Vienna, Austria

ESTRO 2023

Session Item

May 14
10:30 - 11:30
Lehar 4
Tumour radiobiology
Navita Somaiah, United Kingdom;
Pierre Montay-Gruel, Belgium
Proffered Papers
11:10 - 11:20
Interplay of replication stress and immune signaling drives radioresistance in BRCA1 mutated cells
Sandra Classen, Germany


Interplay of replication stress and immune signaling drives radioresistance in BRCA1 mutated cells

Sandra Classen1, Elena Rahlf1, Johannes Jungwirth2, Simon Gehre3, Michael Rückert3, Helmut Pospiech2,4, Kai Rothkamm1, Udo S. Gaipl3, Cordula Petersen5, Kerstin Borgmann1

1University Medical Center Hamburg-Eppendorf, Laboratory for Radiobiology and Experimental Radiooncology, Hamburg, Germany; 2Leibniz Institute on Aging - Fritz Lipmann Institute, Project Group Biochemistry, Jena, Germany; 3Universitätsklinikum Erlangen, Translational Radiobiology, Department of Radiation Oncology, Erlangen, Germany; 4University of Oulu, Faculty of Biochemistry and Molecular Medicine, Oulu, Finland; 5University Medical Center Hamburg-Eppendorf, Department of Radiotherapy and Radiation Oncology, Hamburg, Germany

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Purpose or Objective

Expanding the number of patients that could benefit from immunotherapy is a major aim of many ongoing studies. Lately it has become evident, that an increase in replication stress can lead to accumulation of cytosolic DNA, followed by activation of the intracellular immune response via the cGAS/STING pathway. Subsequently the inflammatory signaling is activated. Enhancing this activation by combined treatment with irradiation, chemotherapeutics or DNA damage response and immune checkpoint inhibitors might lead to new therapeutic options. This project therefore aims to maximize the anti-tumor immune effects by increasing replication stress in BRCA1 mutated breast cancer cell lines using novel combinations of radio-chemotherapy.

Material and Methods

Isogenic MCF7 and MDA-MB231 clones with mutations in BRCA1 were generated using CRISPR/Cas9. Illumina next-generation sequencing was used for analysis of the mutations. HR-capacities were determined by plasmid-reconstruction assay and cell cycle distributions were analyzed by propidium iodide staining. For determination of the radio- and chemoresistance the colony formation assay was used. BRCA1, RAD51, RPA and yH2AX foci formation, micronuclei formation and IRF3 translocation were analyzed by (immuno)fluorescence microscopy. To analyze DNA replication, the DNA fiber assay was conducted. Cytosolic DNA was measured using the PicoGreen Assay. PD-L1 expression on the cell surface was analyzed by multicolor flow cytometry.


To our surprise, mutations in non-functional domains of BRCA1 resulted in resistance, as well as sensitivity against different DNA damaging agents and irradiation (IR) in the generated clones, despite a similar significant reduction of the homologous recombination (HR)-capacity (p ≤ 0.001). One BRCA1 mutated clone showed indeed significantly increased survival after treatment with mitomycin C and the PARP1 inhibitor talazoparib, which both cause damages mainly repaired by HR, compared to the parental cell line. We revealed that this resistance is likely associated with (I) more efficient DNA repair, shown by high RAD51 foci formation (p ≤ 0.05) (II) avoidance of DNA replication stress, indicated by efficient replication fork restart after IR (p ≤ 0.001) and low rates of stalled replication forks after hydroxyurea treatment (p ≤ 0.01) (III) differences in the activation of immune signaling in response to DNA damage, shown by cytosolic DNA after IR, micronuclei formation and IRF3 translocation. Further, a change in PD-L1 surface expression was observed.


Our results indicate that radiochemoresistance may be linked to DNA repair, replication stress and immune signaling in the analyzed cell lines. Targeting at least two of these pathways at the same time may offer a new therapeutic approach to treat tumors that have been shown to be therapy resistant before.