale recurrent copy number alterations (CNAs) in the BWS WT cohort across cancer-associated chr1p36, WT-associated chr16q, and novel chr15q. Two BWS WT presented with an above average number of genomic alterations compared to previous sporadic WT; one had mutations in telomerase-associated genes and the other had mutations in mismatch repair pathway genes. We investigated other known cancer-associated genes affected by CNAs and single nucleotide variants (SNVs) and found BCORL1 mutations in five BWS WT. Cancer-associated pathways, including targets of TP53, CTNNB1, and MYCN, were significantly differentially methylated between BWS WT and NT. Targets of TP53 and CTNNB1 were also differentially methylated between BWS NT and controls. In addition, FOXO-regulated gene promoters were significant when comparing both BWS WT to NT and BWS NT to control methylation. Overall, WES results indicate that BWS WT have similar genomic stability compared to pediatric cancers and sporadic WT, but we did identify two hyper-mutators including a rare mismatch repair defect WT within the cohort. Recurrent mutations in genes associated with sporadic WT, such as TP53, CTNNB1, and MYCN were not present in BWS samples; however, methylation differences were observed in TP53-, CTNNB1- and MYCN-related gene sets. These and other methylation changes in BWS NT and WT may influence the transcriptome in conjunction with 11p15 alterations to promote growth and oncogenesis without coding mutations. Data presented herein suggest one way in which 11p15-altered genomes predispose cells to oncogenic transitions. Further study of non-syndromic WT methylomes as well as BWS NT and WT tissue expression are required to fully understand these effects. Citation Format: Natali Sobel Naveh, Emily Traxler, Snehal Nirgude, Jennifer Kalish. Methylation differences in cancer signaling pathways drive Beckwith-Wiedemann Syndrome Wilms Tumor oncogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6045.

Wilms Tumor (WT), or nephroblastoma, is the most common pediatric renal cancer. The most prevalent epigenetic alterations in WT occur on chromosome 11p15, accounting for at least two-thirds of WT. These same changes on chromosome 11p15 also cause Beckwith-Wiedemann Syndrome (BWS). BWS is a pediatric overgrowth disorder affecting numerous tissues, including the kidney, and up to 28% of patients with BWS develop cancer. As patients with BWS have the same 11p15 epigenetic and structural changes that arise in some sporadic WT, BWS-WT provides a unique opportunity to investigate the specific molecular drivers by which 11p15 alterations induce oncogenesis. Using clinical data and patient samples from the International BWS Registry and Biorepository, we used whole exome sequencing, methylation array, and RNA-sequencing technologies to examine the genomic and epigenomic landscape of BWS-WT compared to control kidney samples. We found several large-scale recurrent copy number alterations (CNAs) in the BWS-WT cohort across cancer-associated chr1p36, WT-associated chr16q, and at a novel chr15q site. Two BWS-WT presented with an above average number of genomic alterations; one had mutations in telomere maintenance genes, and the other had mutations in mismatch repair genes. Recurrent mutations in genes associated with sporadic WT, such as TP53, CTNNB1, and WT1 were not present in BWS samples. We found other single nucleotide variants (SNVs) in BCORL1, ASXL1, ATM and AXL genes in the BWS cohort. We further compared the BWS data to publicly available non-syndromic/nonBWS data, stratified based on 11p15 status (normal or altered), and the molecular signaling differences were investigated. Differential methylation studies showed enrichment of biological processes related to the Wnt signaling, integrin signaling, insulin receptor signaling, and BMP signaling pathways in BWS-WT. Altered-11p15 nonBWS-WT showed enrichment of cell cycle and DNA damage checkpoint processes, as well as TORC1/TOR signaling and Wnt signaling pathways, as assayed by differential methylation. RNA sequencing data showed differential gene expression of Wnt signaling along with other distinct pathways including Notch, BMP, PPAR and, NIK/NF-kappaB signaling pathways in BWS-WT. Based on an interactome study, CTNNB1 exhibited the broadest range of interactions, and its overexpression was observed in BWS-WT, although not specifically mutated in the BWS samples. Weighted gene coexpression network analysis (WGCNA) showed that cell cycle-related processes and DNA repair mechanisms are shared features of WT with alterations in chromosome 11p15. While BWS-WT predisposition and 11p15 alterations in nonBWS-WT are well-established, this study is the first to focus on stratifying tumors by this characteristic. Data presented herein suggest a mechanism in which 11p15-altered genomes predispose cells to oncogenic transitions. Further investigation of our findings may serve to identify novel diagnostic or therapeutic targets in WT oncogenesis. Citation Format: Snehal Dinkar Nirgude, Natali Sobel Naveh, Sanam Kavai, Jennifer Kalish. Cancer predisposition signaling pathways drive Beckwith-Wiedemann syndrome Wilms tumor oncogenesis [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr A008.