Kai Rothkamm

Radiotherapy (RT) is a central treatment for prostate cancer (PCa), relying on the induction of DNA double-strand breaks (DSBs). Tumor ability to repair these breaks limits RT efficacy, making DSB repair inhibitors potential radiosensitizers. However, many of these inhibitors lack tumor specificity and harm normal cells. Therefore, tumor-specific radiosensitization strategies are critically needed for PCa. Approximately 50% of PCa cases harbor the TMPRSS2-ERG gene fusion, leading to overexpression of the ERG transcription factor (ERG ⁺ ). In this study, we demonstrate that ERG ⁺ tumors shift DSB repair toward the poly(ADP-ribose) polymerase 1–dependent end-joining (PARP1-EJ) pathway. Proteomic and Western blot analyses revealed elevated PARP1, XRCC1, and LIG3 levels in ERG ⁺ cells. Notably, PARP inhibition with olaparib increased residual γH2AX/53BP1 foci postirradiation in ERG ⁺ cells, indicating enhanced radiosensitization. In tissue slice cultures (TSCs) from 53 tumors of patients with high-risk PCa, olaparib selectively increased γH2AX/53BP1 foci selectively in ERG ⁺ samples. ERG ⁺ patient–derived organoids also showed significantly delayed growth when treated with olaparib plus RT, compared with either treatment alone. Interestingly, ERG-negative cells within ERG ⁺ TSCs were similarly radiosensitized by olaparib, likely through bystander effect, with residual 53BP1 foci levels comparable to those in ERG ⁺ cells. This was confirmed by medium exchange experiments. These findings suggest that ERG expression promotes dependency on the PARP1-EJ pathway, rendering ERG ⁺ PCa more susceptible to PARP inhibition. This supports combining PARP inhibitors with RT for tumor-selective radiosensitization in ERG ⁺ patients.

Background/Objectives: In recurrent/metastatic head and neck squamous cell carcinoma (R/M HNSCC), the overall prognosis is poor, and systemic treatment options remain limited. While precision therapy approaches have revolutionized treatment strategies in several tumor types, molecularly informed therapies in R/M HNSCC are rare, primarily due to the low number of actionable genetic alterations identified through next-generation sequencing (NGS) panels. There is an urgent need to establish precision therapy approaches in R/M HNSCC using innovative predictive testing. Methods: We report the case of a 43-year-old patient with recurrent oral cancer who was extensively pretreated and comprehensively characterized using both descriptive and functional testing. Results: NGS revealed no targetable alterations. A tumor tissue slice radiosensitivity assay suggested radioresistance, arguing against re-irradiation. Kinome profiling identified upregulated Src-family kinases (SFK), and SFK inhibition reduced kinase activity in vitro. Most notably, mRNA analysis demonstrated high Trop-2 overexpression, confirmed by immunohistochemistry (3+ in 100% of tumor cells). Following six cycles of the Trop-2-directed antibody–drug conjugate Sacituzumab govitecan (SG), the patient had an impressive clinical response. Conclusions: Tumor characterization beyond genetic profiling can identify novel treatment options in therapy-refractory HNSCC. This is the first report of “real-world” data on promising antitumor efficacy of SG in a heavily pretreated oral cancer patient with Trop-2 overexpression. Consistent with the findings of the Basket TROPiCS-03 study, SG appears to be a promising novel therapy option for R/M HNSCC after failure of immunotherapy and chemotherapy, particularly in patients with Trop-2 overexpression.

There is a rising awareness of the toxicity of micro- and nanoplastics (MNPs); however, fundamental precise information on MNP-biodistribution in organisms is currently not available. X-ray fluorescence imaging (XFI) is introduced as a promising imaging modality to elucidate the effective MNP bioavailability and is expected to enable exact measurements on the uptake over the physical barriers of the organism and bioaccumulation in different organs. This is possible because of the ability of XFI to perform quantitative studies with a high spatial resolution and the possibility to conduct longitudinal studies. The focus of this work is a numerical study on the detection limits for a selected XFI-marker, here, palladium, to facilitate the design of future preclinical in vivo studies. Based on Monte Carlo simulations using a 3D voxel mouse model, the palladium detection thresholds in different organs under in vivo conditions in a mouse are estimated. The minimal Pd-mass in the scanning position at a reasonable significance level is determined to be 2 for abdominal organs and 2 for the brain. MNPs labelled with Pd and homogeneously distributed in the organ would be detectable down to a concentration of <1 μg/mL to <2.5 mg/mL in vivo. Long-term studies with a chronic MNP exposure in low concentrations are therefore possible such that XFI measurements could, in the future, contribute to MNP health risk assessment in small animals and humans.

The ubiquitin specific protease 7 (USP7) is a deubiquitinating enzyme with numerous substrates. Aberrant expression of USP7 is associated with tumor progression. This study aims to investigate how a deregulated USP7 expression affects chromosomal instability and prognosis of breast cancer patients in silico and radiosensitivity and DNA repair in breast cancer cells in vitro. The investigations in silico were performed using overall survival and USP7 mRNA expression data of breast cancer patients. The results showed that a high USP7 expression was associated with increased chromosomal instability and decreased overall survival. The in vitro experiments were performed in a luminal and a triple-negative breast cancer cell line. Proliferation, DNA repair, DNA replication stress, and survival after USP7 overexpression or inhibition and irradiation were analyzed. Both, USP7 inhibition and overexpression resulted in decreased cellular survival, distinct radiosensitization and an increased number of residual DNA double-strand breaks in the S phase following irradiation. RAD51 recruitment and base incorporation were decreased after USP7 inhibition plus irradiation and more single-stranded DNA was detected. The results show that deregulation of USP7 activity disrupts DNA repair in the S phase by increasing DNA replication stress and presents USP7 as a promising target to overcome the radioresistance of breast tumors.

Background: In trials using normofractionated regimen, the radiation-induced lymphocyte apoptosis (RILA) and the chromosomal damage assays (CDA) have shown prognostic roles of radiation-induced adverse events. The main objectives here were to validate RILA and CDA in extreme and moderate hypofractionation regimens for breast (FAST) and prostate (CHHiP) cancers. Methods: Blood samples were collected from 400 volunteers included in FAST and CHHiP trials. The primary endpoints (PE) were first change in photographic breast appearance and first grade ≥2 RTOG bladder or bowel toxicity (BBT) in FAST and CHHiP, respectively. The secondary endpoints was first grade ≥2 breast clinical changes (BCC) in FAST and BBT using different scales in CHHiP. Results: 103 FAST and 297 CHHiP patients with lab and clinical data were included. In FAST trial, no significant association of RILA with the primary endpoint was observed. A significant association of higher RILA levels with lower risk of any RIAE was found. The risk of developing grade ≥2 RIAE decreased significantly for patients with RILA≥24% compared to those with RILA≤16% with a HR of 0.50 (95%CI 0.25-1.00, p=0.012). Concerning chromosomic aberrations, no significant associations were found with change in photographic breast appearance nor with the secondary endpoint of any RIAE. In CHHiP trial, a decreased risk of grade≥2 RTOG bladder or bowel RIAE was observed for increasing values of RILA (HR 0.97, 95%CI 0.94-1.01, p=0.11) but did not reach statistical significance. Concerning chromosomic aberrations, we found significant association of higher levels of micronuclei per cell with lower risk of gr2+ RTOGAE (p=0.021 for trend test across tertiles; p=0.023 for upper vs lower tertile). Conclusions: This study is the first to evaluate RILA and CDA as predictors of late effects after breast and prostate RT in the “hypofractionation” era. Development of toxicity biomarkers whatever the RT fractionation are urgently needed.

Einleitung Die aktuellen multimodalen Therapien für HPV-negative HNSCC sind bei nach wie vor nicht-optimalen Heilungsraten mit hoher Morbidität verbunden. Die Kombination der Strahlentherapie mit molekularem Targeting könnte, insbesondere für Cisplatin-ungeeignete Patienten, wirksame Alternativen ermöglichen. In diesem Kontext wurde hier die duale Inhibition von PARP und Wee1 auf ihre radiosensibilisierende Wirkung getestet. Material und Methoden Behandlung dreier strahlenresistenter HPV-negativer Zelllinien (HSC4, SAS, UT-SCC-60a) mittels Olaparib, Adavosertib und Bestrahlung. Analyse des Zellzyklus, G2-Arrest und Replikationsstress mittels Durchflusszytometrie. Analyse des langfristigen Zellüberlebens mittels Kolonieassay und Analyse der Reparatur strahleninduzierter DNA-Schäden über Quantifizierung nukleärer 53BP1-Foci in Zelllinien und Patienten-abgeleiteten Tumorschnittkulturen. Resultate Inhibition von Wee1 und duales Targeting induzieren Replikationsstress, können aber den strahleninduzierten G2-Zellzyklusarrest nicht wirksam inhibieren. Einzelne sowie kombinierte Inhibition erhöht die Strahlenempfindlichkeit und die Anzahl nicht-reparierter, residualer DNA-Doppelstrangbrüche, den toxischsten strahleninduzierten Läsionen. Duales Targeting zeigte hier die höchste Wirksamkeit und erhöhte die Anzahl residualer Doppelstrangbrüche ebenfalls in Patienten-abgeleiteten Schnittkulturen (4/6) HPV-negativer, aber nicht HPV-positiver Tumoren. Schlussfolgerungen Kombinierte PARP und Wee1 Inhibition führt zu wirksamer Radiosensibilisierung strahlenresistenter, HPV-negativer HNSCC Zellen aufgrund verminderter DNA-Reparatur. Ex vivo-Tumorschnittkulturen könnten das Ansprechen individueller HPV-negativer Tumoren vorhersagen und personalisierte Behandlungskonzepte ermöglichen. Deutsche Krebshilfe (Antrag 70113259; SK, KR, TR) & BMBF (Antrag 02NUK032; MK, KR, TR) Article published online: 12 May 2023 Georg Thieme Verlag Rüdigerstraße 14, 70469 Stuttgart, Germany

Introduction HNSCC negative for Human Papillomavirus (HPV) remain a difficult to treat entity and the morbidity of current multimodal treatment is high. Radiotherapy in combination with molecular targeting could represent suitable, less toxic treatment options especially for cisplatin ineligible patients. Therefore, we tested dual targeting of PARP and the intra-S/G2 checkpoint through Wee1 inhibition for its radiosensitizing capacity in radioresistant HPV-negative HNSCC cells. Material and Methods Three radioresistant HPV-negative cell lines (HSC4, SAS, UT-SCC-60a) were treated with olaparib, adavosertib and ionizing irradiation. The impact on cell cycle, G2 arrest and replication stress was assessed through flow cytometry after DAPI, phospho-histone H3 and γH2AX staining. Long term cell survival after treatment was determined through colony formation assay and DNA double-strand break (DSB) levels were assessed through quantification of nuclear 53BP1 foci in cell lines and patient-derived HPV± tumor slice cultures. Results Wee1 and dual targeting induced replication stress but failed to effectively inhibit radiation-induced G2 cell cycle arrest. Single as well as combined inhibition increased radiation sensitivity and residual DSB levels, with the largest effects induced through dual targeting. Dual targeting also enhanced residual DSB levels in patient-derived slice cultures (4/6) from HPV-negative but not HPV+ HNSCC. Conclusion We conclude that the combined inhibition of PARP and Wee1 results in enhanced residual DNA damage levels after irradiation and effectively sensitizes radioresistant HPV-negative HNSCC cells. Ex vivo tumor cultures may predict the response of individual patients with HPV-negative HNSCC to this dual targeting approach. Deutsche Krebshilfe (Antrag 70113259; SK, KR, TR) & BMBF (Antrag 02NUK032; MK, KR, TR) Article published online: 12 May 2023 Georg Thieme Verlag Rüdigerstraße 14, 70469 Stuttgart, Germany

BRCA1 is a well-known breast cancer risk gene, involved in DNA damage repair via homologous recombination (HR) and replication fork protection. Therapy resistance was linked to loss and amplification of the BRCA1 gene causing inferior survival of breast cancer patients. Most studies have focused on the analysis of complete loss or mutations in functional domains of BRCA1. How mutations in non-functional domains contribute to resistance mechanisms remains elusive and was the focus of this study. Therefore, clones of the breast cancer cell line MCF7 with indels in BRCA1 exon 9 and 14 were generated using CRISPR/Cas9. Clones with successful introduced BRCA1 mutations were evaluated regarding their capacity to perform HR, how they handle DNA replication stress (RS), and the consequences on the sensitivity to MMC, PARP1 inhibition, and ionizing radiation. Unexpectedly, BRCA1 mutations resulted in both increased sensitivity and resistance to exogenous DNA damage, despite a reduction of HR capacity in all clones. Resistance was associated with improved DNA double-strand break repair and reduction in replication stress (RS). Lower RS was accompanied by increased activation and interaction of proteins essential for the S phase-specific DNA damage response consisting of HR proteins, FANCD2, and CHK1.