53BP1

Tumor biopsy tissue response to ex vivo irradiation is potentially an interesting biomarker for in vivo tumor response, therefore, for treatment personalization. Tumor response ex vivo can be characterized by DNA damage response, expressed by the large-scale presence of DNA damage foci in tumor nuclei. Currently, characterizing tumor nuclei and DNA damage foci is a manual process that takes hours per patient and is subjective to inter-observer variability, which is not feasible in for clinical decision making. Therefore, our goal was to develop a method to automatically segment nuclei and DNA damage foci in tumor tissue samples treated with radiation ex vivo to characterize the DNA damage response, as potential biomarker for in vivo radio-sensitivity.

Oral cavity tumor tissue of 21 patients was irradiated ex vivo (5 or 0 Gy), fixated 2 h post-radiation, and used to develop our method for automated nuclei and 53BP1 foci segmentation. The segmentation model used both deep learning and conventional image-analysis techniques. The training (22 %), validation (22 %), and test set (56 %) consisted of thousands of manually segmented nuclei and foci. The segmentations and number of foci per nucleus in the test set were compared to their ground truths.

The automatic nuclei and foci segmentations were highly accurate (Dice = 0.901 and Dice = 0.749, respectively). An excellent correlation (R² = 0.802) was observed for the foci per nucleus that outperformed reported inter-observation variation. The analysis took ∼ 8 s per image.

This model can replace manual foci analysis for ex vivo irradiation of head-and-neck squamous cell carcinoma tissue, reduces the image-analysis time from hours to minutes, avoids the problem of inter-observer variability, enables assessment of multiple images or conditions, and provides additional information about the foci size. Thereby, it allows for reliable and rapid ex vivo radio-sensitivity assessment, as potential biomarker for response in vivo and treatment personalization.

DNA polymerase theta (Polθ) has recently emerged as a new attractive synthetic lethal target involved in DNA damage repair. Inactivating Polθ alone or in combination with PARP inhibitors has demonstrated substantial therapeutic potential against tumors with homologous recombination (HR) defects such as alternation of BRCA genes. Herein, we report the design and proof of concept of a highly potent dual Polθ/PARP inhibitor 25d, which exhibited low nanomolar inhibitory activities against both Polθ and PARP1. Compared to combination treatment, 25d demonstrated superior antitumor efficacy in both MDA-MB-436 cells and xenografts by inducing more DNA damage and apoptosis. Importantly, 25d retained sensitivity in PARP inhibitor-resistant MDA-MB-436 cells with 53BP1 defect. Altogether, these findings illustrate the potential advantages of 25d, a first-in-class dual Polθ/PARP inhibitor, over monotherapy in treating HR-deficient tumors, including those with acquired PARP inhibitor resistance.

Dynamic chromatin remodeling requires regulatory mechanisms for its adaptation to different nuclear function, which are likely to be mediated by signaling proteins. In this context, VRK1 is a nuclear Ser-Thr kinase that regulates pathways associated with transcription, replication, recombination, and DNA repair. Therefore, VRK1 is a potential regulatory, or coordinator, molecule in these processes. In this work we studied the effect that VRK1 depletion has on the basal nuclear and chromatin phosphoproteome, and their associated pathways. VRK1 depletion caused an alteration in the pattern of the nuclear phosphoproteome, which is mainly associated with nucleoproteins, ribonucleoproteins, RNA splicing and processing. Next, it was determined the changes in proteins associated with DNA damage that was induced by doxorubicin treatment. Doxorubicin alters the nuclear phosphoproteome affecting proteins implicated in DDR, including DSB repair proteins NBN and 53BP1, cellular response to stress and chromatin organization proteins. In VRK1-depleted cells, the effect of doxorubicin on protein phosphorylation was reverted to basal levels. The nuclear phosphoproteome patterns induced by doxorubicin are altered by VRK1 depletion, and is enriched in histone modification proteins and chromatin associated proteins. These results indicate that VRK1 plays a major role in processes requiring chromatin remodeling in its adaptation to different biological contexts.