IL-15-secreting CAR natural killer cells directed toward the pan-cancer target CD70 eliminate both cancer cells and cancer-associated fibroblasts.

BACKGROUND: It remains challenging to obtain positive outcomes with chimeric antigen receptor (CAR)-engineered cell therapies in solid malignancies, like colorectal cancer (CRC) and pancreatic ductal adenocarcinoma (PDAC). A major obstacle is the lack of targetable surface antigens that are not shared by healthy tissues. CD70 emerges as interesting target, due to its stringent expression pattern in healthy tissue and its apparent role in tumor progression in a considerable amount of malignancies. Moreover, CD70 is also expressed on cancer-associated fibroblasts (CAFs), another roadblock for treatment efficacy in CRC and PDAC. We explored the therapeutic potential of CD70 as target for CAR natural killer (NK) cell therapy in CRC, PDAC, focusing on tumor cells and CAFs, and lymphoma. METHODS: RNA-seq data and immunohistochemical analysis of patient samples were used to explore CD70 expression in CRC and PDAC patients. In addition, CD70-targeting CAR NK cells were developed to assess cytotoxic activity against CD70+ tumor cells and CAFs, and the effect of cytokine stimulation on their efficacy was evaluated. The in vitro functionality of CD70-CAR NK cells was investigated against a panel of tumor and CAF cell lines with varying CD70 expression. Lymphoma-bearing mice were used to validate in vivo potency of CD70-CAR NK cells. Lastly, to consider patient variability, CD70-CAR NK cells were tested on patient-derived organoids containing CAFs. RESULTS: In this study, we identified CD70 as a target for tumor cells and CAFs in CRC and PDAC patients. Functional evaluation of CD70-directed CAR NK cells indicated that IL-15 stimulation is essential to obtain effective elimination of CD70+ tumor cells and CAFs, and to improve tumor burden and survival of mice bearing CD70+ tumors. Mechanistically, IL-15 stimulation resulted in improved potency of CD70-CAR NK cells by upregulating CAR expression and increasing secretion of pro-inflammatory cytokines, in a mainly autocrine or intracellular manner. CONCLUSIONS: We disclose CD70 as an attractive target both in hematological and solid tumors. IL-15 armored CAR NK cells act as potent effectors to eliminate these CD70+ cells. They can target both tumor cells and CAFs in patients with CRC and PDAC, and potentially other desmoplastic solid tumors.

PD-1, PD-L1, IDO, CD70 and microsatellite instability as potential targets to prevent immune evasion in sarcomas.

Background: Soft tissue and bone sarcomas are rare entities, hence, standardized therapeutic strategies are difficult to assess. Materials & methods: Immunohistochemistry was performed on 68 sarcoma samples to assess the expression of PD-1, PD-L1, IDO and CD70 in different tumor compartments and molecular analysis was performed to assess microsatellite instability status. Results: PD-1/PD-L1, IDO and CD70 pathways are at play in the immune evasion of sarcomas in general. Soft tissue sarcomas more often show an inflamed phenotype compared with bone sarcomas. Specific histologic sarcoma types show high expression levels of different markers. Finally, this is the first presentation of a microsatellite instability-high Kaposi sarcoma. Discussion/conclusion: Immune evasion occurs in sarcomas. Specific histologic types might benefit from immunotherapy, for which further investigation is needed.

Sarcomas of the soft tissue and bone are rare cancers. When these cancers spread to other parts of the body, it is hard to find good treatments. Recently, doctors have been using a new type of treatment called immunotherapy to fight several types of cancer. Immunotherapy works by getting one's body's own defense cells to attack the cancer cells. Unfortunately, immunotherapy does not work well for sarcomas and we do not know why. This study was designed to determine if there are certain mechanisms in these tumors that help the cancer cells to hide from defense cells. Determining how to change these mechanisms could make immunotherapy a better treatment for sarcomas in the future.

Tumor Microenvironment in Thymic Epithelial Tumors: A Narrative Review.

The tumor microenvironment (TME) is a complex and constantly changing entity. The TME consists of stromal cells, fibroblasts, endothelial cells, and innate and adaptive immune cells. Cancer development and progression occurs through this interplay between the tumor and the adjacent stroma. Cancer cells are capable of modifying their microenvironment by secreting various message-carrying molecules, such as cytokines, chemokines, and other factors. This action causes a reprogramming of the neighboring cells, which are enabled to play a crucial role in tumor survival and progression. The study of TME has many clinical implications in terms of cancer therapeutics because many new drugs, such as antibodies, kinase inhibitors, and liposome formulations that can encapsulate anti-cancer drugs, can be developed. Although chemotherapy is considered the standard of treatment for advanced disease, recent research has brought to light immunotherapy as a possible systemic alternative. However, the complex structure and function of the thymus hinders its routine use in clinical practice. The aim of this review paper is to discuss the recent advances in the investigation of the unique characteristics of the TME of thymic epithelial tumors that could possibly lead to the development of novel promising therapies.

Prognostic factors and genetic markers in thymic epithelial tumors: A narrative review.

Thymic epithelial tumors (TET) are a group of rare neoplasms of the anterior mediastinum comprising thymomas and thymic carcinomas. The carcinogenesis of TET is mostly unknown. Many studies, mostly retrospective case series, have tried to establish prognostic factors in TET. TET is a very heterogeneous group of tumors with many subtypes for which diagnosis and treatment remains a very challenging task. Despite the disparities among retrospective studies, there are some prognostic factors that are more pertinent such as the completeness of resection, TNM stage and the Masaoka-Koga classification. On the other hand, the identification of different genetic pathways that result in the pathogenesis of TET represents a fascinating field of study that could possibly lead to the development of new targeted therapies. The aim of this review is to discuss the different prognostic factors and genetic markers of TET. The meticulous use of national and international databases could provide sufficient number of patients in order to draw more valid conclusions.

The prognostic impact of the immune signature in head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous group of tumors that retain their poor prognosis despite recent advances in their standard of care. As the involvement of the immune system against HNSCC development is well-recognized, characterization of the immune signature and the complex interplay between HNSCC and the immune system could lead to the identification of novel therapeutic targets that are required now more than ever. In this study, we investigated RNA sequencing data of 530 HNSCC patients from The Cancer Genome Atlas (TCGA) for which the immune composition (CIBERSORT) was defined by the relative fractions of 10 immune-cell types and expression data of 45 immune checkpoint ligands were quantified. This initial investigation was followed by immunohistochemical (IHC) staining for a curated selection of immune cell types and checkpoint ligands markers in tissue samples of 50 advanced stage HNSCC patients. The outcome of both analyses was correlated with clinicopathological parameters and patient overall survival. Our results indicated that HNSCC tumors are in close contact with both cytotoxic and immunosuppressive immune cells. TCGA data showed prognostic relevance of dendritic cells, M2 macrophages and neutrophils, while IHC analysis associated T cells and natural killer cells with better/worse prognostic outcome. HNSCC tumors in our TCGA cohort showed differential RNA over- and underexpression of 28 immune inhibitory and activating checkpoint ligands compared to healthy tissue. Of these, CD73, CD276 and CD155 gene expression were negative prognostic factors, while CD40L, CEACAM1 and Gal-9 expression were associated with significantly better outcomes. Our IHC analyses confirmed the relevance of CD155 and CD276 protein expression, and in addition PD-L1 expression, as independent negative prognostic factors, while HLA-E overexpression was associated with better outcomes. Lastly, the co-presence of both (i) CD155 positive cells with intratumoral NK cells; and (ii) PD-L1 expression with regulatory T cell infiltration may hold prognostic value for these cohorts. Based on our data, we propose that CD155 and CD276 are promising novel targets for HNSCC, possibly in combination with the current standard of care or novel immunotherapies to come.

Blood transcriptomics to facilitate diagnosis and stratification in pediatric rheumatic diseases - a proof of concept study.

BACKGROUND: Transcriptome profiling of blood cells is an efficient tool to study the gene expression signatures of rheumatic diseases. This study aims to improve the early diagnosis of pediatric rheumatic diseases by investigating patients' blood gene expression and applying machine learning on the transcriptome data to develop predictive models. METHODS: RNA sequencing was performed on whole blood collected from children with rheumatic diseases. Random Forest classification models were developed based on the transcriptome data of 48 rheumatic patients, 46 children with viral infection, and 35 controls to classify different disease groups. The performance of these classifiers was evaluated by leave-one-out cross-validation. Analyses of differentially expressed genes (DEG), gene ontology (GO), and interferon-stimulated gene (ISG) score were also conducted. RESULTS: Our first classifier could differentiate pediatric rheumatic patients from controls and infection cases with high area-under-the-curve (AUC) values (AUC = 0.8 ± 0.1 and 0.7 ± 0.1, respectively). Three other classifiers could distinguish chronic recurrent multifocal osteomyelitis (CRMO), juvenile idiopathic arthritis (JIA), and interferonopathies (IFN) from control and infection cases with AUC ≥ 0.8. DEG and GO analyses reveal that the pathophysiology of CRMO, IFN, and JIA involves innate immune responses including myeloid leukocyte and granulocyte activation, neutrophil activation and degranulation. IFN is specifically mediated by antibacterial and antifungal defense responses, CRMO by cellular response to cytokine, and JIA by cellular response to chemical stimulus. IFN patients particularly had the highest mean ISG score among all disease groups. CONCLUSION: Our data show that blood transcriptomics combined with machine learning is a promising diagnostic tool for pediatric rheumatic diseases and may assist physicians in making data-driven and patient-specific decisions in clinical practice.

Expression of SARS-CoV-2-Related Surface Proteins in Non-Small-Cell Lung Cancer Patients and the Influence of Standard of Care Therapy.

In this study, we aimed to study the expression of SARS-CoV-2-related surface proteins in non-small-cell lung cancer (NSCLC) cells and identify clinicopathological characteristics that are related to increased membranous (m)ACE2 protein expression and soluble (s)ACE2 levels, with a particular focus on standard of care (SOC) therapies. ACE2 (n = 107), TMPRSS2, and FURIN (n = 38) protein expression was determined by immunohistochemical (IHC) analysis in NSCLC patients. sACE2 levels (n = 64) were determined in the serum of lung cancer patients collected before, during, or after treatment with SOC therapies. Finally, the TCGA lung adenocarcinoma (LUAD) database was consulted to study the expression of ACE2 in EGFR- and KRAS-mutant samples and ACE2 expression was correlated with EGFR/HER, RAS, BRAF, ROS1, ALK, and MET mRNA expression. Membranous (m)ACE2 was found to be co-expressed with mFURIN and/or mTMPRSS2 in 16% of the NSCLC samples and limited to the adenocarcinoma subtype. TMPRSS2 showed predominantly atypical cytoplasmic expression. mACE2 and sACE2 were more frequently expressed in mutant EGFR patients, but not mutant-KRAS patients. A significant difference was observed in sACE2 for patients treated with targeted therapies, but not for chemo- and immunotherapy. In the TCGA LUAD cohort, ACE2 expression was significantly higher in EGFR-mutant patients and significantly lower in KRAS-mutant patients. Finally, ACE2 expression was positively correlated with ERBB2-4 and ROS1 expression and inversely correlated with KRAS, NRAS, HRAS, and MET mRNA expression. We identified a role for EGFR pathway activation in the expression of mACE2 in NSCLC cells, associated with increased sACE2 levels in patients. Therefore, it is of great interest to study SARS-CoV-2-infected EGFR-mutated NSCLC patients in greater depth in order to obtain a better understanding of how mACE2, sACE2, and SOC TKIs can affect the course of COVID-19.

Prognostic implications of cellular senescence in resected non-small cell lung cancer.

Background: Cure and long-term survival for non-small cell lung cancer (NSCLC) remains hard to achieve. Cellular senescence, an emerging hallmark of cancer, is considered as an endogenous tumor suppressor mechanism. However, senescent cancer cells can paradoxically affect the surrounding tumor microenvironment (TME), ultimately leading to cancer relapse and metastasis. As such, the role of cellular senescence in cancer is highly controversial. Methods: In 155 formalin-fixed paraffin-embedded (FFPE) samples from surgically resected NSCLC patients with pathological tumor-node-metastasis (pTNM) stages I-IV (8th edition), cellular senescence was assessed using a combination of four immunohistochemical senescence markers, i.e., lipofuscin, p16INK4a, p21WAF1/Cip1 and Ki67, and correlated to clinicopathological parameters and outcomes, including overall survival (OS) and disease-free survival (DFS). Results: A tumoral senescence signature (SS) was present in 48 out of 155 NSCLC patients, but did not correlate to any clinicopathological parameter, except for p53 mutation status. In a histologically homogenous patient cohort of 100 patients who fulfilled the following criteria: (I) one type of histology, i.e., adenocarcinoma, (II) without known epidermal growth factor receptor (EGFR) mutation, (III) curative (R0) resection and (IV) no neoadjuvant systemic therapy or radiotherapy, the median OS and DFS for patients with a tumoral SS (n=30, 30.0%) compared to patients without a tumoral SS (n=70, 70.0%) was 53 versus 141 months (P=0.005) and 45 versus 55 months (P=0.25), respectively. In multiple Cox proportional hazards (Cox PH) model analysis correcting for age, pTNM stage I-III and adjuvant therapy, a tumoral SS remained a significant prognostic factor for OS (HR =2.03; P=0.014). Conclusions: The presence of a tumoral SS particularly based on high p16INK4a expression significantly affects OS in NSCLC adenocarcinoma. In this light, adjuvant senolytic therapy could be an interesting strategy for NSCLC patients harboring a tumoral SS, ultimately to improve survival of these patients.

The effect of local non-thermal plasma therapy on the cancer-immunity cycle in a melanoma mouse model.

Melanoma remains a deadly cancer despite significant advances in immune checkpoint blockade and targeted therapies. The incidence of melanoma is also growing worldwide, which highlights the need for novel treatment options and strategic combination of therapies. Here, we investigate non-thermal plasma (NTP), an ionized gas, as a promising, therapeutic option. In a melanoma mouse model, direct treatment of tumors with NTP results in reduced tumor burden and prolonged survival. Physical characterization of NTP treatment in situ reveals the deposited NTP energy and temperature associated with therapy response, and whole transcriptome analysis of the tumor identified several modulated pathways. NTP treatment also enhances the cancer-immunity cycle, as immune cells in both the tumor and tumor-draining lymph nodes appear more stimulated to perform their anti-cancer functions. Thus, our data suggest that local NTP therapy stimulates systemic, anti-cancer immunity. We discuss, in detail, how these fundamental insights will help direct the translation of NTP technology into the clinic and inform rational combination strategies to address the challenges in melanoma therapy.

DOG1-Positive Primary Mesenteric Leiomyosarcoma: Report of a Case and Review of the Literature.

The mesentery constitutes a common location for the metastatic spread of malignant gastrointestinal tumors. Primary mesenteric tumors, on the other hand, are very rare; lymphomas are the most common, followed by benign and malignant mesenchymal tumors. We present a case of a 43-year-old patient operated on for a primary mesenteric leiomyosarcoma with a positive immunostain for DOG1, despite having no KIT or PDGFRa mutations on molecular analysis. Moreover, we review the pertinent literature.

NTRK Fusions in a Sarcomas Series: Pathology, Molecular and Clinical Aspects.

Targeting molecular alterations has been proven to be an inflecting point in tumor treatment. Especially in recent years, inhibitors that target the tyrosine receptor kinase show excellent response rates and durable effects in all kind of tumors that harbor fusions of one of the three neurotrophic tyrosine receptor kinase genes (NTRK1, NTRK2 and NTRK3). Today, the therapeutic options in most metastatic sarcomas are rather limited. Therefore, identifying which sarcoma types are more likely to harbor these targetable NTRK fusions is of paramount importance. At the moment, identification of these fusions is solely based on immunohistochemistry and confirmed by molecular techniques. However, a first attempt has been made to describe the histomorphology of NTRK-fusion positive sarcomas, in order to pinpoint which of these tumors are the best candidates for testing. In this study, we investigate the immunohistochemical expression of pan-TRK in 70 soft tissue and bone sarcomas. The pan-TRK positive cases were further investigated with molecular techniques for the presence of a NTRK fusion. Seven out of the 70 cases showed positivity for pan-TRK, whereas two of these seven cases presented an NTRK3 fusion. Further analysis of the fused sarcomas revealed some unique histological, molecular and clinical findings. The goal of this study is to expand the histomorphological spectrum of the NTRK-fused sarcomas, to identify their fusion partners and to correlate these parameters with the clinical outcome of the disease. In addition, we evaluated the immunohistochemical expression pattern of the pan-TRK and its correlation with the involved NTRK gene.

NTRK Gene Fusion Detection in a Pan-Cancer Setting Using the Idylla GeneFusion Assay.

Recently, approval of tyrosine receptor kinase (TRK) inhibitors by Food and Drug Administration and European Medicines Agency in NTRK fusion-positive cancer types has led to a variety of proposed testing algorithms. In this study, performance of the fully automated Idylla GeneFusion Assay was assessed in a set of clinically relevant cancer types, including glioblastoma, non-small-cell lung cancer, microsatellite instability-positive colorectal cancer, and thyroid carcinoma. Analysis with the Idylla GeneFusion Assay revealed significant differences in baseline RNA expression profile between the different cancer types, which corresponded to both literature and pan-TRK immunohistochemical staining. Compared with the RNA-based Oncomine Focus Assay, the Idylla GeneFusion Assay demonstrated an overall percentage agreement, positive percentage agreement, and negative percentage agreement of 92.7%, 81.8%, and 93.8%, respectively; and the pan-TRK immunohistochemistry demonstrated an overall percentage agreement, positive percentage agreement, and negative percentage agreement of 82.1%, 45.5%, and 85.7%, respectively. These findings highlighted the importance of tailoring NTRK testing algorithms per cancer type. In a small subset, data from the RNA-based Archer FusionPlex Assay were also available. NTRK fusion detection efficiency was compared between the four NTRK testing modalities, with a high concordance between the PCR-based methods. Last, RNA degradation was observed when using the Idylla GeneFusion Assay on snap frozen tissue samples as these are nonfixated. This might be countered by increasing the amount of sample input. To conclude, the Idylla GeneFusion Assay has shown a clear potential in identifying NTRK fusions.

Update on Pulmonary Ossifications in the Differential Diagnosis of Solitary Pulmonary Nodules.

Pulmonary ossifications have often been regarded as rare, post-mortem findings without any clinical significance. We have investigated the occurrence of pulmonary ossifications in patients undergoing thoracic procedures, and how this may affect the differential diagnosis of solitary pulmonary nodules. In addition, we have performed a literature search on the occurrence and possible pathogenesis of these ossifications. From January 2008 until August 2019, we identified pulmonary ossifications in 34 patients who underwent elective pulmonary surgery. Pre-operative imaging was unable to differentiate these ossifications from solid tumors. A definitive diagnosis was made by an experienced pathologist (VS, ML). The PubMed database was researched in December 2019 with the search terms "pulmonary ossifications"; "heterotopic ossifications"; and "solitary pulmonary nodule". In total, 27 patients were male, with a mean age of 63 ± 12 years (age 41 to 82 on diagnosis). All lesions were identified on thoracic CT and marked for resection by a multidisciplinary team. A total of 17 patients were diagnosed with malignancy concurrent with ossifications. There was a clear predilection for the right lower lobe (12 cases, 35.3%) and most ossifications had a nodular form (70.6%). We could not identify a clear association with any other pathology, either cancerous or non-cancerous in origin. Oncologic or pulmonary comorbidities did not influence patient survival. Pulmonary ossifications are not as seldom as thought and are not just a curiosity finding by pathologists. These formations may be mistaken for a malignant space-occupying lesion, both pre-and perioperatively, as they are indistinguishable in imaging. We propose these ossifications as an underestimated addition to the differential diagnosis of a solitary pulmonary nodule.

EWSR1-The Most Common Rearranged Gene in Soft Tissue Lesions, Which Also Occurs in Different Bone Lesions: An Updated Review.

EWSR1 belongs to the FET family of RNA-binding proteins including also Fused in Sarcoma (FUS), and TATA-box binding protein Associated Factor 15 (TAF15). As consequence of the multifunctional role of EWSR1 leading to a high frequency of transcription of the chromosomal region where the gene is located, EWSR1 is exposed to aberrations such as rearrangements. Consecutive binding to other genes leads to chimeric proteins inducing oncogenesis. The other TET family members are homologous. With the advent of widely used modern molecular techniques during the last decades, it has become obvious that EWSR1 is involved in the development of diverse benign and malignant tumors with mesenchymal, neuroectodermal, and epithelial/myoepithelial features. As oncogenic transformation mediated by EWSR1-fusion proteins leads to such diverse tumor types, there must be a selection on the multipotent stem cell level. In this review, we will focus on the wide variety of soft tissue and bone entities, including benign and malignant lesions, harboring EWSR1 rearrangement. Fusion gene analysis is the diagnostic gold standard in most of these tumors. We present clinicopathologic, immunohistochemical, and molecular features and discuss differential diagnoses.

Pterygium-The Good, the Bad, and the Ugly.

Pterygium is a multifaceted pathology that displays apparent conflicting characteristics: benign (e.g., self-limiting and superficial), bad (e.g., proliferative and potentially recurrent) and ugly (e.g., signs of preneoplastic transformation). The natural successive question is: why are we lacking reports showing that pterygium lesions become life-threatening through metastasis, especially since pterygium has considerable similarities with UV-related malignancies on the molecular level? In this review, we consider how our pathophysiological understanding of the benign pterygium pathology overlaps with ocular surface squamous neoplasia and skin cancer. The three UV-related disorders share the same initial insult (i.e., UV radiation) and responsive repair mechanisms to the ensuing (in)direct DNA damage. Their downstream apoptotic regulators and other cellular adaptations are remarkably alike. However, a complicating factor in understanding the fine line between the self-limiting nature of pterygium and the malignant transformation in other UV-related diseases is the prominent ambiguity in the pathological evaluation of pterygium biopsies. Features of preneoplastic transformation (i.e., dysplasia) are used to define normal cellular reactions (i.e., atypia and metaplasia) and vice versa. A uniform grading system could help in unraveling the true nature of this ancient disease and potentially help in identifying the earliest intervention point possible regarding the cellular switch that drives a cell's fate towards cancer.

NTRK Fusions in Sarcomas: Diagnostic Challenges and Clinical Aspects.

Tropomyosin receptor kinase (TK) is encoded by the neurotrophic tyrosine receptor kinase genes (NTRK) 1, 2, and 3, whose activation plays an important role in cell cycle proliferation and survival. Fusions of one of these genes can lead to constitutive activation of TRK, which can potentially be oncogenic. NTRK fusions are commonly present in rare histologic tumor types. Among sarcomas, infantile fibrosarcoma shows NTRK fusion in more than 90% of the cases. Many other sarcoma types are also investigated for NTRK fusions. These fusions are druggable alteration of the agnostic type, meaning that all NTRK fused tumors can be treated with NTRK-inhibitors regardless of tumor type or tissue of origin. TRK-inhibitors have shown good response rates, with durable effects and limited side effects. Resistance to therapy will eventually occur in some cases, wherefore the next-generation TRK-inhibitors are introduced. The diagnosis of NTRK fused tumors, among them sarcomas, remains an issue, as many algorithms but no guidelines exist to date. Given the importance of this diagnosis, in this paper we aim to (1) analyze the histopathological features of sarcomas that correlate more often with NTRK fusions, (2) give an overview of the TRK-inhibitors and the problems that arise from resistance to the therapy, and (3) discuss the diagnostic algorithms of NTRK fused tumors with emphasis on sarcomas.

Prognostic and Predictive Biomarkers in Non-Small Cell Lung Cancer Patients on Immunotherapy-The Role of Liquid Biopsy in Unraveling the Puzzle.

In the last decade, immunotherapy has been one of the most important advances in the non-small cell lung cancer (NSCLC) treatment landscape. Nevertheless, only a subset of NSCLC patients benefits from it. Currently, the only Food and Drug Administration (FDA) approved diagnostic test for first-line immunotherapy in metastatic NSCLC patients uses tissue biopsies to determine the programmed death ligand 1 (PD-L1) status. However, obtaining tumor tissue is not always feasible and puts the patient at risk. Liquid biopsy, which refers to the tumor-derived material present in body fluids, offers an alternative approach. This less invasive technique gives real-time information on the tumor characteristics. This review addresses different promising liquid biopsy based biomarkers in NSCLC patients that enable the selection of patients who benefit from immunotherapy and the monitoring of patients during this therapy. The challenges and the opportunities of blood-based biomarkers such as cell-free DNA (cfDNA), circulating tumor cells (CTCs), exosomes, epigenetic signatures, microRNAs (miRNAs) and the T cell repertoire will be addressed. This review also focuses on the less-studied feces-based and breath-based biomarkers.

Auranofin reveals therapeutic anticancer potential by triggering distinct molecular cell death mechanisms and innate immunity in mutant p53 non-small cell lung cancer.

Auranofin (AF) is an FDA-approved antirheumatic drug with anticancer properties that acts as a thioredoxin reductase 1 (TrxR) inhibitor. The exact mechanisms through which AF targets cancer cells remain elusive. To shed light on the mode of action, this study provides an in-depth analysis on the molecular mechanisms and immunogenicity of AF-mediated cytotoxicity in the non-small cell lung cancer (NSCLC) cell line NCI-H1299 (p53 Null) and its two isogenic derivates with mutant p53 R175H or R273H accumulation. TrxR is highly expressed in a panel of 72 NSCLC patients, making it a valid druggable target in NSCLC for AF. The presence of mutant p53 overexpression was identified as an important sensitizer for AF in (isogenic) NSCLC cells as it was correlated with reduced thioredoxin (Trx) levels in vitro. Transcriptome analysis revealed dysregulation of genes involved in oxidative stress response, DNA damage, granzyme A (GZMA) signaling and ferroptosis. Although functionally AF appeared a potent inhibitor of GPX4 in all NCI-H1299 cell lines, the induction of lipid peroxidation and consequently ferroptosis was limited to the p53 R273H expressing cells. In the p53 R175H cells, AF mainly induced large-scale DNA damage and replication stress, leading to the induction of apoptotic cell death rather than ferroptosis. Importantly, all cell death types were immunogenic since the release of danger signals (ecto-calreticulin, ATP and HMGB1) and dendritic cell maturation occurred irrespective of (mutant) p53 expression. Finally, we show that AF sensitized cancer cells to caspase-independent natural killer cell-mediated killing by downregulation of several key targets of GZMA. Our data provides novel insights on AF as a potent, clinically available, off-patent cancer drug by targeting mutant p53 cancer cells through distinct cell death mechanisms (apoptosis and ferroptosis). In addition, AF improves the innate immune response at both cytostatic (natural killer cell-mediated killing) and cytotoxic concentrations (dendritic cell maturation).