EACR25-0957

Targeting immune signaling to overcome radioresistance by increasing DNA damage in breast cancer cells

S. Classen¹, A. Moustafa^1,2, K. Hintelmann³, N. Zhafirah¹, M. Vogt¹, H. Schlüter², H. Pospiech^4,5,6, K. Rothkamm¹, C. Petersen³, K. Borgmann¹

¹University Medical Center Hamburg-Eppendorf, Department of Radiotherapy and Radiation Oncology, Hubertus Wald Tumor Center - University Cancer Center Hamburg, Hamburg, Germany
²University Medical Center Hamburg-Eppendorf, Section Mass Spectrometry and Proteomics, Hamburg, Germany
³University Medical Center Hamburg-Eppendorf, Department of Radiotherapy and Radiation Oncology, Hamburg, Germany
⁴University Hospital Düsseldorf, Research Laboratory of the Clinic for Gynecology and Obstetrics, Düsseldorf, Germany
⁵Leibniz Institute on Aging - Fritz Lipmann Institute, Project Group Biochemistry, Jena, Germany
⁶University of Oulu, Faculty of Biochemistry and Molecular Medicine, Oulu, Finland

Introduction:

Around 70% of breast cancer (BC) patients receive radiotherapy (RT). However, development of resistance is a frequent cause for therapy failure. One promising strategy to overcome radiation resistance is the use of DNA damage response inhibitors to modulate the immune response and thereby increase the efficacy of immune checkpoint inhibitors in BC. Therefore, this project aims to understand how immune pathways, like cGAS/STING, can be exploited as target to overcome radioresistance by increasing DNA damage.

Material and method:

Bottom-up quantitative proteomics of BRCA1-mutated MCF7 cells was performed using liquid chromatography tandem mass spectrometry. Cellular survival after irradiation (IR) and ATR inhibition (ATRi) by AZD6738 was assessed by colony formation assay. Protein expression was determined by Western Blot. PD-L1 surface expression was measured by flow cytometry. Changes in the secreted chemo- and cytokines were analyzed by real-time PCR. DNA repair capacity was analyzed by γH2AX foci formation. cGAS positive micronuclei and IRF3 translocation were analyzed by immunofluorescence.

Result and discussion:

Proteomic profiling revealed significant upregulation of interferon (IFN) alpha/gamma signaling pathway in radiosensitive compared to radioresistant BRCA1-mutated cell lines. Notably, only upon addition of ATRi, IFN signaling was upregulated in resistant cells, which was accompanied by a radiosensitizing effect. STAT1 was detected to be among the top candidates of the IFN signaling pathway, which was further confirmed by Western Blotting. STAT1 activation led to significantly increased expression of IFN-stimulated genes (IFIT1, IFIT3, OAS3 and ISG15) after IR in sensitive cell lines, whereas in resistant cells, this effect was observed only after combined IR and ATRi treatment. A similar trend was observed for several chemo- and cytokines (IFNβ1, CXCL10 and CCL5). Further, PD-L1 surface expression was upregulated after IR, however reduced after ATRi in all cell lines. Previous studies have described a CHK1-dependant PD-L1 upregulation, and our results indicate that by ATRi this might be disrupted. To identify how addition of ATRi led to an increased immune signaling in radioresistant cells, cGAS/STING activation due to increased DNA damage was investigated. γH2AX foci revealed an increase in DNA damage in the resistant cells after combined treatment. Correspondingly, the number of cGAS-positive micronuclei and IRF3 translocation were both enhanced.

Conclusion:

These results show that immune signaling was enhanced by DNA damage-induced activation of cGAS/STING signaling after combined treatment of IR and ATRi in radioresistant, BRCA1-mutated BC cells. This consequently led to a radiosensitizing effect, which may open up new avenues for the treatment of patients that developed a resistance to RT.