Anticancer nitrogen mustard drugs including cyclophosphamide, mechlorethamine and chlorambucil induce covalent DNA-protein cross-links (DPCs) by sequentially alkylating the N7 position of guanine in DNA and amino acid residues within proteins. In cells, DPCs are proteolytically processed to yield corresponding peptide-DNA cross-links (DpCs), allowing polymerase bypass. In this study, site-specific, hydrolytically stable DNA lesions resembling nitrogen mustard-induced DpCs were generated via oxime ligation of an 11-mer peptide derived from histone H4 (NH2-GGGK*GLGKGGA, K*=oxy-lysine) to 7-deaza-7-(2,3-dihydroprop-1-yl)-dG (DHP) in DNA. DNA sequence was derived from codon 249 of the p53 tumor suppressor gene, a common mutational hotspot in tumors. Translesion synthesis (TLS) experiments of DpC containing plasmids in human HEK 293T cells revealed that 7-deaza-DpC lesion was bypassed most efficiently in wild-type cells (83%) with lower efficiency in Pol ι KO (53%), Pol η KO (59%), and Pol κ KO (63%) cells, with Pol ζ KO unaffected (76%). Mutation frequency for polymerase bypass of 7-deaza-DpC (~9%) was significantly higher than for 7-deaza-DHP-dG control (~5%). Pol η and Pol ι knockouts led to increased error rates, suggesting they play a role in error-free bypass of DpCs. In contrast Pol ζ deficiency reduced mutation rates, implying that it plays a role in error-prone bypass.

Nitrogen mustard (HN2) is a highly reactive bifunctional alkylating agent that causes severe skin injury. To define its impact on keratinocyte bioenergetics and stress responses, we examined mitochondrial function, metabolism, and cell death signaling in human HaCaT cells. Seahorse analysis showed that HN2 caused time- and concentration-dependent suppression of oxidative phosphorylation, including reductions in basal, adenosine 5'-triphosphate-linked, and maximal oxygen consumption. Glycolytic activity was similarly impaired, with decreased extracellular acidification, reduced glucose-stimulated glycolysis, and loss of glycolytic capacity and reserve, indicating broad metabolic dysfunction. HN2-induced bioenergetic impairment triggered rapid nuclear accumulation of nuclear factor erythroid 2-related factor 2 (Nrf2) and upregulation of antioxidant and mitochondrial regulatory genes (HO-1, NQO1, GSTA4, and PGC1α). In parallel, HN2 activated multiple programmed cell death pathways, including apoptosis, autophagy, and ferroptosis, as evidenced by corresponding alterations in Bax, Bcl-xL, LC3-II, SQSTM1/p62, caspase-2, caspase-9, GPx4, TFRC, and ACSL4. Cell cycle analysis identified cells in G2/M as particularly susceptible to HN2, which exhibited enhanced apoptotic signaling. N-acetylcysteine attenuated Nrf2 activation, preserved mitochondrial and glycolytic function, and reduced activation of cell death pathways, demonstrating a central role for oxidative and electrophilic stress in HN2 toxicity. These findings reveal a novel mechanism by which HN2 disrupts keratinocyte bioenergetics to drive stress-dependent cell death and highlight antioxidant intervention as a potential strategy to mitigate HN2-induced skin injury.

Interstrand DNA crosslinks (ICLs) are a highly toxic form of DNA damage. ICL repair in both eukaryotes and bacteria involves unhooking of the two strands by specialized DNA glycosylases. We recently established that the human pathogen Acinetobacter baumannii contains an ICL glycosylase (AlkX) that facilitates pathogenesis and protects the bacteria from DNA damage and acid stress. However, the physical basis for glycosylase-catalyzed ICL unhooking is unknown. Here, we describe a crystal structure of AlkX bound to DNA representing a product of the ICL unhooking reaction. Mutational analysis of ICL unhooking in vitro and A. baumannii sensitivity to the crosslinking agent mechlorethamine enable the identification of several AlkX motifs critical for ICL repair. We also find that a genetic variant from an antibiotic-resistant strain of the human pathogen Salmonella enterica reduces AlkX activity in vitro and increases A. baumannii sensitivity to DNA crosslinking. This work provides a structural basis for how bacterial ICL glycosylases recognize and repair DNA adducts and contributes additional evidence that ICL repair is important for fitness of human pathogens.

Designing efficient nanocarriers for targeted cancer therapy requires a deep understanding of drug-nanomaterial interactions at the molecular level. In this study, the adsorption behavior of four clinically relevant anticancer drugs, 6-mercaptopurine (6-MP), hydroxyurea (HU), chlormethine (CM), and 5-fluorouracil (5-FU), on a CC1 nanocage was systematically investigated using density functional theory (DFT). Geometry optimizations and thermodynamic analyses were performed at the ωB97XD/6-31G(d, p) level, while optical properties were assessed through time-dependent DFT (TD-DFT) calculations at the B3LYP/6-31G(d, p) level. The computed adsorption energies and electronic descriptors revealed that all drug-nanocage interactions are spontaneous and exothermic in an aqueous medium, with 5-FU exhibiting the strongest binding (- 8.68 kcal/mol). TD-DFT spectra displayed redshifts in absorption peaks after adsorption, signifying charge transfer between the drugs and the CC1 framework. Moreover, topological (QTAIM) and Non-Covalent Interaction (NCI) analyses confirmed that van der Waals forces primarily stabilize the complexes. The short recovery times predicted for the drug-loaded nanocage suggest efficient release kinetics under physiological conditions. Overall, this theoretical study provides a molecular-level perspective on CC1 nanocage-drug interactions, offering useful insights for the rational design of carbon-based nanocarriers in targeted anticancer drug delivery.

Nitrogen mustard (NM) is a vesicant, and the respiratory tract is one of its primary target organs. The recovery following NM exposure is protracted. Despite the existence of numerous theories regarding the toxic effects of NM, but to date, no studies have investigated the global cellular response profiles across distinct phases after NM exposure. Herein, we examined alterations in pulmonary function in mice before and after NM exposure, characterized the biological changes in human bronchial epithelial cells (HBEs) during acute NM exposure and the subsequent post-exposure recovery phase, and further analyzed the impact of p97 deficiency on cell injury. Following acute NM exposure, the expiratory time and total respiratory cycle time were prolonged in mice, whereas the expiratory flow at 50% of tidal volume (EF50) and alveolar volume were reduced. NM rapidly perturbs nucleic acid-associated biological processes within the nucleus, including the cell cycle, chromatin dynamics, mitosis, and RNA processing and splicing, with concomitant upregulation of the expression of relevant proteins. Acute NM exposure for 1 h exerted no effect on total p97 expression level, but promoted the accumulation of p97 in the cytoplasm. In contrast, nuclear p97 expression was increased at 24 h after NM removal. Although p97 deficiency exerted minimal effects on cellular stress responses during the acute exposure phase, it disrupted intracellular material transport, elicited a tendency toward the suppression of nuclear protein expression, and impeded the recovery process after the termination of NM exposure. P97 deficiency primarily repressed the expression of nucleoporins and histones, and inhibited aerobic respiration during the cellular recovery phase. Collectively, these findings suggest that the perturbation of nucleic acid-associated biological processes constitutes the initial and critical event underlying the respiratory toxicity of NM. Furthermore, p97 deficiency impairs material transport, small-molecule metabolism, and mitochondrial function, and prolongs the recovery time in the aftermath of NM exposure.

Cancer therapy has long faced a critical bottleneck wherein monotherapy often proves insufficient due to tumor heterogeneity and compensatory mechanisms, while combination chemotherapy frequently leads to localized drug ratio mismatch and exacerbated toxicity owing to differential in vivo drug distribution. To fundamentally address this challenge, we focused on achieving precise combined therapy at the single-molecule level. The core of this strategy involves designing novel bifunctional molecules that integrate the Topo I inhibitor SN-38 and the alkylating agent nitrogen mustard via a covalent linkage, with further prodrug modification using salt forms and phosphate ester groups to optimize their physicochemical properties. Among the candidate molecules, MN33-47 and MN33-63 demonstrated outstanding performance, not only significantly improving the solubility of SN-38 but also exhibiting broad-spectrum antiproliferative activity superior to individual drugs or their physical mixtures across various cancer cell models. Mechanistic studies further revealed that these molecules inhibit Topo I and promote its degradation, while simultaneously inducing DNA crosslinking, thereby combinedly enhancing DNA damage. This cascade of events subsequently leads to G2/M phase cell cycle arrest and inhibition of cell migration. Subsequent downregulation of Bcl-2 protein relieves its suppression of the mitochondrial apoptosis pathway, ultimately activating caspase-3 and effectively initiating the apoptosis program. In CT26 tumor-bearing mouse models, MN33-47 and MN33-63 exhibited significant dose-dependent tumor suppression without causing apparent toxic side effects, demonstrating superior safety compared to combination chemotherapy. In summary, this work provides a practical solution to overcome the clinical limitations of SN-38 and nitrogen mustard, primarily through molecular innovation that "solidifies" combination therapy into a single entity, thereby directly resolving the inherent conflict between enhancing efficacy and exacerbating toxicity.

Effective skin-directed therapies (SDT) are the cornerstone for managing early-stage Cutaneous T-cell Lymphoma (CTCL). However, standard treatments like PUVA phototherapy and topical mechlorethamine carry significant drawbacks, including mutagenic risk and treatment-limiting skin reactions. This unmet need has driven demand for safer options. Topical photodynamic therapy with synthetic hypericin (research name: SGX301; trade name: HyBryte™) has emerged as a novel agent addressing this gap.

The composite index lesion severity (CAILS) score is used to monitor disease and therapeutic response in mycosis fungoides (MF), but is limited by interobserver variability and low sensitivity. Emerging imaging techniques, such as multispectral imaging (MSI), colourimetry and laser speckle contrast imaging (LSCI), offer objective alternatives for quantifying CAILS parameters. The aim of this study was to evaluate non-invasive imaging modalities for objective and reliable quantification of disease extent in MF. Sixty-six participants were enrolled in two prospective studies: a cross-sectional discovery cohort to assess baseline characteristics of 35 MF patients (IA-IVB) and 10 healthy controls using CAILS and MSI, and a longitudinal confirmation cohort including 21 early-stage MF patients (IA-IIA) treated with chlormethine gel 0.016% for 16 weeks, in whom lesional and non-lesional skin were assessed using CAILS, MSI, colourimetry and LSCI at multiple time points. Candidate biomarkers were required to meet five clinical validation criteria: disease discrimination, repeatability, treatment responsiveness, correlation with CAILS and patient acceptability. In the discovery cohort, MSI detected significant differences in erythema, pigmentation, elevation and desquamation between healthy, non-lesional and lesional skin. In the confirmation cohort, four candidate biomarkers met all validation criteria: MSI CIELAB a*, MSI average haemoglobin, and colourimetry CIELAB a* (DSMIII) for quantifying erythema, and MSI individual typology angle (ITA) for pigmentation. These biomarkers reliably discriminated lesional from non-lesional skin (p ≤ 0.001), showed strong test-retest reliability (CV < 10%, ICC > 0.84), detected treatment effects, showed moderate concordance with CAILS, and were associated with low patient burden (mean 3.4/100). These findings show that MSI- and colourimetry-derived biomarkers can objectively monitor disease extent in MF and complement existing clinical assessments.

Vesicants are highly toxic chemicals used as warfare agents, primarily absorbed through the skin, inhalation, or ocular surface. Nitrogen mustard (NM), a potent vesicant, often leads to blindness due to the inadequacies of existing therapies. This study explores the role of peroxisome proliferator-activated receptor-α (PPARα) in NM-induced ocular injury and evaluates the efficacy of fenofibrate (Feno), a PPARα agonist, in preventing corneal injury progression. Feno, an FDA-approved oral medication used to treat dyslipidemia, has shown off-labelled potential in promoting corneal wound healing. However, its limited ocular distribution following oral administration reduces its therapeutic efficacy. We developed Feno-loaded oil-in-water microemulsion (Feno ME) eyedrop formulations to enhance aqueous solubility and ocular bioavailability of Feno, aiming to improve the therapeutic efficacy in treating NM-induced corneal injury. Feno ME exhibited a mean droplet size of approximately 20 nm, remained physically stable for at least twelve months, showed prolonged ocular retention, and demonstrated well-accepted rheological properties without inducing ocular irritation or toxicity. Furthermore, Feno ME demonstrated sustained drug levels in ocular tissues with minimal systemic absorption. A 14-day treatment with 0.5% Feno ME (3×/day) significantly reduced corneal-ulceration, neovascularization (NV), and opacity in NM-induced ocular injury in rats. Histopathological analysis confirmed preserved corneal integrity and reduced inflammation. Immunohistochemistry revealed that NM injury significantly reduced PPARα levels in the corneal epithelium, which was restored with Feno ME eyedrop treatment, suggesting PPARα as a promising drug target for NM-induced corneal injury. Our Feno ME eyedrop formulation represents an effective topical delivery platform that effectively mitigates NM-induced corneal injury and offers a novel therapeutic strategy for vesicant exposure.

Nitrogen mustards are highly potent DNA-alkylating agents, but their clinical utility is limited by systemic toxicity and poor tumor selectivity. Tumor-selective prodrug strategies have emerged to address these challenges, enabling spatiotemporally controlled activation of otherwise inert precursors. These approaches exploit tumor-specific cues such as hypoxia, acidic pH, elevated reactive oxygen species (ROS), and redox imbalance, to achieve precise payload release. Stimulus-induced bond cleavage or structural transformation liberates the masked nitrogen mustard (NM) in situ, facilitating targeted DNA cross-linking. This review highlights molecular design principles underlying diverse activation strategies, emphasizing scaffold modifications and linker chemistries tailored to cancer-associated triggers. We also discuss integration with advanced delivery platforms, including polymeric carriers and nanoplatforms, to enhance tumor accumulation and therapeutic indices while minimizing off-target toxicity. By consolidating recent advances, this review illustrates how bioorthogonal and tumor-selective activation strategies, including those responsive to hypoxia, ROS, and pH, are redefining the precision oncology potential of NM prodrugs.

The Pediatric Hodgkin Consortium hypothesized that by increasing chemotherapeutic dose density for Hodgkin lymphoma (HL) they could increase the complete response (CR) rate among patients with favorable-risk HL after 8 weeks of Stanford V (vinblastine, doxorubicin, vincristine, bleomycin, mechlorethamine, etoposide and prednisone) compared with 8 weeks of VAMP (vinblastine, Adriamycin [doxorubicin], methotrexate, and prednisone). This would translate to a decrease in patients who required radiation therapy (RT) to achieve a cure. The HOD08 study was a phase 2 multicenter, investigator-initiated single-arm trial for patients aged ≤21 years with previously untreated stage 1A or 2A HL without mediastinal bulk or extranodal disease extension and <3 sites of disease. Treatment consisted of a modified 8-week Stanford V regimen. Modified, tailored, field RT was administered only to disease sites achieving less than a CR. The primary objective was to increase CR rate after 8 weeks of chemotherapy by at least 20% (from an estimated 44% to 64%) compared with patients treated on a previous trial (HOD99). HOD08 enrolled 85 patients with HL and 72 were evaluable for the primary objective, of whom 55 (76.4%) achieved a CR at all sites and did not receive RT. The 5-year event-free survival and overall survival rates for the entire cohort were 87.4% (95% confidence interval [CI], 80.4-95.0) and 98.7% (95% CI, 96.2-100), respectively. A dose-dense modified Stanford V regimen reduced the proportion of pediatric patients with low-risk HL who received RT while maintaining excellent outcomes. This trial was registered at www.clinicaltrials.gov as #NCT00846742.