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Abstract Inflammasome assembly is a potent mechanism responsible for the host protection against pathogens, including viruses. When compromised, it can allow viral replication, while when disrupted, it can perpetuate pathological responses by IL-1 signaling and pyroptotic cell death. SARS-CoV-2 infection was shown to activate inflammasome in the lungs of COVID-19 patients, however, potential mechanisms responsible for this response are not fully elucidated. In this study, we investigated the effects of ORF3a, E and M SARS-CoV-2 viroporins in the inflammasome activation in major populations of alveolar sentinel cells: macrophages, epithelial and endothelial cells. We demonstrated that each viroporin is capable of activation of the inflammasome in macrophages to trigger pyroptosis-like cell death and IL-1α release from epithelial and endothelial cells. Small molecule NLRP3 inflammasome inhibitors reduced IL-1 release but weakly affected the pyroptosis. Importantly, we discovered that while SARS-CoV-2 could not infect the pulmonary microvascular endothelial cells it induced IL-1α and IL-33 release. Together, these findings highlight the essential role of macrophages as the major inflammasome-activating cell population in the lungs and point to endothelial cell expressed IL-1α as a potential novel component driving the pulmonary immunothromobosis in COVID-19.

Abstract Background Lymphotoxin β receptor (LTβR) plays important roles in the development of the immune system and immune response. At the cellular level, ligand-bound LTβR activates the pro-inflammatory NF-κB pathway but the detailed mechanisms regulating its signaling remain unknown. Understanding them is of high importance since LTβR and its ligands are promising therapeutic targets. Here, we studied the consequences of perturbed cellular cholesterol content on LTβR-induced NF-κB signaling. Methods To modulate cholesterol availability and/or level in lung carcinoma A549 and H2228, and endothelial HUVEC cells different treatment regimens with filipin, methyl-β-cyclodextrin and simvastatin were applied. LTβR localization was studied by confocal microscopy. The activity of LTβR-induced NF-κB pathway was assessed by measuring the levels of NF-κB pathway inhibitor IκBα and phosphorylation of RelA transcription factor by Western blotting. The NF-κB transcriptional response, production of chemokines and adhesion molecules were examined by qRT-PCR, ELISA, and Western blotting, respectively. Adherence of different types of primary immune cells to epithelial A549 cells and endothelial HUVECs was measured fluorometrically. Interactions of LTβR with its protein partners were investigated by immunoprecipitation. Results We showed that filipin-mediated sequestration of cholesterol or its depletion from the plasma membrane with methyl-β-cyclodextrin impaired LTβR internalization and potentiated LTβR-dependent activation of the canonical branch of the NF-κB pathway. The latter was manifested by enhanced degradation of IκBα inhibitor, elevated RelA phosphorylation, substantial increase in the expression of NF-κB target genes encoding, among others, cytokines and adhesion molecules known to play important roles in immune response. It was followed by robust secretion of CXCL8 and upregulation of ICAM1, that favored the adhesion of immune cells (NK and T cells, neutrophils) to A549 cells and HUVECs. Mechanistically, we showed that cholesterol depletion stabilized interactions of ligand-stimulated LTβR with modified forms of TRAF2 and NEMO proteins. Conclusions Our results showed that the reduction of the plasma membrane content of cholesterol or its sequestration strongly potentiated signaling outcome initiated by LTβR. Thus, drugs modulating cholesterol levels could potentially improve efficacy of LTβR-based therapies. Video abstract.

Resistance to glucocorticosteroids (GCs) is a major adverse prognostic factor in B-ALL, but the molecular mechanisms leading to GC resistance are not completely understood. Herein, we sought to elucidate the molecular background of GC resistance in B-ALL and characterize the therapeutic potential of targeted intervention in these mechanisms. Using exploratory bioinformatic approaches, we found that resistant cells exhibited significantly higher expression of MEK/ERK (MAPK) pathway components. We found that GC-resistant ALL cell lines had markedly higher baseline activity of MEK and small-molecule MEK1/2 inhibitor selumetinib increased GCs-induced cell death. MEK inhibitor similarly increased in vitro dexamethasone activity in primary ALL blasts from 19 of 22 tested patients. To further confirm these observations, we overexpressed a constitutively active MEK mutant in GC-sensitive cells and found that forced MEK activity induced resistance to dexamethasone. Since recent studies highlight the role GC-induced autophagy upstream of apoptotic cell death, we assessed LC3 processing, MDC staining and GFP-LC3 relocalization in cells incubated with either DEX, SEL or combination of drugs. Unlike either drug alone, only their combination markedly increased these markers of autophagy. These changes were associated with decreased mTOR activity and blocked 4E-BP1 phosphorylation. In cells with silenced beclin-1 (BCN1), required for autophagosome formation, the synergy of DEX and SEL was markedly reduced. Taken together, we show that MEK inhibitor selumetinib enhances dexamethasone toxicity in GC-resistant B-ALL cells. The underlying mechanism of this interaction involves inhibition of mTOR signaling pathway and modulation of autophagy markers, likely reflecting induction of this process and required for cell death. Thus, our data demonstrate that modulation of MEK/ERK pathway is an attractive therapeutic strategy overcoming GC resistance in B-ALL patients.

Thiosemicarbazones (TSCs) are an interesting class of ligands that show a diverse range of biological activity, including anti-fungal, anti-viral and anti-cancer effects. Our previous studies have demonstrated the potent in vivo anti-tumor activity of novel TSCs and their ability to overcome resistance to clinically used chemotherapeutics. In the current study, 35 novel TSCs of 6 different classes were designed using a combination of retro-fragments that appear in other TSCs. Additionally, di-substitution at the terminal N4 atom, which was previously identified to be critical for potent anti-cancer activity, was preserved through the incorporation of an N4-based piperazine or morpholine ring. The anti-proliferative activity of the novel TSCs were examined in a variety of cancer and normal cell-types. In particular, compounds 1d and 3c demonstrated the greatest promise as anti-cancer agents with potent and selective anti-proliferative activity. Structure-activity relationship studies revealed that the chelators that utilized "soft" donor atoms, such as nitrogen and sulfur, resulted in potent anti-cancer activity. Indeed, the N,N,S donor atom set was crucial for the formation of redox active iron complexes that were able to mediate the oxidation of ascorbate. This further highlights the important role of reactive oxygen species generation in mediating potent anti-cancer activity. Significantly, this study identified the potent and selective anti-cancer activity of 1d and 3c that warrants further examination.

Our investigation uncovers that nanomolar concentrations of salinomycin, monensin, nigericin, and narasin (a group of potassium/sodium cation carriers) robustly enhance surface expression of CD20 antigen in B-cell-derived tumor cells, including primary malignant cells of chronic lymphocytic leukemia and diffuse large B-cell lymphoma. Experiments in vitro, ex vivo, and animal model reveal a novel approach of combining salinomycin or monensin with therapeutic anti-CD20 monoclonal antibodies or anti-CD20 CAR-T cells, significantly improving non- Hodgkin lymphoma (NHL) therapy. The results of RNA-seq, genetic editing, and chemical inhibition delineate the molecular mechanism of CD20 upregulation, at least partially, to the downregulation of MYC, the transcriptional repressor of the MS4A1 gene encoding CD20. Our findings propose the cation carriers as compounds targeting MYC oncogene, which can be combined with anti-CD20 antibodies or adoptive cellular therapies to treat NHL and mitigate resistance, which frequently depends on the CD20 antigen loss, offering new solutions to improve patient outcomes.

Abstract Multiple myeloma (MM) remains an incurable malignancy of plasma cells despite constantly evolving therapeutic approaches including various types of immunotherapy. Increased arginase activity has been associated with potent suppression of T-cell immune responses in different types of cancer. Here, we investigated the role of arginase 1 (ARG1) in Vκ*MYC model of MM in mice. ARG1 expression in myeloid cells correlated with tumor progression and was accompanied by a systemic drop in ʟ-arginine levels. In MM-bearing mice antigen-induced proliferation of adoptively transferred T-cells was strongly suppressed and T-cell proliferation was restored by pharmacological arginase inhibition. Progression of Vκ*MYC tumors was significantly delayed in mice with myeloid-specific ARG1 deletion. Arginase inhibition effectively inhibited tumor progression although it failed to augment anti-myeloma effects of bortezomib. However, arginase inhibitor completely prevented development of bortezomib-induced cardiotoxicity in mice. Altogether, these findings indicate that arginase inhibitors could be further tested as a complementary strategy in multiple myeloma to mitigate adverse cardiac events without compromising antitumor efficacy of proteasome inhibitors.

Grzywa et al. show that early CD71+ erythroid cells expand in the spleen of anemic mice and in humans and suppress T-cell activation. These results provide insight into the role of CECs in the immune response regulation.

Regulating polyadenylation is important for mRNA stability, which can in turn affect B cell maturation and humoral immune responses. Here the authors use Nanopore poly(A) sequencing to explore the importance of the cytoplasmic poly(A) polymerase TENT5C, particularly in the production of immunoglobulins.

Cancer cells employ a variety of ways to escape the immune system. Here, the authors show that ovarian cancer cells produce small extracellular vescicles containing arginase 1 that are taken up by dendritic cells in the draining lymph nodes, resulting in inhibition of antigen-specific T-cell proliferation.

B‐cell precursor acute lymphoblastic leukemia (BCP‐ALL) is a genetically heterogeneous blood cancer characterized by abnormal expansion of immature B cells. Although intensive chemotherapy provides high cure rates in a majority of patients, subtypes harboring certain genetic lesions, such as MLL rearrangements or BCR‐ABL1 fusion, remain clinically challenging, necessitating a search for other therapeutic approaches. Herein, we aimed to validate antioxidant enzymes of the thioredoxin system as potential therapeutic targets in BCP‐ALL. We observed oxidative stress along with aberrant expression of the enzymes associated with the activity of thioredoxin antioxidant system in BCP‐ALL cells. Moreover, we found that auranofin and adenanthin, inhibitors of the thioredoxin system antioxidant enzymes, effectively kill BCP‐ALL cell lines and pediatric and adult BCP‐ALL primary cells, including primary cells cocultured with bone marrow‐derived stem cells. Furthermore, auranofin delayed the progression of leukemia in MLL‐rearranged patient‐derived xenograft model and prolonged the survival of leukemic NSG mice. Our results unveil the thioredoxin system as a novel target for BCP‐ALL therapy, and indicate that further studies assessing the anticancer efficacy of combinations of thioredoxin system inhibitors with conventional anti‐BCP‐ALL drugs should be continued.

Immunotherapy has demonstrated significant activity in a broad range of cancer types, but still the majority of patients receiving it do not maintain durable therapeutic responses. Amino acid metabolism has been proposed to be involved in the regulation of immune response. Here, we investigated in detail the role of arginase 1 (Arg1) in the modulation of antitumor immune response against poorly immunogenic Lewis lung carcinoma. We observed that tumor progression is associated with an incremental increase in the number of Arg1+ myeloid cells that accumulate in the tumor microenvironment and cause systemic depletion of ʟ-arginine. In advanced tumors, the systemic concentrations of ʟ-arginine are decreased to levels that impair the proliferation of antigen-specific T-cells. Systemic or myeloid-specific Arg1 deletion improves antigen-induced proliferation of adoptively transferred T-cells and leads to inhibition of tumor growth. Arginase inhibitor was demonstrated to modestly inhibit tumor growth when used alone, and to potentiate antitumor effects of anti-PD-1 monoclonal antibodies and STING agonist. The effectiveness of the combination immunotherapy was insufficient to induce complete antitumor responses, but was significantly better than treatment with the checkpoint inhibitor alone. Together, these results indicate that arginase inhibition alone is of modest therapeutic benefit in poorly immunogenic tumors; however, in combination with other treatment strategies it may significantly improve survival outcomes.

Amino acid metabolism is a critical regulator of the immune response, and its modulating becomes a promising approach in various forms of immunotherapy. Insufficient concentrations of essential amino acids restrict T-cells activation and proliferation. However, only arginases, that degrade L-arginine, as well as enzymes that hydrolyze L-tryptophan are substantially increased in cancer. Two arginase isoforms, ARG1 and ARG2, have been found to be present in tumors and their increased activity usually correlates with more advanced disease and worse clinical prognosis. Nearly all types of myeloid cells were reported to produce arginases and the increased numbers of various populations of myeloid-derived suppressor cells and macrophages correlate with inferior clinical outcomes of cancer patients. Here, we describe the role of arginases produced by myeloid cells in regulating various populations of immune cells, discuss molecular mechanisms of immunoregulatory processes involving L-arginine metabolism and outline therapeutic approaches to mitigate the negative effects of arginases on antitumor immune response. Development of potent arginase inhibitors, with improved pharmacokinetic properties, may lead to the elaboration of novel therapeutic strategies based on targeting immunoregulatory pathways controlled by L-arginine degradation.

An increasing body of evidence from both academic and clinical studies shows that time-of-day exposure to antigens might significantly alter and modulate the development of adaptive immune responses. Considering the immense impact of the COVID-19 pandemic on global health and the diminished efficacy of vaccination in selected populations, such as older and immunocompromised patients, it is critical to search for the most optimal conditions for mounting immune responses against SARS-CoV-2. Hence, we conducted an observational study on 435 healthy young adults vaccinated with two doses of BNT162b2 (Pfizer-BioNTech) vaccine to determine whether time-of-day of vaccination influences either the magnitude of humoral response or number of adverse drug reactions (ADR) being reported. We found no significant differences between morning and afternoon vaccination in terms of both titers of anti-Spike antibodies and frequency of ADR in the studied population. In addition, our analysis of data on the occurrence of ADR in 1324 subjects demonstrated that the second administration of vaccine in those with previous SARS-CoV-2 infection was associated with lower incidence of ADR. In aggregate, vaccination against COVID-19 with two doses of BNT162b2 mRNA vaccine is presumed to generate an equally efficient anti-Spike humoral response.

Abstract Background Accumulating evidence suggest that autophagy plays a pivotal role in various anticancer therapies, including photodynamic therapy (PDT), acting as a pro-death or pro-survival mechanism in a context-dependent manner. Therefore, we aimed to determine the role of autophagy in Photofrin-based PDT. Methods In vitro cytotoxic/cytostatic effects of PDT were evaluated with crystal violet cell viability assay. Autophagy induction was analyzed by immunoblotting and immunofluorescence using anti-LC3 antibody. Autophagy was inhibited by shRNA-mediated ATG5 knockdown or CRISPR/Cas9-mediated ATG5 knockout. Apoptosis was assessed by flow cytometry analysis of propidium iodide and anexin V-positive cells as well as by detection of cleaved PARP and caspase 3 proteins using immunoblotting. Protein carbonylation was evaluated by the 2,4-dinitrophenylhydrazine (DNPH) method. Results Photofrin-PDT leads to robust autophagy induction in two cancer cell lines, Hela and MCF-7. shRNA-mediated knockdown of ATG5 only partially blocks autophagic response and only marginally affects the sensitivity of Hela and MCF-7 cells to PDT. ATG5 knockout in HeLa cell line utilizing CRISPR/Cas9 genome editing results in increased PDT-mediated cytotoxicity, which is accompanied by an enhanced apoptotic response and increased accumulation of carbonylated proteins. Conclusions Altogether, these observations imply that autophagy contributes to Photofrin-PDT resistance by enabling clearance of carbonylated and other damaged proteins. Therefore, autophagy inhibition may serve as a strategy to improve PDT efficacy.

Expression of arginase-1 (ARG1) is an immunosuppressive feature of tumor microenvironment that leads to depletion of ʟ-arginine, a nutrient required for T-cells expansion. Ovarian carcinoma cells release extracellular vesicles carrying enzymatically active ARG1, that contributes to local and systemic immune suppression, which can be restored by ARG inhibitor.

L-ascorbate (L-ASC) is a widely-known dietary nutrient which holds promising potential in cancer therapy when given parenterally at high doses. The anticancer effects of L-ASC involve its autoxidation and generation of H2O2, which is selectively toxic to malignant cells. Here we present that thioredoxin antioxidant system plays a key role in the scavenging of extracellularly-generated H2O2 in malignant B-cells. We show that inhibition of peroxiredoxin 1, the enzyme that removes H2O2 in a thioredoxin system-dependent manner, increases the sensitivity of malignant B-cells to L-ASC. Moreover, we demonstrate that auranofin (AUR), the inhibitor of the thioredoxin system that is used as an antirheumatic drug, diminishes the H2O2-scavenging capacity of malignant B-cells and potentiates pharmacological ascorbate anticancer activity in vitro and in vivo. The addition of AUR to L-ASC-treated cells triggers the accumulation of H2O2 in the cells, which results in iron-dependent cytotoxicity. Importantly, the synergistic effects are observed at as low as 200 µM L-ASC concentrations. In conclusion, we observed strong, synergistic, cancer-selective interaction between L-ASC and auranofin. Since both of these agents are available in clinical practice, our findings support further investigations of the efficacy of pharmacological ascorbate in combination with auranofin in preclinical and clinical settings.

Tumor-infiltrating immune cells can impact tumor growth and progression. The inhibitory CD200 receptor (CD200R) suppresses the activation of myeloid cells and lack of this pathway results in a reduction of tumor growth, conversely a tumorigenic effect of CD200R triggering was also described. Here we investigated the role of CD200R activation in syngeneic mouse tumor models. We showed that agonistic CD200R antibody reached tumors, but had no significant impact on tumor growth and minor effect on infiltration of immune myeloid cells. These effects were reproduced using two different anti-CD200R clones. In contrast, we showed that CD200-deficiency did decrease melanoma tumor burden. The presence of either endogenous or tumor-expressed CD200 restored the growth of metastatic melanoma foci. On the basis of these findings, we conclude that blockade of the endogenous ligand CD200 prevented the tumorigenic effect of CD200R-expressing myeloid cells in the tumor microenvironment, whereas agonistic anti-CD200R has no effect on tumor development.

A cute myeloid leukemia is a malignant disease of immature myeloid cells. Despite significant therapeutic effects of differentiation-inducing agents in some acute myeloid leukemia subtypes, the disease remains incurable in a large fraction of patients. Here we show that SK053, a thioredoxin inhibitor, induces differentiation and cell death of acute myeloid leukemia cells. Considering that thioredoxin knock-down with short hairpin RNA failed to exert antiproliferative effects in one of the acute myeloid leukemia cell lines, we used a biotin affinity probe-labeling approach to identify potential molecular targets for the effects of SK053. Mass spectrometry of proteins precipitated from acute myeloid leukemia cells incubated with biotinylated SK053 used as a bait revealed protein disulfide isomerase as a potential binding partner for the compound. Biochemical, enzymatic and functional assays using fluorescence lifetime imaging confirmed that SK053 binds to and inhibits the activity of protein disulfide isomerase. Protein disulfide isomerase knockdown with short hairpin RNA was associated with inhibition of cell growth, increased CCAAT enhancer-binding protein α levels, and induction of differentiation of HL-60 cells. Molecular dynamics simulation followed by the covalent docking indicated that SK053 binds to the fourth thioredoxin-like domain of protein disulfide isomerase. Differentiation of myeloid precursor cells requires the activity of CCAAT enhancer-binding protein α, the function of which is impaired in acute myeloid leukemia cells through various mechanisms, including translational block by protein disulfide isomerase. SK053 increased the levels of CCAAT enhancer-binding protein α and upregulated mRNA levels for differentiation-associated genes. Finally, SK053 decreased the survival of blasts and increased the percentage of cells expressing the maturation-associated CD11b marker in primary cells isolated from bone marrow or peripheral blood of patients with acute myeloid leukemia. Collectively, these results provide a proof-of-concept that protein disulfide isomerase inhibition has potential as a therapeutic strategy for the treatment of acute myeloid leukemia and for the development of small-molecule inhibitors of protein disulfide isomerase.

Diminished overall survival rate of non-Hodgkin lymphoma (NHL) patients treated with a combination regimen of rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP) has been recently linked to recurrent somatic mutations activating FOXO1. Despite of the clinical relevance of this finding, the molecular mechanism driving resistance to R-CHOP therapy remains largely unknown. Herein, we investigated the potential role of FOXO1 in the therapeutic efficacy of rituximab, the only targeted therapy included in the R-CHOP regimen. We found CD20 transcription is negatively regulated by FOXO1 in NHL cell lines and in human lymphoma specimens carrying activating mutations of FOXO1. Furthermore, both the expression of exogenous mutants of FOXO1 and the inhibition of AKT led to FOXO1 activation in lymphoma cells, increased binding to MS4A1 promoter and diminished CD20 expression levels. In contrast, a disruption of FOXO1 with CRISPR/Cas9 genome-editing (sgFOXO1) resulted in CD20 upregulation, improved the cytotoxicity induced by rituximab and the survival of mice with sgFOXO1 tumors. Accordingly, pharmacological inhibition of FOXO1 activity in primary samples upregulated surface CD20 levels. Importantly, FOXO1 was required for the downregulation of CD20 levels by the clinically tested inhibitors of BTK, SYK, PI3K and AKT. Taken together, these results indicate for the first time that the AKT-unresponsive mutants of FOXO1 are important determinant of cell response to rituximab-induced cytotoxicity, and suggest that the genetic status of FOXO1 together with its transcriptional activity need further attention while designing anti-CD20 antibodies based regimens for the therapy of pre-selected lymphomas.

Inhibition of the proteasome offers many therapeutic possibilities in inflammation as well as in neoplastic diseases. However, clinical use of proteasome inhibitors is limited by the development of resistance or severe side effects. In our study we characterized the anti-tumor properties of the novel proteasome inhibitor BSc2118. The sensitivity of tumor lines to BSc2118 was analyzed in comparison to bortezomib using crystal violet staining in order to assess cell viability. The In Vivo distribution of BSc2118 in mouse tissues was tracked by a fluorescent-modified form of BSc2118 (BSc2118-FL) and visualized by confocal microscopy. Inhibition of the 20S proteasome was monitored both in cultured cell lines and in mice, respectively. Finally, safety and efficacy of BSc2118 was evaluated in a mouse melanoma model. BSc2118 inhibits proliferation of different tumor cell lines with a similar potency as compared with bortezomib. Systemic administration of BSc2118 in mice is well tolerated, even when given in a dose of 60 mg/kg body weight. After systemic injection of BSc2118 or bortezomib similar proteasome inhibition patterns are observed within the murine organs. Detection of BSc2118-FL revealed correlation of distribution pattern of BSc2118 with inhibition of proteasomal activity in cells or mouse tissues. Finally, administration of BSc2118 in a mouse melanoma model shows significant local anti-tumor effects. Concluding, BSc2118 represents a novel low-toxic agent that might be alternatively used for known proteasome inhibitors in anti-cancer treatment.

Statins, HMG-CoA reductase inhibitors, are used in the prevention and treatment of cardiovascular diseases owing to their lipid-lowering effects. Previous studies revealed that, by modulating membrane cholesterol content, statins could induce conformational changes in cluster of differentiation 20 (CD20) tetraspanin. The aim of the presented study was to investigate the influence of statins on glucose transporter 1 (GLUT1)-mediated glucose uptake in tumor cells. We observed a significant concentration- and time-dependent decrease in glucose analogs' uptake in several tumor cell lines incubated with statins. This effect was reversible with restitution of cholesterol synthesis pathway with mevalonic acid as well as with supplementation of plasma membrane with exogenous cholesterol. Statins did not change overall GLUT1 expression at neither transcriptional nor protein levels. An exploratory clinical trial revealed that statin treatment decreased glucose uptake in peripheral blood leukocytes and lowered 18F-fluorodeoxyglucose (18F-FDG) uptake by tumor masses in a mantle cell lymphoma patient. A bioinformatics analysis was used to predict the structure of human GLUT1 and to identify putative cholesterol-binding motifs in its juxtamembrane fragment. Altogether, the influence of statins on glucose uptake seems to be of clinical significance. By inhibiting 18F-FDG uptake, statins can negatively affect the sensitivity of positron emission tomography, a diagnostic procedure frequently used in oncology.

Aminolevulinic acid (ALA) is an endogenous metabolite normally formed in the mitochondria from succinyl-CoA and glycine. Conjugation of eight ALA molecules yields protoporphyrin IX (PpIX) and finally leads to formation of heme. Conversion of PpIX to its downstream substrates requires the activity of a rate-limiting enzyme ferrochelatase. When ALA is administered externally the abundantly produced PpIX cannot be quickly converted to its final product - heme by ferrochelatase and therefore accumulates within cells. Since PpIX is a potent photosensitizer this metabolic pathway can be exploited in photodynamic therapy (PDT). This is an already approved therapeutic strategy making ALA one of the most successful prodrugs used in cancer treatment.