The impact of a mixed neutron-gamma beam on the activation of DNA damage response (DDR) proteins and non-coding RNAs (ncRNAs) is poorly understood. Ionizing radiation is characterized by its biological effectiveness and is related to linear energy transfer (LET). Neutron-gamma mixed beam used in boron neutron capture therapy (BNCT) can induce another type of DNA damage such as clustered DNA or multiple damaged sites, as indicated for high LET particles, such as alpha particles, carbon ions, and protons. We speculate that after exposure to a mixed radiation field, the repair capacity might reduce, leading to unrepaired complex DNA damage for a long period and may promote genome instability and cell death. This review will focus on the poorly studied impact of neutron-gamma mixed beams with an emphasis on DNA damage and molecular mechanisms of repair. In case of BNCT, it is not clear which repair pathway is involved, and recent experimental work will be presented. Further understanding of BNCT-induced DDR mechanisms may lead to improved therapeutic efficiency against different tumors.

Boron cluster-modified therapeutic nucleic acids with improved properties are of interest in gene therapy and in cancer boron neutron capture therapy (BNCT). High metallacarborane-loaded antisense oligonucleotides (ASOs) targeting epidermal growth factor receptor (EGFR) were synthesized through post-synthetic Cu (I)-assisted “click” conjugation of alkyne-modified DNA-oligonucleotides with a boron cluster alkyl azide component. The obtained oligomers exhibited increased lipophilicity compared to their non-modified precursors, while their binding affinity to complementary DNA and RNA strands was slightly decreased. Multiple metallacarborane residues present in the oligonucleotide chain, each containing 18 B-H groups, enabled the use of IR spectroscopy as a convenient analytical method for these oligomers based on the diagnostic B-H signal at 2400–2650 cm⁻¹. The silencing activity of boron cluster-modified ASOs used at higher concentrations was similar to that of unmodified oligonucleotides. The screened ASOs, when used in low concentrations (up to 50 μM), exhibited pro-oxidative properties by inducing ROS production and an increase in mitochondrial activities in HeLa cells. In contrast, when used at higher concentrations, the ASOs exhibited anti-oxidative properties by lowering ROS species levels. In the HeLa cells (tested in the MTT assay) treated (without lipofectamine) or transfected with the screened compounds, the mitochondrial activity remained equal to the control level or only slightly changed (±30%). These findings may be useful in the design of dual-action boron cluster-modified therapeutic nucleic acids with combined antisense and anti-oxidant properties.

Nucleic acids are key biomolecules in all life forms. These biomolecules can encode and transfer information via Watson-Crick base-pairing interactions and can form double-stranded structures between complementary sequences with high precision. These properties make nucleic acids extremely successful in applications in materials science as nanoconstruction materials. Herein, we describe a method for the automated synthesis of “oligopeds”, which are building blocks based on the boron cluster structure equipped with short DNA adapters; these building blocks assemble into functional nanoparticles. The obtained, well defined, torus-like structures are the first DNA nanoconstructs based on a boron cluster scaffold. The results indicate the potential of boron clusters in DNA nanoconstruction and open the way for the design of entirely new types of buildings blocks based on polyhedral heteroborane geometry and its unique properties. The use of antisense oligonucleotides as DNA adapters illustrates one of the possible applications of the obtained nanoconstructs as vectors for therapeutic nucleic acids.

New boron carriers with high boron content and targeted cancer-cell delivery are considered the first choice for boron neutron capture therapy (BNCT) for cancer treatment. Previously, we have shown that composites of antisense oligonucleotide and boron clusters are functional nanoparticles for the downregulation of expression of epidermal growth factor receptor (EGFR) and can be loaded into EGFR-overexpressing cancer cells without a transfection factor. In this study, we hypothesize that free cellular uptake is mediated by binding and activation of the EGFR by boron clusters. Proteomic analysis of proteins pulled-down from various EGFR-overexpressing cancer cells using short oligonucleotide probes, conjugated to 1,2-dicarba-closo-dodecaborane (1,2-DCDDB, [C₂B₁₀H₁₂]) and [(3,3′-Iron-1,2,1′,2′-dicarbollide)⁻] (FESAN, [Fe(C₂B₉H₁₁)₂]⁻), evidenced that boron cage binds to EGFR subdomains. Moreover, inductively coupled plasma mass spectrometry (ICP MS) and fluorescence microscopy analyses confirmed that FESANs-highly decorated B-ASOs were efficiently delivered and internalized by EGFR-overexpressing cells. Antisense reduction of EGFR in A431 and U87-MG cells resulted in decreased boron accumulation compared to control cells, indicating that cellular uptake of B-ASOs is related to EGFR-dependent internalization. The data obtained suggest that EGFR-mediated cellular uptake of B-ASO represents a novel strategy for cellular delivery of therapeutic nucleic acids (and possibly other medicines) conjugated to boron clusters.

Introduction: Immune checkpoint blockade (ICB) improved clinical outcomes in renal and bladder cancer patients, but the response rates remain limited especially in metastatic disease. While STAT3 transcription factor is well-known master regulator of tumor immune evasion, little is known about the role of STAT3 in the resistance of renal or bladder cancers to immunotherapy.Methods: To better understand immune alterations associated with ICB resistance, we assessed blood biomarkers in renal cancer patients classified as responders or non-responders to first line nivolumab/ipilimumab immunotherapy.Results: We observed that non-responders showed elevated levels of proinflammatory mediators, such as IL-1RA, IL-6, IL-8 and to lesser extent IL-10, which are associated with STAT3 activation and tumor immunosuppression. In addition, we found STAT3 activation primarily in circulating myeloid immune cells such as tolerogenic MDSCs. To assess whether STAT3 inhibition within these cell subsets can promote antitumor immune responses and/or enhance sensitivity to ICB in vivo, we used an original antisense oligonucleotide (ASO) strategy for myeloid-cell selective STAT3 knockdown (CpG-STAT3ASO). Our results in syngeneic models of renal and bladder cancers in mice demonstrated potent antitumor activity of CpG-STAT3ASO alone in contrast to PD1 blockade alone in both models. The CpG-STAT3ASO/anti-PD1 combination improved therapeutic efficacy especially against bladder tumors. Therapeutic efficacy correlated with activation of dendritic cells (DCs) and M1 macrophages in the tumor microenvironment, reduced percentages of regulatory T cells (Tregs) and the expansion of CD8 T cells in both tumor models.Discussion/Conclusion: Our study underscores the potential of using myeloid-cell targeted CpG-STAT3 inhibitors for genitourinary cancer therapy to disrupt tolerogenic signaling, restore immune cell activity and sensitivity to immune checkpoint inhibitors and/or T cell-based immunotherapies.