Introduction

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.

Our Aims

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.

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.
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.

Previous Work

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.

Project Related Publications

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.
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.
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.
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.
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.

Publications

Publications 2019 (January - August)

Castiglione, R., Alidousty, C., Holz, B., Wagener, S., Baar, T., Heydt, C., Binot, E., Zupp, S., Kron, A., Wolf, J., Merkelbach-Bruse, S., Reinhardt, H.C., Buettner, R., and Schultheis, A.M. (2019). Comparison of the genomic background of MET-altered carcinomas of the lung: biological differences and analogies. Mod Pathol 32, 627-38.

Dalvi, P.S., Macheleidt, I.F., Lim, S.Y., Meemboor, S., Muller, M., Eischeid-Scholz, H., Schaefer, S.C., Buettner, R., Klein, S., and Odenthal, M. (2019). LSD1 Inhibition Attenuates Tumor Growth by Disrupting PLK1 Mitotic Pathway. Mol Cancer Res 17, 1326-37.

Dammert, M.A., Bragelmann, J., Olsen, R.R., Bohm, S., Monhasery, N., Whitney, C.P., Chalishazar, M.D., Tumbrink, H.L., Guthrie, M.R., Klein, S., Ireland, A.S., Ryan, J., Schmitt, A., Marx, A., Ozretic, L., Castiglione, R., Lorenz, C., Jachimowicz, R.D., Wolf, E., Thomas, R.K., Poirier, J.T., Buttner, R., Sen, T., Byers, L.A., Reinhardt, H.C., Letai, A., Oliver, T.G., and Sos, M.L. (2019). MYC paralog-dependent apoptotic priming orchestrates a spectrum of vulnerabilities in small cell lung cancer. Nat Commun 10, 3485.

Grimm, C., Fischer, A., Farrelly, A.M., Kalachand, R., Castiglione, R., Wasserburger, E., Hussong, M., Schultheis, A.M., Altmuller, J., Thiele, H., Reinhardt, H.C., Hauschulz, K., Hennessy, B.T., Herwig, R., Lienhard, M., Buettner, R., and Schweiger, M.R. (2019). Combined Targeted Resequencing of Cytosine DNA Methylation and Mutations of DNA Repair Genes with Potential Use for Poly(ADP-Ribose) Polymerase 1 Inhibitor Sensitivity Testing. J Mol Diagn 21, 198-213.

Howell, J., Atkinson, S.R., Pinato, D.J., Knapp, S., Ward, C., Minisini, R., Burlone, M.E., Leutner, M., Pirisi, M., Buttner, R., Khan, S.A., Thursz, M., Odenthal, M., and Sharma, R. (2019). Identification of mutations in circulating cell-free tumour DNA as a biomarker in hepatocellular carcinoma. Eur J Cancer 116, 56-66.

Mariappan, A., Soni, K., Schorpp, K., Zhao, F., Minakar, A., Zheng, X., Mandad, S., Macheleidt, I., Ramani, A., Kubelka, T., Dawidowski, M., Golfmann, K., Wason, A., Yang, C., Simons, J., Schmalz, H.G., Hyman, A.A., Aneja, R., Ullrich, R., Urlaub, H., Odenthal, M., Buttner, R., Li, H., Sattler, M., Hadian, K., and Gopalakrishnan, J. (2019). Inhibition of CPAP-tubulin interaction prevents proliferation of centrosome-amplified cancer cells. EMBO J 38.

Rossi, A., Dupaty, L., Aillot, L., Zhang, L., Gallien, C., Hallek, M., Odenthal, M., Adriouch, S., Salvetti, A., and Buning, H. (2019). Vector uncoating limits adeno-associated viral vector-mediated transduction of human dendritic cells and vector immunogenicity. Sci Rep 9, 3631.

Zhang, L., Rossi, A., Lange, L., Meumann, N., Koitzsch, U., Christie, K., Nesbit, A., Moore, T., Hacker, U., Morgan, M.A., Hoffmann, D., Zengel, J.R., Carette, J.E., Schambach, A., Salvetti, A., Odenthal, M., and Buning, H. (2019). Capsid engineering overcomes barriers toward Adeno-associated viral (AAV) vector-mediated transduction of endothelial cells. Hum Gene Ther10.1089/hum.2019.027.

Publications 2018

Ackermann S, Cartolano M, Hero B, Welte A, Kahlert Y, Roderwieser A, Bartenhagen C, Walter E, Gecht J, Kerschke L, Volland R, Menon R, Heuckmann JM, Gartlgruber M, Hartlieb S, Henrich KO, Okonechnikov K, Altmuller J, Nurnberg P, Lefever S, de Wilde B, Sand F, Ikram F, Rosswog C, Fischer J, Theissen J, Hertwig F, Singhi AD, Simon T, Vogel W, Perner S, Krug B, Schmidt M, Rahmann S, Achter V, Lang U, Vokuhl C, Ortmann M, Buttner R, Eggert A, Speleman F, O'Sullivan RJ, Thomas RK, Berthold F, Vandesompele J, Schramm A, Westermann F, Schulte JH, Peifer M, and Fischer M (2018). A mechanistic classification of clinical phenotypes in neuroblastoma. Science 362, 1165-1170.

Castiglione R, Alidousty C, Holz B, Wagener S, Baar T, Heydt C, Binot E, Zupp S, Kron A, Wolf J, Merkelbach-Bruse S, Reinhardt HC, Buettner R, and Schultheis AM (2018). Comparison of the genomic background of MET-altered carcinomas of the lung: biological differences and analogies. Mod Pathol10.1038/s41379-018-0182-8.

Drapkin BJ, George J, Christensen CL, Mino-Kenudson M, Dries R, Sundaresan T, Phat S, Myers DT, Zhong J, Igo P, Hazar-Rethinam MH, Licausi JA, Gomez-Caraballo M, Kem M, Jani KN, Azimi R, Abedpour N, Menon R, Lakis S, Heist RS, Buttner R, Haas S, Sequist LV, Shaw AT, Wong KK, Hata AN, Toner M, Maheswaran S, Haber DA, Peifer M, Dyson N, Thomas RK, and Farago AF (2018). Genomic and Functional Fidelity of Small Cell Lung Cancer Patient-Derived Xenografts. Cancer Discov 8, 600-615.

Fassunke J, Muller F, Keul M, Michels S, Dammert MA, Schmitt A, Plenker D, Lategahn J, Heydt C, Bragelmann J, Tumbrink HL, Alber Y, Klein S, Heimsoeth A, Dahmen I, Fischer RN, Scheffler M, Ihle MA, Priesner V, Scheel AH, Wagener S, Kron A, Frank K, Garbert K, Persigehl T, Pusken M, Haneder S, Schaaf B, Rodermann E, Engel-Riedel W, Felip E, Smit EF, Merkelbach-Bruse S, Reinhardt HC, Kast SM, Wolf J, Rauh D, Buttner R, and Sos ML (2018). Overcoming EGFR(G724S)-mediated osimertinib resistance through unique binding characteristics of second-generation EGFR inhibitors. Nat Commun 9, 4655.

Frank R, Scheffler M, Merkelbach-Bruse S, Ihle MA, Kron A, Rauer M, Ueckeroth F, Konig K, Michels S, Fischer R, Eisert A, Fassunke J, Heydt C, Serke M, Ko YD, Gerigk U, Geist T, Kaminsky B, Heukamp LC, Clement-Ziza M, Buttner R, and Wolf J (2018). Clinical and Pathological Characteristics of KEAP1- and NFE2L2-Mutated Non-Small Cell Lung Carcinoma (NSCLC). Clin Cancer Res10.1158/1078-0432.CCR-17-3416.

George J, Walter V, Peifer M, Alexandrov LB, Seidel D, Leenders F, Maas L, Muller C, Dahmen I, Delhomme TM, Ardin M, Leblay N, Byrnes G, Sun R, De Reynies A, McLeer-Florin A, Bosco G, Malchers F, Menon R, Altmuller J, Becker C, Nurnberg P, Achter V, Lang U, Schneider PM, Bogus M, Soloway MG, Wilkerson MD, Cun Y, McKay JD, Moro-Sibilot D, Brambilla CG, Lantuejoul S, Lemaitre N, Soltermann A, Weder W, Tischler V, Brustugun OT, Lund-Iversen M, Helland A, Solberg S, Ansen S, Wright G, Solomon B, Roz L, Pastorino U, Petersen I, Clement JH, Sanger J, Wolf J, Vingron M, Zander T, Perner S, Travis WD, Haas SA, Olivier M, Foll M, Buttner R, Hayes DN, Brambilla E, Fernandez-Cuesta L, and Thomas RK (2018). Integrative genomic profiling of large-cell neuroendocrine carcinomas reveals distinct subtypes of high-grade neuroendocrine lung tumors. Nat Commun 9, 1048.

Golfmann K, Meder L, Koker M, Volz C, Borchmann S, Tharun L, Dietlein F, Malchers F, Florin A, Buttner R, Rosen N, Rodrik-Outmezguine V, Hallek M, and Ullrich RT (2018). Synergistic anti-angiogenic treatment effects by dual FGFR1 and VEGFR1 inhibition in FGFR1-amplified breast cancer. Oncogene 37, 5682-5693.

Grasse S, Lienhard M, Frese S, Kerick M, Steinbach A, Grimm C, Hussong M, Rolff J, Becker M, Dreher F, Schirmer U, Boerno S, Ramisch A, Leschber G, Timmermann B, Grohe C, Luders H, Vingron M, Fichtner I, Klein S, Odenthal M, Buttner R, Lehrach H, Sultmann H, Herwig R, and Schweiger MR (2018). Epigenomic profiling of non-small cell lung cancer xenografts uncover LRP12 DNA methylation as predictive biomarker for carboplatin resistance. Genome Med 10, 55.

Grimm C, Fischer A, Farrelly AM, Kalachand R, Castiglione R, Wasserburger E, Hussong M, Schultheis AM, Altmuller J, Thiele H, Reinhardt HC, Hauschulz K, Hennessy BT, Herwig R, Lienhard M, Buettner R, and Schweiger MR (2018). Combined Targeted Re-Sequencing of Cytosine DNA Methylation and Mutations of DNA Repair Genes with Potential Use for PARP1 Inhibitor Sensitivity Testing. J Mol Diagn10.1016/j.jmoldx.2018.10.007.

Herling CD, Abedpour N, Weiss J, Schmitt A, Jachimowicz RD, Merkel O, Cartolano M, Oberbeck S, Mayer P, Berg V, Thomalla D, Kutsch N, Stiefelhagen M, Cramer P, Wendtner CM, Persigehl T, Saleh A, Altmuller J, Nurnberg P, Pallasch C, Achter V, Lang U, Eichhorst B, Castiglione R, Schafer SC, Buttner R, Kreuzer KA, Reinhardt HC, Hallek M, Frenzel LP, and Peifer M (2018). Clonal dynamics towards the development of venetoclax resistance in chronic lymphocytic leukemia. Nat Commun 9, 727.

Macheleidt IF, Dalvi PS, Lim SY, Meemboor S, Meder L, Kasgen O, Muller M, Kleemann K, Wang L, Nurnberg P, Russeler V, Schafer SC, Mahabir E, Buttner R, and Odenthal M (2018). Preclinical studies reveal that LSD1 inhibition results in tumor growth arrest in lung adenocarcinoma independently of driver mutations. Mol Oncol 12, 1965-1979.

Mariappan A, Soni K, Schorpp K, Zhao F, Minakar A, Zheng X, Mandad S, Macheleidt I, Ramani A, Kubelka T, Dawidowski M, Golfmann K, Wason A, Yang C, Simons J, Schmalz HG, Hyman AA, Aneja R, Ullrich R, Urlaub H, Odenthal M, Buttner R, Li H, Sattler M, Hadian K, and Gopalakrishnan J (2019). Inhibition of CPAP-tubulin interaction prevents proliferation of centrosome-amplified cancer cells. EMBO J10.15252/embj.201899876.

Meder L, Konig K, Dietlein F, Macheleidt I, Florin A, Ercanoglu MS, Rommerscheidt-Fuss U, Koker M, Schon G, Odenthal M, Klein F, Buttner R, Schulte JH, Heukamp LC, and Ullrich RT (2018). LIN28B enhanced tumorigenesis in an autochthonous KRAS(G12V)-driven lung carcinoma mouse model. Oncogene 37, 2746-2756.

Meder L, Schuldt P, Thelen M, Schmitt A, Dietlein F, Klein S, Borchmann S, Wennhold K, Vlasic I, Oberbeck S, Riedel R, Florin A, Golfmann K, Schlosser HA, Odenthal M, Buttner R, Wolf J, Hallek M, Herling M, von Bergwelt-Baildon M, Reinhardt HC, and Ullrich RT (2018). Combined VEGF and PD-L1 blockade displays synergistic treatment effects in an autochthonous mouse model of small cell lung cancer. Cancer Res10.1158/0008-5472.CAN-17-2176.

Meyer MF, Meinrath J, Seehawer J, Lechner A, Odenthal M, Quaas A, Semrau R, Huebbers CU, Marnitz S, Buttner R, and Beutner D (2018). The relevance of the lymph node ratio as predictor of prognosis is higher in HPV-negative than in HPV-positive oropharyngeal squamous cell carcinoma. Clin Otolaryngol 43, 192-198.

Plenker D, Bertrand M, de Langen AJ, Riedel R, Lorenz C, Scheel AH, Muller J, Bragelmann J, Dassler-Plenker J, Kobe C, Persigehl T, Kluge A, Wurdinger T, Schellen P, Hartmann G, Zacherle T, Menon R, Thunnissen E, Buttner R, Griesinger F, Wolf J, Heukamp L, Sos ML, and Heuckmann JM (2018). Structural Alterations of MET Trigger Response to MET Kinase Inhibition in Lung Adenocarcinoma Patients. Clin Cancer Res 24, 1337-1343.

Torgovnick A, Heger JM, Liaki V, Isensee J, Schmitt A, Knittel G, Riabinska A, Beleggia F, Laurien L, Leeser U, Jungst C, Siedek F, Vogel W, Klumper N, Nolte H, Wittersheim M, Tharun L, Castiglione R, Kruger M, Schauss A, Perner S, Pasparakis M, Buttner R, Persigehl T, Hucho T, Herter-Sprie GS, Schumacher B, and Reinhardt HC (2018). The Cdkn1a(SUPER) Mouse as a Tool to Study p53-Mediated Tumor Suppression. Cell Rep 25, 1027-1039 e1026.

Welcker D, Jain M, Khurshid S, Jokic M, Hohne M, Schmitt A, Frommolt P, Niessen CM, Spiro J, Persigehl T, Wittersheim M, Buttner R, Fanciulli M, Schermer B, Reinhardt HC, Benzing T, and Hopker K (2018). AATF suppresses apoptosis, promotes proliferation and is critical for Kras-driven lung cancer. Oncogene 37, 1503-1518.