Direct Cytotoxic Activity Research Articles

Tetrazolium–based colorimetric assays underestimat the direct antitumor effects of anti-VEGF agent bevacizumab

The direct antitumor effect of bevacizumab (BEV) has long been debated. Assessment of the direct cytotoxic activities of drugs is usually conducted via in vitro experiments, of which tetrazolium-based colorimetric assays are widely employed to measure the direct antitumor activity of BEV. This study aimed to investigate whether tetrazolium-based colorimetric assays are applicable when evaluating the cytotoxicity of BEV against tumor cells. Our results showed that BEV significantly augmented tumor-cell mitochondrial metabolism. Enhanced mitochondrial metabolism caused changes in cellular oxidation-and-reduction environment and upregulated succinate dehydrogenase, which in turn promoted the reduction of tetrazolium to produce formazan. Increased formazan formation resulted in underestimation of the in vitro direct antitumor effect of BEV. Furthermore, inhibition of mitochondrial hypermetabolism partially corrected the underestimation of colorimetric assays in evaluating the direct antitumor activity of BEV. Our findings suggest that tetrazolium-based colorimetric assays are unsuitable for accurately assessing the in vitro cytotoxicity of anti-VEGF drugs and may be the methodological reason for the controversial direct antitumor effect of BEV.

Type I interferon signaling in malignant blasts contributes to treatment efficacy in AML patients

While type I interferon (IFN) is best known for its key role against viral infection, accumulating preclinical and clinical data indicate that robust type I IFN production in the tumor microenvironment promotes cancer immunosurveillance and contributes to the efficacy of various antineoplastic agents, notably immunogenic cell death inducers. Here, we report that malignant blasts from patients with acute myeloid leukemia (AML) release type I IFN via a Toll-like receptor 3 (TLR3)-dependent mechanism that is not driven by treatment. While in these patients the ability of type I IFN to stimulate anticancer immune responses was abolished by immunosuppressive mechanisms elicited by malignant blasts, type I IFN turned out to exert direct cytostatic, cytotoxic and chemosensitizing activity in primary AML blasts, leukemic stem cells from AML patients and AML xenograft models. Finally, a genetic signature of type I IFN signaling was found to have independent prognostic value on relapse-free survival and overall survival in a cohort of 132 AML patients. These findings delineate a clinically relevant, therapeutically actionable and prognostically informative mechanism through which type I IFN mediates beneficial effects in patients with AML.

A vector-encoded bispecific killer engager to harness virus-activated NK cells as anti-tumor effectors

Treatment with oncolytic measles vaccines (MV) elicits activation of immune cells, including natural killer (NK) cells. However, we found that MV-activated NK cells show only modest direct cytotoxic activity against tumor cells. To specifically direct NK cells towards tumor cells, we developed oncolytic measles vaccines encoding bispecific killer engagers (MV-BiKE) targeting CD16A on NK cells and carcinoembryonic antigen (CEA) as a model tumor antigen. MV-BiKE are only slightly attenuated compared to parental MV and mediate secretion of functional BiKE from infected tumor cells. We tested MV-BiKE activity in cocultures of colorectal or pancreatic cancer cells with primary human NK cells. MV-BiKE mediate expression of effector cytokines, degranulation and specific anti-tumor cytotoxicity by NK cells. Experiments with patient-derived pancreatic cancer cultures indicate that efficacy of MV-BiKE may vary between individual tumors with differential virus permissiveness. Remarkably, we confirmed MV-BiKE activity in primaryhuman colorectal carcinoma specimens with autochthonous tumor and NK cells.This study provides proof-of-concept for MV-BiKE as a novel immunovirotherapy to harness virus-activated NK cells as anti-tumor effectors.

CD46 protects the bladder cancer cells from cetuximab-mediated cytotoxicity

Epidermal growth factor receptor (EGFR) is an effective target for those patients with metastatic colorectal cancers that retain the wild-type RAS gene. However, its efficacy in many cancers, including bladder cancer, is unclear. Here, we studied the in vitro effects of cetuximab monoclonal antibodies (mAbs) targeting EGFR on the bladder cancer cells and role of CD46. Cetuximab was found to inhibit the growth of both colon and bladder cancer cell lines. Furthermore, cetuximab treatment inhibited AKT and ERK phosphorylation in the bladder cancer cells and reduced the expression of CD46 membrane-bound proteins. Restoration of CD46 expression protected the bladder cancer cells from cetuximab-mediated inhibition of AKT and ERK phosphorylation. We hypothesized that CD46 provides protection to the bladder cancer cells against mAb therapies. Bladder cancer cells were also susceptible to cetuximab-mediated immunologic anti-tumor effects. Further, cetuximab enhanced the cell killing by activating both antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) in bladder cancer cells. Restoration of CD46 expression protected the cells from both CDC and ADCC induced by cetuximab. Together, CD46 exhibited a cancer-protective effect against both direct (by involvement of PBMC or complement) and indirect cytotoxic activity by cetuximab in bladder cancer cells. Considering its clinical importance, CD46 could be an important link in the action mechanism of ADCC and CDC intercommunication and may be used for the development of novel therapeutic strategies.

Targeting fibroblast growth factor (FGF)-inducible 14 (Fn14) for tumor therapy.

Fibroblast growth factor-inducible 14 (Fn14) is a member of the tumor necrosis factor (TNF) receptor superfamily (TNFRSF) and is activated by its ligand TNF-like weak inducer of apoptosis (TWEAK). The latter occurs as a homotrimeric molecule in a soluble and a membrane-bound form. Soluble TWEAK (sTWEAK) activates the weakly inflammatory alternative NF-κB pathway and sensitizes for TNF-induced cell death while membrane TWEAK (memTWEAK) triggers additionally robust activation of the classical NF-κB pathway and various MAP kinase cascades. Fn14 expression is limited in adult organisms but becomes strongly induced in non-hematopoietic cells by a variety of growth factors, cytokines and physical stressors (e.g., hypoxia, irradiation). Since all these Fn14-inducing factors are frequently also present in the tumor microenvironment, Fn14 is regularly found to be expressed by non-hematopoietic cells of the tumor microenvironment and most solid tumor cells. In general, there are three possibilities how the tumor-Fn14 linkage could be taken into consideration for tumor therapy. First, by exploitation of the cancer associated expression of Fn14 to direct cytotoxic activities (antibody-dependent cell-mediated cytotoxicity (ADCC), cytotoxic payloads, CAR T-cells) to the tumor, second by blockade of potential protumoral activities of the TWEAK/Fn14 system, and third, by stimulation of Fn14 which not only triggers proinflammtory activities but also sensitizes cells for apoptotic and necroptotic cell death. Based on a brief description of the biology of the TWEAK/Fn14 system and Fn14 signaling, we discuss the features of the most relevant Fn14-targeting biologicals and review the preclinical data obtained with these reagents. In particular, we address problems and limitations which became evident in the preclinical studies with Fn14-targeting biologicals and debate possibilities how they could be overcome.

Local anesthetics and immunotherapy: a novel combination to fight cancer.

Intratumoral injection of oncolytic agents such as modified herpes simplex virus T-VEC or local administration of non-viral oncolytic therapies (such as radiofrequency, chemoembolization, cryoablation, or radiotherapy) can activate an anticancer immune response and hence trigger abscopal effects reducing secondary lesions. Preliminary data suggested that oncolytic treatments modulate tumor-infiltrating immune effectors and can be advantageously combined with the immune checkpoint inhibitors. Recent findings indicate that local anesthetics, which are usually used in the clinics to control surgical pain, also possess antineoplastic effects mimicking oncolytic treatments if they are injected into malignant lesions. Moreover, the association of local anesthetics with systemic immune checkpoint inhibition significantly improved overall survival in several preclinical tumor models. This may be explained by direct cytotoxic activity of local anesthetics and additional immune-related abscopal effects. We also summarize the molecular and cellular mechanisms by which the combination of local anesthetics and immunotherapy improves tumor control by the immune system.

Macrophage cytotoxic activity and its role in the tumor pathogenesis

Macrophages, natural killers and T cells play the central role in tumor cells destruction. The purpose of this review is to summarize the state-of-the-art perspectives of the interplay between tumor cells and tumor stroma leading both to the formation of a macrophage population incapable of effective antitumor activity and to the selection of tumor cells resistant to macrophage cytotoxicity. Macrophages are highly versatile cells that can both stimulate the inflammatory response (type 1 macrophages, M1) and suppress it (type 2 macrophages, M2). Tumor-associated macrophages (TAMs) are considered the main regulator of the antitumor immune response and usually have anti-inflammatory properties, that is, they belong to M2 type. Tumor cells are able to affect macrophages, "reprogramming" them to perform an immunosuppressive function. In addition, TAMs stimulate angiogenesis and remodelling of the extracellular matrix necessary for metastasis. Recently, more and more studies have been published describing a mixed TAMs phenotype with characteristics of both M2 and M1. M1 is characterized by production of pro-inflammatory cytokines, reactive oxygen species, bactericidal and cytotoxic activity. M1 can destroy tumor cells both directly and indirectly by attracting other cells. Despite the mechanisms of direct cytotoxic activity are quite variable, their effectiveness is largely dependent on the properties of a particular tumor. The cytotoxic activity of macrophages is a powerful factor that inhibits tumor initiation and progression. However, in some cases, it is not sufficient to control the tumor process. Activation of the cytotoxic activity of TAMs is one of the strategies to use macrophages for cancer treatment. Understanding the mechanisms of macrophage cytotoxic activity and specific patterns of its manifestation in a tumor environment is of critical importance for better effectiveness of existing cancer treatments and development of promising methods for tumor immunotherapy.

Pharmacologic Strategies for Post-Transplant Maintenance in Acute Myeloid Leukemia: It Is Time to Consider!

Simple SummaryAllogeneic hematopoietic cell transplantation (allo-HCT) is a potentially curative therapeutic procedure for patients with high risk acute myeloid leukemia. Its anti-leukemic activity is mainly derived from the intensity of the conditioning regimen used and the graft-versus-leukemia effect exerted by the donor T lymphocytes. Despite major progress in the allo-HCT procedure in recent years with the achievement of lower non-relapse mortality and long-term disease control. However, up to 45% of patients still experience relapse. Therapeutic strategies post-transplant aiming at preventing disease relapse are discussed in this review.Patients with high-risk acute myeloid leukemia are offered allogeneic hematopoietic cell transplantation (allo-HCT) in first remission to reduce risk of relapse. However, disease recurrence remains the major reason of allo-HCT failure, occurring in around 35–45% of patients, and leading to dismal outcomes. Strategies to reduce the risk of relapse are greatly needed, especially in the early post-transplant phase where the graft-versus-leukemia (GVL) effect is not yet activated. Some practices include the use of myeloablative conditioning regimens, close monitoring of measurable residual disease and donor chimerism, rapid tapering of immunosuppression, and implementation of pre-emptive strategies as the use of donor lymphocyte infusion. However, it’s time to consider prophylactic pharmacologic interventions post allo-HCT that aim at maintaining leukemic clones under control by both direct cytotoxic activity and by enhancing the GVL effect. In this current review, available data on drugs targeting epigenetic pathways like azacitidine, or actionable mutations like FLT3 and IDH1/2 inhibitors used as maintenance post allo-HCT, will be discussed.

Galectin-9 Triggers Neutrophil-Mediated Anticancer Immunity.

In earlier studies, galectin-9 (Gal-9) was identified as a multifaceted player in both adaptive and innate immunity. Further, Gal-9 had direct cytotoxic and tumor-selective activity towards cancer cell lines of various origins. In the current study, we identified that treatment with Gal-9 triggered pronounced membrane alterations in cancer cells. Specifically, phosphatidyl serine (PS) was rapidly externalized, and the anti-phagocytic regulator, CD47, was downregulated within minutes. In line with this, treatment of mixed neutrophil/tumor cell cultures with Gal-9 triggered trogocytosis and augmented antibody-dependent cellular phagocytosis of cancer cells. Interestingly, this pro-trogocytic effect was also due to the Gal-9-mediated activation of neutrophils with upregulation of adhesion markers and mobilization of gelatinase, secretory, and specific granules. These activation events were accompanied by a decrease in cancer cell adhesion in mixed cultures of leukocytes and cancer cells. Further, prominent cytotoxicity was detected when leukocytes were mixed with pre-adhered cancer cells, which was abrogated when neutrophils were depleted. Taken together, Gal-9 treatment potently activated neutrophil-mediated anticancer immunity, resulting in the elimination of epithelial cancer cells.

Selinexor Enhances NK Cell Activation Against Malignant B Cells via Downregulation of HLA-E.

Selinexor is an FDA approved selective inhibitor of the nuclear export protein exportin-1 (XPO1) and causes specific cancer cell death via nuclear accumulation of tumor suppressor proteins. Design of rational studies for the use of selinexor in combination with other therapeutic agents, such as immunotherapies, requires a fundamental understanding of the effects of selinexor on the immune system. One important emerging area of immunotherapy are natural killer (NK) cell based therapeutics. NK cell function is tightly regulated by a balance of signals derived from multiple activating and inhibitory receptors. Thus in cancer, up-regulation of stress ligands recognised by activating receptors or down-regulation of HLA class I recognised by inhibitory receptors can result in an anti-cancer NK cell response. Changes in XPO1 function therefore have the potential to affect NK cell function through shifting this balance. We therefore sought to investigate how selinexor may affect NK cell function. Selinexor pre-treatment of lymphoma cells significantly increased NK cell mediated cytotoxicity against SU-DHL-4, JeKo-1 and Ramos cells, concurrent with increased CD107a and IFNγ expression on NK cells. In addition, selinexor enhanced ADCC against lymphoma cells coated with the anti-CD20 antibodies rituximab and obinutuzumab. In probing the likely mechanism, we identified that XPO1 inhibition significantly reduced the surface expression of HLA-E on lymphoma cell lines and on primary chronic lymphocytic leukemia cells. HLA-E binds the inhibitory receptor NKG2A and in accordance with this, selinexor selectively increased activation of NKG2A+ NK cells. Our data reveals that selinexor, in addition to its direct cytotoxic activity, also activates an anti-cancer immune response via disruption of the inhibitory NKG2A:HLA-E axis.

The Sumoylation Inhibitor TAK-981 in Combination with the CD19-Targeting Antibody Tafasitamab Shows Enhanced Anti-Tumor Activity in Preclinical B-Cell Lymphoma Models

IntroductionTAK-981 is a first-in-class small molecule inhibitor of the SUMO activating enzyme currently in Phase I/II clinical trials. TAK-981 has been shown to increase NK cell activation and M1 macrophage polarization via upregulation of Type I interferon (IFN) signaling, leading to enhanced antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) in combination with rituximab (Nakamura 2019, AACR).Tafasitamab (MOR208) is a CD19-targeting antibody with enhanced Fc effector function mediating ADCC, ADCP and direct cytotoxic activities against B-lymphoma cells. Based on the Phase II clinical study L-MIND (Salles et al., 2020 and Duell et al., 2021), tafasitamab in combination with lenalidomide received accelerated approval by the Food and Drug Administration for the treatment of transplant-ineligible adult patients with relapsed or refractory (R/R) diffuse large B-cell lymphoma (DLBCL).Due to the potential for TAK-981 to enhance the activity of tafasitamab via activation of innate effector cells, we aimed to investigate the effects of this drug combination on ADCC, ADCP and tumor cell viability in vitro. Additionally, combinatorial activity of TAK-981 plus tafasitamab was evaluated in lymphoma xenograft models.MethodsA panel of 9 aggressive lymphoma cell lines was analyzed (7 DLBCL and 2 Burkitt lymphoma). For ADCC, PBMC effector cells from healthy human donors were pre-treated with 0.1 or 1 µM TAK-981 or dimethyl sulfoxide (DMSO) control for 24 hours. Tumor cells were incubated with/without 1 nM tafasitamab in the presence of TAK-981 pretreated PBMCs at effector-to-target (E:T) ratios of 5:1 to 10:1 for 2 hours. Degranulation of NK cells was determined via CD107a surface expression after co-incubation of TAK-981 pre-treated PBMCs with tumor cells and 0.1 or 10 nM tafasitamab for 3 hours. Cytokine levels in the supernatant were investigated upon incubation of PBMCs with lymphoma cells, 1 µM TAK-981 and/or 10 nM tafasitamab for 24 hours. For the ADCP assays, in vitro differentiated macrophages were treated with 1 µM TAK-981 for 24 hours. Next, macrophages were incubated with lymphoma cells and 1 or 10 nM tafasitamab at an E:T ratio of 2:1 for 3 hours. For cell viability assays, tumor cells were treated with 1-1000 nM TAK-981 and/or 5 nM tafasitamab for 24 hours in the absence of effector cells. Cytotoxicity, phagocytosis, degranulation and cytokine release were analyzed by flow cytometry. Cell viability was assessed by determination of ATP levels. For in vivo analysis, effects of TAK-981 (7.5 mg/kg IV twice weekly) in combination with tafasitamab (3, 10 or 20 mg/kg IP twice weekly) on tumor growth were evaluated in Daudi and WSU-DLCL2 xenograft models of Burkitt lymphoma and DLBCL grown in SCID mice.ResultsIn ADCC experiments, increased cytotoxicity was observed upon combination treatment with TAK-981 and tafasitamab compared to the respective mono treatments in 5/8 tested lymphoma cell lines (Daudi, SU-DHL-2, SU-DHL-6, TMD8, OCI-LY10). Moreover, TAK-981 plus tafasitamab enhanced degranulation of NK cells and cytokine release compared to mono treatments. In ADCP assays, combination of TAK-981 and tafasitamab resulted in increased phagocytosis rates in comparison to mono treatments in 2/2 tested cell lines (Daudi, Ramos). Cell viability analysis revealed a combination benefit by increased direct cytotoxic effects against SU-DHL-6 cells. Finally, TAK-981 and tafasitamab were investigated in Daudi and WSU-DLCL2 xenograft models with 3 weeks of dosing. In the Daudi model, the combination treatments of TAK-981 with 10 or 20 mg/kg tafasitamab performed better than either treatment alone, and in the WSU-DLCL2 model, the combination treatments of TAK-981 with 3, 10 or 20 mg/kg tafasitamab performed better than the single agent treatments.ConclusionsThe combination of TAK-981 with tafasitamab significantly enhanced anti-tumor effects compared to the respective monotherapies in vitro and in vivo. These preclinical data support a clinical evaluation of this drug combination in patients with lymphoma including aggressive subtypes such as Burkitt lymphoma and DLBCL.The study was funded by MorphoSys AG and Takeda Development Center Americas, Inc. DisclosuresPatra-Kneuer: MorphoSys AG: Current Employment. Nakamura: Takeda Development Center Americas, Inc.: Current Employment. Song: Takeda Pharmaceuticals International Co.: Current Employment. Grossman: Takeda Development Center, Cambridge MA: Current Employment. Polzer: MorphoSys: Current Employment. Ginzel: MorphoSys: Current Employment. Steidl: MorphoSys AG: Current Employment. Berger: Takeda Development Center Americas, Inc.: Current Employment. Proscurshim: Takeda Pharmaceuticals: Current Employment, Current holder of individual stocks in a privately-held company. Heitmüller: MorphoSys AG: Current Employment.

MiR-22 Modulates Lenalidomide Activity by Counteracting MYC Addiction in Multiple Myeloma.

Simple SummaryMYC-driven deregulation of microRNAs represents a critical event in human malignancies, including multiple myeloma (MM). Although the introduction of new therapeutic strategies has prolonged survival of patients, MM remains an incurable disease, often due to the onset of drug resistance. MYC hyperactivation is involved in the development of resistance to immunomodulatory imide drugs (IMiDs), but the mechanism is still unclear. Here, we report that MYC represses the transcription of tumor suppressor miR-22 in MM, and that low miR-22 expression is associated with IMiD resistance in MM patients. By in silico and in vitro analysis, we show that miR-22 mimics affect MYC signaling, leading to MM cell death in MYC proficient cells. Furthermore, we demonstrate here that lenalidomide treatment enhances miR-22 activity by reducing the MYC inhibitory effect, and that the combination of lenalidomide with miR-22 mimics restores drug sensitivity, leading to synergistic anti-MM activity.Background: MYC is a master regulator of multiple myeloma (MM) by orchestrating several pro-tumoral pathways, including reprograming of the miRNA transcriptome. MYC is also involved in the acquirement of resistance to anti-MM drugs, including immunomodulatory imide drugs (IMiDs). Methods: In silico analysis was performed on MM proprietary and on public MMRF-CoMMpass datasets. Western blot and chromatin immunoprecipitation (ChIP) experiments were performed to validate miR-22 repression induced by MYC. Cell viability and apoptosis assays were used to evaluate lenalidomide sensitization after miR-22 overexpression. Results: We found an inverse correlation between MYC and miR-22 expression, which is associated with poor outcome in IMiD-treated MM patients. Mechanistically, we showed that MYC represses transcription of miR-22, which, in turn, targets MYC, thus establishing a feed-forward loop. Interestingly, we found that IMiD lenalidomide increases miR-22 expression by reducing MYC repression and, most importantly, that the combination of lenalidomide with miR-22 mimics results in a synergistic direct and NK-mediated cytotoxic activity. Conclusions: Taken together, our findings indicate that: (1) low miR-22 expression could represent a potential predictive biomarker of poor lenalidomide response in MM patients; and (2) miR-22 reduces MYC oncogenic activity, thus triggering a novel synthetic lethality loop, which sensitizes MM cells to lenalidomide.

Icaritin Induces Anti-tumor Immune Responses in Hepatocellular Carcinoma by Inhibiting Splenic Myeloid-Derived Suppressor Cell Generation

Recent studies have demonstrated that splenic extramedullary hematopoiesis (EMH) is an important mechanism for the accumulation of myeloid-derived suppressor cells (MDSCs) in tumor tissues, and thus contributes to disease progression. Icaritin, a prenylflavonoid derivative from plants of the Epimedium genus, has been implicated as a novel immune-modulator that could prolong the survival of hepatocellular carcinoma (HCC) patients. However, it is unclear whether icaritin achieves its anti-tumor effects via the regulation of MDSCs generated by EMH in HCC. Here, we investigated the anti-tumor potential of icaritin and its mechanism of action in murine HCC. Icaritin suppressed tumor progression and significantly prolonged the survival of mice-bearing orthotopic and subcutaneous HCC tumors. Rather than exerting direct cytotoxic activity against tumor cells, icaritin significantly reduced the accumulation and activation of tumoral and splenic MDSCs, and increased the number and activity of cytotoxic T cells. Mechanistically, icaritin downregulates the tumor-associated splenic EMH, thereby reducing the generation and activation of MDSCs. The inhibitory effects of icaritin on human MDSCs in vitro were verified in short-term culture with cord-blood derived hematopoietic precursors. Furthermore, icaritin synergistically enhanced the therapeutic efficacy of immune checkpoint blockade therapy in HCC mice. These findings revealed that icaritin dampens tumoral immunosuppression to elicit anti-tumor immune responses by preventing MDSC generation via the attenuation of EMH. Thus, icaritin may serve as a novel adjuvant or even a stand-alone therapeutic agent for the effective treatment of HCC.

HAMLET a human milk protein‐lipid complex induces a pro‐inflammatory phenotype of myeloid cells

HAMLET is a protein‐lipid complex with a specific and broad bactericidal and tumoricidal activity, that lacks cytotoxic activity against healthy cells. In this study, we show that HAMLET also has general immune‐stimulatory effects on primary human monocyte‐derived dendritic cells and macrophages (Mo‐DC and Mo‐M) and murine RAW264.7 macrophages. HAMLET, but not its components alpha‐lactalbumin or oleic acid, induces mature CD14low/–CD83+ Mo‐DC and M1‐like CD14+CD86++ Mo‐M surface phenotypes. Concomitantly, inflammatory mediators, including IL‐2, IL‐6, IL‐10, IL‐12 and MIP‐1α, were released in the supernatant of HAMLET‐stimulated cells, indicating a mainly pro‐inflammatory phenotype. The HAMLET‐induced phenotype was mediated by calcium, NFκB and p38 MAPK signaling in Mo‐DCs and calcium, NFκB and ERK signaling in Mo‐M as inhibitors of these pathways almost completely blocked the induction of mature Mo‐DCs and M1‐like Mo‐M. Compared to unstimulated Mo‐DCs, HAMLET‐stimulated Mo‐DCs were more potent in inducing T cell proliferation and HAMLET‐stimulated macrophages were more efficient in phagocytosis of Streptococcus pneumoniae in vitro. This indicates a functionally activated phenotype of HAMLET‐stimulated DCs and macrophages. Combined, we propose that HAMLET has a two‐fold anti‐bacterial activity; one inducing direct cytotoxic activity, the other indirectly mediating elimination of bacteria by activation of immune cells of the myeloid lineage.

Calcium Channel Blockers Impair the Antitumor Activity of Anti-CD20 Monoclonal Antibodies by Blocking EGR-1 Induction.

Direct cell death induction, in addition to immune-effector cell-mediated mechanisms, is one of the key mechanisms of action of anti-CD20 antibodies, and yet the signaling pathways implicated remain poorly investigated. Here we show that the transcription factor EGR-1 is rapidly induced by anti-CD20 antibodies and is a key mediator for CD20-induced cell death. EGR-1 induction results from an increased calcium influx induced by anti-CD20 antibodies. We show that both rituximab and obinutuzumab induce calcium influx, albeit through different mechanisms, and this influx is crucial for cell death induction. Inhibition of the calcium flux with calcium channel blockers (CCB) abolished EGR-1 induction and impaired the efficacy of anti-CD20 antibodies in preclinical in vitro and in vivo models. Finally, we investigated the impact of CCBs in patients treated with anti-CD20 antibodies included in the clinical trials GOYA and REMARC, and found that patients simultaneously receiving CCBs and anti-CD20 therapy have a shorter progression-free survival and overall survival. These results reveal EGR-1 as a key mediator of the direct cytotoxic activity of anti-CD20 antibodies and provide a rationale to evaluate EGR-1 expression as a new biomarker to predict response to anti-CD20 treatment. In addition, our findings show that calcium influx is required for anti-CD20-mediated tumor cell death and suggest that simultaneous administration of calcium channel blocking agents could be deleterious in patients receiving anti-CD20-based immunotherapy.

Exosomes derived from Vδ2-T cells control Epstein-Barr virus-associated tumors and induce T cell antitumor immunity.

Treatment of life-threatening Epstein-Barr virus (EBV)-associated tumors remains a great challenge, especially for patients with relapsed or refractory disease. Here, we found that exosomes derived from phosphoantigen-expanded Vδ2-T cells (Vδ2-T-Exos) contained death-inducing ligands (FasL and TRAIL), an activating receptor for natural killer (NK) cells (NKG2D), immunostimulatory ligands (CD80 and CD86), and antigen-presenting molecules (MHC class I and II). Vδ2-T-Exos targeted and efficiently killed EBV-associated tumor cells through FasL and TRAIL pathways and promoted EBV antigen-specific CD4 and CD8 T cell expansion. Administration of Vδ2-T-Exos effectively controlled EBV-associated tumors in Rag2-/-γc-/- and humanized mice. Because expanding Vδ2-T cells and preparing autologous Vδ2-T-Exos from cancer patients ex vivo in large scale is challenging, we explored the antitumor activity of allogeneic Vδ2-T-Exos in humanized mouse cancer models. Here, we found that allogeneic Vδ2-T-Exos had more effective antitumor activity than autologous Vδ2-T-Exos in humanized mice; the allogeneic Vδ2-T-Exos increased the infiltration of T cells into tumor tissues and induced more robust CD4 and CD8 T cell-mediated antitumor immunity. Compared with exosomes derived from NK cells (NK-Exos) with direct cytotoxic antitumor activity or dendritic cells (DC-Exos) that induced T cell antitumor responses, Vδ2-T-Exos directly killed tumor cells and induced T cell-mediated antitumor response, thus resulting in more effective control of EBV-associated tumors. This study provided proof of concept for the strategy of using Vδ2-T-Exos, especially allogeneic Vδ2-T-Exos, to treat EBV-associated tumors.

Abstract 4053: Mucin 5ac as a potential therapeutic target for KRAS-driven lung adenocarcinoma

Abstract Lung cancer continues to be the leading cause of cancer death in the United States, with more than 140,000 deaths per year. One of the main types of lung cancer is non-small cell lung cancer with lung adenocarcinoma (LUAD) being the most prominent subtype and is mainly due to oncogenic mutations like KRAS, which offers limited and unsuccessful treatments. The mucus barrier produced by mucins, particularly Mucin 5ac (Muc5ac) which is mainly expressed by lung epithelial cells, is essential for airway homeostasis. However, its overproduction can promote respiratory diseases such as chronic obstructive pulmonary disease (COPD), which is known to increase the risk of lung cancer by 3-10 fold. We have previously shown that Muc5ac is commonly expressed in patients with KRAS-driven LUAD and is associated with poor prognosis. Additionally, we found that lack of Muc5ac (genetic deletion) in mice with LUAD either induced chemically by urethane or genetically engineered with mutant KRAS presented with significantly reduced tumor burden. Therefore, we sought a translational approach by inhibiting Muc5ac in our KRAS-driven lung cancer mutant mouse model using an antisense oligonucleotide (ASO) against Muc5ac and hypothesized that the administration of this drug would result in decreased Muc5ac mRNA expression and reduced tumor burden. This was tested by intrapharyngeal administration of either ASO (5mg/kg) or vehicle (saline) twice a week to a cohort of six-week-old KRASG12D mutant mice for a total duration of eight weeks. Our results showed a 5-fold decrease in Muc5ac mRNA expression and a 42% reduction in overall tumor burden when compared to age and sex matched control mice. Additionally, analysis of bronchoalveolar lavage fluid showed a predominance of macrophages with significant downregulation of the protumorigenic M2 type macrophage polarization markers Fizz1 and Arg1 as well as significant upregulation of TNF-a, which suggests a mechanism mediated by direct tumor cytotoxic activity or apoptosis associated with a reduction in macrophage mediated immunosuppressive tumor microenvironment. In conclusion, our study suggests that administration of Muc5ac ASO effectively inhibits mucin production and ameliorates tumor progression, which ultimately could provide insight in both treatments and prevention against KRAS-driven LUAD. Citation Format: Walter V. Velasco Torrez, Shanshan Deng, Marco A. Ramos-Castaneda, Segundo W. Del Aguila-Soto, Seyed Moghaddam. Mucin 5ac as a potential therapeutic target for KRAS-driven lung adenocarcinoma [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 4053.

Differential Cytotoxic Function of Resident and Non-resident CD8+ T Cells in the Human Female Reproductive Tract Before and After Menopause.

The functional characterization and regulation of tissue resident and non-resident CD8+ T cells in the human female reproductive tract (FRT) as women age remains a gap in our knowledge. Here we characterized the cytotoxic activity and granular contents of CD8+ T cells from the FRT in pre- and postmenopausal women. We found that under steady-state conditions, CD8+ T cells from endometrium (EM), endocervix and ectocervix displayed direct cytotoxic activity, and that cytotoxicity increased in the EM after menopause. Cytotoxic activity was sensitive to suppression by TGFβ exclusively in the EM, and sensitivity to TGFβ was reduced after menopause. Under steady-state conditions, cytotoxic activity (measured as direct killing activity), cytotoxic potential (measured as content of cytotoxic molecules) and proliferation are enhanced in non-resident CD8+ (CD103−) T cells compared to tissue resident (CD103+) T cells. Upon activation, CD103+ T cells displayed greater degranulation compared to CD103− T cells, however the granular content of perforin, granzyme A (GZA) or granzyme B (GZB) was significantly lower. After menopause, degranulation significantly increased, and granular release switched from predominantly GZB in premenopausal to GZA in postmenopausal women. Postmenopausal changes affected both CD103+ and CD103− subpopulations. Finally, CD103+ T cells displayed reduced proliferation compared to CD103− T cells, but after proliferation, cytotoxic molecules were similar in each population. Our results highlight the complexity of regulation of cytotoxic function in the FRT before and after menopause, and are relevant to the development of protective strategies against genital infections and gynecological cancers as women age.

The Evaluation of Potential Cytotoxic Effect of Different Proton Pump Inhibitors on Different Human Cancer Cell Lines.

To assess the differential cytotoxic activity of PPIs on different human cancer cell lines; namely A549 lung cancer, CACO-2 colorectal cancer, MCF-7 breast cancer, and PANC-1 pancreatic cancer, A375 skin melanoma. In this study, the five human cancer cell lines and human non-cancerous fibroblasts were treated with increasing concentration of PPIs Omeprazole (OMP), Esomeprazole (ESOM), and Lansoprazole (LANSO) (50-300μM), over 24h, 48h, and 72h. Cell viability was determined using 3-(4,5- Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay and the IC50 values of PPIs were measured. The most sensitive cell line A375 was used for further investigation. The cytotoxic effects of LANSO on these cells were assessed using Annexin-V Propidium Iodide (AV-PI) flow cytometry. As of action mechanism; anti-inflammatory effects of each PPIs and PPIs-DOXO combination therapy on LPS-stimulated RAW 264.7 mouse macrophages were assessed. Dose and time dependence cytotoxic activity of PPIs on human cancer cell lines was founded. Unlike DOXO; All PPIs had a selective cytotoxic effect in the normal fibroblasts. Unlike the equipotent OMP and ESOM; LANSO was the most potent drug with IC50 values at 72h of 99, 217, 272, 208, 181μM against A375, A549, CACO-2, MCF-7, and PANC-1, respectively. AV-PI flow cytometry revealed dose-dependent apoptotic effects of LANSO alone and substantially enhanced in DOXO-co-treatments. Interestingly unlike ESOM and OMP, LANSO proved more effective than indomethacin in LPS-stimulated RAW 264.7 macrophages. None of the tested compounds, as well as indomethacin, exerted any cytotoxicity against RAW 264.7 macrophages. PPIs-DOXO lacked potential synergistic combination antiinflammation therapies. This study provides the evidence that PPIs induce a direct and differential cytotoxic activity against human cancer cell line by the induction of the apoptosis. Moreover, PPIs increase cancer cell lines sensitivity to doxorubicin via apoptosis augmentation. Nevertheless, PPIs-DOXO lacked potential synergistic combination therapies in either antiproliferation or anti-inflammation.

The terminal sialic acid of stage-specific embryonic antigen-4 has a crucial role in binding to a cancer-targeting antibody

The terminal sialic acid of stage-specific embryonic antigen-4 has a crucial role in binding to a cancer-targeting antibody