Abstract 401: The impact of DNA structural variants on PRKN gene function and colorectal cancer pathogenesis

Abstract Background: Cancer is caused by somatic DNA alterations, such as single nucleotide variants, somatic copy number alterations and structural variants (SVs). PRKN is among the genes most frequently affected by SVs in colorectal cancer (CRC), with 37% of primary cancers and 56% of metastatic lesions having focal deletions in PRKN. PRKN encodes the E3 ubiquitin ligase Parkin, which regulates the biological activity and degradation of substrates via post-translational modifications. The genomic location of PRKN is within a common fragile site. Therefore, it remains unclear whether the high frequency of SVs in PRKN are passenger events due to genomic instability or driver events of CRC pathogenesis. Aim: Our study aims to investigate the impact of SVs on the functionality of Parkin in CRC pathogenesis. Methods: We used publicly available DNA deep whole genome sequencing (WGS) and tumor-matched RNA sequencing data (n=394) to estimate the impact of SVs in PRKN on tumor biology. For this, the transcriptome-wide p-value distribution generated by differential expression analysis was used as a measure of biological impact. The HCT116-MLH1-repaired cell line and patient-derived adenoma organoids were genetically modified using CRISPR/Cas9 to mimic the SVs observed in patient material and analyze their impact. RNA-sequencing, whole proteome and ubiquitin remnant-specific proteomics were used to assess the effect of SVs on gene function and identify altered biological processes. Results: Analysis of WGS and RNA sequencing of patient material showed that SVs in PRKN have a large impact on CRC biology, comparable to the impact of mutations in TP53 (impact score 0.379 vs 0.332). The most pronounced effects are seen in the ubiquitin-like, RING0 and RING1 domains. These domains are important for recognition and/or binding to ubiquitin, E2 ubiquitin-conjugating enzymes and substrates. In the HCT116-MLH1-repaired CRC cell line, hemizygous introduction of an SV in PRKN had minimal effects on genome-wide gene expression. In contrast, homozygous deletion altered the expression of nearly 2000 genes. Analyses of ubiquitin motif remnants showed differential ubiquitination of proteins involved in DNA repair following the introduction of an SV in PRKN in the CRC cell line. These proteins include TOP2A (Log2 fold change (L2FC) = -3.69, P = 5.71−5), RAD18 (L2FC = -4.55, P = 1.35−4) and Spartan (SPRTN; L2FC = -2.57, P = 7.43−4). Conclusion: SVs in PRKN have a high impact on tumor biology and result in aberrant gene expression and post-translational modifications of proteins related to DNA repair. Further research will expand to the patient-derived adenoma organoids and focus on understanding if these recurrent somatic alterations contribute to genomic instability and the development of CRC pathogenesis. Citation Format: Elisabeth J. van Bree, Soufyan Lakbir, Carmen Rubio-Alarcón, Renske de Wit, Anne Bolijn, Pien Delis-van Diemen, Marianne Tijssen, Ellen Stelloo, Harma Feitsma, Gerrit A. Meijer, Beatriz Carvalho, Sanne Abeln, Remond J. Fijneman. The impact of DNA structural variants on PRKN gene function and colorectal cancer pathogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 401.