Cellular

2nd field of activity

Prof. Dr. Ulrike A. Nuber (TU Darmstadt, Biologie)

ATP-dependent SWI/SNF chromatin remodeling complexes change the interaction between genomic DNA and histone proteins and thereby control chromatin accessibility and structure. These complexes play a pivotal role in the spatial and temporal control of gene expression and ultimately determine normal and diseased cell and tissue states. Close to 20% of all human cancers harbor SWI/SNF complex gene mutations thus constituting the most frequently mutated genes in human tumors. In addition to their role in regulating gene expression, recent data support a function in maintaining genome stability by transcription-independent pathways (Brownlee et al., DNA Repair 2015).

In this project, we focus on the tumor suppressor SMARCB1 (also referred to as BAF47, INI1, hSNF5), a core subunit of mammalian SWI/SNF complexes. Mutations in the human SMARCB1 gene are associated with and assumed to be causally involved in two distinct diseases: malignant tumors and Coffin-Siris syndrome (the latter being characterized by intellectual disability, facial coarseness, microcephaly of varying degree, and hypoplasia/aplasia of the fifth digit or finger-/toenail).

Although published results support a function of SMARCB1 in promoting nucleotide excision repair (Gong et al., Nature Struc & Mol Biology 2006; Ray et al., Mol Cell Biol 2009) and DNA double strand break repair (Watanabe et al., 2014), tumors that lack SMARCB1 function are exceptional as they possess extremely few mutations with loss of SMARCB1 being essentially the sole recurrent event (Kenna et al., Mol Cell Biology 2008; Hasselblatt et al., Genes Chromosomes Cancer 2013, Lee et al. J Clin Invest 2012). Moreover, no genomic instability is detected in SMARCB1-deficient cells (Kenna et al., Mol Cell Biology 2008).

Most of the biallelic SMARCB1 mutations in rhabdoid tumors are somatic ones, however germline mutations in one SMARCB1 allele are estimated to be present in up to one third of patients with rhabdoid tumors (Sredni and Tomita, 2015). In addition to sporadic tumors, familial cases are known and occur in the context of the so-called rhabdoid tumor predisposition syndrome 1.

A major limitation of many cellular systems used to study human tumor biology, in particular the function of tumor suppressor genes, is their failure to recapitulate the temporal changes that normally occur during tumor development and that are associated with adaptive cellular alterations, finally leading to a tumorigenic cell state. In this project, we will develop a cell system that allows us to recapitulate temporal changes occurring in SMARCB1-associated human tumors – namely the sequential occurrence of loss-of-function mutations in the two SMARCB1 alleles. This system will be used to study the role of SMARCB1 in DNA repair processes.

Prof. Dr. Franz Rödel (Klinikum der Goethe-Universität, Frankfurt)

The impact of the Inhibitor of Apoptosis Protein (IAP) Survivin on the cellular radiation response is not restricted to the inhibition of apoptotic pathways but further comprises modulation of DNA damage response. By using colorectal SW480 cancer cells stably expressing a variety of deletion and phosphorylation mutants, we recently indicated the importance of the X-linked IAP (XIAP) binding and T34 phosphorylation site for radiation survival and modulation of DNA repair as indicated by increased numbers of gammaH2AX/53BP1 foci detection. Functionally, in wildtype (wt) Survivin and phosphomimetic T34D expressing cells the protein co-immuno- precipitated with DNA-dependent protein kinase catalytic subunit (DNA-PKCS) and more specific with the kinase (PIK3) domain of DNA-PKcs, while mutants did not show complexation or rescue of an impaired DNA-PKcs kinase activity upon knockdown of endogenous Survivin by siRNA. These data indicate the XIAP binding and T34 phosphorylation site of Survivin to be essential for the modulation of the non-homologous end-joining repair (NHEJ) pathway probably by interfering with the activity of DNA-PKcs. Moreover, SILAC (stable isotope labeling with amino acids in cell culture) and mass spectroscopic analyses further revealed an interaction of survivin with Poly(ADP-ribose)-Polymerase 1 (PARP-1) and X-ray repair cross-complementing protein 1 (XRCC1). Consequently, using the above mentioned wt/mutant constructs the project aims at further analyzing the functional impact of Survivin expression on NHEJ and on additional DNA damage repair mechanisms like alternative end-joining repair and base excision repair pathways.

PD Dr. Michael Scholz (GSI Biophysik)

In previous work we could demonstrate the relevance of DSB-damage classification based on DSB clustering in chromatin loops by means of biophysical models like the Local Effect Model (LEM) and the Giant-LOop Binary LEsion (GLOBLE) model. This work comprised validation by application to different radiation qualities (high energy photon beams, ultrasoft X-rays and ion beams) and different biological endpoints (cell killing after acute and protracted irradiation, DSB rejoining, cell cycle dependent sensitivity).

In the new project the models will be extended to applications on more complex endpoints like e.g. radiation induced cell transformation and cancer induction. Therefore, cell killing and transformation need to be characterized as competing processes using adequately chosen dose-response curves for the two components. Observed transformation and cancer induction probabilities will then be derived by appropriate combination of both endpoints.

These methods need to be implemented in the LEM computer programs and consistence checks will be performed. The accuracy of the model predictions will be tested by detailed comparison to published experimental data. Furthermore, uncertainties will be characterized by the corresponding sensitivity analyses.

The model will then be used e.g. for risk estimates of high energetic ion beams, as they occur in galactic cosmic rays. Implementation in the treatment planning program TRiP98 will also allow performing corresponding risk estimates in the framework of ion beam tumor therapy.

Prof. Dr. Alexander Loewer (TU Darmstadt, Biologie)

The tumor suppressor p53 is activated by cellular stress and controls target genes that mediate cellular response programs such as cell cycle arrest, repair and apoptosis. One of the most dangerous forms of cellular stress is the induction of DNA double strand breaks (DSBs) by ionizing radiation. The cellular response to DSBs is coordinated by the PI3K-like kinases ATM, ATR and DNA-PKcs, which also relay the damage signal to p53. We recently identified a new regulatory interplay in which loss of DNA-PKcs function leads to hyper-activation of ATM and amplification of the p53 response, sensitizing cells for damage-induced senescence (Finzel et al Mol Biol Cell 2016).

The present project aims to dissect the dynamics of damage signaling from the time of break induction to its repair and to identify molecular mechanisms mediating the interplay of PI3K-like kinases. To this end, time-resolved quantitative measurements in fluorescent reporter cells will be combined with targeted genetic or pharmacological perturbations and biochemical analysis. Using this approach, a dynamic profile of kinase activation during break recognition and repair will be determined for damage of different complexity depending on the cellular state. The temporal requirements for known activators will be probed by perturbation experiment to reveal when and how ATM is hyper-activated upon DNA-PKcs inhibition. Finally, the consequences of kinase activation on p53 activation and cellular fate will be determined.