Inflammation-Driven Tumorigenesis

In one of our projects published within the last year, we extended our understanding of the role of H2B monoubiquitination in chronic intestinal inflammation (Kosinsky et al. 2021). The  monoubiquitination of the histone H2B at lysine 120 (H2Bub1) represents an epigenetic modification which is linked to tumorigenesis in several cancer types. A previous study examining 1,800 CRC tissue samples reported that a decrease in H2Bub1 was associated with advanced tumor grade and stage. The monoubiquitination of histone H2B is mediated by the E3 ubiquitin ligase activity of the RING finger proteins RNF20 and RNF40, which form an obligate heterodimer (Figure 1). Notably, H2Bub1 can promote transcriptional elongation by facilitating the recruitment of the Facilitates Chromatin Transcription (FACT) complex that displaces an H2A/H2B dimer to ease the passage of elongating RNA Polymerase II. The Ubiquitin-Specific Protease 22 (USP22) was shown to remove the monoubiquitination of H2B in a context-dependent manner.

We and others previously demonstrated that the ubiquitin ligases RNF20 and RNF40 play context- dependent roles in the development of inflammation (Kosinsky et al. 2021; Kosinsky et al. 2019). Therefore, to test for a putative function of the RNF20/40/H2Bub1 axis in controlling intestinal  inflammation, we performed immunohistochemical staining of H2Bub1 using a translational model  for colitis as well as in a panel of human IBD samples with corresponding uninflamed adjacent tissue  from resection margins as a control. Notably, when quantifying nuclear staining intensity, we  observed a significant decrease in H2Bub1 levels in inflamed epithelium (Figure 2A, B). In fact, when  comparing inflamed to healthy tissue within the same individual, we discovered that 80% of IBD patients displayed a strong reduction in H2Bub1 staining in inflamed epithelial cells, while only 20% of patients displayed no changes in H2Bub1 levels.

In agreement with this finding, the intestinal deletion of Rnf20 or Rnf40 in vivo resulted in the development of spontaneous colorectal inflammation. To gain insight into the molecular mechanisms  by which perturbation of the H2B monoubiquitination axis leads to intestinal inflammation, we performed next-generation sequencing approaches using intestinal epithelial cells (IECs). ChIP-seq for the histone mark H3K4me3, an established mark of actively transcribed transcription start sites, revealed that the loss of Rnf20 or Rnf40 resulted in a significant reduction of H3K4me3 occupancy in the gene body. Intriguingly, we found a profound H3K4me3 peak narrowing on the Vitamin D Receptor (Vdr) gene (Figure 2C) as well as VDR-bound regions in Rnf20- and Rnf40-deficient IECs. Accordingly, H2Bub1 reduction resulted in decreased mRNA and protein levels of VDR and its targets.

Given the established association of Vitamin D biology as a risk factor to IBD activity, a cohort of Crohn’s disease (CD) patients was analyzed for gene expression and H3K27ac occupancy. Genes differentially marked with H3K27ac between patients with low and high expression of RNF20/40- dependent genes were overlapped with genes which lost H3K4me3 occupancy in Rnf20- and Rnf40- deficient IECs. Intriguingly, 45% of genes differentially bound by H3K27ac in CD patients also displayed decreased H3K4me3 in Rnf20/40-deficient IECs (Figure 2D).

Together, our findings demonstrate that RNF20 and RNF40 not only regulate the IBD susceptibility gene VDR but also a high proportion of VDR target genes. Importantly, we identified a subset of IBD patients that displays deregulated expression and epigenetic marking of RNF20/40-dependent VDR targets.
 

During the next years, our group will mainly focus on epigenetic mechanisms associated with inflammation-associated and sporadic colorectal cancer. As IBD patients possess an increased risk to develop CRC, tissues isolated from ulcerative colitis as well as Crohn’s disease patients will be analyzed as well (Figure 3).

One part of our group will evaluate the H2Bub1-dependent epigenetic landscape promoting CRC progression and therapy resistance. Based on differences in colitis burden we observed in Rnf20- and Rnf40-deficient mice, we hypothesize that these E3 ligases exert differential effects on inflammation-driven and sporadic CRC and that tumorigenesis is influenced by the dosage of RNF20 and RNF40 expression. Therefore, we aim to elucidate their individual functions and identify their H2Bub1-independent ubiquitination targets. In addition, other team members will mainly focus on the transition from the inflammatory to the tumorous state. These projects are based on the hypothesis that the tumor microenvironment contains subsets of pro-inflammatory immune cells  whose expression level is actively influenced by the tumor. In order to test this, the interaction between epithelial and immune regulatory cells will be examined to determine how immune cells contribute to tumorigenesis as well as how different cellular compartments influence each other. There will be a strong emphasis on state-of-the-art next-generation sequencing approaches (such as spatial transcriptomics as well as single cell RNA and ATAC sequencing), histology, various in vitro and ex vivo culture techniques using primary patient material (including organoid and tissue slice cultures) as well as in vivo approaches (Saul & Kosinsky 2022; Kosinsky et al. 2021; Kosinsky et al. 2019).

Due to their major role in disease development, molecular changes in epithelial cells will be closely characterized. In addition, their interaction with and the contribution of immune regulatory cells will be examined. We aim to explore the targetability of cell populations contributing to the  abovementioned diseases taking tissue heterogeneity and resistance mechanisms into consideration. First collaborations with physicians at the RBK have been established to ensure the possibility to perform research covering clinical needs and to obtain primary tumor and control samples to work close to patients. Our long-term goal is the translation of our findings to the clinic and confirm our in vitro, ex vivo and in vivo results in clinical trials.

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