Testicular germ cell tumors (TGCTs) are the most common cancers in men aged 15–39 years and are divided into two major groups, seminomas and nonseminomas. Novel treatment options are required for these patients, to limit side effects of chemotherapy. We hypothesized that promoter methylation of relevant homologous recombination (HR) genes might be predictive of response to poly‐ADP ribose polymerase inhibitors (PARPis) in TGCTs. We report a study pipeline combining in silico, in vitro, and clinical steps. By using several databases and in silico tools, we identified BRCA1, RAD51C, PALB2, RAD54B, and SYCP3 as the most relevant genes for further investigation and pinpointed specific CpG sites with pronounced negative correlation to gene expression. Nonseminomas displayed significantly higher methylation levels for all target genes, where increased methylation was observed in patients with more differentiated subtypes and higher disease burden. We independently performed second‐line targeted validation in tissue series from TGCT patients. A moderate and/or strong anti‐correlation between gene expression (assessed by RNA‐sequencing) and promoter methylation (assessed by 450k array) was found, for all of the targets. As a proof of concept, we demonstrated the sensitivity of TGCT cell lines to Olaparib, which associated with differential methylation levels of a subset of targets, namely BRCA1 and RAD51C. Our findings support the use of HR genes promoter methylation as a predictor of the therapeutic response to PARPis in patients with TGCT.

Pancreatic ductal adenocarcinoma (PDAC) is among the most lethal cancers. Its poor prognosis is predominantly due to the fact that most patients remain asymptomatic until the disease reaches an advanced stage, alongside the lack of early markers and screening strategies. A better understanding of PDAC risk factors is essential for the identification of groups at high risk in the population. Genome-wide association studies (GWAS) have been a powerful tool for detecting genetic variants associated with complex traits, including pancreatic cancer. By exploiting functional and GWAS data, we investigated the associations between polymorphisms affecting gene function in the pancreas (expression quantitative trait loci, eQTLs) and PDAC risk. In a two-phase approach, we analysed 13 713 PDAC cases and 43 784 controls and identified a genome-wide significant association between the A allele of the rs2035875 polymorphism and increased PDAC risk (P=7.14×10 -10). This allele is known to be associated with increased expression in the pancreas of the keratin genes KRT8 and KRT18, whose increased levels have been reported to correlate with various tumor cell characteristics. Additionally, the A allele of the rs789744 variant was associated with decreased risk of developing PDAC (P=3.56×10 -6). This SNP is situated in the SRGAP1 gene and the A allele is associated with higher expression of the gene, which in turn inactivates the cyclin-dependent protein 42 (CDC42) gene expression, thus decreasing the risk of PDAC. In conclusion, we present here a functional-based novel PDAC risk locus and an additional strong candidate supported by significant associations and plausible biological mechanisms.

Epigenetic cancers lead to tumor progression via oncogene activation, tumor suppressor silencing, cell fate transitions, and genomic instability. Approximately 50% of tumors have alterations in Chromatin regulators. One group of Chromatin regulators, the mSWI/SNF complexes, are mutated in 20% of tumors making them one of the most mutated chromatin regulator complexes in cancer. In the past, the mechanism of action of ATP-dependent chromatin regulators, such as mSWI/SNF was largely explored using in vitro methods based on measurements of nucleosome mobility. These studies were limited by the fact that these assays were not sensitive to tissue-specific chromatin modifications, topology, long-range interactions, and complex patterns of histone modifications as well as patterns of DNA methylation and human disease mutational backgrounds. To circumvent these problems, we developed the FIRE-Cas9 system that allows one to recruit a specific chromatin regulator and follow the consequences with minute-by-minute kinetics on physiologic chromatin. I can examine any locus of biological or medical importance in virtually any cell type using this technique. We can recruit endogenous proteins or protein complexes such as different mSWI/SNF complexes, histone methyltransferases, or histone demethylases and assay changes in chromatin state and transcriptional state. Because chromatin regulator complexes have alterations in many human cancers, it is imperative to understand the mechanism of action of these complexes with unparalleled precision as a basis for therapeutic development. The FIRE-Cas9 system allows one to do this for the first time. Citation Format: Mary Bergwell, JinYoung Park, Laura Pistoni, Jacob G. Kirkland. Chromatin engineering using a dCas9-based inducible dimerization system. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr B001.

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