Mass cytometry analysis reveals a distinct immune environment in peritoneal fluid in endometriosis: a characterisation study.

BACKGROUND: Endometriosis is a gynaecological condition characterised by immune cell infiltration and distinct inflammatory signatures found in the peritoneal cavity. In this study, we aim to characterise the immune microenvironment in samples isolated from the peritoneal cavity in patients with endometriosis. METHODS: We applied mass cytometry (CyTOF), a recently developed multiparameter single-cell technique, in order to characterise and quantify the immune cells found in peritoneal fluid and peripheral blood from endometriosis and control patients. RESULTS: Our results demonstrate the presence of more than 40 different distinct immune cell types within the peritoneal cavity. This suggests that there is a complex and highly heterogeneous inflammatory microenvironment underpinning the pathology of endometriosis. Stratification by clinical disease stages reveals a dynamic spectrum of cell signatures suggesting that adaptations in the inflammatory system occur due to the severity of the disease. Notably, among the inflammatory microenvironment in peritoneal fluid (PF), the presence of CD69+ T cell subsets is increased in endometriosis when compared to control patient samples. On these CD69+ cells, the expression of markers associated with T cell function are reduced in PF samples compared to blood. Comparisons between CD69+ and CD69- populations reveal distinct phenotypes across peritoneal T cell lineages. Taken together, our results suggest that both the innate and the adaptive immune system play roles in endometriosis. CONCLUSIONS: This study provides a systematic characterisation of the specific immune environment in the peritoneal cavity and identifies cell immune signatures associated with endometriosis. Overall, our results provide novel insights into the specific cell phenotypes governing inflammation in patients with endometriosis. This prospective study offers a useful resource for understanding disease pathology and opportunities for identifying therapeutic targets.

Rogaratinib: A potent and selective pan-FGFR inhibitor with broad antitumor activity in FGFR-overexpressing preclinical cancer models.

Aberrant activation in fibroblast growth factor signaling has been implicated in the development of various cancers, including squamous cell lung cancer, squamous cell head and neck carcinoma, colorectal and bladder cancer. Thus, fibroblast growth factor receptors (FGFRs) present promising targets for novel cancer therapeutics. Here, we evaluated the activity of a novel pan-FGFR inhibitor, rogaratinib, in biochemical, cellular and in vivo efficacy studies in a variety of preclinical cancer models. In vitro kinase activity assays demonstrate that rogaratinib potently and selectively inhibits the activity of FGFRs 1, 2, 3 and 4. In line with this, rogaratinib reduced proliferation in FGFR-addicted cancer cell lines of various cancer types including lung, breast, colon and bladder cancer. FGFR and ERK phosphorylation interruption by rogaratinib treatment in several FGFR-amplified cell lines suggests that the anti-proliferative effects are mediated by FGFR/ERK pathway inhibition. Furthermore, rogaratinib exhibited strong in vivo efficacy in several cell line- and patient-derived xenograft models characterized by FGFR overexpression. The observed efficacy of rogaratinib strongly correlated with FGFR mRNA expression levels. These promising results warrant further development of rogaratinib and clinical trials are currently ongoing (ClinicalTrials.gov Identifiers: NCT01976741, NCT03410693, NCT03473756).

Pan-mutant IDH1 inhibitor BAY 1436032 for effective treatment of IDH1 mutant astrocytoma in vivo.

Mutations in codon 132 of isocitrate dehydrogenase (IDH) 1 are frequent in diffuse glioma, acute myeloid leukemia, chondrosarcoma and intrahepatic cholangiocarcinoma. These mutations result in a neomorphic enzyme specificity which leads to a dramatic increase of intracellular D-2-hydroxyglutarate (2-HG) in tumor cells. Therefore, mutant IDH1 protein is a highly attractive target for inhibitory drugs. Here, we describe the development and properties of BAY 1436032, a pan-inhibitor of IDH1 protein with different codon 132 mutations. BAY 1436032 strongly reduces 2-HG levels in cells carrying IDH1-R132H, -R132C, -R132G, -R132S and -R132L mutations. Cells not carrying IDH mutations were unaffected. BAY 1436032 did not exhibit toxicity in vitro or in vivo. The pharmacokinetic properties of BAY 1436032 allow for oral administration. In two independent experiments, BAY 1436032 has been shown to significantly prolong survival of mice intracerebrally transplanted with human astrocytoma carrying the IDH1R132H mutation. In conclusion, we developed a pan-inhibitor targeting tumors with different IDH1R132 mutations.

Neuropeptide S receptor 1 is a nonhormonal treatment target in endometriosis.

Endometriosis is a common chronic inflammatory condition causing pelvic pain and infertility in women, with limited treatment options and 50% heritability. We leveraged genetic analyses in two species with spontaneous endometriosis, humans and the rhesus macaque, to uncover treatment targets. We sequenced DNA from 32 human families contributing to a genetic linkage signal on chromosome 7p13-15 and observed significant overrepresentation of predicted deleterious low-frequency coding variants in NPSR1, the gene encoding neuropeptide S receptor 1, in cases (predominantly stage III/IV) versus controls (P = 7.8 × 10-4). Significant linkage to the region orthologous to human 7p13-15 was replicated in a pedigree of 849 rhesus macaques (P = 0.0095). Targeted association analyses in 3194 surgically confirmed, unrelated cases and 7060 controls revealed that a common insertion/deletion variant, rs142885915, was significantly associated with stage III/IV endometriosis (P = 5.2 × 10-5; odds ratio, 1.23; 95% CI, 1.09 to 1.39). Immunohistochemistry, qRT-PCR, and flow cytometry experiments demonstrated that NPSR1 was expressed in glandular epithelium from eutopic and ectopic endometrium, and on monocytes in peritoneal fluid. The NPSR1 inhibitor SHA 68R blocked NPSR1-mediated signaling, proinflammatory TNF-α release, and monocyte chemotaxis in vitro (P < 0.01), and led to a significant reduction of inflammatory cell infiltrate and abdominal pain (P < 0.05) in a mouse model of peritoneal inflammation as well as in a mouse model of endometriosis. We conclude that the NPSR1/NPS system is a genetically validated, nonhormonal target for the treatment of endometriosis with likely increased relevance to stage III/IV disease.

TGF-ß1 potentiates Vγ9Vδ2 T cell adoptive immunotherapy of cancer.

Despite its role in cancer surveillance, adoptive immunotherapy using γδ T cells has achieved limited efficacy. To enhance trafficking to bone marrow, circulating Vγ9Vδ2 T cells are expanded in serum-free medium containing TGF-ß1 and IL-2 (γδ[T2] cells) or medium containing IL-2 alone (γδ[2] cells, as the control). Unexpectedly, the yield and viability of γδ[T2] cells are also increased by TGF-ß1, when compared to γδ[2] controls. γδ[T2] cells are less differentiated and yet display increased cytolytic activity, cytokine release, and antitumor activity in several leukemic and solid tumor models. Efficacy is further enhanced by cancer cell sensitization using aminobisphosphonates or Ara-C. A number of contributory effects of TGF-ß are described, including prostaglandin E2 receptor downmodulation, TGF-ß insensitivity, and upregulated integrin activity. Biological relevance is supported by the identification of a favorable γδ[T2] signature in acute myeloid leukemia (AML). Given their enhanced therapeutic activity and compatibility with allogeneic use, γδ[T2] cells warrant evaluation in cancer immunotherapy.

Therapy of thyroid carcinoma with the histone deacetylase inhibitor MS-275.

PURPOSE: Dysregulation of histone acetylation associated with an up-regulation of histone deacetylase (HDAC) activity is common in malignant tumours. Therefore, HDAC inhibitors were developed whose effects on proliferation and apoptosis have been shown in different tumour entities. Since non-iodide-concentrating thyroid carcinomas represent a therapeutic problem, this study addressed the effects of the HDAC inhibitor MS-275 on thyroid carcinoma cells. METHODS: After the antiproliferative effect of MS-275 had been proven in different human and rat thyroid carcinoma cell lines, FRO82-2, SW1736 and FTC133 cells were further investigated with respect to changes in apoptosis, cell cycle and metabolism by the annexin V/propidium iodide assay, FACS analysis and uptake experiments employing 3-O-methyl-D-(3H)glucose, fluoro-2-deoxy-D-glucose2 [5,6-(3)H] and 14C-aminoisobutyric acid (AIB). The induction of iodide transport and gene expression were investigated in 125iodide uptake experiments and real-time polymerase chain reaction (PCR). RESULTS: MS-275 induced a concentration- and time-dependent inhibition of proliferation in the thyroid carcinoma cell lines with varying IC50 values. In FRO82-2, SW1736 and FTC133 cells characterized by low, moderate and high sensitivity an up-regulation of p21CIP/WAF1 expression and G1 and/or G2 phase arrest were observed upon MS-275 exposure corresponding to the sensitivity of individual cell lines. In addition, high MS-275 concentrations increased the apoptotic cell fraction of FTC133 and SW1736 cells, whereas resistance to apoptosis and simultaneous up-regulation of Bcl-2 gene expression were observed in FRO82-2 cells. MS-275 treatment also mediated a concentration-dependent decrease of 3H-FDG uptake and an increased 3-O-methyl-D-(3H)glucose uptake in all thyroid carcinoma cell lines after 24 h, an increased uptake of both tracers in FTC133 cells after 48 h, and restored the functional activity of the sodium-iodide symporter in SW1736 and FTC133 cells up to 20- and 45-fold. CONCLUSION: MS-275 exerts dose-dependent antiproliferative effects including growth arrest, differentiation and apoptosis in some thyroid carcinoma cell lines and might, therefore, be considered for the treatment of anaplastic and non-iodide-concentrating thyroid carcinomas.

Author Correction: Amine oxidase 3 is a novel pro-inflammatory marker of oxidative stress in peritoneal endometriosis lesions.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Amine oxidase 3 is a novel pro-inflammatory marker of oxidative stress in peritoneal endometriosis lesions.

Endometriosis is a common gynaecological disease of women in reproductive age, and is thought to arise from retrograde menstruation and implantation of endometrial tissue, mostly into the peritoneal cavity. The condition is characterized by a chronic, unresolved inflammatory process thereby contributing to pain as cardinal symptom in endometriosis. Elevated reactive oxygen species (ROS) and oxidative stress have been postulated as factors in endometriosis pathogenesis. We here set out for a systematic study to identify novel mechanisms and pathways relating to oxidative stress in ectopic peritoneal lesions. Using combined proteomic and transcriptomic approaches, we identified novel targets including upregulated pro-oxidative enzymes, such as amine oxidase 3/vascular adhesion protein 1 (AOC3/VAP1) as well as downregulated protective factors, in particular alkenal reductase PTGR1 and methionine sulfoxide reductase. Consistent with an altered ROS landscape, we observed hemoglobin / iron overload, ROS production and lipid peroxidation in ectopic lesions. ROS-derived 4-hydroxy-2-nonenal induced interleukin IL-8 release from monocytes. Notably, AOC3 inhibitors provoked analgesic effects in inflammatory pain models in vivo, suggesting potential translational applicability.

Sagopilone crosses the blood-brain barrier in vivo to inhibit brain tumor growth and metastases.

The aim of this study was to determine the efficacy of sagopilone (ZK-EPO), a novel epothilone, compared with other anticancer agents in orthotopic models of human primary and secondary brain tumors. Autoradiography and pharmacokinetic analyses were performed on rats and mice to determine passage across the blood-brain barrier and organ distribution of sagopilone. Mice bearing intracerebral human tumors (U373 or U87 glioblastoma, MDA-MB-435 melanoma, or patient-derived non-small-cell lung cancer [NSCLC]) were treated with sagopilone 5-10 mg/kg, paclitaxel 8-12.5 mg/kg (or temozolomide, 100 mg/kg) or control (vehicle only). Tumor volume was measured to assess antitumor activity. Sagopilone crossed the blood-brain barrier in both rat and mouse models, leading to therapeutically relevant concentrations in the brain with a long half-life. Sagopilone exhibited significant antitumor activity in both the U373 and U87 models of human glioblastoma, while paclitaxel showed a limited effect in the U373 model. Sagopilone significantly inhibited the growth of tumors from CNS metastasis models (MDA-MB-435 melanoma and patient-derived Lu7187 and Lu7466 NSCLC) implanted in the brains of nude mice, in contrast to paclitaxel or temozolomide. Sagopilone has free access to the brain. Sagopilone demonstrated significant antitumor activity in orthotopic models of both glioblastoma and CNS metastases compared with paclitaxel or temozolomide, underlining the value of further research evaluating sagopilone in the treatment of brain tumors. Sagopilone is currently being investigated in a broad phase II clinical trial program, including patients with glioblastoma, NSCLC, breast cancer, and melanoma.

Lethal Poisoning of Cancer Cells by Respiratory Chain Inhibition plus Dimethyl α-Ketoglutarate.

Inhibition of oxidative phosphorylation (OXPHOS) by 1-cyclopropyl-4-(4-[(5-methyl-3-(3-[4-(trifluoromethoxy)phenyl]-1,2,4-oxadiazol-5-yl)-1H-pyrazol-1-yl)methyl]pyridin-2-yl)piperazine (BAY87-2243, abbreviated as B87), a complex I inhibitor, fails to kill human cancer cells in vitro. Driven by this consideration, we attempted to identify agents that engage in synthetically lethal interactions with B87. Here, we report that dimethyl α-ketoglutarate (DMKG), a cell-permeable precursor of α-ketoglutarate that lacks toxicity on its own, kills cancer cells when combined with B87 or other inhibitors of OXPHOS. DMKG improved the antineoplastic effect of B87, both in vitro and in vivo. This combination caused MDM2-dependent, tumor suppressor protein p53 (TP53)-independent transcriptional reprogramming and alternative exon usage affecting multiple glycolytic enzymes, completely blocking glycolysis. Simultaneous inhibition of OXPHOS and glycolysis provoked a bioenergetic catastrophe culminating in the activation of a cell death program that involved disruption of the mitochondrial network and activation of PARP1, AIFM1, and APEX1. These results unveil a metabolic liability of human cancer cells that may be harnessed for the development of therapeutic regimens.