HUT-78 Cells Research Articles

The HIV-1 vpr R77Q Mutant Induces Apoptosis, G2 Cell Cycle Arrest, and Lower Production of Pro-Inflammatory Cytokines in Human CD4+ T Cells.

Acquired immunodeficiency syndrome (AIDS) occurs when HIV depletes CD4+ helper T cells. Some patients develop AIDS slowly or not at all, and are termed long-term non-progressors (LTNP), and while mutations in the HIV-1 Viral Protein R (vpr) gene such as R77Q are associated with LTNP, mechanisms for this correlation are unclear. This study examines the induction of apoptosis, cell cycle arrest, and pro-inflammatory cytokine release in the HUT78 T cell line following infection with replication-competent wild-type strain NL4-3, the R77Q mutant, or a vpr Null mutant. Our results show a significant enhancement of apoptosis and G2 cell cycle arrest in HUT78 cells infected with R77Q, but not with WT NL4-3 or the vpr Null strain. Conversely, HUT78 cells infected with the WT virus show higher levels of necrosis. We also detected lower TNF and IL-6 release after infection with R77Q vs. WT. The apoptotic phenotype was also seen in the CEM cell line and in primary CD4+ T cells. Protein expression of the R77Q vpr variant was low compared to WT vpr, but expression levels alone cannot explain these phenotypes because the Null virus did not show apoptosis or G2 arrest. These results suggest that R77Q triggers a non-inflammatory apoptotic pathway that attenuates inflammation, possibly contributing to LTNP.

Enhanced anti-cancer potency of sustainably synthesized anisotropic silver nanoparticles as compared with L-asparaginase

Acute lymphoblastic leukemia (ALL), a hematologic cancer that involves the production of abnormal lymphoid precursor cells, primarily affects children aged 2 to 10 years. The bacterial enzyme L-asparaginase produced from Escherichia coli is utilised as first-line therapy, despite the fact that 30 % of patients have a treatment-limiting hypersensitivity reaction. The current study elucidates the biosynthesis of extremely stable, water-dispersible, anisotropic silver nanoparticles (ANI Ag NPs) at room temperature and investigation of its anti-tumor potency in comparison to L-asparaginase. The optical, morphological, compositional, and structural properties of synthesized nanoparticles were evaluated using UV–Vis-NIR spectroscopy, Transmission Electron Microscopy (TEM), Fourier Transform Infrared Spectroscopy, and X-ray Diffractometer. The UV–Vis-NIR spectra revealed the typical Surface Plasmon Resonance (SPR) at 423 nm along with additional NIR absorption at 962 nm and 1153 nm, while TEM images show different shapes and sizes of Ag nanoparticles ranging from 6.81 nm to 46 nm, together confirming their anisotropic nature. Further, the MTT assay demonstrated promising anticancer effects of ANI Ag NPs with an IC50 value of ∼7 μg/mL against HuT-78 cells. These sustainable anisotropic silver nanoparticles exhibited approximately four times better cytotoxic ability (at and above 10 μg/mL concentrations) than L-asparaginase against HuT-78 cells (a human T lymphoma cell line). Apoptosis analysis by Wright-Geimsa, Annexin-V, and DAPI staining indicated the role of apoptosis in ANI Ag NPs-mediated cell death. The measurement of NO, and Bcl2 and cleaved caspase-3 levels by colorimetric method and immunoblotting, respectively suggested their involvement in ANI Ag NPs-elicited apoptosis. The findings indicate that the biogenic approach proposed herein holds tremendous promise for the rapid and straightforward design of novel multifunctional nanoparticles for the treatment of T cell malignancies.

Inter-alpha-trypsin inhibitor (IαI) and hyaluronan modifications enhance the innate immune response to influenza virus in the lung

The inter-alpha-trypsin inhibitor (IαI) complex is composed of the bikunin core protein with a single chondroitin sulfate (CS) attached and one or two heavy chains (HCs) covalently linked to the CS chain. The HCs from IαI can be transferred to hyaluronan (HA) through a TNFα-stimulated gene-6 (TSG-6) dependent process to form an HC•HA matrix. Previous studies reported increased IαI, HA, and HC•HA complexes in mouse bronchoalveolar lavage fluid (BALF) post-influenza infection. However, the expression and incorporation of HCs into the HA matrix of the lungs during the clinical course of influenza A virus (IAV) infection and the biological significance of the HC•HA matrix are poorly understood. The present study aimed to better understand the composition of HC•HA matrices in mice infected with IAV and how these matrices regulate the host pulmonary immune response. In IAV infected mice bikunin, HC1–3, TSG-6, and HAS1–3 all show increased gene expression at various times during a 12-day clinical course. The increased accumulation of IαI and HA was confirmed in the lungs of infected mice using immunohistochemistry and quantitative digital pathology. Western blots confirmed increases in the IαI components in BALF and lung tissue at 6 days post-infection (dpi). Interestingly, HCs and bikunin recovered from BALF and plasma from mice 6 dpi with IAV, displayed differences in the HC composition by Western blot analysis and differences in bikunin's CS chain sulfation patterns by mass spectrometry analysis. This strongly suggests that the IαI components were synthesized in the lungs rather than translocated from the vascular compartment. HA was significantly increased in BALF at 6 dpi, and the HA recovered in BALF and lung tissues were modified with HCs indicating the presence of an HC•HA matrix. In vitro experiments using polyinosinic-polycytidylic acid (poly(I:C)) treated mouse lung fibroblasts (MLF) showed that modification of HA with HCs increased cell-associated HA, and that this increase was due to the retention of HA in the MLF glycocalyx. In vitro studies of leukocyte adhesion showed differential binding of lymphoid (Hut78), monocyte (U937), and neutrophil (dHL60) cell lines to HA and HC•HA matrices. Hut78 cells adhered to immobilized HA in a size and concentration-dependent manner. In contrast, the binding of dHL60 and U937 cells depended on generating a HC•HA matrix by MLF. Our in vivo findings, using multiple bronchoalveolar lavages, correlated with our in vitro findings in that lymphoid cells bound more tightly to the HA-glycocalyx in the lungs of influenza-infected mice than neutrophils and mononuclear phagocytes (MNPs). The neutrophils and MNPs were associated with a HC•HA matrix and were more readily lavaged from the lungs. In conclusion, this work shows increased IαI and HA accumulation and the formation of a HC•HA matrix in mouse lungs post-IAV infection. The formation of HA and HC•HA matrices could potentially create specific microenvironments in the lungs for immune cell recruitment and activation during IAV infection.

Blockade of phosphodiesterase 5 by sildenafil reduces tumor growth and potentiates tumor-killing ability of cisplatin in vivo against T cell lymphoma: Implication of modulated apoptosis, reactive oxygen species homeostasis, glucose metabolism, and pH regulation.

In the past years, PDE5 has emerged as a promising therapeutic target for many cancers due to its highly upregulated expression. Interestingly, a recent in vitro study by our group has shown the antitumor and chemopotentiating action of sildenafil against T cell lymphoma. Our study showed that lower doses of sildenafil (50 μM) and cisplatin (0.5 μg/mL) exhibited 4% and 23% cytotoxicity against HuT78 cells, respectively, which was dramatically increased up to 50% when treated with both. Hence, the present study was designed to evaluate the antitumor and chemo-potentiating action of sildenafil in a murine model of T cell lymphoma (popularly called as Dalton's lymphoma [DL]). In the present study, DL-bearing mice were administered with vehicle (PBS), sildenafil (5 mg/kg bw), cisplatin (5 mg/kg bw), and sildenafil and cisplatin followed by evaluation of their impact on tumor growth by analyzing various parameters. The apoptosis was assessed by Wright-Giemsa, annexin-V, and DAPI staining. Reactive oxygen species (ROS) level was examined through DCFDA staining. The expression of genes and proteins were estimated by RT-PCR and Western blotting, respectively. The experimental findings of the study demonstrate for the first time that sildenafil inhibits tumor growth and potentiates tumor inhibitory ability of cisplatin by altering apoptosis, glycolysis, ROS homeostasis, and pH regulation in T cell lymphoma-carrying host. In addition, our investigation also showed amelioration of tumor-induced liver and kidney damage by sildenafil. Overall, the experimental data of our study strongly advocate the use and repurposing of SDF in designing promising chemotherapeutic regimens against malignancies of T cells.

Abstract B131: NXC03, an AI-designed, affinity-attenuated IL-21 mutein with half-life extension enhances antitumor immunity

Abstract Background:IL-21 is a pleotropic cytokine with actions including functional programming of CD8+ T cells towards a central memory phenotype with enhanced activation, expansion and survival. Indeed, recombinant IL-21 exhibited antitumor activity in preclinical and clinical studies, but its clinical development has been hindered due to short serum half-life and high dose-limiting toxicities such as hepatotoxicity and neutropenia. We hypothesized that an IL-21 mutein with weaker binding to IL-21 receptor (IL-21R) and increased stability and half-life may achieve an optimal therapeutic index for development. Here we describe the discovery and preclinical profile of such an AI-designed IL-21-Fc fusion mutein, NXC03. Methods:We computationally evaluated ~400,000 combinatorial mutations of IL-21 to look for those with desired affinity and stability profile via multiple techniques, including direct coupling analysis and physical energy functions. Following iterative in silico and in vitro screening, we selected NXC03-9804 out of the top-20 hits and fashioned it as an IgG4 Fc fusion protein (NXC03). We measured the affinity of NXC03 to human IL-21R by biolayer interferometry and its melting temperature (Tm) by label-free nano differential scanning fluorimetry. We determined the bioactivity of NXC03 by assessing human primary T-cell proliferation and STAT3 phosphorylation (pSTAT3) in huT78 cells. We evaluated the antitumor efficacy of NXC03 alone or in combination with a murine PD-1 mAb (aPD1) in MC38 and anti-PD-1 resistant B16-F10 syngeneic tumor models in hIL21R knock-in mice. We also studied the safety profiles and pharmacokinetics of NXC03 following subcutaneous (SC) administrations in monkeys. Results:NXC03 differs from wildtype (WT) IL-21 by 4 amino acid residues and displays a unique fast-on fast-off binding kinetics to IL-21R with a 1000-fold larger KD than that of WT IL-21. It has a Tm of 62.0 °C, notably higher than that of WT IL-21 (48.5 °C). Despite a ~10-fold reduction in potency in inducing pSTAT3 in huT78 cells, NXC03 was comparable to WT IL-21 in enriching CCR7+CD45RO-CD8+ stem memory T cells (TSCM) in PBMC culture. In both MC38 and B16-F10 mouse models, NXC03 monotherapy demonstrated strong, dose-dependent antitumor activity and was more potent than WT IL-21. Combination treatment of NXC03 with aPD1 further increased efficacy compared to either alone. Fusion of NXC03 and aPD1 had similar effect. Moreover, NXC03 treatment produced lasting memory response that protected against tumor rechallenge following drug cessation. NXC03 exhibited a half-life in monkeys (14.1 h, IV; 55.5h, SC) much greater than that of WT IL-21 (0.8 h, IV). NXC03 was well tolerated with no neutropenia or hepatotoxicity observed in monkeys. Conclusion:NXC03, an AI-designed attenuated IL-21 mutein, exhibits strong antitumor activity and holds promise as an immunostimulatory agent for combination with checkpoint inhibitors. Given its excellent drug developability profile, NXC03 is being fast-tracked for development towards clinical testing. Citation Format: Puwei Yuan, Junli Liu, Fei Zhang, Ying Lei, Yanan Zhu, Songsong Gao, Fan Liu, Taylor B Guo. NXC03, an AI-designed, affinity-attenuated IL-21 mutein with half-life extension enhances antitumor immunity [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B131.

CD84 (SLAMF5) As a Potential Target for Immunomodulation in Cutaneous T-Cell Lymphoma (CTCL)

Background: Cutaneous T-cell lymphoma (CTCL) develops from clonally expanded, skin-homing CD4 + T cells within a background of chronic inflammation. Mycosis fungoides (MF) and Sézary syndrome (SS) are the most common subtypes of CTCLs. Patients with advanced-stage MF/SS experience progressively worsening disease and decreased overall survival (Scarisbrick et al., 2015). Immune cells and immune checkpoints (ICs) are essential factors for tumor growth and the host immune response in CTCL. Tumor-associated macrophages (TAMs [M2-like]) play a key role in CTCL growth, and neoplastic T cells escape immune surveillance via the PD-L1/PD1 axis (Wu et al., 2014). However, the approach of ICs inhibition with monoclonal antibodies (mAbs) in CTCL has been challenged due to modest efficacy and remaining immunosuppression. Hence, novel therapeutic strategies are needed to improve anti-tumor immunity in CTCL. We previously characterized CD84 (SLAMF5) as one of the key players in the tumor microenvironment (TME) by regulating multiple ICs, including PD-L1 and PD1, and modulating myeloid cell fractions and T cells in our preclinical MM models. Our CD84 targeting strategy using anti-CD84 mAb significantly reduced PD1 on effector CD8 + T cells and PD-L1 on the tumor and myeloid cells, decreased tumor burden, and increased the survival of the mice with myeloma (Lewinsky and Gunes et al., 2021). The role of CD84 in ICs regulation and anti-tumor immunity in CTCL remains to be explored. Methods and Results: In this study, we evaluated the role of CD84 in CTCL and, for the first time, reported CD84 as a potential target to reduce immunosuppression through the PD-L1/PD1 axis in CTCL. We found upregulated CD84 CD274 (PD-L1) , and PDCD1 (PD1) gene expressions in CTCL patients (n=5) compared to healthy individuals (n=4). We also noted higher CD84 geneexpression in SS patients (n=8) than in MF patients (n=6). We next evaluated CD84 expression on the formalin-fixed paraffin-embedded (FFPE) tumor sections derived from SS patients' skin lesions. We noted that CD84 is highly expressed in the TME and co-expressed in Ki67 + Sézary cells. We further evaluated the CD84 expression in immune cell clusters in SS patients and healthy individuals using publicly available single-cell RNA sequencing (scRNA-seq) datasets. We noted a profound CD84 expression in CD4 + T cells and CD163 + M2-like macrophages in SS patients. We also noted a strong correlation between CD84 and CD274 (p=0.0086) and CD84 and PDCD1 (p=0.0002) in SS patients. We next assessed the expressions of CD84, PD-L1 on CD163 + M2-like macrophages, and PD1 on T cell subsets in a coculturing setting with human SS cell line, Hut78, to determine the impact of Sézary cells on these cell compartments in the TME. We observed a significant increase in the percentages of CD163 + M2-like macrophages and CD4 + T cells and elevated CD84, PD-L1, and PD1 expressions on these cells following the coculturing of peripheral blood mononuclear cells (PBMCs) derived from healthy donors (HDs) with Hut78 cells (n=3, *P < 0.05, **P < 0.01). While the percentages of CD8 + T cells were slightly reduced, CD84 and PD1 expressions were elevated in these cells following the coculturing with Hut78 cells (n=3, *P < 0.05, ***P < 0.001). Dysregulation of STAT proteins, particularly STAT3, has been shown in MF/SS (Olszewska et al., 2020). To explore whether Sézary cells regulate STAT3 in CD163 + M2-like macrophages, we assessed pSTAT3 levels in CD163 + M2-like macrophages following the coculturing of HDs PBMCs with Hut78 cells. We found that Hut78 cellssignificantly increased intracellular pSTAT3 levels inCD163 + M2-like macrophages (n=3, *P < 0.05). Furthermore, we noted the inhibition of STAT signalingusing a pan-STAT inhibitor decreased PD-L1 and PD1 expression onCD163 + M2-like macrophages and T-cell subsets, respectively. Notably, we found the stimulation of CD84 signaling with an agonist anti-CD84 mAb enhanced pSTAT3 levels in CD163 + M2-like macrophages, suggesting the STAT pathway might regulate PD-L1 expression in immunosuppressive CD163 + M2-like macrophages through CD84 signaling in SS. Conclusion: Our preliminary data from this exploratory study indicate that CD84 may be a potential target to reduce immunosuppression in CTCL. Further studies are needed to explore the functions of CD84 as an immune receptor in CTCL TME and its therapeutic potential in CTCL.

LW-213 induces immunogenic tumor cell death via ER stress mediated by lysosomal TRPML1

Dying tumor cells release biological signals that exhibit antigenicity, activate cytotoxic T lymphocytes, and induce immunogenic cell death (ICD), playing a key role in immune surveillance. We demonstrate that the flavonoid LW-213 activates endoplasmic reticulum stress (ERS) in different tumor cells and that the lysosomal calcium channel TRPML1 mediates the ERS process in human cellular lymphoma Hut-102 cells. Apoptotic tumor cells induced by ERS often possess immunogenicity. Tumor cells treated with LW-213 exhibit damage-associated molecular patterns (DAMPs), including calreticulin translocation to the plasma membrane and extracellular release of ATP and HMGB1. When co-cultured with antigen-presenting cells (APCs), LW-213-treated tumor cells activated APCs. Two groups of C57BL/6J mice were inoculated with Lewis cells: a “vaccine group”, which demonstrated that LW-213-treated tumor cells promote the maturation of dendritic cells and increase CD8+ T cells infiltration in the tumor microenvironment and a “pharmacodynamic group”, treated with a combination of LW-213 and PD1/PD-L1 inhibitor (BMS-1), which reduced tumor growth and significantly prolonged the survival time of mice in the “pharmacodynamic group”. Therefore, LW-213 can be developed as a novel ICD inducer, providing a new concept for antitumor immunotherapy.

Mechanochemical synthesis and antiproliferative activity of novel ferrocene quinoline/quinolone hybrids

The synthesis of novel 1,1′‐disubstituted ferrocene conjugates appended with N‐1 and O‐4 alkylated quinolone and quinoline was reported. The target compounds were synthesized employing a liquid‐assisted grinding (LAG) mechanochemical method in copper‐catalyzed azide‐alkyne cycloaddition (CuAAC) to obtain ferrocene−quinoline/quinolone hybrids (5a–5d, 6a–6d, 7a, 7b, 8a, and 8b) in higher yields and shorter reaction time compared to a conventional method, thus proving superiority of mechanochemistry versus conventional synthesis. Bis‐quinoline and bis‐quinolone ferrocene derivatives were evaluated for their antiproliferative effects on five selected tumor and two non‐tumor cell lines. Bis‐6‐methylquinolone–ferrocene conjugate 8b showed the best antiproliferative effect on T‐cell lymphoma (HuT78) cells (IC50 = 14.8 μM) and no cytotoxicity on both non‐tumor MDCK1 and BJ cells. Results obtained after mitochondrial membrane potential (∆Ψm) measurement using flow cytometry showed that compound 8b caused accumulation of HuT78 cells in subG0/G1 phase, disturbance of mitochondrial membrane potential, and apoptosis. The structure of the quinolone and ferrocene hybrid 8b can be further optimized to obtain candidates with better inhibitory effects on HuT78 cells.

HUVECs affect HuT-78 cell apoptosis and cytokine production via the HIF-1α-PD-L1/PD-1 pathway under hypoxia.

We investigated whether human umbilical vein endothelial cells (HUVECs) under hypoxic conditions can suppress the production of cytokines in Hut-78 cells via the HIF-1α/PD-L1/PD-1 pathway, and the intervention effect of Nivolumab. HUVECs and HuT-78 cells were monocultured or cocultured in a tri-gas incubator with or without Nivolumab pretreatment. Real-time PCR, western blotting, and protein chips were used. Transcriptional regulation of PD-L1 and PD-1 by HIF-1α was analyzed by ChIP-qPCR and luciferase reporter gene assays. Apoptosis was assessed by flow cytometry. In HuT-78 cells, hypoxic monoculture significantly increased the expression of HIF-1α, PD-1, IL-2, IL-4, IL-6, IL-8, IL-10, TNF-α, IFN-α, and Bax, decreased the expression of Bcl-2, and resulted in increased apoptosis. In comparison to hypoxic monoculture, hypoxic coculture significantly reduced the expression of IL-2, IL-4, IL-6, IL-8, IL-10, TNF-α, and IFN-α, as well as Bcl-2, in HuT-78 cells. Meanwhile, Bax expression was significantly increased with elevated apoptosis in HuT-78 cells. However, pretreatment with Nivolumab significantly antagonized the reduction in cytokines and the elevation in apoptosis in HuT-78 cells. Chip-qPCR and luciferase reporter gene assays demonstrated that hypoxia significantly increased the binding of HIF-1α to the upstream regulatory regions of PD-1 at -63 and -66bp and PD-L1 at -571bp, promoting their transcription. Therefore, HUVECs under hypoxia can reduce cytokine production and inhibit their own apoptosis in co-culture with HuT-78 cells via the HIF-1α/PD-L1/PD-1 pathway. These findings provide new clues for exploring the combined use of immune checkpoint inhibitors and anti-angiogenic drugs in clinical settings.

Cost effective and reliable cell based ELISA as an alternative method of flow cytometry for assessment of binding activity of Vedolizumab

Vedolizumab is a humanized monoclonal antibody used for inflammatory bowel disease treatment. Vedolizumab binds to the α4β7 integrin complex and inhibits its binding to mucosal addressin cell adhesion molecule-1 (MAdCAM-1). To evaluate the binding efficacy and quality control check of Vedolizumab, flow cytometry is performed by using HuT78 cells. As we know, flow cytometer is costly and require high equipment maintenance with a designated technical manpower to handle it. In this regard, the aim of study was to develop and validate an economical, simple and efficient cell based ELISA assay for potency estimation of Vedolizumab which has not been reported in any pharmacopoeia. The proposed bioassay method was optimized by investigating Vedolizumab binding to α4β7 integrin which is expressed by HuT78 cells. The validation of this method was done at different parameters including specificity, linearity, range, repeatability, precision, and accuracy. The Vedolizumab binding by ELISA results were found specific for Vedolizumab with linearity (R2 = 0.99) and precision (%Geometric Coefficient of variance) observed for repeatability and intermediate precision were 3.38% and 2.6% respectively. The relative bias was calculated as 8.68% for repeated performances by different analysts and found in accordance with parameter of accuracy as per various pharmacopoeial guidelines. The developed method is established as robust, effective, and less expensive than high maintenance setup like flow cytometry based assay.

Development and validation of a duplex real-time PCR for the rapid detection and quantitation of HTLV-1

BackgroundThe HTLV-1 prevalence in China varies geographically, while HTLV-2 infection has rarely been found so far. Proviral load is one of the determining factors of pathogenesis and progression of HTLV-1 related diseases. However, neither molecular assays nor commercial kits are available for HTLV-1 diagnosis in China. The objective of the present study was to develop and validate a TaqMan qPCR assay for HTLV-1 proviral load quantification.ResultsA plasmid containing both the HTLV-1 of interest and a fragment of the RNase P (RPPH1) gene was constructed and used to establish the standard curves. The assay has a wide dynamic range (2.5 × 108 copies/reaction ~ 25 copies/reaction) and sensitive to 1 copy for HTLV-1 and RPPH1. The limit of detection for Hut102 cell concentration was 0.0218% (95% confidence interval 0.0179–0.0298%). The assay gave coefficient of variation (CV) for both the HTLV-1 and RPPH1 Ct values. All of the HTLV-1 sero-negative samples and MOT cell line (infected with HTLV-2) amplified only the RPPH1 gene by our method, presenting 100% specificity. 85 Samples confirmed positive or indeterminate by LIA were performed by established qPCR assay and WB. 90.0% (27/30) of LIA-HTLV-1-positive, 33% (2/6) of LIA-untypeable and 2% (1/49) of LIA-indeterminate samples were defined as qPCR-positive. The median PVL of LIA-positive samples (n = 27, 1.780 copies/100 cells) was much higher than that of LIA-untypeable and (n = 2, 0.271 copies/100 cells) indeterminate samples (n = 1, 0.017 copies/ 100 cells). Additionally, compared to WB, the duplex qPCR verified more positive samples, demonstrating a better sensitivity.ConclusionThe duplex qPCR developed here with high sensitivity, good specificity and reproducibility could accurately and quantitatively detect the HTLV-1 PVLs, which can be used to confirm the initial reactive samples for an improved cost/benefit ratio as well as to monitor the clinical progression and efficacy of therapy in patients with HTLV-1 related disease.

Novel 7-Chloro-4-aminoquinoline-benzimidazole Hybrids as Inhibitors of Cancer Cells Growth: Synthesis, Antiproliferative Activity, in Silico ADME Predictions, and Docking.

In this study, new 7-chloro-4-aminoquinoline-benzimidazole compounds were synthesized and characterized by NMR, MS, and elemental analysis. These novel hybrids differ in the type of linker and in the substituent on the benzimidazole moiety. Their antiproliferative activities were evaluated on one non-tumor (MDCK1) and seven selected tumor (CaCo-2, MCF-7, CCRF-CEM, Hut78, THP-1, and Raji) cell lines by MTT test and flow cytometry analysis. The compounds with different types of linkers and an unsubstituted benzimidazole ring, 5d, 8d, and 12d, showed strong cytotoxic activity (the GI50 ranged from 0.4 to 8 µM) and effectively suppressed the cell cycle progression in the leukemia and lymphoma cells. After 24 h of treatment, compounds 5d and 12d induced the disruption of the mitochondrial membrane potential as well as apoptosis in HuT78 cells. The drug-like properties and bioavailability of the compounds were calculated using the Swiss ADME web tool, and a molecular docking study was performed on tyrosine-protein kinase c-Src (PDB: 3G6H). Compound 12d showed good solubility and permeability and bound to c-Src with an energy of -119.99 kcal/mol, forming hydrogen bonds with Glu310 and Asp404 in the active site and other residues with van der Waals interactions. The results suggest that compound 12d could be a leading compound in the further design of effective antitumor drugs.

Synthesis, Antiproliferative Evaluation and QSAR Analysis of Novel Halogen- and Amidino-Substituted Benzothiazoles and Benzimidazoles.

Syntheses of 6-halogen-substituted benzothiazoles were performed by condensation of 4-hydroxybenzaldehydes and 2-aminotiophenoles and subsequent O-alkylation with appropriate halides, whereas 6-amidino-substituted benzothiazoles were synthesized by condensation of 5-amidino-2-aminothiophenoles and corresponding benzaldehydes. While most of the compounds from non-substituted and halogen-substituted benzothiazole series showed marginal antiproliferative activity on tested tumor cell lines, amidino benzazoles exhibited stronger inhibitory activity. Generally, imidazolyl benzothiazoles showed pronounced and nonselective activity, with the exception of 36c which had a strong inhibitory effect on HuT78 cells (IC50 = 1.6 µM) without adverse cytotoxicity on normal BJ cells (IC50 >100 µM). Compared to benzothiazoles, benzimidazole structural analogs 45a-45c and 46c containing the 1,2,3-triazole ring exhibited pronounced and selective antiproliferative activity against HuT78 cells with IC50 < 10 µM. Moreover, compounds 45c and 46c containing the methoxy group at the phenoxy unit were not toxic to normal BJ cells. Of all the tested compounds, benzimidazole 45a with the unsubstituted phenoxy central core showed the most pronounced cell growth inhibition on THP1 cells in the nanomolar range (IC50 = 0.8 µM; SI = 70). QSAR models of antiproliferative activity for benzazoles on T-cell lymphoma (HuT78) and non-tumor MDCK-1 cells elucidated the effects of the substituents at position 6 of benzazoles, demonstrating their dependence on the topological and spatial distribution of atomic mass, polarizability, and van der Waals volumes. A notable cell cycle perturbation with higher accumulation of cells in the G2/M phase, and a significant cell increase in subG0/G1 phase were found in HuT78 cells treated with 36c, 42c, 45a-45c and 46c. Apoptotic morphological changes, an externalization of phosphatidylserine, and changes in the mitochondrial membrane potential of treated cells were observed as well.

TCL-318 Evaluation of the Antitumor Activity of Amiloride in Peripheral T-Cell Lymphomas Using In-Vitro Models.

Peripheral T-cell lymphomas (PTCL) have an aggressive clinical behavior and poor response to treatment; therefore, the search for new drugs is required. We have previously demonstrated the antitumor activity of amiloride in multiple myeloma. This prompted us to investigate the possibility of a cytotoxic effect of amiloride in this kind of T-cell lymphoma. To evaluate the antitumor activity of amiloride in different PTCL using in-vitro assays and identify the possible mechanisms involved. Two anaplastic large-cell lymphoma (ALCL) cell lines, KARPAS-299 and SR-786, and two cutaneous T-cell lymphoma cell lines, HH and HuT-78 (Sézary syndrome), were used. Cell viability and apoptosis were studied after exposure to different increasing concentrations of the drug at 24, 48, and 72 hours by colorimetric MTT and annexin V/propidium iodide double-labeling assays, respectively. Amiloride significantly inhibited cell growth in the PTCL cell lines, depending on increasing drug concentration and time. Inhibition was statistically significant (P<0.05) from 0.15 mM for KARPAS-299, SR-786, and HH at 24, 48, and 72 hours and from 0.3 mM for HuT-78 at 48 and 72 hours. KARPAS-299 and SR-786 were the most sensitive cell lines to amiloride exposure, whereas HuT-78 cells were less sensitive, although with the highest concentration of amiloride, its cell viability decreased to levels below 50% and 40% at 48 and 72 hours, respectively. Induction of cell death by apoptosis was detected in all cell lines, with apoptosis levels up to 25% at 24 hours and more than 80% at 48 and 72 hours using intermediate doses of the drug. 1) Amiloride significantly decreased the cell viability of PTCL. 2) ALCL cell lines were the most sensitive to amiloride exposure. 3) The cytotoxic effect induced by amiloride in PTCL was mediated by apoptosis. Ongoing Objectives: To evaluate apoptosis as the possible mechanism of cell death induction through the analysis of caspases, synergism with drugs commonly used in the treatment of PTCL, and changes in gene and protein expression induced by amiloride. Regional Health Management of Castilla y León (GRS 2142/A/2020);); Carlos III Health Institute European Union (PI19/00674); AECC (PROYE20047GUTI).

LB900 Role of p38β in cutaneous T cell lymphoma

Cutaneous T cell lymphoma (CTCL) is an incurable cancer; understanding its underlying molecular mechanisms may unlock a cure. In patient samples of CTCL, we observed a significant increase in gene expression of p38β while that of p38α decreased, compared to normal healthy CD4+ T cells. This prompted us to further dissect the role of p38β in CTCL to inform the application of small molecule inhibitors that specifically target p38β. Current well-developed small molecule p38 inhibitors target both p38α as well as p38β, as they share ∼80% structural similarity. However, multiple clinical trials have shown adverse effects and development of drug resistance when patients with cancer are treated with potent p38 inhibitors alone. Such side effects likely occur because p38α is an essential protein in many cell types; indeed, p38α gene knock-out mice exhibit embryonic lethality. Therefore, any prolonged treatment using p38α inhibition may cause adverse effects. Using Hut78 CTCL cells in which we silenced p38β using lentiviral siRNA, we tested for possible mechanisms of drug resistance that could explain why patients who participated in p38α/β inhibitor clinical trials experience adverse effects. Gene silencing of p38β in Hut78 cells did not decrease cell proliferation; instead, proliferation slightly increased compared to that of WT cells. This aligned with increased IL-17 RA and p38γ which is a driver for cell proliferation in Hut78 cells. Our hypothesis is that p38β-depleted CTCL cells increase survival by elevating the MAPK12-NFAT-IL17 signaling pathway axis, which increases proliferation and propagandas inflammation in the surrounding regions resulting in drug resistance and adverse effects. We used confocal immuno-fluorescence microscopy analyses of p38β-depleted Hut78 cells to reveal a novel molecular mechanism, in which depleting p38β offset cytoskeleton formation in the cytosol. This suggests p38β is important for maintaining the shape or frame of CTCL cells, and may explain why CTCL, a malignant T cell, infiltrate skin, from which novel revenues of drug development may be invented that are complementary to p38β inhibition.

Abstract 5196: CD47 Blockade potentiates immunotherapy of durvalumab against cutaneous T cell lymphoma

Abstract Immune checkpoint (IC) blockade for programmed cell death 1 (PD1) and PD-Ligand 1 (PD-L1) has shown promising and durable therapeutic outcomes in cutaneous T-cell lymphoma (CTCL), but resistance and/or relapses are common. While research focused on improving adaptive immune functions in CTCL, we investigated the role of 1) the “do not eat me signal” (CD47), and PD-L1, as a dual (innate and adaptive IC)-targeting strategy in CTCL. RNA sequencing and flow cytometric analysis showed that CTCL tumor cells and CTCL cell lines (MyLa, Hut78, HH, and H9 cells) overexpressed CD47 and PD-L1 compared with healthy control, and CD47 positively correlated with PD-L1 in CTCL patients. Overexpression of CD47 and PD-L1 was induced by high MYC expression in CTCL tumor cells lines. In addition, the signal-regulatory protein (SIRP)α receptor for CD47 and PD-L1 were significantly more abundant on macrophages, dendritic cells and natural killer cells in CTCL patients than healthy control by flow cytometric analysis. Furthermore, the RNA sequencing data indicated that the M2 macrophages, immature dendritic cells, and inhibitory receptors expressed natural killer cells in CTCL patients were higher than in healthy control. Notably, TTI-621 (SIRPαFc) treatment decreased M2 macrophage, immature dendritic cells, and inhibitory receptors expressed natural killer cells in CTCL patients at the end of the treatment, compared to baseline. In vitro, TTI-621 treatment increased the macrophage phagocytic activity compared to untreated control. Moreover, TTI-621 synergized with an anti-PD-L1 antibody (durvalumab) to reprogram M2-like TAMs, induced by MyLa supernatant, to M1-like phenotypes. The simultaneous blockade of both CD47 and PD-L1 inhibited growth of CTCL cell lines more potently than each single antibody alone or blank control in vitro. Enhanced CD8+ T cell mediated killing was detected using a chromium-release assay following dual blockade of in CTCL cell lines, suggesting anti-CD47 and anti-PD-L1 may synergistically facilitate elimination of CTCL tumor cells through specific pathways. RNA-sequencing analysis indicated that these effects were mediated by cell death related pathways such as apoptosis, autophagy, and necroptosis. Collectively, our findings demonstrated that CD47 and PD-L1 are critical regulators of innate and adaptive immune surveillance in CTCL and that dual targeting of CD47 and PD-L1 will provide insight into tumor immunotherapy to improve tumor control in CTCL. Citation Format: Zhen Han, Mingye Feng, Xiwei Wu, Chingyu Su, Yate-Ching Yuan, Hanjun Qin, James F. Sanchez, Jasmine Zain, Steven T. Rosen, Christiane Querfeld. CD47 Blockade potentiates immunotherapy of durvalumab against cutaneous T cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5196.

BPTF activates the MAPK pathway through coexpression with Raf1 to promote proliferation of T-cell lymphoma.

The aim of the present study was to explore the role and biological function of bromodomain PHD finger transcription factor (BPTF) in T-cell lymphoma. Reverse transcription-quantitative PCR (RT-qPCR), western blotting, immunohistochemistry and bioinformatics analysis were used to determine the expression levels of BPTF and Raf1 in T-cell lymphoma tissues and matched adjacent normal tissues. RT-qPCR and western blot analyses were used to examine the role of BPTF in the activation of MAPK signaling. The function of BPTF and Raf1 in T-cell lymphoma was investigated through in vitro and in vivo assays (MTT assay, colony formation assay, flow cytometry, western blotting, tumor xenograft model and TUNEL assay) following silencing and overexpression experiments in Hut-102 cells. The results demonstrated that BPTF and Raf1 were overexpressed in T-cell lymphoma tissues compared with normal tissues, and high expression of BPTF or Raf1 was associated with advanced clinical stage. BPTF promoted the activation of the MAPK pathway and was coexpressed with Raf1 in T-cell lymphoma tissues. Functional assays demonstrated that silencing of BPTF or Raf1 in Hut-102 cells suppressed cell proliferation and induced apoptosis. Furthermore, the carcinogenic effect of BPTF was confirmed by xenograft experiments in nude mice. The present findings suggested that BPTF may function as a crucial oncogenic factor and may serve as a novel therapeutic target in T-cell lymphoma.

DNA/Protein binding and anticancer activity of Zn(II) complexes based on azo-Schiff base ligands

Synthesis of the azo–Schiff base ligands 2-((E)-((4-(diethylamino)phenyl)imino)methyl)-4-((E)-(2-(phenylthio)phenyl)diazenyl)phenol (L1) and-((E)-((4-(diethylamino)phenyl)-imino)methyl)-4-((E)-(2-phenoxyphenyl)diazenyl)phenol (L2) and Zn(II) complexes (C1-C2) containing these ligands have been described. Characterization of the ligands and complexes has been achieved by spectral studies (IR, 1H, 13C, ESI–MS, UV/vis., emission) and structures of C1 and C2 verified by X-ray single crystal analyses. Absorption titration studies on the complexes revealed that these strongly interact with calf thymus DNA (CT-DNA). Calculated equilibrium binding constants (Kb) for C1 and C2 converged to 4.51 × 104 and 3.74 × 104. Protein binding studies of the complexes with BSA have been performed by UV/vis, fluorescence, synchronous and 3D fluorescence spectroscopy. MTT assay toward Hut-78 cell line suggested high cytotoxicity for C1 (IC50: 70 μM) relative to C2 (IC50: 110 µM). Upon irradiation (UV light; λ, 365 nm) the complexes C1 and C2 exhibited cis–trans photo isomerization and after withdrawal of the light these settled back to more stable trans form. Rate constant (k) for photo isomerization of C1 and C2 has been worked out using electronic absorption data (2.29 × 10−2,C1; 2.12 × 10−2 s−1, C2) and supported by theoretical studies (DFT).

Vedolizumab Antagonizes MAdCAM-1-Dependent Human Placental Cytotrophoblast Adhesion and Invasion In Vitro.

Anti-α4β7 (Vedolizumab) treats inflammatory bowel disease (IBD) by blocking the interaction between integrin α4β7 on leukocytes and mucosal addressin cell-adhesion molecule-1 (MAdCAM-1) on the gut endothelium. Women with IBD often require continuing biologic therapy during pregnancy to avoid disease flare. To date, there have been no reports of an increase in adverse events with Vedolizumab use during pregnancy. Notably, integrins play a major role in human placental development during pregnancy. It is unknown whether Vedolizumab disrupts placental cell (cytotrophoblast) invasion and/or adhesion by blocking interactions with MAdCAM-1. We therefore investigated human placental expression of MAdCAM-1, the role of MAdCAM-1/α4β7 interactions in cytotrophoblast invasion/adhesion in vitro, and whether Vedolizumab administration in vivo alters the placental structure. Histological sections of placentas from normal pregnancies were evaluated for MAdCAM-1 expression by immunofluorescence. The impacts of Vedolizumab or anti-integrin β7 on human cytotrophoblast invasion and adhesion were assessed. Histology results from term placentas of 2 patients with IBD receiving Vedolizumab were compared to those of untreated healthy controls. Placental MAdCAM-1 expression was predominantly associated with invading extravillous cytotrophoblasts at the maternal-fetal interface. Treatment of isolated primary cytotrophoblasts with Vedolizumab or anti-integrin β7 significantly reduced Matrigel invasion, adherence to a MAdCAM-1-coated substrate, and interactions with HuT-78 cells. Placentas from 2 Vedolizumab-treated patients with IBD exhibited pronounced pathologic features as compared to healthy control specimens. This study revealed a previously unrecognized role for α4β7 and MAdCAM-1 in human placentation. More clinical and histological data from Vedolizumab-treated pregnant patients will be necessary to determine whether this medication poses any risk to the mother and fetus.

A novel intramolecular negative regulation of mouse Jak3 activity by tyrosine 820.

Jak3, a member of the Janus kinase family, is essential for the cytokine receptor common gamma chain (γc)-mediated signaling. During activation of Jak3, tyrosine residues are phosphorylated and potentially regulate its kinase activity. We identified a novel tyrosine phosphorylation site within mouse Jak3, Y820, which is conserved in human Jak3, Y824. IL-2-induced tyrosine phosphorylation of Jak3 Y824 in human T cell line HuT78 cells was detected by using a phosphospecific, pY824, antibody. Mutation of mouse Jak3 Y820 to alanine (Y820A) showed increased autophosphorylation of Jak3 and enhanced signal transducer and activator of transcription 5 (STAT5) tyrosine phosphorylation and transcriptional activation. Stably expressed Jak3 Y820A in F7 cells, an IL-2 responsive mouse pro-B cell line Ba/F3, exhibited enhanced IL-2-dependent cell growth. Mechanistic studies demonstrated that interaction between Jak3 and STAT5 increased in Jak3 Y820A compared to wild-type Jak3. These data suggest that Jak3 Y820 plays a role in negative regulation of Jak3-mediated STAT5 signaling cascade upon IL-2-stimulation. We speculate that this occurs through an interaction promoted by the tyrosine phosphorylated Y820 or a conformational change by Y820 mutation with either the STAT directly or with the recruitment of molecules such as phosphatases via a SH2 interaction. Additional studies will focus on these interactions as Jak3 plays a crucial role in disease and health.