Cell-based Cytotoxicity Assay Research Articles

Abstract 6741: Targeting glutamine metabolism potentiates antigen presentation in colorectal cancer

Abstract Colorectal cancer (CRC) ranks among the most prevalent cancers worldwide, causing substantial mortality. The urgent need for effective treatments has driven research into immune checkpoint blockade (ICB) therapies, which have demonstrated significant clinical benefits across various cancer types. Despite the success of ICB therapies, a significant proportion of CRC patients exhibit resistance to treatment, prompting the identification of mechanisms that hinder therapy resistance or enhance treatment response. CRC cells display remarkable adaptability, orchestrating metabolic changes that confer growth advantages, pro-tumor microenvironment, and therapeutic resistance. One such metabolic change occurs in glutamine metabolism. In this study, we found that colorectal tumors with high glutaminase (GLS) expression exhibit reduced T-cell infiltration and cytotoxicity, leading to poor clinical outcomes. To validate these findings, we applied the ovalbumin (OVA)/OT-I T cell-based cytotoxicity assay and the CRC patient-derived organoids (PDOs)/autologous T cell killing assay. Our results indicated that inhibition of GLS significantly enhanced T-cell cytotoxicity in vitro and ex vivo. Mechanistically, we elucidated that inhibition of GLS activated reactive oxygen species-related signaling pathways in tumor cells, thereby potentially increasing tumor immunogenicity by promoting antigen presentation. The combinational therapy of GLS inhibitor and ICB exhibited a superior tumor growth inhibitory effect in comparison with either of the monotherapies. These findings shed new light on clinical therapeutic strategies for CRC patients, offering a promising approach to enhance the efficacy of ICB therapy. Citation Format: Tao Yu, Kevin Van der Jeught, Haiqi Zhu, Zhuolong Zhou, Samantha Sharma, Sheng Liu, Ka Man So, Haniyeh Eyvani, Xiyu Wang, George E. Sandusky, Yunlong Liu, Mateusz Opyrchal, Sha Cao, Jun Wan, Chi Zhang, Xinna Zhang. Targeting glutamine metabolism potentiates antigen presentation in colorectal cancer [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 6741.

Discovery of new DHA ethanolamine derivatives as potential anti-inflammatory agents targeting Nur77

Docosahexaenoic acid (DHA) has a strong anti-inflammatory effect and is reported to bind to the ligand-binding domain (LBD) of the anti-inflammatory modulator Nur77. Recently, we have found that DHA ethanolamine (DHA-EA) exerts anti-inflammatory activity as a Nur77 modulator. Herein, using a fragment splicing-based drug design strategy, nineteen new DHA-EA derivatives were synthesized starting from DHA algae oil and then evaluated for their anti-inflammatory activity. The cell-based cytotoxicity assays showed that compounds J2, J9, and J18 had no noticeable effect on the cell morphology and viability of RAW 264.7, LO2, and MCR-5 cells. Meanwhile, J9 was identified as the most potent anti-inflammatory molecule in LPS-stimulated RAW 264.7 cells. Also, the molecular docking study and SPR assay demonstrated that J9 exhibited in vitro Nur77-binding affinity (KD = 8.58 × 10-6 M). Moreover, the mechanism studies revealed that the anti-inflammatory activity of J9 was associated with its inhibition of NF-κB activation in a Nur77-dependent manner. Notably, J9 could attenuate LPS-induced inflammation in the mouse acute lung injury (ALI) model. Overall, the DHA-EA derivative J9 targeting Nur77 is a potential candidate for developing anti-inflammatory and ALI-treating agents.

Design, synthesis, and evaluation of novel benzoylhydrazone derivatives as Nur77 modulators with potent antitumor activity against hepatocellular carcinoma

Nur77 modulators have emerged as a promising therapeutic approach for hepatocellular carcinoma. In this study, a structure-based rational drug design approach was used to design and synthesise a series of 4-((8-hydroxy-2-methylquinolin-4-yl)amino)benzoylhydrazone derivatives based on the binding characteristics of our previously reported 10g and the native ligand 3NB at the binding Site C of Nur77. Cell-based cytotoxicity assays revealed that compound TMHA37 demonstrated the highest cytotoxicity against all tested cancer cells. The induced fit docking and binding pose metadynamics simulation suggested that TMHA37 was the most promising Nur77 binder at Site C. Molecular dynamics simulation validated the stable binding of TMHA37 to Nur77’s Site C but not to Sites A or B. Specifically, TMHA37 bound strongly to Nur77-LBD (K D = 445.3 nM) and could activate Nur77’s transcriptional activity. Furthermore, TMHA37 exhibited antitumor effects by blocking the cell cycle at G2/M phase and inducing cell apoptosis in a Nur77-dependent manner.

Sequestration of zearalenone using microorganisms blend in vitro.

Zearalenone (ZEN) is an estrogenic mycotoxin produced by the Fusarium species and induces severe reproductive disorders in animals thus a major concern in the livestock industry. Probiotic bacteria treatments have been shown to inactivate mycotoxins, therefore, in this study, we investigated the effect of two commercial probiotic feed additives on the sequestration of ZEN. Commercial probiotic blends containing clay-based binder with Aspergillus niger, Bacillus licheniformis, Bacillus pumilus, and Bacillus subtilis at various proportions from BioMatrix International were incubated with ZEN in a time-dependent manner and then analyzed by Enzyme-Linked Immunosorbent Assay (ELISA) to quantify unbound ZEN. Sequestration of ZEN was further verified by using MCF-7 cell-based cytotoxicity and/or cell proliferation assays. ZEN, or probiotic mix, was nontoxic to MCF-7 cells. Probiotic blends decreased ZEN concentration by 45% (∼100 μg L-1) and prevented ZEN from inducing MCF-7 cell proliferation (20%-28% reduction). The probiotic feed supplements tested show a potential utility in ZEN neutralization.

Gefitinib conjugated PEG passivated graphene quantum dots incorporated PLA microspheres for targeted anticancer drug delivery

In the present study, polyethylene Glycol passivated Graphene Quantum Dots (PEG-GQDs) were successfully synthesized via the hydrothermal method. Furthermore, for the synthesis of anticancer drug loaded GQD embedded microspheres, the anticancer drug was mixed with synthesized PEG-GQD. As prepared, Gefitinib-PEG-GQDs were incorporated into poly-lactic acid (PLA) microspheres using poly-vinyl-acetate (PVA) as surfactant via solvent evaporation technique and single emulsification method. The successful synthesis of anticancer drug loaded microspheres was confirmed by several characterization techniques, including Field-Emission Scanning Electron Microscopy (FE-SEM), which shows the morphology of microspheres, Fourier Transform Infrared Spectroscopy (FTIR) analysis gives an idea about functional group present in the microspheres. X-ray diffraction (XRD) provides information about the crystallinity of the samples respectively. The drug release characteristics were determined by UV-Vis spectrophotometric analysis. Moreover, the in-vitro cell-based cytotoxicity assay indicated almost insignificant cytotoxicity of the NCI–H522 cell line (Human, Lung, Non-small cell lung cancer).

The Antioxidant and Anti-inflammatory Activities of Echinacea angustifolia Hot Water Extract

Purpose: This study attempted to investigate the antioxidant and anti-inflammatory effects of <i>Echinacea angustifolia</i> extract.Methods: For analysis of radical scavenging activities, DPPH, ABTS and FRAP were performed. To analyze antioxidant properties, in addition, polyphenol and flavonoid concentrations were measured. In cell-based assays, cytotoxicity and anti-inflammatory assays were performed, using RAW 264.7.Results: The results found the followings: DPPH and ABTS assays revealed 27.98% and 20.29% respectively in terms of antioxidant activities when compared to ascorbic acid. In the FRAP assay, <i>E. angustifolia</i> extract (1 mg) showed 0.412±0.013 μg of reducing power (ascorbic acid). Polyphenol and flavonoid concentrations were 104.42±3.21 mg/g and 62.86±2.26 mg/g each. In terms of cytotoxicity, more than 80% cells survived after the assays, confirming the low toxicity of <i>E. angustifolia</i> extract. In anti-inflammatory activities, the extract reduced the inflammatory response and, at the same time, showed 51.07±4.58% inflammation-inhibiting effects at 200 μg/mL, confirming the potential of E. angustifolia extract as an ingredient of cosmeceuticals.Conclusion: These results confirmed the potential of <i>E. angustifolia</i> extract as an ingredient of cosmeceuticals.

One of the 5-aminosalicylates drug, mesalamine as a drug repurposing lead against breast cancer

BackgroundBreast cancer is the world's second leading cause of death in women. The problem of chemoresistance in breast cancer is proving to be a challenge for researchers and several oncologists all around the world. Current treatment modalities are associated with severe toxicities and lower efficiency. Hence, there is an unmet need for the development of novel drugs that can be used as adjuvants in breast chemotherapy. One of the strategies used to overcome this problem and that has received scientific coverage over the years is ‘Drug Repurposing’. For this purpose, a list of 5-aminosalicylates drugs were evaluated for their drug repurposing potential in breast cancer. Mesalamine, sulfasalazine, balsalazide, and olsalazine were docked with high expression signatures in cancer cells such as EGFR (epidermal growth factor receptor), ERα (Estrogen Receptor alpha), Aromatase, mTOR (mammalian target of rapamycin), ALOX5 (Arachidonate 5-lipoxygenase), and Topoisomerase II.ResultsDocking analysis revealed that the selected ligands (drug) exhibited good binding affinity for all receptors. Based on the specificity with receptors, mesalamine was further selected for in vitro functional validation in a breast cancer cell line. Cell-based cytotoxicity assay in MCF-7 (Michigan Cancer Foundation-7) cells demonstrated the anticancer potential of mesalamine in breast cancer with IC-50 (Inhibitory Concentration) of 6.358 µM.ConclusionsSignificant morphological alterations were observed in breast cells treated with mesalamine. Further studies are warranted to explore the anticancer effect of mesalamine in breast cancer and its role in combination therapies to be used as an adjuvant in chemotherapy.

Development of Ultrapure and Potent Tannic Acids as a Pan-coronal Antiviral Therapeutic.

The rampageous transmission of SARS-CoV-2 has been devastatingly impacting human life and public health since late 2019. The waves of pandemic events caused by distinct coronaviruses at present and over the past decades have prompted the need to develop broad-spectrum antiviral drugs against them. In this study, our Pentarlandir ultrapure and potent tannic acids (UPPTA) showed activities against two coronaviral strains, SARS-CoV-2 and HCoV-OC43, the earliest-known coronaviruses. The mode of inhibition of Pentarlandir UPPTA is likely to act on 3-chymotrypsin-like protease (3CLpro) to prevent viral replication, as supported by results of biochemical analysis, a 3CLpro assay, and a “gain-of-function” 3CLpro overexpressed cell-based method. Even in the 3CLpro overexpressed environment, Pentarlandir UPPTA remained its antiviral characteristic. Utilizing cell-based virucidal and cytotoxicity assays, the 50% effective concentrations (EC50) and 50% cytotoxicity concentration (CC50) of Pentarlandir UPPTA were determined to be ∼0.5 and 52.5 μM against SARS-CoV-2, while they were 1.3 and 205.9 μM against HCoV-OC43, respectively. In the pharmacokinetic studies, Pentarlandir UPPTA was distributable at a high level to the lung tissue with no accumulation in the body, although the distribution was affected by the food effect. With further investigation in toxicology, Pentarlandir UPPTA demonstrated an overall safe toxicology profile. Taking these findings together, Pentarlandir UPPTA is considered to be a safe and efficacious pancoronal antiviral drug candidate that has been advanced to clinical development.

Discovery of Isatin-Based Carbohydrazones as Potential Dual Inhibitors of Fatty Acid Amide Hydrolase and Monoacylglycerol Lipase.

Using ligand-based design strategy, a set of isatin-3-carbohydrazones was designed, synthesized and evaluated for dual fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) inhibition properties. Compound 5-chloro-N'-(5-chloro-2-oxoindolin-3-ylidene)-2-hydroxybenzohydrazide (13 b) emerged as a potent MAGL inhibitor with nanomolar activity (IC50 =3.33 nM), while compound 5-chloro-N'-(1-(4-fluorobenzyl)-2-oxoindolin-3-ylidene)-2-hydroxybenzohydrazide (13 j) was the most potent selective FAAH inhibitor (IC50 =37 nM). Compound 5-chloro-N'-(6-chloro-2-oxoindolin-3-ylidene)-2-hydroxybenzohydrazide (13 c) showed dual FAAH-MAGL inhibitory activity with an IC50 of 31 and 29 nM respectively. Enzyme kinetics studies revealed that the isatin-based carbohydrazones are reversible inhibitors for both FAAH and MAGL. Further, blood-brain permeability assay confirmed that the lead compounds (13 b, 13 c, 13 g, 13 m and 13 q) are suitable as CNS candidates. Molecular dynamics simulation studies revealed the putative binding modes and key interactions of lead inhibitors within the enzyme active sites. The lead dual FAAH-MAGL inhibitor 13 c showed significant antioxidant activity and neuroprotection in the cell-based cytotoxicity assay. In summary, the study yielded three potent FAAH/MAGL inhibitor compounds (13 b, 13 c and 13 j) with acceptable pharmacokinetic profile and thus can be considered as promising candidates for treating neurological and mood disorders.

Abstract 1373: Primary or iPSC-derived cell-based cytotoxicity assays to assess potential safety risks of engineered T cell therapies in vitro

Abstract Engineered T cell therapies, such as Chimeric Antigen Receptor (CAR) and T Cell Receptor (TCR) T cells, but also bi-specific antibody therapies, play a major role in the field of immuno-oncology and offer great promise in becoming a new wave of highly specific therapies against solid tumors. Solid tumors generally lack the expression of tumor specific target antigens, posing a significant safety challenge. Selected solid tumor target antigens are often expressed at low levels in healthy tissues throughout the human body, risking the activation of the engineered T cells against these tissues. This can potentially result in life-threatening side effects, as such, assessing the safety of engineered T cell therapies is a critical step during early development and before filing for Investigational New Drug (IND) status. We have generated an in vitro safety profile for CAR-T cells targeting the ‘Human Epidermal growth factor Receptor 2' (HER2). The gene encoding this receptor is found to be amplified and/or overexpressed in 20-30% of invasive breast carcinomas and ovarian cancers. To determine which tissues are most at risk for unwanted reactivity by the CAR-T cells, in silico analysis for expression of the HER2 gene was performed. Subsequently, HER2 protein expression in various tissues was validated by staining cells with a HER2 antibody and determining the percentage of positive cells by flow cytometry. Primary tissues and iPSC-derived cell with high and low HER2 protein expression were selected, characterized and utilized for in vitro co-culture assays to evaluate on-target off-tumor and/or off-target cytotoxicity of the HER2 targeting CAR-T cells. Readouts for the in vitro cytotoxicity assays included measuring target cell viability by flow cytometry and/or HCA and T cell activation by cytokine release. Our study generated high quality data that provided insight into the safety of the HER2 targeting CAR-T cells. Moreover, we were able to demonstrate the value of using iPSC-derived cells in de-risking selected tissues against unwanted reactivity of engineered T cell therapies. Our strategy to generate a safety profile for T cell therapies and bi-specific antibodies is robust can be applied during both early stage development and late stage testing of the therapeutic product. Engineered T cell therapies have the capacity to fill tremendous unmet medical needs and are moving into the clinic at a high rate. This expedited timetable, although good news for patients with late stage disease, results in less time to conduct the rigorous safety tests required before FDA approval. The use of primary tissues and iPSC derived cells to test the safety of these therapies in vitro has been and will be essential in getting these novel drugs on the market. Citation Format: Sanne L. Holt, Sophie Vermond, Monique Hazenoot, Rene McLaughlin, Marco Guadagnoli, Marijn Vlaming. Primary or iPSC-derived cell-based cytotoxicity assays to assess potential safety risks of engineered T cell therapies in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1373.

Bioluminescence Measurement of Time-Dependent Dynamic Changes of CYP-Mediated Cytotoxicity in CYP-Expressing Luminescent HepG2 Cells.

We sought to develop a cell-based cytotoxicity assay using human hepatocytes, which reflect the effects of drug-metabolizing enzymes on cytotoxicity. In this study, we generated luminescent human hepatoblastoma HepG2 cells using the mouse artificial chromosome vector, in which click beetle luciferase alone or luciferase and major drug-metabolizing enzymes (CYP2C9, CYP2C19, CYP2D6, and CYP3A4) are expressed, and monitored the time-dependent changes of CYP-mediated cytotoxicity expression by bioluminescence measurement. Real-time bioluminescence measurement revealed that compared with CYP-non-expressing cells, the luminescence intensity of CYP-expressing cells rapidly decreased when the cells were treated with low concentrations of aflatoxin B1 or primaquine, which exhibits cytotoxicity in the presence of CYP3A4 or CYP2D6, respectively. Using kinetics data obtained by the real-time bioluminescence measurement, we estimated the time-dependent changes of 50% inhibitory concentration (IC50) values in the aflatoxin B1- and primaquine-treated cell lines. The first IC50 value was detected much earlier and at a lower concentration in primaquine-treated CYP-expressing HepG2 cells than in primaquine-treated CYP-non-expressing cells, and the decrease of IC50 values was much faster in the former than the latter. Thus, we successfully monitored time- and concentration-dependent dynamic changes of CYP-mediated cytotoxicity expression in CYP-expressing luminescent HepG2 cells by means of real-time bioluminescence measurement.

Impact of Drug Conjugation and Loading on Target Antigen Binding and Cytotoxicity in Cysteine Antibody-Drug Conjugates.

Antibody-drug conjugates (ADCs) consist of a target-specific antibody that is covalently conjugated to a drug via a linker. ADCs are designed to deliver cytotoxic drugs (payloads), specifically to cancer cells, while minimizing systemic toxicity. Conventional cysteine conjugation typically results in the formation of ADC molecules containing a heterogeneous mixture of 2, 4, 6, and 8 drug-loaded species. The drug-to-antibody ratio (DAR) of the mixture represents the weighted average of these species. In this report, we have investigated the impact of the hydrophobicity of payloads and the overall drug loading on the in vitro binding and cytotoxicity of ADC species. Several ADCs were prepared by conventional cysteine conjugation using different payloads. ADC species with different DAR values were purified from the ADC mixture and characterized by standard analytical techniques. These ADC species were evaluated for target antigen binding using an immunoassay, enzyme-linked immunosorbent assay (ELISA). The potency was assessed using a cell-based cytotoxicity assay. These structure-function studies lead to a better understanding of factors that impact the in vitro target binding and cytotoxicity of ADC species. ADC species containing hydrophobic payloads with high DAR were found to have lower target binding by ELISA compared to that of the unconjugated antibody or the heterogeneous reference ADC with DAR ∼4. Under similar assay conditions, the ADCs conjugated to hydrophilic payloads did not show a significant impact on the target binding. The cytotoxic potency of ADC species increased with increasing level of drug loading in the cell-based cytotoxicity assay.

HIV-1 Env-Dependent Cell Killing by Bifunctional Small-Molecule/Peptide Conjugates.

A strategy has been established for the synthesis of a family of bifunctional HIV-1 inhibitor covalent conjugates with the potential to bind simultaneously to both the gp120 and gp41 subunits of the HIV-1 envelope glycoprotein trimeric complex (Env). One component of the conjugates is derived from BNM-III-170, a small-molecule CD4 mimic that binds to gp120. The second component, comprised of the peptide DKWASLWNW ("Trp3"), was derived from the N-terminus of the HIV-1 gp41 Membrane Proximal External Region (MPER) and found previously to bind to the gp41 subunit of Env. The resulting bifunctional conjugates were shown to inhibit virus cell infection with low micromolar potency and to induce lysis of the HIV-1 virion. Crucially, virolysis was found to be dependent on the covalent linkage of the BNM-III-170 and Trp3 domains, as coadministration of a mixture of the un-cross-linked components proved to be nonlytic. However, a significant magnitude of lytic activity was observed in Env-negative and other control pseudoviruses, suggesting parallel mechanisms of action of the conjugates involving Env interaction and direct membrane disruption. Computational modeling suggested strong membrane-binding activity of BNM-III-170, which may underly the nonspecific virolytic effects of the conjugates. To investigate the scope of the membrane effect, cell-based cytotoxicity and membrane permeability assays were performed employing flow cytometry. Here, we observed a dose-dependent and specific cytotoxic effect on HIV-1 Env-expressing cells by the small-molecule bifunctional inhibitor. Most importantly, Env-negative cells were not susceptible to the cytotoxic effect upon exposure to this construct at concentrations where cell-killing effects were observed for Env-positive cells. Computational structural modeling supports a mechanism in which the bifunctional inhibitors bind to the gp120 and gp41 subunits in tandem in open-state Env trimers and induce relative motion of the gp120 subunits consistent with models of Env inactivation. This observation supports the idea that the cell-killing effect of the small-molecule bifunctional inhibitor is due to specific Env conformational triggering. This work lays important groundwork to advance a small-molecule bifunctional inhibitor approach for eliminating Env-expressing infected cells and the eradication of HIV-1.

Comparative analytical profiling of bevacizumab biosimilars marketed in India: a national control laboratory study

In this study, analytical profiling of the bevacizumab (BVZ) biosimilars (N = 3) approved in India were evaluated for charge heterogeneity, isoelectric focusing, aggregation and in vitro potency analysis. The charge variants were characterized using high performance cation-exchange chromatography (CEX-HPLC), capillary zone electrophoresis (CZE) and capillary isoelectric focusing (cIEF). cIEF was also used for estimation of isoelectric point (pI value). In addition, aggregate analysis was done using size exclusion high performance chromatography (SEC-HPLC). The cell-based inhibition of proliferation assay using HUVEC cells, indirect ELISA and Western blot were performed for in vitro biological activity. In addition of cell-based cytotoxicity assay was also performed and found no cytotoxic effect on both HuT78 and WIL2S cells by bevacizumab biosimilars. The significant variations in acidic (p < 0.0001) and basic variants (p < 0.0001), pI value (p = 0.0035), aggregates (p = 0.0306) of biosimilars were found as compared to innovator product; however, cell-based potency analysis (p = 0.6047) and indirect ELISA (p = 0.1611) have shown no significant difference in the biological activity. The banding patterns of all biosimilars in western blot were found similar to the innovator product. The comparatively higher basic variants in the biosimilars were attributing to the high pI value of biosimilars to that of innovator product, although these variations were not affecting the biological activity of the biosimiars. This is a unique study, wherein the independent analysis by a National Control Laboratory (NCL) will not only help the National Regulatory Authority (NRA) to assess the quality and consistency in manufacturing of BVZ biosimilars marketed in India but also facilitate the uptake of BVZ biosimilars, and sustainable access to new medicines against the anti-angiogenic therapy.

In silico and in vitro screening for potential anticancer candidates targeting GPR120

The G-protein coupled receptor - GPR120 has recently been implicated as a novel target for colorectal cancer (CRC) and other cancer managements. In this study, a homology model of GPR120S (short isoform) was generated to identify potential anti-cancer compounds targeting the GPR120 receptor using a combined in silico docking-based virtual screening (DBVS), structure–activity relationships (SAR) and in vitro screening approach. SPECS database of synthetic chemical compounds (~350,000) was screened using the developed GPR120S model to identify molecules binding to the orthosteric binding pocket followed by an AutoDock SMINA rigid-flexible docking protocol.The best 13 hit molecules were then tested in vitro to evaluate their cytotoxic activity against SW480 – human CRC cell line expressing GPR120. The test compound 1 (3-​(4-​methylphenyl)​-​2-​[(2-​oxo-​2-​phenylethyl)​sulfanyl]​-​5,6-​dihydrospiro(benzo[h]​quinazoline-​5,1′-​cyclopentane)​-​4(3H)​-​one) showed ~ 90% inhibitory effects on cell growth with micromolar affinities (IC50 = 23.21–26.69 µM). Finally, SAR analysis of compound 1 led to the identification of a more active compound from the SPECS database showing better efficacy during cell-based cytotoxicity assay –5 (IC50 = 5.89–6.715 µM), while a significant reduction in cytotoxic effects of 5 was observed in GPR120-siRNA pre-treated SW480 cells.The GPR120S homology model generated, and SAR analysis conducted by this work discovered a potential chemical scaffold, dihydrospiro(benzo[h]quinazoline-5,1′-cyclopentane)-4(3H)-one, which will aid future research on anti-cancer drug development for CRC management.

DNA polymerase theta repression enhances the docetaxel responsiveness in metastatic castration-resistant prostate cancer

ObjectiveDocetaxel remains a main treatment for metastatic castration-resistant prostate cancer (mCRPC); however, the development of docetaxel resistance has been found in some mCRPC patients. The aim of this work is to identify an effective biomarker for predicting therapeutic effectiveness of docetaxel in mCRPC patients. MethodsWe examined DNA polymerase theta (POLQ) expression in The Cancer Genome Atlas (TCGA) database and Tissue microarray. Kaplan-Meier analyses were performed to estimate the prognostic significance of POLQ. A series of functional analyses were conducted in cell lines and xenograft models. Regulated pathways were predicted by Geneset Enrichment Analysis (GSEA) software and further investigated by luciferase reporter and RT-PCR assays. ResultsWe found that POLQ mRNA levels in CRPC tissues was significantly higher than that of other DNA polymerases in non-CRPC prostate tissues. POLQ upregulation was extensively detected in mCRPC and strongly predicted a poor prognosis. POLQ knockdown enhanced docetaxel sensitivity in a cell-based cytotoxicity assay and promoted the therapeutic effect on the tumor growth of metastatic PC-3M cells in xenograft models. The computational simulation by GSEA software significantly predicted the association between POLQ upregulation and the activation of E2F/G2M checkpoint-related pathways. Moreover, luciferase reporter and RT-PCR assays demonstrated that POLQ knockdown downregulated the transcriptional regulatory activity of E2F and repressed E2F/G2M checkpoint-regulated CDK1 in mCRPC cells. ConclusionOur results suggest that POLQ serves as a predictive factor for poor docetaxel response and provide a novel strategy to enhance the anticancer effects of docetaxel therapy by targeting POLQ in mCRPC patients.

Abstract 2209: Rapidly switchable universal CAR-T cells with improved safety profile allow for active targeting of PD-L1 expressing solid tumors

Abstract Despite remarkable therapeutic success using chimeric antigen receptor modified T-cells (CAR-T) in hematologic malignancies, targeting solid tumors still remains challenging, given their heterogeneity and immunosuppressive milieu, including the expression of inhibitory immune checkpoints such as PD-1/PD-L1. Preclinical models demonstrate that combining CAR-T therapy with checkpoint inhibitors targeting the PD-1/PD-L1 axis could be a promising strategy to enhance anti-cancer activity of CAR-T in solid tumors and on-going phase I trials are clinically evaluating this strategy. However, primary and acquired resistance to checkpoint blockade is frequently observed limiting its broad applicability and efficacy. In addition, lack of specificity results in frequent occurrence of immune-related adverse events affecting several organs. Alternatively, expression of PD-L1 by solid tumor cells could be an attractive target for CAR-T therapy, especially in order to prevent or reverse tumor re-growth and relapse after successful initial tumor cell lysis. The development of a rapidly switchable universal CAR-T platform (UniCAR) with enhanced safety features allowed us to explore the possibility to directly attack PD-L1 expressing solid tumor cells. An inherent key feature of the UniCAR-T platform is a rapid and reversible turn off mechanism (less than 4 hours) determined by the short pharmacokinetic half-life of soluble targeting modules (TMs) and fast internalization of cell-bound TMs. TMs provide the antigen-specificity for UniCAR gene-modified T-cells (UniCAR-T) and consist of an antigen-binding moiety, e.g. an scFv, linked to a small peptide motif recognized by the UniCAR. For redirecting UniCAR-T against the PD-1/PD-L1 axis, a PD-L1 specific TM (TM-PD-L1) was developed and its functionality confirmed in binding assays to human and murine PD-L1 using surface plasmon resonance as well as in cell-based cytotoxicity assays. Half maximal killing efficacy (EC50) mediated by TM-PD-L1 redirected UniCAR-T was in the low picomolar range. As PD-L1 is also expressed on healthy tissue, toxicity studies were performed in a humanized mouse model with human UniCAR-T. Humanized mice treated with TM-PD-L1 twice a day for ten consecutive days did not reveal any signs of toxicity. In a prostate cancer (PCa) xenograft model efficient suppression of tumor growth could be demonstrated by administration of TM-PD-L1. Our data are the first to demonstrate safety and feasibility of active targeting of PD-L1 expressed by solid tumors with CAR-T and that this approach is capable of inducing a significant anti-tumor response. Citation Format: Josephine Dietrich, Simon Loff, Johannes Spehr, Julia Riewaldt, Cordula Gründer, Maria Schreiber, Michael Bachmann, Gerhard Ehninger, Michael Pehl, Marc Cartellieri, Armin Ehninger. Rapidly switchable universal CAR-T cells with improved safety profile allow for active targeting of PD-L1 expressing solid tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2209.

Abstract 5658: A homogenous PBMC ADCC bioassay enables bridging studies with ADCC reporter bioassays in immunotherapy monoclonal antibody development

Abstract Measurement of antibody-dependent cellular cytotoxicity (ADCC) is critical for characterizing antibody Fc effector functions during monoclonal antibody development. Classic ADCC assays measure the short-term cytotoxicity of target cells, typically pre-loaded with radioactive or fluorescent dyes, after exposure to antibody bound primary PBMCs or NK cells. These assays are widely used in antibody discovery and characterization during early drug development. However, these assays are relatively low throughput, present a biosafety risk due to radioactivity, and are prone to donor variability and weak assay signal. Therefore, these assays are not well-suited for implementation in quality-controlled drug development environments. Previously, we developed a mechanism of action (MOA)-based ADCC reporter bioassay using genetically engineered Jurkat effector cells. When co-cultured with antigen-expressing target cells and a relevant antigen-specific antibody, engagement of FcγRIIIa expressed on the effector cells leads to NFAT-mediated expression of luciferase and a bioluminescent signal. The effector cells are optimized for use in thaw-and-use format and can accurately measure antibody potency for a wide range of therapeutic antibodies targeted to hematopoietic or solid tumors. The bioassay can distinguish effector functions for different antibody IgG isotypes, is stability indicating, and prequalified according to ICH guidelines (precision, accuracy, linearity, and robustness). The ADCC reporter bioassay can be developed as a QC lot release assay for antibody therapeutics that function via an ADCC MOA. However, prior to implementation, a bridging study with a primary cell-based cytotoxicity assay must be performed. To enable this analysis, we developed a PBMC-based ADCC assay that overcomes limitations of traditional primary cell ADCC assays. First, we established a workflow to prequalify donor PBMCs for ADCC activity, significantly reducing the level of variability introduced with random donor PBMCs. We then developed a platform to genetically engineer target cells with a cytosolic protein (HaloTag) fused to HiBiT, an 11 amino acid protein tag. When cultured with a relevant antibody and ADCC-qualified PBMCs, the target cells are killed and HaloTag-HiBiT is released into the extracellular medium containing the protein LgBiT. Binding of HiBit and LgBiT reconstitutes a luciferase enzyme and produces a bioluminescent signal. The use of HaloTag as a binding partner offers the additional opportunity to image the target cells using fluorescent HaloTag ligands. This improved PBMC ADCC assay is simple, homogenous, highly sensitive, and yields a robust assay window. Importantly, we have demonstrated that this assay accurately measures the potency of a range of antibody therapeutics including rituximab, cetuximab, and trastuzumab, and correlates with the surrogate ADCC reporter bioassay. Citation Format: Pete Stecha, Denise Garvin, Julia Gilden, Jun Wang, Jamison Grailer, Jim Hartnett, Vanessa Ott, Frank Fan, Mei Cong, Zhijie Jey Cheng. A homogenous PBMC ADCC bioassay enables bridging studies with ADCC reporter bioassays in immunotherapy monoclonal antibody development [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5658.

Design, synthesis, biological evaluation of 6-(2-amino-1H-benzo[d]imidazole-6-yl)quinazolin-4(3H)-one derivatives as novel anticancer agents with Aurora kinase inhibition

Aurora A kinase, a member of the Aurora kinase family, is frequently overexpressed in various human cancers. In addition, Overexpression of Aurora A kinase is associated with drug resistance and poor prognosis in many cancers including breast cancer. Therefore, Aurora A kinase has been considered as an attractive anticancer target for the treatment of human cancers. Herein, A series of 6-(2-amino-1H-benzo[d]imidazole-6-yl)quinazolin-4(3H)-one derivatives were designed, synthesized, and evaluated as Aurora A kinase inhibitors. The cell-based cytotoxicity assays showed that compound 16h was the most potent cytotoxic agent against all tested cancer cells and had a lower IC50 value than ENMD-2076 against MDA-MB-231 cells. Meanwhile, Aurora A kinase assay and Western blot analysis showed that 16h inhibited Aurora A kinase with an IC50 value of 21.94 nM and suppressed the phosphorylation of Histone H3 on Ser10 and Aurora A kinase on Thr288, which were consistent with the activation of Aurora A kinase. Accordingly, 16h caused aberrant mitotic phenotypes and obvious G2/M phase arrest in MDA-MB-231 cells and induced caspase-dependent apoptosis in MDA-MB-231 cells. These results demonstrated that 16h is a potential candidate for the development of anticancer agents targeting Aurora A kinase.

Polyhydroxylated sterols from Monascus purpureus-fermented rice

Using a cell-based cytotoxicity assay, two new polyhydroxylated sterols, 16(S),22(S)-epoxy-3β,5α,6β,20(R),23(R),25-hexahydroxy-7-ergostene and 3β,7β,8α,25-tetrahydroxy-5,22E-ergostadiene were isolated from the ethyl acetate portion of the ethanolic extract of Monascus purpureus. Their structures were elucidated by spectroscopic analysis and in comparison with those reported in the literature. Both compounds showed cytotoxic activity against the lung adenocarcinoma (A549) with IC50 values of 12.6 and 18.5 μM, exhibited moderate activities against human ovarian cancer (A2780), with IC50 values of 8.8 and 9.4 μM.