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.

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.

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.

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.

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).

Suppression of antitumor T cell immunity by the oncometabolite (R)-2-hydroxyglutarate.

The oncometabolite (R)-2-hydroxyglutarate (R-2-HG) produced by isocitrate dehydrogenase (IDH) mutations promotes gliomagenesis via DNA and histone methylation. Here, we identify an additional activity of R-2-HG: tumor cell-derived R-2-HG is taken up by T cells where it induces a perturbation of nuclear factor of activated T cells transcriptional activity and polyamine biosynthesis, resulting in suppression of T cell activity. IDH1-mutant gliomas display reduced T cell abundance and altered calcium signaling. Antitumor immunity to experimental syngeneic IDH1-mutant tumors induced by IDH1-specific vaccine or checkpoint inhibition is improved by inhibition of the neomorphic enzymatic function of mutant IDH1. These data attribute a novel, non-tumor cell-autonomous role to an oncometabolite in shaping the tumor immune microenvironment.

Discovery of Rogaratinib (BAY 1163877): a pan-FGFR Inhibitor.

Rogaratinib (BAY 1163877) is a highly potent and selective small-molecule pan-fibroblast growth factor receptor (FGFR) inhibitor (FGFR1-4) for oral application currently being investigated in phase 1 clinical trials for the treatment of cancer. In this publication, we report its discovery by de novo structure-based design and medicinal chemistry optimization together with its pharmacokinetic profile.

Acetyl-CoA Carboxylase 1-Dependent Protein Acetylation Controls Breast Cancer Metastasis and Recurrence.

Breast tumor recurrence and metastasis represent the main causes of cancer-related death in women, and treatments are still lacking. Here, we define the lipogenic enzyme acetyl-CoA carboxylase (ACC) 1 as a key player in breast cancer metastasis. ACC1 phosphorylation was increased in invading cells both in murine and human breast cancer, serving as a point of convergence for leptin and transforming growth factor (TGF) ß signaling. ACC1 phosphorylation was mediated by TGFß-activated kinase (TAK) 1, and ACC1 inhibition was indispensable for the elevation of cellular acetyl-CoA, the subsequent increase in Smad2 transcription factor acetylation and activation, and ultimately epithelial-mesenchymal transition and metastasis induction. ACC1 deficiency worsened tumor recurrence upon primary tumor resection in mice, and ACC1 phosphorylation levels correlated with metastatic potential in breast and lung cancer patients. Given the demonstrated effectiveness of anti-leptin receptor antibody treatment in halting ACC1-dependent tumor invasiveness, our work defines a "metabolocentric" approach in metastatic breast cancer therapy.

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.

SIAH ubiquitin ligases regulate breast cancer cell migration and invasion independent of the oxygen status.

Seven-in-absentia homolog (SIAH) proteins are evolutionary conserved RING type E3 ubiquitin ligases responsible for the degradation of key molecules regulating DNA damage response, hypoxic adaptation, apoptosis, angiogenesis, and cell proliferation. Many studies suggest a tumorigenic role for SIAH2. In breast cancer patients SIAH2 expression levels correlate with cancer aggressiveness and overall patient survival. In addition, SIAH inhibition reduced metastasis in melanoma. The role of SIAH1 in breast cancer is still ambiguous; both tumorigenic and tumor suppressive functions have been reported. Other studies categorized SIAH ligases as either pro- or antimigratory, while the significance for metastasis is largely unknown. Here, we re-evaluated the effects of SIAH1 and SIAH2 depletion in breast cancer cell lines, focusing on migration and invasion. We successfully knocked down SIAH1 and SIAH2 in several breast cancer cell lines. In luminal type MCF7 cells, this led to stabilization of the SIAH substrate Prolyl Hydroxylase Domain protein 3 (PHD3) and reduced Hypoxia-Inducible Factor 1α (HIF1α) protein levels. Both the knockdown of SIAH1 or SIAH2 led to increased apoptosis and reduced proliferation, with comparable effects. These results point to a tumor promoting role for SIAH1 in breast cancer similar to SIAH2. In addition, depletion of SIAH1 or SIAH2 also led to decreased cell migration and invasion in breast cancer cells. SIAH knockdown also controlled microtubule dynamics by markedly decreasing the protein levels of stathmin, most likely via p27(Kip1). Collectively, these results suggest that both SIAH ligases promote a migratory cancer cell phenotype and could contribute to metastasis in breast cancer.

BAY 87-2243, a novel inhibitor of hypoxia-induced gene activation, improves local tumor control after fractionated irradiation in a schedule-dependent manner in head and neck human xenografts.

BACKGROUND: The transcription factor hypoxia-inducible factor-1 (HIF-1) pathway plays an important role in tumor response to cytotoxic treatments. We investigated the effects of a novel small molecule inhibitor of mitochondrial complex I and hypoxia-induced HIF-1 activity BAY-87-2243, on tumor microenvironment and response of human squamous cell carcinoma (hSCC) to clinically relevant fractionated radiotherapy (RT) with and without concomitant chemotherapy. METHODS: When UT-SCC-5 hSCC xenografts in nude mice reached 6 mm in diameter BAY-87-2243 or carrier was administered before and/or during RT or radiochemotherapy with concomitant cisplatin (RCT). Local tumor control was evaluated 150 days after irradiation and the doses to control 50% of tumors (TCD50) were compared between treatment arms. Tumors were excised at different time points during BAY-87-2243 or carrier treatment for western blot and immunohistological investigations. RESULTS: BAY-87-2243 markedly decreased nuclear HIF-1α expression and pimonidazole hypoxic fraction already after 3 days of drug treatment. BAY-87-2243 prior to RT significantly reduced TCD50 from 123 to 100 Gy (p=0.037). Additional BAY-87-2243 application during RT did not decrease TCD50. BAY-87-2243 before and during radiochemotherapy did not improve local tumor control. CONCLUSIONS: Pronounced reduction of tumor hypoxia by application of BAY-87-2243 prior to RT improved local tumor control. The results demonstrate that radiosensitizing effect importantly depends on treatment schedule. The data support further investigations of HIF-1 pathway inhibitors for radiotherapy and of predictive tests to select patients who will benefit from this combined treatment.

Inhibition of hypoxia-induced gene transcription by substituted pyrazolyl oxadiazoles: initial lead generation and structure-activity relationships.

The transcription factors hypoxia-inducible factor-1 and -2 (HIF-1 and HIF-2) orchestrate a multitude of processes that allow tumor cells to survive under conditions of low oxygen and nutrients, and that lead to resistance to some apoptotic pathways and facilitate invasion and metastasis. Therefore, inhibition of transactivation by HIF has become an attractive target in cancer research. Herein we present the results of a cell-based screening approach that led to the discovery of substituted 1H-pyrazole-3-carboxamides. Chemical optimization of the hit class with respect to potency and metabolic stability is described; it resulted in novel 5-(1H-pyrazol-3-yl)-1,2,4-oxadiazoles that inhibit the hypoxia-induced accumulation of HIF-1α and HIF-2α. The HIF inhibitory potency in the screening cell system was improved from IC50 190 to 0.7 nM, and significant parts of the SAR are disclosed. For a key compound, the ability to suppress the hypoxia-induced expression of HIF target genes was studied in A549 human lung adenocarcinoma cells. The same compound shows a favorable pharmacokinetic profile in rats after i.v. and p.o. administration.

Hypoxia-inducible factor pathway inhibition resolves tumor hypoxia and improves local tumor control after single-dose irradiation.

PURPOSE: To study the effects of BAY-84-7296, a novel orally bioavailable inhibitor of mitochondrial complex I and hypoxia-inducible factor 1 (HIF-1) activity, on hypoxia, microenvironment, and radiation response of tumors. METHODS AND MATERIALS: UT-SCC-5 and UT-SCC-14 human squamous cell carcinomas were transplanted subcutaneously in nude mice. When tumors reached 4 mm in diameter BAY-84-7296 (Bayer Pharma AG) or carrier was daily administered to the animals. At 7 mm tumors were either excised for Western blot and immunohistologic investigations or were irradiated with single doses. After irradiation animals were randomized to receive BAY-84-7296 maintenance or carrier. Local tumor control was evaluated 150 days after irradiation, and the dose to control 50% of tumors (TCD50) was calculated. RESULTS: BAY-84-7296 decreased nuclear HIF-1α expression. Daily administration of inhibitor for approximately 2 weeks resulted in a marked decrease of pimonidazole hypoxic fraction in UT-SCC-5 (0.5% vs 21%, P<.0001) and in UT-SCC-14 (0.3% vs 19%, P<.0001). This decrease was accompanied by a significant increase in fraction of perfused vessels in UT-SCC-14 but not in UT-SCC-5. Bromodeoxyuridine and Ki67 labeling indices were significantly reduced only in UT-SCC-5. No significant changes were observed in vascular area or necrosis. BAY-84-7296 before single-dose irradiation significantly decreased TCD50, with an enhancement ratio of 1.37 (95% confidence interval [CI] 1.13-1.72) in UT-SCC-5 and of 1.55 (95% CI 1.26-1.94) in UT-SCC-14. BAY-84-7296 maintenance after irradiation did not further decrease TCD50. CONCLUSIONS: BAY-84-7296 resulted in a marked decrease in tumor hypoxia and substantially reduced radioresistance of tumor cells with the capacity to cause a local recurrence after irradiation. The data suggest that reduction of cellular hypoxia tolerance by BAY-84-7296 may represent the primary biological mechanism underlying the observed enhancement of radiation response. Whether this mechanism contributes to the improved outcome of fractionated chemoradiation therapy warrants further investigation.

BAY 87-2243, a highly potent and selective inhibitor of hypoxia-induced gene activation has antitumor activities by inhibition of mitochondrial complex I.

The activation of the transcription factor hypoxia-inducible factor-1 (HIF-1) plays an essential role in tumor development, tumor progression, and resistance to chemo- and radiotherapy. In order to identify compounds targeting the HIF pathway, a small molecule library was screened using a luciferase-driven HIF-1 reporter cell line under hypoxia. The high-throughput screening led to the identification of a class of aminoalkyl-substituted compounds that inhibited hypoxia-induced HIF-1 target gene expression in human lung cancer cell lines at low nanomolar concentrations. Lead structure BAY 87-2243 was found to inhibit HIF-1α and HIF-2α protein accumulation under hypoxic conditions in non-small cell lung cancer (NSCLC) cell line H460 but had no effect on HIF-1α protein levels induced by the hypoxia mimetics desferrioxamine or cobalt chloride. BAY 87-2243 had no effect on HIF target gene expression levels in RCC4 cells lacking Von Hippel-Lindau (VHL) activity nor did the compound affect the activity of HIF prolyl hydroxylase-2. Antitumor activity of BAY 87-2243, suppression of HIF-1α protein levels, and reduction of HIF-1 target gene expression in vivo were demonstrated in a H460 xenograft model. BAY 87-2243 did not inhibit cell proliferation under standard conditions. However under glucose depletion, a condition favoring mitochondrial ATP generation as energy source, BAY 87-2243 inhibited cell proliferation in the nanomolar range. Further experiments revealed that BAY 87-2243 inhibits mitochondrial complex I activity but has no effect on complex III activity. Interference with mitochondrial function to reduce hypoxia-induced HIF-1 activity in tumors might be an interesting therapeutic approach to overcome chemo- and radiotherapy-resistance of hypoxic tumors.

LDH-A influences hypoxia-inducible factor 1α (HIF1 α) and is critical for growth of HT29 colon carcinoma cells in vivo.

Serum lactate dehydrogenase (LDH) is a well-known clinical surrogate parameter. A high activity of LDH is associated with a poor prognosis in different tumor types. Here we demonstrate by a gene silencing approach that LDH-A is critical for in vivo but not in vitro growth of HT29 colon carcinoma cells. We provide evidence that the suppression of the LDH-A gene leads to an increased level of hypoxia inducible factor 1α (HIF1α) but in consequence not to an increase of HIF1 regulated proteins such as carbonic anhydrase IX (CAIX), vascular endothelial growth factor (VEGF), prolyl-hydroxylase 2 (PHD2), and factor-inhibiting HIF (FIH) in cell cultures and tumor lysates. This effect is independent of LDH activity in vivo. We conclude that LDH-A has an influence on the activity of HIF1α and thus on the adaptation of cells to a hypoxic tumor microenvironment in HT29 colon cells. We suggest the use of LDH-M as a potential therapeutic target for anticancer treatment.

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.

Efficacy of MS-275, a selective inhibitor of class I histone deacetylases, in human colon cancer models.

N-(2-aminophenyl)-4-[N-(pyridine-3yl-methoxy-carbonyl) aminomethyl] benzamide (MS-275) is a second generation histone deacetylase (HDAC) inhibitor with significant anti-tumor efficacy currently in clinical development. We investigated the effect of MS-275 treatment on various colon cancer cell lines, as well as on mouse xenograft models derived from human colorectal cancer. MS-275 exerted strong anti-proliferative effects in five cell lines and increased the acetylation of histones 3 and 4. In vivo testing of the compound in eight different models of human colon cancer derived from primary colorectal cancers or from established cell lines revealed that five models were responders, two non-responders and one an anti-responder. Gene expression profiles were determined in order to identify genes and pathways differentially regulated upon MS-275 treatment in responder versus non-responder models. Principle component analysis revealed a correlation of the anti-tumor efficacy with the sub-clustering of the MS-275 treatment groups in 7 out of 8 models. Although the overall gene expression pattern was rather unique for each individual model, 129 genes were significantly up- and 58 genes significantly down-regulated in at least 2 out of 5 responder models in response to MS-275 treatment. We identified potential biomarkers for response to MS-275, such as PRA1, MYADM and PALM2-AKAP2 which were up-regulated in all responder models and down-regulated or unchanged in all non-responder models. Our results provide a starting point for the development of clinically relevant biomarkers for predicting a response to MS-275 and the understanding of the mode of action of this HDAC inhibitor.

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.