Non-homologous end joining (NHEJ) is a critical DNA double-strand break (DSB) repair pathway that operates throughout the cell cycle to maintain the genomic stability of the cell. Unlike homologous recombination (HR), NHEJ is capable of repairing DSBs without the need for a homologous template, making it a rapid response mechanism, but potentially prone to errors. Central to NHEJ function and essential for the ligation through the recruitment and activation of additional repair factors, such as Artemis, XRCC4, and DNA ligase IV, is the DNA-dependent protein kinase (DNA-PK) complex. Dysregulation in the NHEJ pathway contributes to genomic instability, oncogenesis, and resistance to genotoxic therapies. Consequently, inhibitors of DNA-PK have emerged as promising therapeutic agents to sensitize tumor cells to radiation and DNA-damaging chemotherapeutics. Inhibiting the DNA-PK ability to recruit the protein complex needed for successful DSB repair promotes cell death through apoptosis or mitotic catastrophe. While inhibitors of DNA-PK can be used to enhance the effects of genotoxic therapies, the field still struggles to address critical problems: how to best exploit the differential DNA repair capacities among tumor subtypes, how to maximize radiosensitization of cancerous cells while sparing normal tissues, and how to translate preclinical studies into clinical benefits. Given that NHEJ constitutes the primary line of defense against radiation-induced damage, rapidly repairing the majority of double-strand breaks throughout the cell cycle, this review concentrates on targeting the DNA-PK complex, as the master regulator of this rapid-response mechanism, highlighting why its inhibition represents a strategic action to overcome intrinsic radioresistance. The implementation of DNA-PK inhibitors into medical practice can enable the stratification of oncologic patients into two categories, based on the tumors’ vulnerability to NHEJ disruptions. Thus, the therapeutic pathways of patients with NHEJ tumors could branch, combining traditional genotoxic therapies (radiation and DNA-damaging chemotherapeutics) with DNA-PK inhibitors to achieve an enhanced effect and improved survival outcomes.

Background: The ⁸⁹Zr radioisotope is increasingly vital in positron emission tomography (PET), especially immuno-PET, due to its long half-life of 78.4 h, allowing extended tracking of biological processes. This makes it particularly suitable for researching medicines with slow pharmacokinetics and enhances the precision of molecular imaging, especially in oncology. Despite zirconium’s potential for skeletal accumulation, effective chelation with agents like deferoxamine (DFO) enables high-resolution imaging of antigen-specific tumours, such as HER2-positive breast cancer, offering insights into tumour biology and treatment response. Methods: ⁸⁹Zr was produced at the ACSI TR-19 cyclotron via ⁸⁹Y(p,n)⁸⁹Zr reaction. Natural yttrium foils (250 μm) were irradiated with 12.9 MeV protons on target, with 100 μA·h. An HER2-targeting affibody was synthesized and conjugated with p-NCS-Bz-DFO (1:4 mass ratio) at 37 °C for 60 min (pH 9.2 ± 0.2), then purified on a PD-10 column. Radiolabelling was performed with [⁸⁹Zr]Zr-oxalate at pH ranging from 7.0 to 9.0, with concentrations from 110 to 460 MBq/mL. Results: Final activity reached 2.95 ± 0.31 GBq/batch (EOB corrected), with ≥ 99.9% radionuclide and ≥95% radiochemical purities. The anti-HER2 affibody was successfully radiolabelled with ⁸⁹Zr, resulting in a radiochemical purity of over 85% with molar activity of 26.5 ± 4.4 and 11.45 MBq/nmol at pH 7.0–7.5. In vitro tests on BT-474 and MCF-7 cell lines confirmed high uptake in HER2-positive cells, validating specificity and stability. Conclusions: The successful synthesis and labelling of the [⁸⁹Zr]Zr-p-NCS-Bz-DFO-anti-HER2 affibody are promising achievements for its further application in targeted immuno-PET imaging for HER2-positive malignancies. Further in vivo studies are needed to support its clinical translation.

NHEJ is one of the preferred DNA-damage repairing mechanisms for human cells, due to its rapid onset and activity throughout the cell cycle. It is, nevertheless, error prone and highly relevant in numerous oncological malignancies. In the recent years, it gained a lot of attention as a target either for cancer treatment or synergetic strategies, as pharmacological- and radio-sensitization approaches. This study evaluated the genotoxicity and radiosensitization potential of three DNA-PK inhibitors (PIK-75, M3814, and CC-115) in breast cancer by comparing MDA-MB-231 (NHEJ reliant) and MCF-7 (NHEJ and HR reliant) cell lines. Cells were preincubated with the DNA-PK inhibitors, using four different concentrations either in monotherapy or in combination with a DNA damage-inducer (doxorubicin or [64Cu]CuCl2). Viability was measured by MTS assays at 24, 48, and 72 h, while the DNA damage by γH2AX and flow cytometry. DNA-PK blockade preferentially sensitized NHEJ-reliant breast cancer cells to doxorubicin and 64Cu effect. Combination treatments generally reduced viability relative to inhibitor’s monotherapy, with clearer concentration dependence and stronger effects in MDA-MB-231. In the case of [64Cu]CuCl2 alone, MDA-MB-231 viability was reduced to 70–75% at 24 h, whereas several inhibitor combinations reduced it further; MCF-7 showed weaker or inconsistent potentiation. Delayed cytotoxicity was most obvious at 72 h, showing persistence of unrepaired DNA damage after DNA-PK inhibition. Overall, the results highlight the potential of exploiting repair-pathway dependence to improve targeted radiotherapy in breast cancer.

NHEJ is one of the preferred DNA-damage-repairing mechanisms for human cells, due to its rapid onset and activity throughout the cell cycle. It is, nevertheless, error-prone and highly relevant in numerous oncological malignancies. In recent years, it has gained a lot of attention as a target either for cancer treatment or as pharmacological- and radio-sensitization approaches. This study evaluated the genotoxicity and radiosensitization potential of three DNA-PK inhibitors (PIK-75, M3814, and CC-115) in breast cancer by comparing MDA-MB-231 and MCF-7 breast cancer cell lines, selected based on reported differences in DNA-repair characteristics and relative NHEJ dependence. Cells were preincubated with the DNA-PK inhibitors, using four different concentrations either in monotherapy or in combination with a DNA damage inducer (doxorubicin or [⁶⁴Cu]CuCl₂). Viability was measured by MTS assays at 24, 48, and 72 h, while the DNA damage was measured by γH2AX and flow cytometry. DNA-PK inhibitor combinations produced greater reductions in MTS viability in MDA-MB-231 than in MCF-7 under experimental conditions when combined with doxorubicin and ⁶⁴Cu. Combination treatments generally reduced viability relative to inhibitor monotherapy, with clearer concentration dependence and stronger effects in MDA-MB-231. In the case of [⁶⁴Cu]CuCl₂ alone, MDA-MB-231 viability was reduced to 70–75% at 24 h, whereas several inhibitor combinations reduced it further; MCF-7 showed weaker or inconsistent potentiation. The reduction in metabolic viability became more pronounced at 72 h, particularly in MDA-MB-231 cells. Overall, the results highlight the potential of exploiting repair-pathway dependence to improve targeted radiotherapy in breast cancer.

Background: The current study integrates AlphaFold2-based structural modeling of DNA-PKcs with an experimentally resolved DNA-PKcs structure, large-scale molecular docking of over 1500 known DNA-PK inhibitors, aiming to identify clinically relevant candidates for next-generation pharmacological delivery methods, evaluate receptor model suitability, and predict how potential radio-halogenation may influence binding behavior and translational radiotherapeutic potential of current clinically relevant candidates. AlphaFold2-based prediction of the DNA-PKcs structure was the starting point, followed by comparison of the AI-derived model with an experimentally resolved structure to evaluate the model’s accuracy and docking suitability. A large database of 1369 known DNA-PK inhibitors was then screened by molecular docking, after which six clinically relevant compounds were analyzed for molecular dynamics. Finally, a focused docking study was conducted on 67 possible radio-halogenated derivatives derived from these clinically relevant scaffolds to investigate the effect of potential iodination, bromination, or astatination on binding behavior. These findings indicate that large-scale docking can be used to comparatively rank DNA-PK-targeting compounds and explore the impact of theoretical radiohalogenation on predicted binding behavior. However, the results also underline the limitations of current receptor models and molecular simulation workflows, supporting the need for experimental validation before translational conclusions are drawn.