DNA damage responses and cancer

Development of novel (geno)toxicity test systems

Stem cell-based reporter assays            

Interaction of newly developed materials, chemicals and drugs with biomolecules may disrupt cellular homeostasis and can ultimately lead to severe tissue damage or induction of cancer. Inflicted cellular damage is recognized by specialized sensor proteins that trigger a complex network of cellular signaling pathways resulting in activation or inactivation of specific enzymes and altered expression of distinct gene networks. Visualization of damage-specific cellular stress response pathways provides insight into the type and extent of cellular damage that has been induced and thus the biological (re)activity of compounds. Following extensive whole-genome transcription profiling of mouse embryonic stem (mES) cells after exposure to over 40 different carcinogenic chemicals we generated the ToxTracker assay. This assay consists of a panel of six GFP-based mES reporter cell lines that are preferentially induced by specific types of damage and represent four distinct biological responses i.e. general cellular stress (Tp53), DNA damage, oxidative stress and the unfolded protein response (Hendriks et al., 2016). In 2014 the LUMC spin-off company Toxys, was founded that markets the Toxtracker assay for commercial purposes (https://toxys.com/). Together with Toxys, we are currently developing novel reporter cell lines for specific classes of toxicants. 

Human bronchial epithelial cell model

Primary human bronchial epithelial cells that are cultured at the air-liquid interface (PBEC-ALI) differentiate into a multi-cellular layer that contains various cell-types, including basal, club, ciliated and mucus producing cells. The most important characteristics of differentiated PBEC-ALI cultures encompass: a proper barrier function, formation of cilia beating cells and potent drug metabolism (Boei et al., 2017). We have established dedicated protocols for various toxicity-related endpoints such as the induction of cytotoxicity (TEER, LDH leakage), genotoxicity (micronucleus assay, comet assay and γH2AX measurements) or altered proliferation (EdU incorporation) that make these human 3D lung cultures a promising vehicle for the detection of toxic properties of (air-borne) chemicals.

We performed single cell RNAseq analysis of PBEC-ALI cultures to improve the identification and quantification of the various differentiated cell types in these cultures. Currently, we are investigating how the cell type composition may be (permanently) altered following exposure to (cell type-specific) toxicants in relation to the occurrence of lung diseases in patients.

Translating fundamental knowledge on DDR pathways to the clinic

Functional testing of BRCA2 variants

Genetic testing for pathogenic variants in BRCA1 and BRCA2 has become a routine procedure in the medical management of women with a family history of breast and ovarian cancer. Mutation carriers have a steeply increased risk for the development of breast cancer (26-87% cumulative risk) and ovarian cancer (11-60% cumulative risk) before the age of 70. Besides classical pathogenic mutations that truncate or inactivate the protein, the recent implementation of high-throughput sequence analysis is yielding a rapidly increasing number of genetic variants in cancer-predisposing genes for which the clinical significance in terms of cancer risk is unknown. As a consequence these so called “variants of uncertain clinical significance” (VUS) represent a major challenge for genetic counseling and clinical management of these patients since the pathogenicity of these VUS is unclear. Therefore, there is a strong demand for reliable tests to rapidly assess the clinical significance of VUS, providing VUS carriers with the necessary information to make an informed clinical decision and in addition to improve cancer treatment by personalized therapy strategies. To this end, we have developed efficient pipelines for functional testing of BRCA1 and BRCA2 variants which will improve VUS classification and contribute to informed clinical decision-making. More than 100 BRCA2 variants have been functionally evaluated and a high concordance between the impairment of the major tumor suppressor activity of BRCA2, i.e. homology-directed repair, and cancer risk has been established (Shimelis et al., 2017, Mesman et al., 2018). Recently we have shown that nonsense and canonical splice site variants in BRCA2 that are generally considered to represent loss-of-function (LoF) alleles and consequently are being classified as pathogenic, may sometimes be rescued through the production of alternative in-frame transcripts (Meulemans et al., 2020, Mesman et al., 2020). These findings emphasize the need for caution in the interpretation of putative LoF variants that are located in non-essential parts of the BRCA2 gene.

Functional assessment of the HR pathway in breast, ovarian and endometrial tumors

Recently, it has been established that BRCA1 and BRCA2-related breast and ovarian tumors respond very well to treatment with PARP inhibitors (PARPi) because of their deficiency in homology-directed repair (HDR). In addition, a substantial fraction of remaining tumors might contain deficiencies in HDR unrelated to BRCA1/2 deficiency. To determine functionality of the HDR repair pathway in fresh (e.g. breast, ovarian, endometrium) tumor tissue we have set up an ex vivo culture system that allows us to assess RAD51 foci formation in replicating tumor cells following irradiation (de Jonge et al., 2019, van Wijk et al., 2020). Identification of HDR-deficient tumors may facilitate the selection of additional patients eligible for treatment with PARP inhibitors.

Biological consequences of low dose ionizing radiation

Low dose IR-responses in stem cells

Epidemiological studies have clearly demonstrated ionizing radiation to be a human carcinogen. However, the cancer risk from the more commonly encountered (chronic) low dose IR (<100 mSv) exposures is unclear and is primarily based on risk extrapolations from populations exposed to higher levels of radiation using a linear no-threshold (LNT) model. Since stem cells are considered to be the cells of origin for many cancers, understanding their responses to radiation will improve knowledge of the processes that contribute to radiation carcinogenesis. To gain more insight into the cellular responses that are uniquely activated after low- and high-dose radiation, we performed extensive genome-wide analysis at both the transcriptional (RNA-seq, Bru-seq) and the translational (phospho)proteome) level in mESC comparing high (1 Gy) and low dose IR (100 mGy) exposure. In addition, we established that the induction of genetic damage at low IR doses (>20 mGy) follows a linear-dose relationship fully congruent with the LNT model.