Search Results (273)

Antiretroviral therapy (ART) effectively suppresses HIV-1 replication but does not purge the latent HIV-1 reservoir. Strategies aimed at HIV-1 latency reversal and subsequent elimination of infected cells are being explored. Targeting the inhibitor of apoptosis proteins (IAP) and DEAD-box polypeptide 3 (DDX3) RNA helicase reduces the HIV-1 reservoir ex vivo. However, the mechanisms driving apoptosis of HIV-1 infected cells remain unclear. Here, we uncovered the mechanism regarding HIV-1 transcriptional activation and induction of apoptosis specific for HIV-1 infected cells using an acute in vitro infection model. Inhibition of IAP by second mitochondrial-derived activator of caspases mimetic (SMACm; AZD5582) resulted in activation of non-canonical NF-κB pathway (RelB/p52) that induced HIV-1 transcription, confirming previous reports, whereas inhibition of DDX3 sensitized HIV-1 infected cells for apoptosis (DDX3i; FH1321). Transcriptome analysis revealed that HIV-1 actively suppressed apoptosis-related genes in HIV-1 infected cells. SMACm treatment resulted in a broad induction of these genes irrespective of infection. Notably, DDX3 inhibition specifically restored the expression of the majority of HIV-1 suppressed genes, and when combined with SMACm, restored almost all HIV-1 downregulated genes, thereby rendering HIV-1 infected cells sensitive to apoptosis. Thus, our data strongly suggest that inhibition of host factors IAP and DDX3 not only induces activation of HIV-1 transcription but also restores HIV-1 suppressed apoptotic processes in infected cells. Full article

The use of proton beam therapy (PBT), as a more precision-targeted radiotherapy technique, is increasing in the treatment of head and neck squamous cell carcinoma (HNSCC). PBT benefits from the precise delivery of the radiation dose to the tumour via the Bragg peak. However, challenges still remain in the treatment of HNSCC with radiotherapy, particularly with tumour radioresistance and recurrence, requiring strategies leading to radiosensitisation. There are added complexities with the use of PBT given the increase in linear energy transfer (LET) at and around the Bragg peak, which can cause an altered cellular response compared to low-LET radiation. Nevertheless, targeting the cellular DNA damage response is considered an important strategy to enhance tumour cell killing caused by radiotherapy. Therefore, using specific inhibitors against the protein kinases ataxia telangiectasia mutated (ATM), ataxia telangiectasia and Rad3-related (ATR) and the DNA-dependent protein kinase catalytic subunit (DNA-Pkcs), we investigated their impact in radiosensitising HPV-negative HNSCC cells to PBT of increasing LET. We demonstrate that inhibitors against ATR (AZD6738), and particularly ATM (AZD1390) and DNA-Pkcs (AZD7648), could significantly decrease clonogenic survival of HNSCC cell lines following PBT at both low and relatively high LET (~2 keV/µm and ~8 keV/µm, respectively). We confirmed that the inhibitors in combination with PBT led to DSB persistence through neutral comet assays and monitoring γH2AX/53BP1 foci. We also show that this strategy can enhance the sensitivity of patient-derived organoids of HNSCC to PBT of both low and high LET, highlighting this as a strategy which should be exploited further. Full article

Background/Objectives: Our previous study demonstrated that while the third SARS-CoV-2 booster effectively enhanced immunity against the Delta subvariant, its protection declined over time. This study aimed to evaluate and compare the humoral and cellular immune responses, as well as reactogenicity, of the mRNA-1273 vaccine administered as a fourth booster in healthy Thai adults previously vaccinated with two doses of CoronaVac (CV) followed by a third dose of either AZD1222 (AZ) or BNT162b2 (BNT). Methods: Participants received a single 100 µg (0.5 mL) intramuscular dose of mRNA-1273. Blood samples were collected at baseline (D0), D14, D90, and D180 to assess anti-RBD IgG, conduct a surrogate virus neutralization test (sVNT) against the Delta and Omicron variants, and assess IFN-γ levels and reactogenicity. Results: Both 2CV/AZ- and 2CV/BNT-primed groups exhibited comparable local and systemic reactogenicity. The fourth mRNA-1273 dose markedly increased Delta variant inhibition within 14 days in both groups and remained at high levels at Days 90 and 180. sVNT inhibition against Omicron rose similarly in both groups at Day 14; it declined sharply by Days 90 and 180, with the 2CV/AZ-primed group showing significantly lower levels than the 2CV/BNT-primed group. Baseline anti-RBD IgG levels were lower in the 2CV/AZ group (p = 0.003) but surpassed those of the 2CV/BNT group by Day 14, with no significant differences at later time points. IFN-γ responses followed a similar pattern to anti-RBD IgG Conclusions: A heterologous fourth mRNA-1273 booster in both 2CV/AZ- and 2CV/BNT-primed groups effectively enhances B-cell and T-cell responses against SARS-CoV-2. However, emerging variants such as Omicron may still pose challenges. The trial was registered with the Thai Clinical Trials Registry: the name of the registry: “The comparison of immune response to the 4th dose booster with mRNA-1273 COVID-19 vaccine in individuals who had received 2 doses of CoronaVac and booster with ChAdOx-1 or BNT162b2 COVID-19 vaccine”, TCTR20220205002 on 5 February 2022. Full article

Introduction: PMBCL is an aggressive type of lymphoma characterized by high heterogeneity in clinical, molecular, and genetic features. In PMBCL, disturbances in the NFkB pathway and deregulation of BCL-2 and mTOR family proteins are observed, which may contribute to impaired apoptosis. Therefore, many strategies have been established to target the functioning of these pathways. Early clinical trials of mTOR, NFkB and Bcl-2 inhibitors suggest their activity in many hematological cancers, but their activity as monotherapy agents may still be insufficient; therefore, combinations of these compounds with other molecules acting on those active in a given cancer subtype are being sought. Materials and Methods: In vitro studies were conducted on a single PMBCL cell line, Karpas 1106P. We administered three novel drugs: AZD2014 (vistusertib), an inhibitor of the serine-threonine kinase mTOR; IMD-0354, an NFκB inhibitor; and ABT-199 (venetoclax), a highly selective inhibitor for BCL-2. Drugs were administered alone, in pairs and in combination of all three agents. Results: Based on the results of our own research, for the Karpas cell line individually, ABT-199 had the strongest pro-apoptotic effect on cancer cells, while in pairs the most potent induction of apoptosis occurred following treatment with AZD2014+ABT-199. The combination of three drugs did not have a stronger effect than either a single drug used alone or any two-drug combination. Conclusions: These results provide preliminary in vitro evidence that targeting the BCL-2 and mTOR pathways may enhance pro-apoptotic activity in a PMBCL cell model; however, further validation in additional cell lines and in vivo models is needed before translational implications can be considered. Full article

Objectives: Using high-performance liquid chromatography (HPLC), we developed and validated an in vitro assay for the quantitative determination of beta-site amyloid precursor protein cleaving enzyme 1 (BACE1) activity, supplementing limited current methodologies to assess the efficacy of BACE1 inhibitor compounds. A hexa-histidine tagged peptide substrate of BACE1 was used as the analyte for the determination of in vitro BACE1 activity; it was validated according to ICH guidelines. Methods: The HPLC analysis was performed on the Agilent 1290 Series Infinity II UHPLC System equipped with a Phenomenex Kinetex EVO C18 (100 × 3 mm) 5 µm column. The method was developed using a gradient programme comprising 10% aqueous acetonitrile (0.02 M TFA) to 30% aqueous acetonitrile (0.02 M TFA) for 5 min at a flow rate of 0.6 mL/min. Results: The method showed linearity over the range of 14.92 to 72 µM with ${\mathrm{r}}^2=0.9997$. The accuracy of the method in terms of mean recovery ranged between 96.62 and 98.38%. The %RSD for intra- and inter-day precision was less than 5%. Two commercial inhibitors, AZD3839 and OM99-2, were used to evaluate the performance of the method at their respective IC₅₀, resulting in inhibition of 53.46 and 50.74%, respectively. The described method addresses the void for a practical and cheap alternative to quantitatively determine the activity of BACE1 compared to current commercially available detection assays. Conclusions: We have successfully developed an HPLC method to measure the inhibitory function of two commercial inhibitors of BACE1, indicating the suitability of the method for the identification and characterisation of novel BACE1 inhibitors. Full article

Background/Objectives: Non-small cell lung cancer (NSCLC) is the most frequent type of lung cancer, and its main treatments include chemotherapy with genotoxic drugs and immunotherapy. Central to the cellular response to genotoxic stress is the DNA damage response (DDR) network, regulated by key kinases such as ataxia-telangiectasia mutated and Rad3-related (ATR). Herein, we tested the hypothesis that inhibition of ATR enhances the cytotoxicity of genotoxic agents and the antitumor immune response. Methods: DDR-related parameters and redox status, expressed as GSH/GSSG ratio, and apurinic/apyrimidinic lesions, were evaluated in human (A549, H1299) and murine (LLC) NSCLC cell lines after co-exposure to ATR inhibitor (AZD6738) and ultraviolet C (UVC) irradiation or cisplatin. Using a syngeneic LLC model, treatments of AZD6738 alone or in combination with cisplatin and/or anti-programmed cell death 1 antibody (anti-PD1) were examined. Results: In all cell lines, combined treatment with AZD6738 and cisplatin or UVC irradiation markedly decreased cell viability, DNA repair efficiency, and GSH/GSSG ratios; increased drug-induced DNA damage; and augmented apurinic/apyrimidinic lesions. In vivo, following treatment with AZD6738 and cisplatin, flow cytometry analysis performed in tumor cells revealed an increased infiltration of CD3⁺ and CD8⁺ T cells, with the triple combination of AZD6738, cisplatin, and anti-PD1 achieving the strongest antitumor effect. The CD3⁺CD4⁻CD8⁻ double-negative (DN) T cell population in tumor samples also emerged as a contributing factor in this context. Conclusions: These results demonstrate that ATR blockade concurrently enhances the efficacy of genotoxic agents and immune checkpoint inhibitors, thus paving the way for combination therapies in NSCLC. Full article

Background/Objectives: The aberrant epigenetic landscape of cancer cells has attracted wide attention, motivating the search for new epigenetically active drugs both for anticancer therapy and for overcoming the drug resistance promoted by epigenetic changes. The use of epi-drugs in cancer therapy requires consideration of the influence of applied treatment on epigenetic regulation of gene expression. Therefore, it is reasonable to screen epigenetically active compounds among the drugs widely used in clinical oncology. Methods: We applied the HeLa TI cell-based assay to analyze the epigenetic activity of 40 drugs including 22 chemotherapeutic, 2 immunotherapeutic, 13 targeted, and 3 palliative agents. Reactivation of the epigenetically silenced GFP reporter gene integrated into the genome of HeLa TI cells was assessed using flow cytometry. Results: Statistically significant increases in the proportions of GFP-positive cells were demonstrated for the alkylating agent chlorambucil; the antimetabolites cytarabine, fluorouracil, gemcitabine, and pemetrexed; the platinum-based compounds cisplatin, and oxaliplatin; the topoisomerase inhibitor topotecan; and the antimicrotubule agents docetaxel, vincristine, and eribulin. Epigenetic activity was also detected for the targeted-therapy agents AZD8055, wortmannin, and cetuximab, as well as for the corticosteroid dexamethasone. Thus, epigenetic activity was revealed for 15 drugs widely used in cancer therapy, which possess different modes of action. Conclusions: Our findings show that many anticancer therapy agents modulate the epigenetic landscape of cancer cells, providing a rationale for expanding their therapeutic applications and enhancing the efficacy of combination strategies by overcoming epigenetically driven chemoresistance. Full article

DLBCLs constitute an aggressive type of lymphoma with varied clinical, molecular and genetic features. The cells are characterized by NFkB pathway disturbances and BCL-2 and mTOR protein deregulation, which significantly inhibit apoptosis. Hence, many treatment strategies have been established to target the functioning of these pathways. While early clinical trials have found mTOR, NFkB and Bcl-2 inhibitors to have activity in many hematological cancers, their activity as monotherapy agents may still be insufficient; therefore, combinations of these compounds with other molecules demonstrating activity in a given cancer subtype are under evaluation. In vitro studies were conducted on the Riva (ABC subtype) and Toledo (GCB subtype) cell lines. Three novel drugs were administered: AZD2014 (vistusertib)—an inhibitor of the serine–threonine kinase mTOR; IMD-0354—an NFκB inhibitor; and ABT-199 (venetoclax)—a highly selective inhibitor for BCL-2. Drugs were administered alone, in pairs and as a combination of all three agents. For the Riva cell line, ABT-199 had the strongest pro-apoptotic effect on cancer cells as monotherapy. As pairs, AZD2014+ABT-199 and ABT-199+IMD0354 demonstrated similar effects. The combination of the three drugs did not have a stronger effect than the drug pairs. For the Toledo cell line, no significant differences were noted between the drugs when used as monotherapy. In pairs, the strongest effect was observed for AZD2014+ABT-199; furthermore, this effect was not intensified by the combination of the three drugs. Our findings, including those for the BCL-2 and mTOR inhibitors, indicate that there is a need for further in vivo studies to evaluate these drugs as potentially effective treatments for DLBCL of the ABC and GCB subtypes. Full article

Background/Objectives: Poly(ADP-ribose) polymerase inhibitors (PARPis) have significantly transformed the treatment landscape for ovarian cancer; however, their clinical efficacy is often limited by poor response rates and the emergence of resistance. Recent studies have revealed that in ovarian cancer cells resistant to PARPi, the expression levels of adenosine receptors are upregulated. Accumulation of adenosine activates adenosine A2A receptor (A2AR) on immune cells, leading to immune suppression and immune escape. We hypothesize that this is a key factor limiting the efficacy of PARPi and driving the development of resistance. Therefore, the rational combination of PARPi with A2AR antagonists (A2ARas) may represent a highly promising anticancer strategy. Methods: To assess the effects of the PARPi AG14361 and the A2ARa AZD4635 on ovarian cancer growth and the immune microenvironment, we conducted in vitro and in vivo experiments and utilized single-cell RNA sequencing (scRNA-seq) to construct a high-resolution immune landscape. Results: AG14361 significantly inhibited ovarian cancer growth both in vitro and in vivo, accompanied by the accumulation of cyclic adenosine monophosphate (cAMP) and activation of the cAMP/cAMP response element-binding protein (CREB) pathway in mouse cells and tumor tissues. However, compared to monotherapy, the combination of AG14361 and AZD4635 significantly enhanced antitumor activity by inhibiting cAMP accumulation and the cAMP/CREB pathway. More importantly, the combination therapy of PARPi and A2ARa reduced the infiltration of immunosuppressive cells (such as regulatory T cells and M2 macrophages) while increasing the infiltration of cytotoxic T cells and granzyme B-positive cells, thereby creating a more favorable immune microenvironment for tumor clearance. Single-cell analysis revealed distinct functional subpopulations of macrophages and T cells, highlighting the complexity of immune heterogeneity and the potential for targeting specific immune cell subpopulations to enhance therapeutic efficacy. Conclusions: These findings suggest that the combination therapy of PARPi and A2ARa is a highly promising strategy that overcomes PARPi-induced immune escape by targeting the cAMP/CREB axis, thereby synergistically enhancing antitumor effects and holding promise as an effective treatment for solid tumors. Full article

The prompt and extensive use of COVID-19 vaccines has dramatically reduced both cases and casualties, but it has also underscored a number of critical issues that need to be addressed for their full exploitation at global level. This Special Issue was launched to gather fresh data to improve the way we use vaccines that are currently available to contribute to the design and the development of new ones and to elucidate social, ethical and psychological concerns that might hamper vaccine acceptance and use. It includes 15 articles six of which address, under various aspects, the level and durability of protective immunity; five deal with the highly debated field of vaccine use, efficacy and safety, in persons with an impaired/dysregulated immune system; one paper reports on adjuvants’ role in immune stimulation; one on the interference of natural adenovirus immunity with DNA vaccine response; one is a review on the cellular mechanisms of vaccine-dependent myocarditis; and one explores social attitudes to vaccines in different ethnic groups during early phases of the pandemic. Far from being complete and exhaustive, this Special Issue provides the reader with fresh insights into several critical questions raised by or connected to the advent of COVID-19 vaccines. This introductory article provides an overview of their contribution, while offering a quick glance at the current state of the art. Full article

Pancreatic cancer (PC) remains a highly lethal malignancy with limited treatment options and poor survival. Targeting DNA damage response (DDR) pathways has emerged as a promising therapeutic strategy, particularly the ATR-CHK1 and ATM-CHK2 axes. Preclinical studies demonstrate that ATR inhibition disrupts replication stress tolerance, impairs homologous recombination, and disables checkpoint control, enhancing cytotoxicity from standard therapies including gemcitabine, FOLFIRINOX, fluoropyrimidines, and radiotherapy. Synergistic effects have also been observed with other DDR-targeted agents, such as PARP and WEE1 inhibitors. Genomic contexts, including ATM deficiency, ARID1A alterations, and oncogene-driven replication stress, refine therapeutic sensitivity, supporting precision patient stratification. Early-phase clinical trials of ATR inhibitors (ART0380, AZD6738, BBI-355) alone or in combination show promising safety, tolerability, and preliminary efficacy. In this review, we summarize current literature on targeting the ATM-CHK2 and ATR-CHK1 pathways in PC, highlighting preclinical evidence, clinical developments, and strategies for biomarker-driven, precision oncology approaches. Full article

Fyn is a Src-family tyrosine kinase implicated in synaptic dysfunction and neuroinflammation across multiple neurodegenerative disorders, including Alzheimer’s disease (AD) and Parkinson’s disease (PD). Saracatinib (AZD0530) is a potent Src-family inhibitor that has been explored as a repurposed therapeutic; however, its clinical utility is limited by poor kinase selectivity caused by high sequence conservation within Src-family ATP-binding sites. Here, we combine surface plasmon resonance (SPR) and X-ray crystallography to define saracatinib recognition by the Fyn kinase domain (KD). SPR single-cycle kinetics shows that saracatinib binds the isolated Fyn KD and full-length Fyn with low-nanomolar affinity, whereas dasatinib binds with subnanomolar affinity and markedly slower dissociation. We determined the crystal structure of the Fyn KD-saracatinib complex at 2.22 Å resolution. The kinase adopts an active-like conformation with the DFG motif and αC-helix in the ‘in’ state and a conserved β3 αC Lys-Glu salt bridge. Saracatinib occupies the adenine and ribose pockets, and engages the hinge through direct and water-mediated hydrogen bonding while complementing a hydrophobic back pocket by van der Waals contacts. Comparison with reported saracatinib-bound structures of other kinases suggests that the active-state geometry observed for Fyn creates a pocket not observed in inactive-like complexes, providing a structural handle for designing Fyn-selective inhibitors. Comparison with all saracatinib-bound kinase co-structures currently available in the PDB (ALK2 and PKMYT1) indicates a conserved monodentate hinge binding mode but kinase-dependent αC-helix conformations, providing a structural rationale for designing Fyn-selective analogues. Full article

Background/Objectives: Approved for treatment of acute leukemia, gemtuzumab ozogamicin (GO) and inotuzumab ozogamicin (InO) are antibody–drug conjugates (ADCs) that deliver a toxic calicheamicin (CLM) derivative. The resistance mechanisms to GO/InO remain incompletely understood. Methods: We performed a genome-wide clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 screen for CLM sensitivity genes, and then performed confirmatory cytotoxicity assays. Results: Several DNA damage pathway regulation genes were identified, most notably TP53. Across 13 acute leukemia cell lines, the six TP53-mutant cell lines (TP53^MUT) were indeed 10- to 1000-fold less sensitive to CLM than the seven TP53^WT cell lines. In five TP53^WT/KO syngeneic cell line pairs we generated, TP53^KO cells were significantly less sensitive to CLM than their TP53^WT counterparts. In TP53^WT but not TP53^MUT cells, the MDM2 inhibitor and p53 activator, idasanutlin, enhanced CLM cytotoxicity, demonstrating that decoupling of cells from MDM2-p53 regulation sensitizes leukemia cells to CLM. The ATM inhibitors AZD1390 and lartesertib also significantly enhanced CLM efficacy but did so independent of the TP53 status. In contrast, neither an ATR inhibitor, Chk1/Chk2 inhibitor, Chk2 inhibitor, or a PARP inhibitor significantly impacted CLM-induced cytotoxicity across the thirteen cell lines. Together, our studies identify ATM, MDM2, and TP53—which are in the same cellular response to DNA damage pathway—as key modulators of CLM-induced cytotoxicity in acute leukemia cells. Conclusions: These results support further evaluation of combination therapies with corresponding small-molecule inhibitors (currently pursued for therapy of other cancers) toward clinical testing as novel strategies to increase the efficacy of CLM-based ADCs such as GO and InO. Full article

Cathepsin C (CTSC) mediates neutrophil serine protease (NSP) maturation, contributing to inflammatory cascades, making it a key therapeutic target. In this study, through large-scale screening of a natural product library, marrubiin, a diterpenoid lactone compound, was identified as a potent CTSC inhibitor, which holds potential value in the treatment of inflammatory diseases. It inhibited human recombinant CTSC (IC₅₀ = 57.5 nM) and intracellular CTSC (IC₅₀ = 51.6 nM) with acceptable cytotoxicity, and reduced the activity and protein levels of downstream NSPs in vitro. Functionally, marrubiin inhibited lipopolysaccharide-induced nitric oxide release and regulated the levels of cytokines and chemokines. Docking result predicted marrubiin may achieve CTSC activity inhibition by using lactone structure as a covalent unit to target Cys234. In vivo study indicated that high-dose marrubiin (IC₅₀ = 30 mg/kg) reduced CTSC and NSPs activities in blood and bone marrow in mice without toxicity, and its efficacy was comparable to that of positive compound AZD7986. In the adjuvant-induced arthritis model, high-dose marrubiin (IC₅₀ = 60 mg/kg) exerted a therapeutic effect by reducing the activities of CTSC and NSPs. These findings indicated marrubiin is a promising natural CTSC inhibitor, which can be used for the treatment of neutrophil-related inflammatory diseases. Full article

Enhancer RNAs (eRNAs) are abundant in most human cells and tissues, and quantifying eRNAs has become a robust approach for biomarker discovery. While eRNAs play crucial roles in regulating biological processes and cancer progression, their functions in lung adenocarcinoma (LUAD) remain poorly understood. Here, we developed a LUAD prognostic model based on eRNA expression data from The Cancer Genome Atlas (TCGA). Through rigorous validation, a 7-eRNA signature was identified, which robustly stratified LUAD patients into high-risk and low-risk groups in both training and testing sets. Functional analyses revealed distinct enrichment of pathways related to amino acid biosynthesis, ribosome biogenesis, and proteasome activity in high-risk patients. Somatic mutation profiling highlighted TP53 and TTN as frequently mutated genes, while drug sensitivity prediction identified four potential therapeutic agents (including AZD4547 and Nutlin-3a) for high-risk individuals. Collectively, this study constructed a 7-eRNA prognostic model for LUAD, providing a powerful tool for clinical risk assessment and uncovering eRNA-mediated regulatory mechanisms. Full article