Reinhard Büttner | Maria Anokhina | Margarete Odenthal - A 01

Alterations in alternative splicing (AS) profiles are crucial for cancer initiation, progression and metastasis as well as immune defense. RNA splicing and transcription is a coupled process, subjected to epigenetic modifications.

Histone modifying complexes impact the outcome of splicing by regulating the histone marks that play a role in splice site recognition and exon definition, affecting the recruitment of the splicing machinery.

The lysine-specific demethylase 1 is a histone modifier that is overexpressed in a wide variety of cancer types. In this study, we propose to address the mechanistic linkage of the lysine-specific demethylase 1 to cancer associated alternative splicing.

In the present project, we study the mechanistic links of histone modification to oncogenic alternative splic-ing on liver and non-small cell lung cancer, using representative cell systems and transgenic mouse models, both carrying cancer relevant, tumor driving mutations. In addition, we investigate the clinical impact of epigenetically regulated alternative splicing on human liver and lung cancer samples.

1. In order to examine the impact of the H3K4/K9 methylation signature on alternative splicing, the lysine-specific demethylase 1 will be in¬hibited either pharmacologically or by means of mutations in the catalytic or protein interactive domains, and the alternative spliced transcriptome will be analysed.
2. Notably, the oncogenic splicing signature of tumor driving key factors will be further examined by minigene reporter construction, by splice site blockade using morpholinos, and additionally by in vivo studies using established transgenic mouse models as well as human lung and liver cancer specimens.

Alternative splicing is a major source of protein diversity involved in many cellular processes. Constitutive splicing is carried out to remove introns by usage of conserved canonical splice sites, whereas alternative splicing (AS) is controlled by additional cis-regulatory elements within the pre-mRNA.

The exonic and intronic splicing enhancer (ESE, ISE) and silencer (ESS, ISS) elements are critical for correct exon recognition and splicing outcome. They define the strength of the splice site by recruitment of the trans-acting splicing factors, mainly including serine/arginine-rich splicing factors (SRSFs) and heterogeneous nuclear ribonucleoproteins (hnRNPs). In response to oncogenic stress, aberrant splicing is observed, strongly promoting cancer progression. Our current findings indicate that epi­genetic dysregulation contributes to a cancer associated, oncogenic alternative splicing pattern.

Chromatin modifying complexes are suggested to be involved in regulating both transcription and splicing by modifying the DNA and histone proteins. Post-translational modifications on the histone tails  are assumed to affect alternative splicing by their influence on the transcription rate, which in turn affects the establishment of the splicing complexes, or by their influence on the recruitment of the splicing machinery, itself. The lysine-specific demethylase 1 is known to be crucial for chromatin remodeling by demethylation of histone 3, namely mono- and di-methylated lysine 4 (H3K4) and lysine 9 (H3K9), resulting in transcriptional gene repression or activation, respectively.

Lysine-specific demethylase 1 is highly overexpressed in a wide variety of cancer types (1-3). Its expression is known to correlate with tumor grade and is associated with malignancy (3) (Figure 1).  Our recent studies reveal that cancer associated overexpression of the lysine-specific demethylase 1 leads to transcriptional control of mediators controlling cell cycle progression (5-6). Moreover, our novel findings provide evidence that expression of the splicing trans-acting machinery is epigenetically controlled by the H3K4 und H3K9 methylation marks.

1. Lim, S., A. Janzer, A. Becker, A. Zimmer, R. Schule, R. Buettner, and J. Kirfel, Lysine-specific demethylase 1 (LSD1) is highly expressed in ER-negative breast cancers and a biomarker predicting aggressive biology. Carcinogenesis, 2010. 31(3): p. 512-20.
2. Janzer, A., S. Lim, F. Fronhoffs, N. Niazy, R. Buettner, and J. Kirfel, Lysine-specific demethyl-ase 1 (LSD1) and histone deacetylase 1 (HDAC1) synergistically repress proinflammatory cy-tokines and classical complement pathway components. Biochem Biophys Res Commun, 2012. 421(4): p. 665-70.
3. Lim, S.Y., I. Macheleidt, P. Dalvi, S.C. Schafer, M. Kerick, L. Ozretic, S. Ortiz-Cuaran, J. George, S. Merkelbach-Bruse, J. Wolf, B. Timmermann, R.K. Thomas, M.R. Schweiger, R. Buettner, and M. Odenthal, LSD1 modulates the non-canonical integrin beta3 signaling pathway in non-small cell lung carcinoma cells. Sci Rep, 2017. 7(1): p. 10292.
4. Macheleidt, I.F., P.S. Dalvi, S.Y. Lim, S. Meemboor, L. Meder, O. Kasgen, M. Muller, K. Klee-mann, L. Wang, P. Nurnberg, V. Russeler, S.C. Schafer, E. Mahabir, R. Buttner, and M. Odenthal, Preclinical studies reveal that LSD1 inhibition results in tumor growth arrest in lung adenocarcinoma independently of driver mutations. Mol Oncol, 2018. 12(11): p. 1965-1979.
5. Dalvi, P.S., I.F. Macheleidt, S.Y. Lim, S. Meemboor, M. Muller, H. Eischeid-Scholz, S.C. Schaefer, R. Buettner, S. Klein, and M. Odenthal, LSD1 Inhibition Attenuates Tumor Growth by Disrupting PLK1 Mitotic Pathway. Mol Cancer Res, 2019. 17(6): p. 1326-1337.