Center for Molecular Medicine Cologne

Michal-Ruth Schweiger | Simon Poepsel | Jens Klußmann - A 12

Structure of the BRD4 stress complex and implications in HPV maintenance and therapy response

Inroduction

BRD4 is a potent target of chemotherapeutic treatments. To further improve the targeted therapeutic strategies a better knowledge of its structure and function in discrete contexts such as e.g. nuclear stress bodies (nSBs) is required.

Together with Dr. Poepsel and Prof. Klußmann we aim to perform mass spectrometry and structural analyses to explore context specific BRD4 interactions and BRD4 containing regulatory complexes, and investigate the consequences of a de-regulation of BRD4 on HNSCC tumor pathophysiology through papillomaviruses.

In the last decade the epigenetic sensor protein bromodomain protein 4 (BRD4) has been discovered as a valuable drug target in many tumor entities, including leukemia, lung cancer, multiple myeloma (MM) and melanoma. However, the exact mechanism of BRD4 inhibition as a powerful anti-cancer treatment is still unclear. Our work is based on the rationale that the ubiquitous epigenetic regulator BRD4 performs distinct biological functions depending on the local molecular context. Therefore, specifying the determinants of such contexts may help identifying interactions and functions that are particularly important in clinical scenarios.

Recently, we have shown that BRD4 interacts with heat shock factor 1 (HSF1), induces SatIII expression during heat shock and chemotherapeutic interventions and co-localizes with SatIII at nuclear stress bodies (nSBs).

We also found that an increase in SatIII expression induces chemotherapy resistance and inhibition of BRD4 with chemical compounds can revert this effect. These findings are clear indications of molecular players and processes involved in specific cancer- and stress-associated functions of BRD4. 

The aim of this project is to better understand BRD4’s functions at sites of nSB formation. We will use mass spectrometry, cryo-electron microscopy (cryo-EM) and functional analyses to gain insight into structural features of BRD4 interactions that define its function within nSB. In particular, we will investigate whether and how papillomaviruses can modify these structures and use it for its propagation. Consequences of this will be investigated in primary head and neck squamous cell carcinoma (HNSCC) tumors.

Knowledge about BRD4 function during the stress response will provide valuable means to optimize chemotherapeutic agents targeting BRD4 and to develop predictive biomarkers for HNSCC.

Our Aims

  1. Mass spectrometric analyses of BRD4 complexes under stress exposure and functional characterization of their effects on chromatin structure.
  2. Structural analysis of BRD4 containing complexes localized at nuclear stress bodies.
  3. Clinical consequences of disruption of the BRD4 complexes on i) therapy resistance, ii) papillomavirus infection and iii) head and neck squamous cell carcinomas (HNSCCs).

Previous Work

During our work on BRD4 we showed that BRD4 is a central part of the cellular stress response. It regulates the expression of the kelch like ECH associated protein 1 (KEAP1) and downstream nuclear factor erythroid 2 like 1 (NFE2L1) cytoprotective target genes. Furthermore, we found that during the heat stress response BRD4 interacts with the heat shock factor 1 (HSF1), co-localizes with HSF1 in nuclear stress bodies (nSB) and regulates Satellite SatIII RNA expression in an HSF1 dependent manner.

Satellites are repetitive sequences located at pericentromeric regions. An aberrant overexpression of SatIII together with decondensation and demethylation of pericentromeric DNA is found in numerous cancer entities and in genetic disorders such as Hutchinson-Gilford progeria. Furthermore, pericentromeric transcripts are involved in heterochromatin formation and genomic stability and might thus be a driving force in malignant transformation. During the heat and proteotoxic stress response, BRD4, HSF1 and SatIII RNA are localized at nSBs.

Within an independent string of research we had identified the papillomaviral protein E2 as BRD4 interacting partner. E2 binds to the C-terminal domain of BRD4, targets E2 to mitotic chromosomes and thereby ensures a faithful distribution of PV genomes to daughter cells. This might be in particular relevant for the development of HVP-related cancers such as cervical cancer and head and neck squamous cell carcinomas (HNSCC). HNSCC arise in the upper aerodigestive tract and infections with HPV have been recently implicated in its pathogenesis.

Specifically, carcinomas arising from the lymphoid tissue of the oropharynx are HPV-associated and they differ in clinical and molecular aspects compared to classical HNSCC. These differences are found for oncogenic pathways driven by genetic but also epigenetic changes. The incidence of this new virus-associated tumor entity is strongly increasing and now 50-80% of all oropharyngeal carcinomas are HPV16 positive. These patients have a significantly improved overall and disease-free survival compared with patients with HPV-negative diseases. They also present with an improved response rate to chemotherapies (including platin-derived chemotherapies).

The overall goal of the proposed project is to understanding BRD4’s function in HNSCC and to explore it as potential therapeutic target. This will open up new ways towards more rational BRD4-directed therapeutic interventions and the development of specific predictive biomarkers.

Description of the groups and their role within the project

The project benefits from the collaboration between three groups: The Department of Otorhinolaryngology, Head and Neck Surgery (Klussmann), the Laboratory of Structure and Biochemistry of Epigenetic Regulators (Poepsel) and the Institute for Translational Epigenetics (Schweiger).

The Schweiger group has long-standing experience in molecular biology and biochemistry as well as in the development and establishment of diverse epigenetic technologies including large-scale experiments and data analyses. A focus of the group are epigenetic regulations in cancer and their targeted disruption for cancer treatments.

The research of the Klussmann group focus on translational and clinical research on HPV-related head and neck cancer for many years. They have established large biobanks with detailed clinical data from patients with HPV-related and unrelated HNSCC.

The Poepsel Lab focuses on the regulation of chromatin modifying enzymes and the read-out of chromatin modifications such as histone acetylation and methylation, with a particular emphasis on how multi-protein complexes are targeted to and locally regulated at their genomic sites of action. Their expertise relevant for this project lies in the isolation, characterization and structural elucidation of chromatin associated complexes.

Project Related Publications

  1. Grimm C, Fischer A, Farrelly AM, Kalachand R, Castiglione R, Wasserburger E, Hussong M, Schultheis AM, Altmüller J, Thiele H, Reinhardt HC, Hauschulz K, Hennessy BT, Herwig R, Lienhard M, Buettner R, Schweiger MR.: 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. 2019 Mar;21(2):198-213.
  2. Herberg M, Siebert S, Quaas M, Thalheim T, Rother K, Hussong M, Altmüller J, Kerner C, Galle J*, Schweiger MR*, Aust G.*: Loss of Msh2 and a single-radiation hit induce common, genome-wide, and persistent epigenetic changes in the intestine. Clin Epigenetics. 2019 Apr 27;11(1):65. *equally contributed
  3. 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, Grohé C, Lüders H, Vingron M, Fichtner I, Klein S, Odenthal M, Büttner R, Lehrach H, Sültmann H, Herwig R, Schweiger MR.: Epigenomic profiling of non-small cell lung cancer xenografts uncover LRP12 DNA methylation as predictive biomarker for carboplatin resistance. Genome Med. 2018 Jul 20;10(1):55.
  4. Lienhard M, Grasse S, Rolff J, Frese S, Schirmer U, Becker M, Börno S, Timmermann B, Chavez L, Sültmann H, Leschber G, Fichtner I, Schweiger MR*, Herwig R.*: QSEA-modelling of genome-wide DNA methylation from sequencing enrichment experiments. Nucleic Acids Res. 2017 Apr 7;45(6) *equally contributed
  5. Hussong M, Kaehler C, Kerick M, Grimm C, Franz A, Timmermann B, Welzel F, Isensee J, Hucho T, Krobitsch S, Schweiger MR.: The bromodomain protein BRD4 regulates splicing during heat shock. Nucleic Acids Res. 2017 Jan 9;45(1):382-394.
  6. Hussong M, Börno ST, Kerick M, Wunderlich A, Franz A, Sültmann H, Timmermann B, Lehrach H, Hirsch-Kauffmann M, Schweiger MR:The bromodomain protein BRD4 regulates the KEAP1/NRF2-dependent oxidative stress response.Cell Death Dis. 2014 Apr 24;5:e1195.
  7. Schweiger, M. R., You, J. & Howley, P. M. Bromodomain protein 4 mediates the papillomavirus E2 transcriptional activation function. Journal of virology80, 4276-4285, doi:10.1128/JVI.80.9.4276-4285.2006 (2006).
  8. You, J., Schweiger, M. R. & Howley, P. M. Inhibition of E2 binding to Brd4 enhances viral genome loss and phenotypic reversion of bovine papillomavirus-transformed cells. Journal of virology79, 14956-14961, doi:10.1128/JVI.79.23.14956-14961.2005 (2005).
  9. Reder H, Wagner S, Gamerdinger U, Sandmann S, Wuerdemann N, Braeuninger A, Dugas M, Gattenloehner S, Klussmann JP, Wittekindt C. Genetic alterations in human papillomavirus-associated oropharyngeal squamous cell carcinoma of patients with treatment failure. Oral Oncol. 2019 Jun;93:59-65.
  10. Wittekindt C, Wagner S, Bushnak A, Prigge ES, von Knebel Doeberitz M, Würdemann N, Bernhardt K, Pons-Kühnemann J, Maulbecker-Armstrong C, Klussmann JP. Increasing Incidence rates of Oropharyngeal Squamous Cell Carcinoma in Germany and Significance of Disease Burden Attributed to Human Papillomavirus. Cancer Prev Res (Phila). 2019 Jun;12(6):375-382.
  11. Huebbers CU, Verhees F, Poluschkin L, Olthof NC, Kolligs J, Siefer OG, Henfling M, Ramaekers FCS, Preuss SF, Beutner D, Seehawer J, Drebber U, Korkmaz Y, Lam WL, Vucic EA, Kremer B, Klussmann JP, Speel EM. Upregulation of AKR1C1 and AKR1C3 expression in OPSCC with integrated HPV16 and HPV-negative tumors is an indicator of poor prognosis. Int J Cancer. 2019 May 15;144(10):2465-2477.
  12. Knuth J, Sharma SJ, Würdemann N, Holler C, Garvalov BK, Acker T, Wittekindt C, Wagner S, Klussmann JP. Hypoxia-inducible factor-1α activation in HPV-positive head and neck squamous cell carcinoma cell lines. Oncotarget. 2017 Sep 11;8(52):89681-89691.
  13. Poepsel, S., Kasinath, V., & Nogales, E. (2018). Cryo-EM structures of PRC2 simultaneously engaged with two functionally distinct nucleosomes. Nature Structural & Molecular Biology,25,154–162.
  14. Kasinath, V., Faini, M., Poepsel, S., Reif, D., Feng, X. A., … Nogales, E. (2018). Structures of human PRC2 with its cofactors AEBP2 and JARID2. Science, 359(6378), 940–944
Prof. Dr. Dr. Michal-Ruth Schweiger CMMC Cologne
Prof. Dr. Dr. Michal-Ruth Schweiger

Institute for Translational Epigenomics

Principal Investigator - A 12

Executive Board Member

+49 221 478 96846

+49 221 478 7789

Institute for Translational Epigenomics

Weyertal 115b

50931 Cologne

CMMC Profile Page

Curriculum Vitae (CV)

Publications - Michal-Ruth Schweiger

Link to PubMed

Dr. Simon Poepsel CMMC Cologne
Dr. Simon Poepsel

Laboratory of Structure and Biochemistry of Epigenetic Regulators - JRG location CMMC

Principal Investigator - CMMC JRG 11

Co-Principal Investigator -  A 12

+49 221 47896987

Laboratory of Structure and Biochemistry of Epigenetic Regulators - JRG location CMMC

Robert-Koch-Str. 21

50931 Cologne

https://www.poepsel-lab.cmmc-uni-koeln.de

CMMC Profile Page

Publications on PubMed

Publications - Simon Pöpsel

Link to PubMed

Prof. Dr. Peter Jens Klussmann CMMC Cologne
Prof. Dr. Peter Jens Klussmann

Dept. of Otorhinolaryngology, Head and Neck Surgery

Co-Principal Investigator -  A 12

Executive Board Member

+49 221 478 85772

+49 221 478 62042

Dept. of Otorhinolaryngology, Head and Neck Surgery

Kerpener Str. 62

50937 Cologne

https://hno.uk-koeln.de

Publications - Jens Klußmann

Link to PubMed

Group Members

Dr. Maria Grosheva
Christian Hübbers
Christopher Kopp
Margaret Maltseva
Dr. Shachi Jenny Sharma
Oliver Siefer
Malte Suchan
Nora Würdemann
Philipp Zimmermann 

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