Epigenetic reprogramming of pancreatic cancer cells as a new therapeutic option

Pichlmeier, Svenja (2023): Epigenetic reprogramming of pancreatic cancer cells as a new therapeutic option. Dissertation, LMU München: Medizinische Fakultät

Abstract

Large-scale gene expression analyses have demonstrated that pancreatic ductal adenocarcinoma can be classified into different molecular subtypes with clinical significance (Collisson et al., 2011, Moffitt et al., 2016, Bailey et al., 2016). So far, great effort has been put into unveiling the factors responsible for tumor heterogeneity in pancreatic cancer. Since epigenetic modifiers are, besides the four driver gene mutations KRAS, p16, p53, and SMAD4, among the most frequently mutated genes in PDAC, this study aimed at investigating the role of epigenetic changes in the two molecular PDAC cancer subtypes, represented by a classical and basal phenotype, as well as their therapeutic potential in pancreatic cancer cell lines (Bailey et al., 2016). The data showed that subtype-specific gene expression of cellular differentiation marker genes, such as EpCAM and GATA6, is epigenetically regulated. Chromatin-immunoprecipitation results demonstrated that the expression of these epithelial differentiation marker genes is activated through histone acetylation marks in the classical subtype. In contrast, their expression is repressed in the basal or quasimesenchymal subtype through increased levels of histone ubiquitination as well as a loss of histone acetylation marks. DNA methylation seemed to only play a minor part in regulating subtype-specific gene expression profiles of EpCAM and GATA6. Despite subtype-specific histone acetylation levels, single-drug treatment with chemical inhibitors targeting histone acetylation and deacetylation marks only showed limited effects in vitro. Classical and basal PDAC cell lines were almost completely resistant to HAT inhibitor treatment with A485. Only one of the basal cell lines, MIAPaca-2, reached a 50 % survival rate at the maximum dosage of 10 µM A485 (see Figure 12A, left panel). High doses of the HDAC inhibitor Vorinostat were able to inhibit cell survival to a greater extent, but the response was independent of the transcriptomic subtypes. It is possible that a compensatory upregulation of other epigenetic modifications limits the therapeutic effects. Hence, a multiplex CRISPR/Cas9 knockout plasmid targeting a combination of epigenetic modifiers (HDAC2, DNMT3A, RING1B) was constructed to induce simultaneous genetic knockout of all three target genes. However, transfection of a basal pancreatic cancer cell line with this plasmid did not yield a successful knockout cell line. Most likely, the combinatory knockout impaired critical cellular functions to such an extent that cell death occurred. To overcome the limitations of a multiplex CRISPR/Cas9 knockout strategy, a successive knockout of one target gene after the other might be a more successful strategy to analyze the effect of a combinatory loss of different epigenetic modifiers. Furthermore, a selection marker should be included in the plasmids to ensure successful transfection. The generated knockout cell line can then be used for transcriptome analysis by RNA sequencing as well as for basic cell assays and drug sensitivity tests. In order to translate preclinical data with epigenetic inhibitors into successful clinical trials, further studies are needed to determine subtype-specific changes after epigenetic targeting. For instance, unpublished data within the working group showed that HAT inhibitor treatment of cell lines with a classical PDAC subtype strongly downregulated the expression of GATA6 and decreased Gemcitabine drug sensitivity indicating a poorer outcome. These results emphasize the importance of establishing patient stratification systems in order to maximize the success of current treatment strategies. Overall, this thesis showed that the transcriptomic profiles defining molecular PDAC subtypes are in part regulated through epigenetic modifications. Although the targeting of single epigenetic modifiers showed some success in tumor cell reprogramming, the therapeutic targeting with epigenetic drugs remains limited. Thus, the precise effects of combination therapies with multiple epigenetic inhibitors need further investigation.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: pancreatic cancer, epigenetics, molecular subtypes
• Themengebiete: 600 Technik, Medizin, angewandte Wissenschaften ; 600 Technik, Medizin, angewandte Wissenschaften > 610 Medizin und Gesundheit
• Fakultäten: Medizinische Fakultät
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 12. Januar 2023
• 1. Bericht­erstatter:in: Mayerle, Julia