SARS-CoV-2 RNA contains guanine-rich sequences that form secondary structures known as G quadruplexes (G4s). The SARS-CoV-2 nonstructural protein (NSP13) resolves G4s due to its helicase and ATPase activity, a process essential for viral replication. Here, we tested the effects of synthetic G4s on SARS-CoV-2 replication. In agreement, a synthetic G4 DNA 20 mer, consisting exclusively of guanines linked by a phosphorothioate backbone (designated GQ20-PTO), inhibited the replication of various SARS-CoV-2 variants in human lung cell cultures. Mechanistically, GQ20-PTO bound to NSP13 and inhibited its helicase and ATPase activity. Independent of its antiviral effects, GQ20-PTO additionally suppressed IFNβ and IL-6 (but not TNFα) signaling and the formation of reactive oxygen species, processes known to contribute to hyperinflammation in severe COVID-19. Hence, G4 quadruplexes like GQ20-PTO represent a novel class of DNA-based compounds for COVID-19 treatment with the potential to interfere with both SARS-CoV-2 replication and the uncontrolled inflammation associated with life-threatening COVID-19.

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Background/Objectives: Cutaneous T-cell lymphomas (CTCL), most commonly mycosis fungoides and Sézary syndrome, are rare non-Hodgkin lymphomas. Advanced disease responds poorly to current treatments, highlighting the need for new molecularly targeted therapies. WEE1 is a central regulator of the G2/M checkpoint and S-phase progression and has emerged as a therapeutic target in several malignancies, yet it has not been systematically explored in CTCL. Methods: We screened a library of more than 2200 kinase inhibitors in CTCL cell lines and selected adavosertib for further study. Its effects were tested in four CTCL lines; primary keratinocytes and fibroblasts; patient-derived malignant CD4+ T cells and healthy donor CD4+ T cells; and a MyLa xenograft model, using viability, apoptosis, cell-cycle, Western blot, and phospho-protein array assays. Results: Adavosertib reduced viability at submicromolar IC50 values (0.26–0.56 µM) across all four CTCL lines while largely sparing primary skin cells and was more active in malignant than in healthy donor CD4+ T cells. It induced apoptosis and cell-line-specific S-phase and/or G2/M accumulation, lowered WEE1 and phospho-CDK1 (Tyr15), and increased phospho-H2A.X. A phospho-protein array showed activation of checkpoint and stress signalling. In a preliminary xenograft experiment, adavosertib slowed MyLa tumour growth. Conclusions: These preclinical data identify WEE1 as a therapeutic target in CTCL and support further preclinical and early-phase clinical evaluation of adavosertib in this disease.

BACKGROUND:mTORC1 activity is oncogenic. However, in the presence of chemotherapy, suppression of mTORC1 is cytoprotective. mTOR suppression requires an intact tuberous sclerosis complex (TSC), composed of TSC1, TSC2 and TBC1D7. Small molecules that activate mTOR by blocking the TSC are lacking. METHODS:We applied in silico docking and medicinal chemistry to generate AcTor, a potential first-of-its-kind TSC2 inhibitor. Because inhibition of TSC2 results in increased sensitivity to proteasome inhibitors, we combined AcTor and the proteasome inhibitor ixazomib (IXZ) in various cancer cell types. RESULTS:Potentiation of cytotoxic activity of IXZ by AcTor was observed across multiple acute myeloid leukemia (AML) cell lines and primary patient samples. The combination triggered a collapse of mitochondrial respiratory capacity, loss of mitochondrial membrane potential, accumulation of ROS and apoptosis. These attributes increased in drug-resistant AML. Transcriptomic profiling revealed that AcTor alone induced anabolic and oxidative phosphorylation programs, whereas AcTor/IXZ redirected the signaling towards stress-associated and pro-apoptotic transcriptional states, including a p53 pathway signature. In vivo studies revealed reduction in AML burden, depletion of blasts and of leukemic stem cells, and retention of activity upon relapse. AcTor/IXZ was equally potent in a TP53-mutated patient-derived xenograft model, exceeding the efficacy of standard-of-care. CONCLUSIONS:As a TSC2 inhibitor, AcTor should not be used alone in cancer. When combined with proteasome inhibitors, the pharmacodynamics of AcTor shifts towards the development of a mitochondrial catastrophe in AML, which is durable, broad range, agnostic to TP53 mutations and to the acquisition of resistance to common clinical anti-AML drugs.

Aggressive metastatic progression often develops in bladder cancer patients with acquired cisplatin or gemcitabine resistance. The potential of the natural isothiocyanates allyl-isothiocyanate (AITC), butyl-isothiocyanate (BITC), and phenylethyl-isothiocyanate (PEITC) to inhibit adhesion and migration of cisplatin- or gemcitabine-resistant and sensitive RT112, T24, and TCCSUP bladder cancer cell lines was investigated. Parameters determined were: cell interaction with collagen or fibronectin, chemotaxis, and membrane receptors involved in adhesion (total and activated integrins β1, β4, β5, CD44s, and CD44v3-v7). CD44s’ location and adhesion- and migration-related signaling proteins were determined. AITC blocked adhesion of almost all sensitive and resistant cancer cells. PEITC and BITC suppressed fibronectin interaction of sensitive and resistant RT112. All three isothiocyanates diminished chemotaxis in all cell lines. Integrin expression was differentially altered but CD44s and CD44v were not altered. BITC and PEITC translocated CD44s from the cell membrane to cytoplasm. The tumor suppressor E-cadherin increased, whereas focal adhesion kinase (FAK), linked to integrin signaling, was deactivated after isothiocyanate treatment. Blocking FAK, β1, β4, or β5 was associated with reduced chemotaxis. Thus, AITC, BITC, and PEITC blocked adhesion and migration in cisplatin- and gemcitabine-resistant bladder cancer cells. This was associated with altered integrin expression and signaling, CD44s translocation, and enhanced E-cadherin.

Mpox is a re-emerging zoonosis caused by monkeypox virus (MPXV), with recent outbreaks driven by sexual transmission. How semen contributes to MPXV infection, however, remains unclear. Here, we show that human seminal fluid potently enhances MPXV infection in susceptible target cells. Pre-exposure of MPXV to seminal fluid from multiple donors markedly increased infection of primary human fibroblasts and keratinocytes. Semen-derived peptide fragments that assemble into cationic amyloid fibrils (SEVI and SEM) enhanced infection of epithelial cells, primary monocytes, and ex vivo human skin, as well as clinical MPXV isolates and other orthopoxviruses. SEVI increased virion attachment and uptake, and fibril-targeting molecular tweezer (CLR01) disrupted seminal amyloids and abrogated enhancement. Notably, tecovirimat and brincidofovir retained activity under SEVI-enhanced conditions. These findings identify seminal amyloids as host factors that amplify MPXV infectivity and broaden cell tropism, suggesting that semen actively promotes sexual transmission of mpox.

Background: Rhabdomyosarcoma (RMS) is the most common type of soft-tissue sarcoma in children, and it remains a challenging cancer with poor outcomes in high-risk and metastatic patients. This study reports the use of epidermal growth factor receptor (EGFR)-targeted chimeric antigen receptor (CAR) natural killer (NK) cells in combination with radiotherapy as a novel immunotherapeutic approach for RMS treatment.Methods: Primary human NK cells from healthy donors were engineered using lentiviral transduction to express a cetuximab-based EGFR-specific CAR. The ability of the engineered NK cells to lyse RMS cells was then assessed in vitro in RMS monolayers and spheroids, as well as against chemotherapy-resistant and primary patient-derived RMS cells. Migratory properties of NK cells were observed in a subcutaneous RMS xenograft model using in vivo imaging, and the efficacy of EGFR-CAR NK cells in combination with localized fractionated radiotherapy was analyzed.Results: Primary human EGFR-CAR NK cells demonstrated enhanced cytotoxicity against multiple RMS cell lines in both two-dimensional culture and three-dimensional spheroid models. Furthermore, EGFR-CAR NK cells were highly efficient against chemotherapy-resistant RMS cells and patient-derived samples. Importantly, EGFR-CAR NK cells also exhibited improved tumor homing compared with non-transduced NK cells in an in vivo RMS xenograft model. Notably, the combination of EGFR-CAR NK cell therapy with fractionated radiotherapy further enhanced NK cell infiltration into the tumor and reduced tumor growth.Conclusion: This study provides a proof-of-concept for EGFR-CAR NK cells as a promising immunotherapy for RMS, particularly when combined with radiotherapy to overcome barriers of solid tumors. This combinatorial approach may hold potential to improve outcomes for patients with RMS and other EGFR-expressing malignancies.

Papaverine (PV) has been previously identified as a promising candidate in SARS-CoV-2 repurposing screens. In this study, we further investigated both its antiviral and immunomodulatory properties. PV displayed antiviral efficacy against SARS-CoV-2 and influenza A viruses H1N1 and H5N1 in single infection as well as in co-infection. We demonstrated PV's activity against various SARS-CoV-2 variants and identified its action at the post-entry stage of the viral life cycle. Notably, treatment of air-liquid interface (ALI) cultures of primary bronchial epithelial cells with PV significantly inhibited SARS-CoV-2 levels. Additionally, PV was found to attenuate interferon (IFN) signaling independently of viral infection. Mechanistically, PV decreased the activation of the IFN-stimulated response element following stimulation with all three IFN types by suppressing STAT1 and STAT2 phosphorylation and nuclear translocation. Furthermore, the combination of PV with approved COVID-19 therapeutics molnupiravir and remdesivir demonstrated synergistic effects. Given its immunomodulatory effects and clinical availability, PV shows promising potential as a component for combination therapy against COVID-19.

Here, we introduce novel sublines of the EGFR‐mutant non‐small cell lung cancer (NSCLC) cell lines HCC827 and HCC4006 adapted to the EGFR kinase inhibitors gefitinib (HCC827 r GEFI 2μ m , HCC4006 r GEFI 1μ m ), erlotinib (HCC827 r ERLO 2μ m , HCC4006 r ERLO 1μ m ) and afatinib (HCC827 r AFA 50n m , HCC4006 r AFA 100n m ). All sublines displayed resistance to gefitinib, erlotinib, afatinib and the third‐generation EGFR kinase inhibitor osimertinib that overcomes T790M‐mediated resistance. HCC4006 r ERLO 1μ m displayed a spindle‐like morphology in agreement with previous findings that had detected epithelial–mesenchymal transition (EMT) in its precursor cell line HCC4006 r ERLO 0.5μ m . EMT had also been reported for the HCC4006 r GEFI 1μ m precursor cell line HCC4006 r GEFI 0.5μ m and for HCC4006 r AFA 100n m , but the morphologies of HCC4006 r GEFI 1μ m or HCC4006 r AFA 100n m did not support this, suggesting plasticity in EMT regulation during the drug adaptation process and in established resistant cell lines. Accordingly, HCC4006 r ERLO 1μ m displayed resistance to MEK and AKT inhibitors in contrast to its precursor HCC4006 r ERLO 0.5μ m . We also detected metabolic plasticity, that is a temporary Warburg metabolism, in HCC4006 and HCC827 r GEFI 2μ m . Response profiles to cytotoxic anticancer drugs, kinase inhibitors and HDAC inhibitors resulted in complex patterns that were specific for each individual subline, indicating individual resistance phenotypes. All resistant sublines remained sensitive or displayed collateral sensitivity to at least one of the investigated drugs. In conclusion, the comparison of EGFR kinase‐resistant NSCLC sublines with their precursor cell lines that had been previously characterised at a lower resistance level and metabolic investigations indicated phenotypic plasticity during the resistance formation process and in established cell lines. This plasticity may contribute to the well‐known variability in cell line phenotypes observed between different laboratories and in intra‐laboratory experiments.

High-risk neuroblastoma remains a clinically challenging pediatric cancer, with an approximate five-year survival rate of ~60%. Frontline therapy for this group of patients includes surgery and intensive chemotherapy that involves combinations of the tubulin inhibitor vincristine with several other chemotherapeutics. Unfortunately, unresponsiveness to therapy and relapse are common, with tumors often displaying resistance to vincristine. Recently, we characterized a novel set of tubulin inhibitors that are distinct from vincristine and bind within the colchicine binding site present on tubulin monomers. Colchicine binding site inhibitors (CBSIs) have gained traction as improved chemotherapeutics due to their potential to overcome tubulin inhibitor-induced resistance. In this study, we investigate the functional impact of CBSI treatment on multiple neuroblastoma cell lines, including those that are vincristine-resistant. We demonstrate that our newly developed compounds are effective at disrupting cell division in non-resistant and resistant cells and have cellular activity against vincristine-resistant cell lines. Interestingly, we find that vincristine-resistant cell lines differ in their ability to undergo apoptotic cell death in response to CBSI treatment. Taken together, these findings provide a solid foundation to further investigate the utility of CBSIs for neuroblastoma treatment, while highlighting the distinct resistance mechanisms that can emerge in these childhood cancers.

The survival of patients with locally advanced and metastatic bladder cancer (BCa) is persistently low. Hence, new treatment options are urgently needed. Artesunate (ART) a derivative of artemisinin, used in Traditional Chinese Medicine, shows anti-tumor activity extending over a broad spectrum of human cancers. As we have previously shown, ART inhibits growth in cisplatin-sensitive (parental) and cisplatin-resistant BCa cells. However, how ART acts on the metastatic potential of BCa remained unclear. To clarify, we applied ART to parental and cisplatin-resistant RT4, RT112, T24, and TCCSup BCa cell lines. We examined tumor cell adhesion to vascular endothelium and immobilized collagen and evaluated chemotactic activity, migration, and invasive activity of the BCa cells. Adhesion receptors, integrin α and β subtypes, integrin-linked kinase (ILK), and focal adhesion kinase (FAK) were investigated. The functional relevance of integrin expression altered by ART was determined by blocking studies. ART significantly reduced tumor cell adhesion to vascular endothelium and immobilized collagen in parental as well as in cisplatin-resistant BCa cells. Depending on cell type, ART suppressed tumor cell motility and diminished integrin expression (surface and total). Functional blocking of integrins altered by ART reduced cell adhesion and invasion of the BCa cells. Thus, the metastatic potential of parental and cisplatin-resistant BCa cells was significantly inhibited by ART, making it a promising treatment option for patients with advanced or therapy-resistant BCa.

PARP inhibitors (PARPi) are approved for the treatment of platinum-based therapy-responsive ovarian cancer. However, this severely restricts their therapeutic potential, since there is only limited knowledge on the efficacy of PARPi in platinum drug-resistant ovarian cancer cells. Here, we studied three approved PARPi, niraparib, olaparib, and rucaparib in three ovarian cancer cell lines and their cisplatin-resistant sublines. Complex response profiles demonstrated that cisplatin resistance was not consistently associated with cross-resistance to PARPi. The combination of PARPi with inhibitors of relevant DNA damage response kinases which are potentially involved in PARPi resistance, such as ATR, ATM, CHK1, and WEE1 again resulted in complex activity patterns, but also identified ATR and ATM as the most promising targets for increasing PARPi activity. Cell adhesion-mediated resistance via collagen I is known to mediate cisplatin resistance. Here, we show that collagen I can also mediate PARPi resistance, which can also be tackled by ATR and ATM inhibition in cisplatin-sensitive and cisplatin-resistant ovarian cancer cell lines. In conclusion, our findings revealed complex, cell line-specific PARPi response profiles. This complexity is in line with other studies investigating drug-resistant cancer cell lines and with the complex evolutionary processes in tumors from cancer patients. Notably, cisplatin resistance was not directly correlated with PARPi resistance, and ATM and ATR inhibitors can increase PARPi activity against cisplatin-sensitive and -resistant ovarian cancer cells. Moreover, we demonstrated for the first time that cell adhesion-mediated resistance can contribute to PARPi resistance, which can also be alleviated by ATR and ATM.

Aim: Urinary bladder cancer (UBC) often develops chemoresistance, reducing treatment effectiveness. This study aimed to investigate diverse molecular mechanisms underlying acquired resistance by establishing and characterizing a comprehensive panel of UBC cell lines resistant to common chemotherapeutics. Methods: Fifteen UBC cell lines were examined: three parental lines (RT-112, TCC-SUP, UMUC-3) and twelve derived sublines adapted to cisplatin, vinblastine, or gemcitabine. Drug sensitivity was assessed using the SRB assay. Resistance mechanisms were explored via quantitative real-time PCR (targeting genes including ABCB1, dCK, hENT1, ECHDC1, TUBB3), Western blotting (assessing proteins such as p21, Cyclin B, and Mcl-1), and biochemical assessment of glutathione levels and redox state. Results: The adapted sublines exhibited distinct resistance profiles and cross-resistance patterns. Gene expression and protein analyses revealed drug- and lineage-specific alterations, involving factors such as p21, Cyclin B, and Mcl-1. Changes in glutathione metabolism were also associated with resistance. Notably, no single, universal mechanism accounted for resistance across the entire panel. Conclusion: UBC cells develop diverse, context-dependent adaptive strategies to resist cisplatin, vinblastine, and gemcitabine. These findings highlight the complexity of chemoresistance mechanisms. The characterized cell line panel represents a valuable resource for future studies aimed at understanding and overcoming drug resistance in bladder cancer, suggesting that personalized therapeutic approaches may be necessary.

Ethnopharmacological relevance: Plants and fungi have a long tradition in ethnopharmacology for the treatment of infectious diseases including viruses. Many of these natural products have also been used to combat SARS-CoV-2 infections or symptoms of the post- and long-COVID form, owing to the scarcity of clinically approved therapeutics. Aim of the study: The ongoing threat posed by SARS-CoV-2, along with the rapidly evolving new variants, requires the development of new antiviral compounds. The aim of this study was to identify anti-SARS-CoV-2 herbal and fungal extracts used in traditional medicine against acute respiratory infection, inflammation, and related symptoms. Additionally, we sought to characterize their bioactive constituents. Materials and methods: The antiviral activity and cell cytotoxicity of 179 herbal and fungal extracts were evaluated using two SARS-CoV-2 infection assays in Caco-2 cells. 19 plant extracts with and without anti-SARS-CoV-2 activity underwent detailed dereplication using molecular networking.Results: Extracts from Angelica sinensis (Oliv.) Diels roots, Annona squamosa L. seeds, Azadirachta indica A. Juss. fruits, Buddleja officinalis Maxim. flowers, Burkea africana Hook. bark and Clinopodium menthifolium (Host) Stace aerial parts showed a potent anti SARS-CoV-2 activity (IC50 < 5 mu g/ml) with only moderate cytotoxicity (CC50 > 60 mu g/ml, Caco-2). By performing the dereplication with a bioactivity-featured molecular network (MN) on the extract library level, rather than on the level of individual extracts, we could pinpoint compounds characteristic for active extracts. Thus, a straight-forward identification of potential anti-SARS-CoV-2 natural compounds was achieved prior to any fractionation or isolation efforts.Conclusions: A sophisticated hyphenation of empirical knowledge with MS-based bioinformatics and automated compound annotation was applied to decipher the chemical space of the investigated extracts. The correlation with experimentally assessed anti-SARS-CoV-2 activities helped in predicting compound classes and structural elements relevant for the antiviral activities. Consequently, this accelerated the identification of constituents from the investigated mixtures with inhibitory effects against SARS-CoV-2.

The oral consumption of alcohol (ethanol) has a long tradition in humans and is an integral part of many cultures. The causal relationship between ethanol consumption and numerous diseases is well known. In addition to the well-described harmful effects on the liver and pancreas, there is also evidence that ethanol abuse triggers pathological skin conditions, including acne. In the present study, we addressed this issue by investigating the effect of ethanol on the energy metabolism in human SZ95 sebocytes, with particular focus on qualitative and quantitative lipogenesis. It was found that ethanol is a strong trigger for lipogenesis, with moderate effects on cell proliferation and toxicity. We identified the non-oxidative metabolism of ethanol, which produced fatty acid ethyl esters (FAEEs), as relevant for the lipogenic effect—the oxidative metabolism of ethanol does not contribute to lipogenesis. Correspondingly, using the Seahorse extracellular flux analyzer, we found an inhibition of the mitochondrial oxygen consumption rate as a measure of mitochondrial ATP production by ethanol. The ATP production rate from glycolysis was not affected. These data corroborate that ethanol-induced lipogenesis is independent from oxygen. In sum, our results give a causal explanation for the prevalence of acne in heavy drinkers, confirming that alcoholism should be considered as a systemic disease. Moreover, the identification of key factors driving ethanol-dependent lipogenesis may also be relevant in the treatment of acne vulgaris.

There is clinical evidence showing that the Pelargonium sidoides root extract EPs 7630 is a safe and effective treatment for a range of acute infectious respiratory illnesses. Moreover, EPs 7630 has been shown to reduce the use of antibiotics, which is important in the context of rising antibiotic resistance levels. A wide range of mechanisms appears to contribute to the beneficial effects of EPs 7630, e.g. antibacterial, antiviral, immunomodulatory, and epithelial barrier effects. This broad spectrum of pharmacological activities seems to enable the clinical activity of EPs 7630 against multiple respiratory infections. In particular, the combination of antiviral and immunomodulatory effects may enable EPs 7630 to tackle acute viral respiratory infections both in early stages of the disease process, which are driven by virus replication, as well as in later stages, which are caused by an overshooting immune response. Hence, EPs 7630 is a prime example of a plant extract with evidence-based clinical efficacy, including a solid understanding of the underlying mechanisms of action. The example of EPs 7630 demonstrates that plant extracts have a potential role as evidence-based clinical treatments and that they deserve pre-clinical and clinical testing and investigation in the same way as any other drug class.

Here, we introduce a novel set of drug-adapted triple-negative breast cancer (TNBC) cell lines consisting of the parental cell lines MDA-MB-468, HCC38, and HCC1806 and their sublines adapted to cisplatin, doxorubicin, eribulin, paclitaxel, gemcitabine, or 5-fluorouracil. Whole exome sequencing in combination with the analysis of TCGA-derived patient data resulted in the identification of 135 biomarker candidates for the guidance of personalized TNBC therapies for further investigation, including 58 novel ones that had not been associated with drug resistance before. The analysis of exome sequencing data showed remarkably few overlaps among the resistant sublines, suggesting that each resistance formation process follows an individual, unpredictable route. This complexity was confirmed by cancer cell line drug sensitivity profiles to cytotoxic anti-cancer drugs and DNA damage repair inhibitors. Drug-adapted sublines of the same parental cell line and sublines adapted to the same drug substantially differed in their drug response patterns. Cross-resistance levels were lowest for the CHK2 inhibitor CCT241533, the PLK1 inhibitor SBE13, and the RAD51 recombinase inhibitor B02, making CHK2, PLK1, and RAD51 promising drug targets for therapy-refractory TNBC. In conclusion, we present novel preclinical models of acquired drug resistance in TNBC and 58 novel candidate biomarkers for further investigation. Whole exome data and drug sensitivity profiles showed that each cancer cell line adaptation process follows an unpredictable route, which reflects recent findings on cancer cell evolution in patients, supporting the relevance of drug-adapted cancer cell lines as preclinical models of acquired resistance.### Competing Interest StatementThe authors have declared no competing interest.

This poster demonstrates the development of drug-resistant cancer cell lines and their application in cancer research.

Knowledge of the molecular pathogenesis of acute myeloid leukemia has advanced in recent years. Despite novel treatment options, acute myeloid leukemia remains a survival challenge for elderly patients. We have recently shown that the triphosphohydrolase SAMHD1 is one of the factors determining resistance to Ara-C treatment. Here, we designed and tested novel and simpler virus-like particles incorporating the lentiviral protein Vpx to efficiently and transiently degrade SAMHD1 and increase the efficacy of Ara-C treatment. The addition of minute amounts of lentiviral Rev protein during production enhanced the generation of virus-like particles. In addition, we found that our 2nd generation of virus-like particles efficiently targeted and degraded SAMHD1 in AML cell lines with high levels of SAMHD1, thereby increasing Ara-CTP levels and response to Ara-C treatment. Primary AML blasts were generally less responsive to VLP treatment. In summary, we have been able to generate novel and simpler virus-like particles that can efficiently deliver Vpx to target cells.