Potential Sensitivity Research Articles

TRIB3 inhibition by palbociclib sensitizes prostate cancer to ferroptosis via downregulating SOX2/SLC7A11 expression

Palbociclib is a CDK4/6 inhibitor approved for the treatment of breast cancer by suppressing cell proliferation. However, monotherapy with palbociclib was discouraging in prostate cancer, calling for a mechanism-based effective therapy. In this study, we reported in prostate cancer that palbociclib is a potent sensitizer of ferroptosis, which is worked out by downregulating the expression of TRIB3, a gene highly expressed in prostate cancer. Specifically, TRIB3 knockdown augmented the response of prostate cancer cells to ferroptosis inducers, whereas, TRIB3 overexpression rescued prostate cancer cells from palbociclib-induced ferroptosis. Mechanistically, TRIB3 inhibition by palbociclib resulted in downregulation of SOX2, which subsequently led to compromised expression of SLC7A11, a cystine/glutamate antiporter that counteracts ferroptosis. Functionally, a combined treatment of palbociclib with ferroptosis inducer significantly suppressed prostate cancer growth in a xenograft tumor model. Together, these results uncover an essential role of TRIB3/SOX2/SLC7A11 axis in palbociclib-induced ferroptosis, suggesting palbociclib a promising targeted therapy in combine with ferroptosis induction for the treatment of prostate cancer.

High TNF and NF-ĸB pathway dependency are associated with AZD5582 sensitivity in OSCC via CASP8-dependent apoptosis.

Drug repurposing can accelerate the development of novel therapeutic strategies. Oral squamous cell carcinoma (OSCC) is prevalent in Asians and is associated with a poor prognosis. Here, we integrate newly generated sensitivity data for 339 anticancer drugs with genome-wide CRISPR‒Cas9 gene essentiality data for 21 cell lines mostly derived from Asian OSCC patients to identify drug repurposing candidates. We observe potent activity of AZD5582 (an antagonist of the inhibitor of apoptosis (IAP) family of proteins) in a subset of OSCC cells, which was associated with dependency on NF-ĸB pathway genes (RNF31, MAP3K7 and IKBKG). We confirm the on-target specificity and potency of AZD5582 in vitro and its ability to suppress cell growth in xenograft models. Furthermore, we identify tumor necrosis factor (TNF) as a key mediator of AZD5582 sensitivity in OSCC. CASP8-dependent apoptotic and CASP8-independent necroptotic cellular programs mediate AZD5582-induced cell death. In summary, through the systematic integration of pharmacological and CRISPR data, we identified a subset of OSCC with potent sensitivity to AZD5582 mediated through the NF-κB and TNF signalling pathways.

Predicting points of departure and potency categories for fragrance ingredients by integrating OECD in vitro models

Continuous potency assessment is crucial for conducting quantitative risk assessment (QRA) of sensitizers. Quantitative regression models, based on in vitro methods, have been developed to calculate points of departure for use in skin sensitization QRA. These models calculate a point of departure as a predicted value for Local Lymph Node Assay (LLNA) EC3 or potency value (PV), integrating data from the kinetic Direct Peptide Reactivity Assay (kDPRA), KeratinoSens (KS) assay, and human Cell Line Activation Test (h-CLAT). The goal of this study was to determine how in vitro predicted EC3s and PVs compare to published reference data. In vitro data were combined in point of departure regression models to predict EC3s and PVs. These points of departure were then grouped into sensitization potency categories, such as extreme, strong, moderate, weak, very weak, or non-sensitizer, as previously described. Trends in potency distribution and high concordance between predicted EC3 and PV categories and published potency categories were observed. Furthermore, the median absolute fold-misprediction ranged between 1.8 and 2.5 for models predicting EC3 and between 1.7 and 3.4 for those predicting PVs. These regression models are a promising animal alternative for determining sensitization quantitative potency for fragrance ingredients, thereby facilitating QRA.

Increasing Accessibility of Bayesian Network-Based Defined Approaches for Skin Sensitisation Potency Assessment.

Skin sensitisation is a critical adverse effect assessed to ensure the safety of compounds and materials exposed to the skin. Alongside the development of new approach methodologies (NAMs), defined approaches (DAs) have been established to promote skin sensitisation potency assessment by adopting and integrating standardised in vitro, in chemico, and in silico methods with specified data analysis procedures to achieve reliable and reproducible predictions. The incorporation of additional NAMs could help increase accessibility and flexibility. Using superior algorithms may help improve the accuracy of hazard and potency assessment and build confidence in the results. Here, we introduce two new DA models, with the aim to build DAs on freely available software and the newly developed kDPRA for covalent binding of a chemical to skin peptides and proteins. The new DA models are built on an existing Bayesian network (BN) modelling approach and expand on it. The new DA models include kDPRA data as one of the in vitro parameters and utilise in silico inputs from open-source QSAR models. Both approaches perform at least on par with the existing BN DA and show 63% and 68% accuracy when predicting four LLNA potency classes, respectively. We demonstrate the value of the Bayesian network's confidence indications for predictions, as they provide a measure for differentiating between highly accurate and reliable predictions (accuracies up to 87%) in contrast to low-reliability predictions associated with inaccurate predictions.

Updating Reaction Mechanistic Domains for Skin Sensitization: 1. Nucleophilic Skin Sensitizers.

It has long been recognized that skin sensitizers either are electrophilic or can be activated to electrophilic species. Several nonanimal assays for skin sensitization are based on this premise. In the course of a project to update dermal sensitization thresholds (DST), we found a substantial number of sensitizers, with no electrophilic or pro-electrophilic alerts, that could be simply explained in terms of the sensitizer acting as a nucleophile. In some cases, the nucleophilic center is a sulfur or phosphorus atom, while in others, it is an aromatic carbon atom. For carbon-centered nucleophiles, a quantitative mechanistic model based on a combination of Hammett σ+ and logP values has been derived. This has been applied to rationalize several groups of known sensitizers with no electrophilic or pro-electrophilic alerts, including anacardic acids and cardols, which are known human sensitizers associated with, inter alia, cashew nut oil, mango, and Ginkgo biloba. The possibility of nucleophilic sensitization needs to be considered when evaluating new chemicals for skin sensitization potential and potency by nonanimal assays, particularly those based on the premise that skin sensitization is dependent upon reactions of electrophiles with skin protein-based nucleophiles.

Identification of STAM-binding protein as a target for the treatment of gemcitabine resistance pancreatic cancer in a nutrient-poor microenvironment

Pancreatic cancer (PC) is a highly malignant solid tumor whose resistance to gemcitabine (GEM) chemotherapy is a major cause of poor patient prognosis. Although PC is known to thrive on malnutrition, the mechanism underlying its chemotherapy resistance remains unclear. The current study analyzed clinical tissue sample databases using bioinformatics tools and observed significantly upregulated expression of the deubiquitinase STAMBP in PC tissues. Functional experiments revealed that STAMBP knockdown remarkably increases GEM sensitivity in PC cells. Multiple omics analyses suggested that STAMBP enhances aerobic glycolysis and suppresses mitochondrial respiration to increase GEM resistance in PC both in vitro and in vivo. STAMBP knockdown decreased PDK1 levels, an essential regulator of the aerobic glycolytic process, in several cancers. Mechanistically, STAMBP promoted the PDK1-mediated Warburg effect and chemotherapy resistance by modulating E2F1 via direct binding to E2F1 and suppressing its degradation and ubiquitination. High-throughput compound library screening using three-dimensional protein structure analysis and drug screening identified the FDA drug entrectinib as a potent GEM sensitizer and STAMBP inhibitor, augmenting the antitumor effect of GEM in a patient-derived xenograft (PDX) model. Overall, we established a novel mechanism, via the STAMBP–E2F1–PDK1 axis, by which PC cells become chemoresistant in a nutrient-poor tumor microenvironment.

Apoptosis of Pancreatic Cancer Cells after Co-Treatment with Eugenol and Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand.

Pancreatic cancer is a refractory cancer with limited treatment options. Various cancer types are resistant to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Eugenol, the main component of clove oil, exhibits anticancer, anti-inflammatory, and antioxidant effects. However, no studies have reported that eugenol increases TRAIL sensitivity by upregulating death receptor (DR) expression. Here, we aimed to investigate eugenol as a potent TRAIL sensitizer. Increased apoptosis and inhibition of cell proliferation was observed in pancreatic cancer cells treated with eugenol and TRAIL compared with those treated with eugenol alone. Eugenol upregulated the expression of DR5, inhibited the FLICE-inhibitory protein (FLIP), an anti-apoptotic protein, and increased p53, a tumor suppressor protein. In addition, eugenol induced the generation of reactive oxygen species (ROS) and caused endoplasmic reticulum (ER) stress. C/EBP-homologous protein (CHOP) knockdown using siRNA decreased the expression of DR5 and reduced the combined effects of eugenol and TRAIL. These results demonstrate that eugenol enhances TRAIL-induced apoptosis by upregulating DR5 through the ROS-mediated ER stress-CHOP pathway, which enhances ER stress by inducing p53 and downregulating FLIP expression. This suggests that eugenol has the potential to treat pancreatic cancer by increasing cell sensitivity to TRAIL.

P19-57 Improved Confidence of Quantitative Sensitizing Potency Assessment for Point of Departure Using GARDskin Dose-Response

P19-57 Improved Confidence of Quantitative Sensitizing Potency Assessment for Point of Departure Using GARDskin Dose-Response

P19-41 The SENS-IS in vitro assay to predict and classify the skin sensitization potency of finished cosmetic products

P19-41 The SENS-IS in vitro assay to predict and classify the skin sensitization potency of finished cosmetic products

Quantum-Mechanics Calculations Elucidate Skin-Sensitizing Pharmaceutical Compounds.

Skin sensitization is a critical end point in occupational toxicology that necessitates the use of fast, accurate, and affordable models to aid in establishing handling guidance for worker protection. While many in silico models have been developed, the scarcity of reliable data for active pharmaceutical ingredients (APIs) and their intermediates (together regarded as pharmaceutical compounds) brings into question the reliability of these tools, which are largely constructed using publicly available nonspecialty chemicals. Here, we present the quantum-mechanical (QM) Computer-Aided Discovery and REdesign (CADRE) model, which was developed with the bioactive and structurally complex chemical space in mind by relying on the fundamentals of chemical interactions in key events (versus structural attributes of training-set data). Validated in this study on 345 APIs and intermediates, CADRE achieved 95% accuracy, sensitivity, and specificity and a combined 79% accuracy in assigning potency categories compared to the mouse local lymph node assay data. We show how historical outcomes from CADRE testing in the pharmaceutical space, generated over the past 10 years on ca. 2500 chemicals, can be used to probe the relationships between sensitization mechanisms (or the underlying chemical classes) and the probability of eliciting a sensitization response in mice of a given potency. We believe this information to be of value to both practitioners, who can use it to quickly screen and triage their data sets, as well as to model developers to fine-tune their structure-based tools. Lastly, we leverage our experimentally validated subset of APIs and intermediates to show the importance of dermal permeability on the sensitization potential and potency. We demonstrate that common physicochemical properties used to assess permeation, such as the octanol-water partition coefficient and molecular weight, are poor proxies for the more accurate energy-pair distributions that can be computed from mixed QM and classical simulations using model representations of the stratum corneum.

Deriving a Continuous Point of Departure for Skin Sensitization Risk Assessment Using a Bayesian Network Model.

Regulations of cosmetic ingredients and products have been the most advanced in embracing new approach methodologies (NAMs). Consequently, the cosmetic industry has assumed a forerunner role in the development and implementation of animal-free next-generation risk assessment (NGRA) that incorporates defined approaches (DAs) to assess the skin sensitization potency of ingredients. A Bayesian network DA predicting four potency categories (SkinSens-BN) was constructed against reference Local Lymph Node Assay data for a total of 297 substances, achieving a predictive performance similar to that of other DAs. With the aim of optimally informing risk assessment with a continuous point of departure (PoD), a weighted sum of the SkinSens-BN probabilities for four potency classes (non-, weak, moderate, and strong/extreme sensitizer) was calculated, using fixed weights based on associated LLNA EC3-values. The approach was promising, e.g., the derived PoDs for substances classified as non-sensitizers did not overlap with any others and 77% of PoDs were similar or more conservative than LLNA EC3. In addition, the predictions were assigned a level of confidence based on the probabilities to inform the evaluation of uncertainty in an NGRA context. In conclusion, the PoD derivation approach can substantially contribute to reliable skin sensitization NGRAs.

Apigenin is an anoikis sensitizer with strong anti-metastatic properties in experimental breast cancer

Loss of susceptibility to anoikis signals is a crucial step in metastasis. Anoikis resistance therefore represents a promising adjuvant therapeutic target for cancer management. In this study, we have conducted a rationalized screening to search for novel leading anoikis sensitizer from daily foods. Among 19 tested dietary phytochemicals, the best results were obtained with apigenin, a natural component of celery. Phenotypically, apigenin sensitized breast cancer cells to anoikis, lowered the number of circulating tumor cells, and protected against breast cancer metastasis to lung in mice. Mechanistically, we demonstrated that the thromboxane A2 (TXA2)-TXA2 receptor (TP) axis has a critical role in acquired anoikis resistance by activating PI3K-Akt signaling pathway. Blockage of TXA2 signaling up-regulated p53 as well as its target gene p21, caused a G1 phase arrest, and finally led to apoptosis in breast cancer cells. TXA2 level was positively correlated with breast cancer cell anoikis rate, and apigenin significantly inhibited TXA2 biosynthesis in vitro and in vivo. Collectively, we identified apigenin as a potent anoikis sensitizer with anti-metastatic properties in a mouse model of breast cancer, and these findings might provide a rationale for introducing apigenin supplementation to breast cancer patients.

Aqueous photolysis of naproxen exposed to UV and natural sunlight: Formation of excited triplet and photosensitizing product

Photochemical transformation is an important attenuation process for the non-steroidal anti-inflammatory drug naproxen (NPX) in both engineered and natural waters. Herein, we investigated the photolysis of NPX in aqueous solution exposed to both ultraviolet (UV, 254 nm) and natural sunlight irradiation. Results show that N2 purging significantly promoted NPX photolysis under UV irradiation, suggesting the formation of excited triplet state (3NPX*) as a critical transient. This inference was supported by benzophenone photosensitization and transient absorption spectra. Sunlight quantum yield of NPX was only one fourteenth of that under UV irradiation, suggesting the wavelength-dependence of NPX photochemistry. 3NPX* formed upon irradiation of NPX underwent photodecarboxylation leading to the formation of 2-(1-hydroxyethyl)-6-methoxynaphthalene (2HE6MN), 2-(1-hydroperoxyethyl)-6-methoxynaphthalene (2HPE6MN), and 2-acetyl-6-methoxynaphthalene (2A6MN). Notably, the conjugation and spin-orbit coupling effects of carbonyl make 2A6MN a potent triplet sensitizer, therefore promoting the photodegradation of the parent NPX. In hospital wastewater, the photolysis of NPX was influenced because the photoproduct 2A6MN and wastewater components could competitively absorb photons. Bioluminescence inhibition assay demonstrated that photoproducts of NPX exhibited higher toxicity than the parent compound. Results of this study provide new insights into the photochemical behaviors of NPX during UV treatment and in sunlit surface waters.

E3 ubiquitin ligase UBR5 promotes gemcitabine resistance in pancreatic cancer by inducing O-GlcNAcylation-mediated EMT via destabilization of OGA.

Pancreatic cancer (PC) is among the deadliest malignancies, with an extremely poor diagnosis and prognosis. Gemcitabine (GEM) remains the first-line drug for treating PC; however, only a small percentage of patients benefit from current immunotherapies or targeted therapies. Resistance to GEM is prevalent and affects long-term survival. We found that ubiquitin-protein ligase E3 module N-recognition 5 (UBR5) is a therapeutic target against GEM resistance. UBR5 was markedly upregulated in clinical GEM-resistant PC samples and GEM-resistant PC cells. UBR5 knockdown markedly increased GEM sensitivity in GEM-resistant PC cell lines. UBR5-mediated GEM resistance was accompanied by activation of epithelial-mesenchymal transition (EMT) and could be mitigated by inhibiting EMT. Further analysis revealed that UBR5 promoted GEM resistance in PC cells by enhancing O-GlcNAcylation-mediated EMT. In addition, UBR5 knockdown resulted in increased O-GlcNAase (OGA) levels, an essential negatively regulated enzyme in the O-GlcNAcylation process. We identified a negative association between OGA and UBR5 levels, which further supported the hypothesis that O-GlcNAcylation-mediated GEM resistance induced by UBR5 is OGA-dependent in PC cells. Mechanistic studies revealed that UBR5 acts as an E3 ubiquitin ligase of OGA and regulates O-GlcNAcylation by binding and modulating OGA, facilitating its degradation and ubiquitination. Additionally, high-throughput compound library screening using three-dimensional protein structure analysis and drug screening identified a Food and Drug Administration drug, Y-39983 dihydrochloride, as a potent GEM sensitiser and UBR5 inhibitor. The combination of Y-39983 dihydrochloride and GEM attenuated tumour growth in a mouse xenograft tumour model. Collectively, these data demonstrated that UBR5 plays a pivotal role in the sensitisation of PC to GEM and provides a potential therapeutic strategy to overcome GEM resistance.

Deriving a point of departure for assessing the skin sensitization risk of wearable device constituents with in vitro methods

Wearable devices are in contact with the skin for extended periods. As such, the device constituents should be evaluated for their skin sensitization potential, and a Point of Departure (PoD) should be derived to conduct a proper risk assessment. Without historical in vivo data, the PoD must be derived with New Approach Methods (NAMs). To accomplish this, regression models trained on LLNA data that use data inputs from OECD-validated in vitro tests were used to derive a predicted EC3 value, the LLNA value used to classify skin sensitization potency, for three adhesive monomers (Isobornyl acrylate (IBOA), N, N- Dimethylacrylamide (NNDMA), and Acryloylmorpholine (ACMO) and one dye (Solvent Orange 60 (SO60)). These chemicals can be used as constituents of wearable devices and have been associated with causing allergic contact dermatitis (ACD). Using kinetic DPRA and KeratinoSens™ data, the PoDs obtained with the regression model were 180, 215, 1535, and 8325 μg/cm2 for IBOA, SO60, ACMO, and NNDMA, respectively. The PoDs derived with the regression model using NAMs data will enable a proper skin sensitization risk assessment without using animals.

Central inhibition of HDAC6 re-sensitizes leptin signaling during obesity to induce profound weight loss

Leptin resistance during excess weight gain significantly contributes to the recidivism of obesity to leptin-based pharmacological therapies. The mechanisms underlying the inhibition of leptin receptor (LepR) signaling during obesity are still elusive. Here, we report that histone deacetylase 6 (HDAC6) interacts with LepR, reducing the latter's activity, and that pharmacological inhibition of HDAC6 activity disrupts this interaction and augments leptin signaling. Treatment of diet-induced obese mice with blood-brain barrier (BBB)-permeable HDAC6 inhibitors profoundly reduces food intake and leads to potent weight loss without affecting the muscle mass. Genetic depletion of Hdac6 in Agouti-related protein (AgRP)-expressing neurons or administration with BBB-impermeable HDAC6 inhibitors results in a lack of such anti-obesity effect. Together, these findings represent the first report describing a mechanistically validated and pharmaceutically tractable therapeutic approach to directly increase LepR activity as well as identifying centrally but not peripherally acting HDAC6 inhibitors as potent leptin sensitizers and anti-obesity agents.

Abstract 7579: Dual inhibition of enhancer of zeste homolog 1 2 induces pyroptosis, modulates oncogenic signaling pathways & sensitizes hepatocellular carcinoma to valemetostat & docetaxel treatments

Abstract HCC is a global health concern, with chemotherapy resistance and patient recurrence posing significant challenges to liver cancer treatment. EZH2 is a crucial part of the polycomb repressive complex 2 (PRC2). It, along with its homolog EZH1, is a focus for cancer therapeutic targets. Dysregulation of EZH1/2 can cause cancer development due to significant changes in geneexpression. The current study aims to investigate the interaction between EZH1/2 and oncogenic signals responsible for HCC progression and pyroptosis induction. Two cell lines, HepG2 and SK-Hep1, were subjected to two FDA-approved drugs, Valemetostat (Val) and Docetaxel (Doc), to determine the impact of EZH1/2 on tumorigenicity and drug resistance. The WST-1 assay was utilized to gauge cell proliferation. The results indisputablydemonstrated a synergistic effect of the drugs after 48 h of treatment. The inhibition of viability was attributed to apoptosis induction in both tested cell lines, and the maximum effect was observed in the combined treatment as compared to either the control or each drug alone. Furthermore, our datashowed activation of caspase-3, upregulation of BAX, indicating apoptotic induction, and downregulation of Bcl-2, Bcl-XL, AKT, S6k, oncogenic β-catenin, and Stat3 protein expression levels, suggesting a modulation of oncogenic signaling pathways at the protein level. After 24 h of Val/Doc treatment, pyroptosis was observed, characterized by cell swelling and bubbles on themembrane and a significant release of lactate dehydrogenase, indicating pyroptotic cell cytotoxicity. The antitumor effects and pyroptosis were enhanced by using a dual inhibitor, UNC1999, for EZH1/2, resulting in a marked increase in the sensitivity of tested cells to the Val/Doc treatment invitro. The present study unequivocally demonstrated that dual inhibition of EZH1/2 acts as a potent sensitizer of HCC cells to Val/Doc treatment, which might be a promising therapeutic approach for inducing cellular pyroptosis in HCC cell lines. Citation Format: Asmaa Elkhder Harira, Adham Maher, Ahmed Sultan. Dual inhibition of enhancer of zeste homolog 1 2 induces pyroptosis, modulates oncogenic signaling pathways & sensitizes hepatocellular carcinoma to valemetostat & docetaxel treatments [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7579.

Abstract 706: Advancing radiotherapy strategies for high-risk rhabdomyosarcoma: Unveiling BCL-xL inhibition as a potent sensitizer to radiotherapy

Abstract Pediatric soft-tissue sarcomas are a group of solid tumors that account for about 7% of childhood cancer cases. Rhabdomyosarcoma (RMS), the most common subtype, further stratifies into PAX3 or PAX7 and FOXO1 fusion-negative (FN) or fusion-positive (FP) RMS. FP RMS is the higher risk subtype and is more aggressive, metastatic, and less responsive to treatment. Front-line treatments for RMS FP patients have had limited progress in the recent years and existing treatments such as radiotherapy lacks tumor cell selectivity, resulting in toxic side effects. To address this, we conducted a high-throughput drug screen to identify radiosensitizers to enhance efficacy of RMS radiotherapy. Using three patient-derived FP RMS tumoroids, we screened a comprehensive drug library with and without radiation, comprising 528 drugs at varying stages of pre-clinical and clinical trials. A validation screen of the top 29 drugs was conducted on a larger cohort of samples including eight patient-derived FP RMS tumoroids and three non-cancer models. The screening revealed that drugs targeting the anti-apoptotic protein B-cell lymphoma-extra-large (BCL-xL) exhibited both potent radiosensitization and cytotoxicity selectively for FP RMS. Notably, the sensitivity to BCL-xL inhibitors correlated with elevated BCL2L1 expression and PAX3 fusion status. Additionally, drugs targeting polo-like kinase 1 (PLK1) and other cell cycle-related genes exhibited robust radioprotective effects across both RMS and non-cancer models. Our findings propose the therapeutic potential of BCL-xL inhibition, either as a standalone treatment or in combination with radiotherapy. In parallel, we show the protective potential of PLK1 inhibition to mitigate radiation toxicity. In the long term, we aspire to integrate the identified radiosensitizing and radioprotecting compounds with existing treatment regiments for RMS, maximizing precision and efficacy in RMS radiotherapy. Citation Format: Sammy Se Whee Park, Nona Struyf, Tom Erkers, Sören Lehmann, Michael T. Meister, Jarno Drost, Frank C. Holstege, Nikolas Herold, Päivi Östling, Jakob Stenman, Olli Kallioniemi, Jan-Inge Henter, Brinton Seashore-Ludlow, Kasper Karlsson. Advancing radiotherapy strategies for high-risk rhabdomyosarcoma: Unveiling BCL-xL inhibition as a potent sensitizer to radiotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 706.

Abstract A035: Genome-wide CRISPRi screening reveals determinants of platinum sensitivity and resistance in high-grade serous ovarian cancer

Abstract A035: Genome-wide CRISPRi screening reveals determinants of platinum sensitivity and resistance in high-grade serous ovarian cancer

Diagnosis of thrombotic thrombocytopenic purpura: easy-to-use fiber optic surface plasmon resonance immunoassays for automated ADAMTS-13 antigen and conformation evaluation

BackgroundLaboratory diagnosis of immune-mediated thrombotic thrombocytopenic purpura (iTTP) remains challenging when ADAMTS-13 activity ranges between 10% and 20%. To prevent misdiagnosis, open ADAMTS-13 conformation gained clinical attention as a novel biomarker, especially to diagnose acute iTTP in patients with diagnostic undecisive ADAMTS-13 activity. Plasma ADAMTS-13 conformation analysis corrects for ADAMTS-13 antigen, with both parameters being characterized in enzyme-linked immunosorbent assay (ELISA)-based reference assays requiring expert technicians. ObjectivesTo design ADAMTS-13 antigen and conformation assays on automated, easy-to-use fiber optic surface plasmon resonance (FO-SPR) technology to promote assay accessibility and diagnose challenging iTTP patients. MethodsADAMTS-13 antigen and conformation assays were designed on FO-SPR technology. Plasma of 20 healthy donors and 20 acute iTTP patients were quantified, and data from FO-SPR and ELISA reference assays were compared. ResultsFollowing assay design, both antigen and conformation FO-SPR assays were optimized and characterized, presenting strong analytical sensitivity (detection limit of 0.001 μg/mL) and repeatability (interassay variation of 14.4%). Comparative analysis suggested positive correlation (Spearman r of 0.92) and good agreement between FO-SPR and ELISA assays. As expected, FO-SPR assays showed a closed or open ADAMTS-13 conformation in healthy donors and acute iTTP patients, respectively. ConclusionBoth ADAMTS-13 antigen and conformation assays were transferred onto automated, easy-to-use FO-SPR technology, displaying potent analytical sensitivity and reproducibility. ADAMTS-13 antigen and conformation were determined for healthy donors and acute iTTP patients showing strong correlation with ELISA reference. Introducing FO-SPR technology in clinical context could support routine diagnosis of acute iTTP patients, notably when ADAMTS-13 activity fluctuates between 10% and 20%.