The biological links between cancer and pregnancy are of interest due to parallel proliferative, immunosuppressive, and invasive mechanisms between tumour and placental cells. However, the proliferation and invasion of placental cells are strictly regulated. The understanding of this regulation is largely unknown. Placental extracellular vesicles (EVs) may play an important role in this regulation, as placental EVs are known to contribute to maternal adaptation, including adaptation of the vascular and immune systems. We have previously reported that placental EVs significantly inhibited ovarian cancer cell proliferation by delaying the progression of the cell cycle. We, therefore, performed this pilot in vivo study to investigate whether placental EVs can also inhibit ovarian tumour growth in a SKOV-3 human tumour xenograft model. A single intraperitoneal injection of placental EVs at 15 days post tumour implantation, significantly inhibited the growth of the tumours in our in vivo model. Signs of cellular necrosis were observed in the ovarian tumour tissues, but not in other organs collected from mice that had been treated with placental EVs. Expression of receptor-interacting kinase 1 (RIPK1) and mixed linkage kinase domain-like (MLKL), which are mediators of necroptosis were not observed in our xenografted tumours. However, extensive infiltration of CD169+ macrophages and NK cells in ovarian tumour tissues collected from placental micro-EVs treated mice were observed. We demonstrate here that inhibition of ovarian tumour growth in our xenograft model by placental EVs involves cellular necrosis and infiltration of CD169+ macrophages and NK cells into the tumour tissues.

Indoleamine 2, 3-dioxygenase 1 (IDO1) is commonly expressed by cancers as a mechanism for evading the immune system. Preclinical and clinical studies have indicated the potential of combining IDO1 inhibitors with immune therapies for the treatment of cancer, strengthening an interest in the discovery of novel dioxygenase inhibitors for reversing tumour-mediated immune suppression. To facilitate the discovery, development and investigation of novel small molecule inhibitors of IDO1 and its hepatic isozyme tryptophan dioxygenase (TDO2), murine tumour cells were engineered to selectively express either murine or human IDO1 and TDO2 for use as tools to dissect both the species specificity and isoenzyme selectivity of newly discovered inhibitors. Lewis lung carcinoma (LLTC) lines were engineered to express either murine or human IDO1 for use to test species selectivity of the novel inhibitors; in addition, GL261 glioma lines were engineered to express either human IDO1 or human TDO2 and used to test the isoenzyme selectivity of individual inhibitors in cell-based assays. The 20 most potent inhibitors against recombinant human IDO1 enzyme, discovered from a commissioned screening of 40,000 compounds in the Australian WEHI compound library, returned comparable IC50 values against murine or human IDO1 in cell-based assays using the LLTC-mIDO1 and LLTC-hIDO1 line, respectively. To test the in vivo activity of the hits, transfected lines were inoculated into syngeneic C57Bl/6 mice. Individual LLTC-hIDO1 tumours showed variable expression of human IDO1 in contrast to GL261-hIDO1 tumours which were homogenous in their IDO1 expression and were subsequently used for in vivo studies. W-0019482, the most potent IDO1 inhibitor identified from cell-based assays, reduced plasma and intratumoural ratios of kynurenine to tryptophan (K:T) and delayed the growth of subcutaneous GL261-hIDO1 tumours in mice. Synthetic modification of W-0019482 generated analogues with dual IDO1/TDO2 inhibitory activity, as well as inhibitors that were selective for either TDO2 or IDO1. These results demonstrate the versatility of W-0019482 as a lead in generating all three subclasses of tryptophan dioxygenase inhibitors which can be applied for investigating the individual roles and interactions between IDO1 and TDO2 in driving cancer-mediated immune suppression.

The expression of tryptophan catabolising enzyme indoleamine 2,3-dioxygenase 1 (IDO1) or tryptophan 2,3-dioxygenase 2 (TDO2) in cancers is associated with suppressed immunity and poor patient prognosis. Results from human clinical trials of IDO1 inhibitors have been disappointing. There is now a strong interest in the development of TDO2-selective or dual IDO1/TDO2 inhibitors that may surpass IDO1 inhibitors by providing broader efficacy and blocking constitutively-expressed hepatic TDO2. To expedite the discovery of novel TDO2-specific and dual inhibitors, an assay that enabled the efficient and accurate measurement of the inhibitory activity of compounds against both IDO1 and TDO2 enzymes, concurrently in the same experiment was established to screen 5,682 compounds that included the National Cancer Institute Diversity set 5, for inhibition of IDO1 and TDO2 activity. This screen identified 82 compounds that inhibited either IDO1, TDO2 or both enzymes > 50% at 20 µM. Thirty Pan Assay Interference compounds were removed from the list and the IC50 of the remaining 52 compounds against IDO1 and TDO2 was subsequently determined using the newly-developed concurrent assay. Ten compounds were confirmed as dual IDO1/TDO2 inhibitors having IC50 values under 50 µM against both enzymes and within 2-fold of each other. Six compounds with IC50 values between 1.39 and 8.41 µM were identified as potential TDO2-selective leads. The use of this concurrent protocol is anticipated to expedite the discovery of novel leads for dual and selective inhibitors against IDO1 and or TDO2 and speed the evaluation of novel analogues that will ensue.

Breast cancer is the most common cancer in women worldwide. Accurate early diagnosis of breast cancer is critical in the management of the disease. Although mammogram screening has been widely used for breast cancer screening, high false-positive and false-negative rates and radiation from mammography have always been a concern. Over the last 20 years, the emergence of “omics” strategies has resulted in significant advances in the search for non-invasive biomarkers for breast cancer diagnosis at an early stage. Circulating carcinoma antigens, circulating tumor cells, circulating cell-free tumor nucleic acids (DNA or RNA), circulating microRNAs, and circulating extracellular vesicles in the peripheral blood, nipple aspirate fluid, sweat, urine, and tears, as well as volatile organic compounds in the breath, have emerged as potential non-invasive diagnostic biomarkers to supplement current clinical approaches to earlier detection of breast cancer. In this review, we summarize the current progress of research in these areas.

Objective Ovarian cancer is a common gynecological cancer, and parity is negatively associated with the incidence of this disease. This negative association is hypothesized to be due in part to shifting the balance of estrogen and progesterone toward more progesterone and reduced ovulation during pregnancy. However, studies suggested that parity is also associated with estrogen-independent gynecological cancers suggesting balance of hormones may not be the only protective factor. Extracellular vesicles (EVs) play an important role in cell-to-cell communication in physiological and pathological conditions. During pregnancy, large amounts of EVs are extruded from the placenta, and they seem to be involved in the remarkable adaptation of a woman's body to normal pregnancy. We hypothesized that EVs extruded from the placenta play a role in this protective effect. Methods Placental EVs were collected from first-trimester placentae, and cancer cell EVs were isolated from ovarian cancer cells. The EVs were exposed to ovarian cancer cells for 48 hours. The proliferation of cancer cells and the cell cycle were measured. In addition, phagocytosis of deported placental EVs by cancer cells was also measured. Results The proliferation of cancer cells was significantly reduced by treatment with placental EVs (P = 0.001, analysis of variance), but not EVs from monocytes (P = 0.195), compared with untreated cancer cells. Furthermore, placental EVs also prevented the proliferation of cancer cells induced by cancer cell-derived EVs (P = 0.001). This inhibition of proliferation of ovarian cancer cells was partially due to phagocytosis of placental EVs by cancer cells. Phagocytosis of placental EVs delayed progression through the cell cycle. Calreticulin, a phagocytic eat me signal carried by placental EVs significantly inhibited ovarian cancer growth (P = 0.001). Conclusions Our data demonstrated that EVs extruded from the placenta prevented ovarian cancer cell growth by a mechanism that involved delaying progression of the cell cycle after phagocytosis of the EVs.

BACKGROUND:Depletion of tryptophan and the accumulation of tryptophan metabolites mediated by the immunosuppressive enzyme indoleamine 2,3-dioxygenase 1 (IDO1), trigger immune cells to undergo apoptosis. However, cancer cells in the same microenvironment appear not to be affected. Mechanisms whereby cancer cells resist accelerated tryptophan degradation are not completely understood. We hypothesize that cancer cells co-opt IMPACT (the product of IMPrinted and AnCienT gene), to withstand periods of tryptophan deficiency.METHODS:A range of bioinformatic techniques including correlation and gene set variation analyses was applied to genomic datasets of cancer (The Cancer Genome Atlas) and normal (Genotype Tissue Expression Project) tissues to investigate IMPACT's role in cancer. Survival of IMPACT-overexpressing GL261 glioma cells and their wild type counterparts cultured in low tryptophan media was assessed using fluorescence microscopy and MTT bio-reduction assay. Expression of the Integrated Stress Response proteins was measured using Western blotting.RESULTS:We found IMPACT to be upregulated and frequently amplified in a broad range of clinical cancers relative to their non-malignant tissue counterparts. In a subset of clinical cancers, high IMPACT expression associated with decreased activity of pathways and genes involved in stress response and with increased activity of translational regulation such as the mTOR pathway. Experimental studies using the GL261 glioma line showed that cells engineered to overexpress IMPACT, gained a survival advantage over wild-type lines when cultured under limiting tryptophan concentrations. No significant difference in the expression of proteins in the Integrated Stress Response pathway was detected in tryptophan-deprived GL261 IMPACT-overexpressors compared to that in wild-type cells. IMPACT-overexpressing GL261 cells but not their wild-type counterparts, showed marked enlargement of their nuclei and cytoplasmic area when stressed by tryptophan deprivation.CONCLUSIONS:The bioinformatics data together with our laboratory studies, support the hypothesis that IMPACT mediates a protective mechanism allowing cancer cells to overcome microenvironmental stresses such as tryptophan deficiency.

Cancers co-opt indoleamine 2,3-dioxygenase 1 (IDO1) as a mechanism for evading the immune system, and IDO1 inhibitors have emerged as a promising new approach for the treatment cancer through their potential to restore antitumor immunity and to synergise with existing therapies. As part of our investigations into novel small molecule inhibitors of IDO1, we screened a number of human and murine tumor lines for expression of tryptophan dioxygenases. All the human and murine tumor lines that we tested were found not to express tryptophan dioxygenases unless induced with IFN-gamma. Our screen included testing 50 primary human melanoma lines developed from tumor samples obtained New Zealand Melanoma patients (NZMel lines) for expression of IDO1 and IDO2. After co-culture with IFN-gamma, 84% of the NZMel lines were IDO1+ and IDO2+; 4% were IDO1+ only; 2 % were IDO2+ only; whilst 10% did not express either IDO1 or IDO2. Tryprophan dioxygenase (TDO) was not detected in any of the NZMel lines before or after IFN-gamma exposure. To overcome the need for IFN-gamma induction for our cell based assays of IDO1 inhibitory activity of our novel compounds, we transfected the wild-type murine Lewis Lung carcinoma line (LLTC) to constitutively express murine IDO1 (LLTC-mIDO1) or human IDO1 (LLTC-hIDO1), and used these engineered lines to assay for potential species selectivity of our inhibitors. IDO1 expression in the lines was consistent and stable, but when LLTC-hIDO1 cells were implanted subcutaneously into syngeneic mice for in vivo testing of inhibitors, we found considerable heterogeneity in IDO1 expression between tumors. Approximately half of the tumors from the same batch of implants were completely negative for IDO1 expression by Western blot analysis. More intriguingly, when fragments of an IDO1+ or an IDO1- donor tumor were implanted into new recipients, the same degree of intertumoral heterogeneity in IDO1 expression was seen in the subsequent generation of tumors. In contrast, subcutaneous tumors developing from murine GL261 glioma cells similarly engineered to over-express hIDO1 (GL261-hIDO1) were homogeneous in their expression of hIDO1, and were used for subsequent in vivo studies of intratumoral IDO1expression on its effects on immune cell infiltrates and tumor growth. GL261-hIDO1 tumors had a significantly faster growth rate than the wild-type tumors, and 24 days after implantation, had reached a mean tumor volume of 3600 mm3 compared to a mean tumor volume of 500 mm3 of wild-type tumors. Analysis of the immune cell infiltrates, showed that 14-day GL261 wild-type tumors had a 3-fold higher percentage of CD3+ T cells than that in GL261-hIDO1 tumors. CD8+ cells made up 55% and 25% of the CD3+ cells in wild-type and GL261-hIDO1 tumors, respectively. Percentage of Foxp3+ (Treg) cells was higher in GL261-hIDO1 tumors compared to that in wild-type tumors. Citation Format: Lai-Ming Ching, Petr Tomek, Brian D. Palmer, Sofian Tijono, Kimiora Henare, Lukas M. Braun. In vitro and in vivo models for evaluation of inhibitors of tryptophan dioxygenases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4138. doi:10.1158/1538-7445.AM2017-4138

Abstract A broad range of human malignancies overexpress an enzyme called indoleamine 2,3-dioxygenase 1 (IDO1) to suppress antitumor immunity which associates with poor patient prognosis. One mechanism whereby IDO1 activity suppresses the host’s immune cells is mediated by deprivation of the essential amino acid tryptophan (Trp) which raises an intriguing question. How do the cancer cells overcome low levels of the essential amino acid when the immune T-cells are triggered to self-destruct? A protein called IMPACT (product of IMPrinted gene with AnCienT domain) was reported to confer skin cells and neuronal cells increased survival in Trp-deprived environments. Based on these reports, we hypothesise that cancer cells hijack IMPACT to gain survival advantage in Trp-deprived environments. To obtain evidence for this hypothesis, we analysed publicly available genomics datasets and performed experimental assays. Murine GL261 glioblastoma cells overexpressing a full-length mouse IMPACT gene (GL261-IMPACT) were generated by lipofection. IMPACT overexpression was confirmed by Western blot. Metabolic activity and survival of the GL261 cells in Trp deprivation experiments were evaluated using MTT assay and staining with live/dead indicators fluorescein diacetate and propidium iodide, respectively. Meta-analysis of the publicly available RNA-sequencing datasets, The Cancer Genome Atlas (TCGA) and Genotype Tissue Expression (GTEx), provided evidence that IMPACT was overexpressed in the majority of the 24 analysed human cancers compared to their non-malignant tissue counterparts. Consistent with the high IMPACT expression in prostate cancer patients, 12-25% prostate tumors show amplification of the IMPACT gene (source: cBioPortal for Cancer Genomics). In addition, we found an association of high IMPACT expression with poorer survival for certain leukemia and lung cancer patients in the Gene Expression Omnibus (GEO) portal. To obtain experimental evidence that IMPACT protects cancer cells during Trp deprivation, GL261-IMPACT cells were cultured in media containing 2.5 - 50 μM Trp. Metabolic activity of GL261-IMPACT cells decreased at a slower rate than that of GL261-wild-type cells after 3 to 5 days in media containing limiting Trp concentration (2.5 - 10 μM). On day 5, GL261-wild-type and GL261-IMPACT cells cultivated in 10 μM Trp showed 4.5% and 23% metabolic activity (p = 2.8x10-8), respectively, relative to the same cells grown in 50 μM Trp. Fluorescence microscopy utilising live/dead staining showed that after 5 days of incubation at limiting Trp concentrations (5 - 10 μM), the majority of GL261-wild-type cells were non-viable whereas GL261-IMPACT cells were predominantly viable. Our initial results support the hypothesis that IMPACT aids cancer cells to overcome periods of tryptophan deprivation. Citation Format: Petr Tomek, Ariane Hallermayr, Michael Kilian, Cristin Gregor Print, Lai-Ming Ching. The role of IMPACT in survival of cancer cells during tryptophan deprivation by immunosuppressive enzyme indoleamine 2,3-dioxygenase (IDO1) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4338. doi:10.1158/1538-7445.AM2017-4338

Abstract The functional plasticity of macrophages and their ability to support tumour growth or promote potent antitumor immunity make them an attractive target cancer therapy. Macrophages have been implicated in the complex antitumor activity of murine STING agonist, DMXAA, including disruption of tumor vasculature, induction of cytokine production, and promotion of durable antitumor immune responses. Many of these activities are lost in STING-/- mice, and DMXAA does not bind to human STING, providing a pertinent explanation for why the anti-tumour effects of DMXAA observed in mice were not recapitulated human clinical trials. Using PMA-differentiated THP-1 macrophages, we sought to identify human active analogues of DMXAA in vitro, comparing their capacity to induce cytokines indicative of a classically activated antitumour phenotype; the non-canonical STING agonist 2'3'-cGAMP was also included in our investigation. IP-10 (CXCL10) production, measured by ELISA, was used as an initial marker for activity, followed by multiplex cytokine analysis for broader characterisation compared to M(IFN-γ/LPS), M(IL-4/IL-13), and ‘resting’ M(media alone) macrophage phenotypes. Parallel experiments were also conducted with RAW264.7 murine macrophages to compare the DMXAA-induced cytokine profile directly with that of 2'3'-cGAMP. 2'3'-cGAMP induced 22-fold and 18-fold increases in IP-10 production in resting and M(IL-4/IL-13) THP-1 macrophages, respectively. By comparison, THP-1 macrophages were much less responsive to DMXAA and analogues tested to date. Furthermore, 2'3'-cGAMP induced a spectrum of cytokines similar to that observed in M(IFN-γ/LPS) macrophages, and markedly different to that of M(IL-4/IL-13) THP-1 macrophages. DMXAA, and analogues previously shown to have cytokine-inducing activity in human leukocytes did not produce cytokine spectra indicative of a classic M(IFN-γ/LPS) phenotype. Unlike human cells, however, cytokine spectra produced by RAW264.7 murine macrophages were comparable between DMXAA- and 2'3'-cGAMP-treated cells. These findings support previous data that suggest that STING agonists can promote macrophage polarisation towards an antitumour phenotype. While DMXAA is active in murine macrophages, only 2'3'-cGAMP can induce in human THP-1 cells, an M(IFN-γ/LPS)-like spectrum of cytokines often attributed to an antitumour macrophage phenotype. Citation Format: Kimiora L. Henare, Sofian M. Tijono, Lai-Ming Ching. Polarization of macrophages with STING agonists [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4691. doi:10.1158/1538-7445.AM2017-4691

Background: Virtual screening of compound libraries by molecular docking can help narrow down a large list of compounds to a more manageable size for testing.Fitness functions within molecular docking programme's software calculate how a compound fits the target site of the protein and assigns a numerical value quantifying how well it fits.Different molecules can be compared and ranked based on these values.As fitness functions calculate 'fitness' based on different parameters and perform differently depending on the properties of the target site, the success of a virtual screen can depend on the choice of the fitness function and the conformation of the target protein used for docking.We evaluated Goldscore, Astex Statistical Potential (ASP), Chemscore, ChemPLP fitness functions within the GOLD molecular docking suite to find the combination of fitness functions that performs the best for enrichment of inhibitors of the immunoregulatory enzyme indoleamine 2,3dioxygenase (IDO1).Methods: IDO1 inhibitory compounds were identified from a fragment library by sequential screening using differential scanning fluorimetry followed by enzyme inhibition assays.This same library was docked into three conformations of the IDO1 active site and rescored with combinations of the four fitness functions in the GOLD suite.The performance of the different scoring function combinations was evaluated by comparison to the empirical screen.Results: For binding mode calculations, all fitness functions in GOLD, except ASP, reproduced the known binding mode of 4-phenylimidazole to within 1.0Å RMSD.A larger active site conformation was important in reproducing known binding of the inhibitor Amg-1.With respect to early enrichment of inhibitors, the Chemscore fitness function performed the best when used to rank compounds.The top 5% of Chemscore-ranked libraries contained >25% of the inhibitors in the library.Only 5-15% of the inhibitors were found in the top 5% of Goldscore-ranked libraries.We also noted that different fitness functions selected different types of compounds.Chemscore ranked inhibitory naphthalene compounds highly, but failed to rank inhibitory benzoxazole and some benzothiazole compounds favourably.In contrast Goldscore ranks inhibitory naphthalene compounds inconsistently, but performs well with benzoxazole and benzothiazole inhibitors.Although apparently worse than Chemscore overall in this study, Goldscore is valuable as it is able to capture inhibitory compounds missed by Chemscore.The active site conformation had little effect on enrichment of fragments. Conclusions:A small pilot screen, such as the one presented here, can be useful for deciding on the docking parameters before embarking on more extensive screens.

High expression of the immunosuppressive enzyme, indoleamine 2,3-dioxygenase 1 (IDO1) for a broad range of malignancies is associated with poor patient prognosis, and the enzyme is a validated target for cancer intervention. To identify novel IDO1 inhibitors suitable for drug development, 1597 compounds in the National Cancer Institute Diversity Set III library were tested for inhibitory activity against recombinant human IDO1. We retrieved 35 hits that inhibited IDO1 activity >50% at 20 μM. Five structural filters and the PubChem Bioassay database were used to guide the selection of five inhibitors with IC50 between 3 and 12 μM for subsequent experimental evaluation. A pyrimidinone scaffold emerged as being the most promising. It showed excellent cell penetration, negligible cytotoxicity and passed four out of the five structural filters applied. To evaluate the importance of Ser167 and Cys129 residues in the IDO1 active site for inhibitor binding, the entire NCI library was subsequently screened against alanine-replacement mutant enzymes of these two residues. The results established that Ser167 but not Cys129 is important for inhibitory activity of a broad range of IDO1 inhibitors. Structure-activity-relationship studies proposed substituents interacting with Ser167 on four investigated IDO1 inhibitors. Three of these four Ser167 interactions associated with an increased IDO1 inhibition and were correctly predicted by molecular docking supporting Ser167 as an important mediator of potency for IDO1 inhibitors.

Abstract A broad range of cancers express the tryptophan catabolising enzyme indoleamine 2,3-dioxygenase 1 (IDO1) as a mechanism of suppressing the endogenous anti-tumour immunity. High IDO1 expression by their tumours is associated with poor patient prognosis and has prompted the discovery of small molecule inhibitors of the enzyme for development as anti-cancer agents. Towards this end, we carried out a high throughput screen of 42,000 small molecules in the Walter and Eliza Hall Institute for Medical Research compound library and retrieved over 30 good hits with diverse structures that inhibited recombinant human IDO1 with potencies in the low μM range. Initial structure activity relationship studies of the best hits led us to focus on one chemical class, which, although less potent than some of the other hits on the list, exhibited a number of features that make it more favourable for development as a therapeutic agent. SN35837, the non-optimised hit from the high throughput screen is highly cell permeable and displays improved potency in inhibiting cytoplasmic hIDO1 in cell-based assays, compared to that for enzymatic assays. Complete inhibition of IDO1 function is seen at concentrations that has no measurable effects on cell viability. The imidazole lead from New Links Genetics, NLG919 has decreased activity in cells, whilst Incyte's hydroxyamidine inhibitor INCB024360 has similar potency in cellular and enzymatic assays. The maximum tolerated dose SN35837 in C57Bl/6 mice given as a single intraperitoneal bolus dose was 500 mg/kg. Mice tolerated daily intraperitoneal or subcutaneous administrations of SN35837 at 150 mg/kg. Statistically significant inhibition of growth of subcutaneously implanted GL261 gliomas and Lewis Lung carcinomas in syngeneic hosts were achieved after daily administration of 75 mg/kg SN35837. Preliminary studies indicate that the non-optimised hit also synergises when given in combination with anti-PD1 and anti-CTLA-4 antibodies. We conclude that derivatives of SN35837 may provide effective and potent inhibitors of IDO1 suitable for development as anti-cancer agents. Citation Format: Lai-Ming Ching, Brian D. Palmer, Petr Tomek, Jack U. Flanagan, Kimiora Henare. A novel class of inhibitors of the immunosuppressive enzyme indoleamine 2,3-dioxygenase 1 (IDO1) with potential for the treatment of cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4469. doi:10.1158/1538-7445.AM2015-4469

Abstract The stromal cells of the tumor microenvironment play a critical role in tumor progression, and are therefore attractive targets for cancer therapy. We previously used the stromal targeting agent, 5,6-dimethylxanthenone-4-acetic acid (DMXAA), to demonstrate how tumor and stromal interactions can be manipulated to favor anti-tumor activity against human melanoma xenografts in immunodeficient mice, mediated by neutrophils and macrophages. An ideal stromal targeting agent would be effective against established metastases as well as primary tumors. In order to design such therapies, and to assess their potential, a better understanding of the tumor microenvironment at the various sites is required. The aim of this study was to characterize the tumor stroma of established B16-F10 murine melanoma tumors at subcutaneous, pulmonary, and intracranial sites, in syngeneic C57BL/6 mice. Immunofluorescence-immunohistochemistry and flow cytometry were used to identify infiltrating leukocyte populations within B16-F10 tumors at these different sites. Furthermore, the antitumor activity of DMXAA against this model was investigated. In subcutaneous tumors, monocytic myeloid cells (CD11b+ F4/80-/dim) and macrophages (CD11b+ F4/80+) were found predominantly around the tumor periphery. Lymphocyte infiltration was variable, with B-cells (CD19+ B220+) and T-cells (CD3+ CD4+ and CD3+ CD8+) rarely detected in some tumors, but scattered diffusely or formed dense lymphoid structures throughout necrotic regions in other tumors. Lung resident alveolar macrophages (CD11c+ CD11bdim F4/80+) appeared to be the most common leukocyte associated with metastatic foci, along with interstitial macrophages (CD11b+ F4/80+). In the presence of metastases, the total leukocyte abundance in the lungs was increased by nearly 70% (p < 0.05) compared to healthy lungs, owing largely to an increase in B-cells and T-cells scattered throughout the lung tissue; however these cells were not specifically associated with metastatic foci. In contrast, lymphocytes were scarcely detected in intracranial tumors or in brain tissue, where resident microglia (CD45dim CD11b+ CD11c+) were the most abundant. Microglia were found mainly at the invasive edge of intracranial tumors, while peripheral macrophages (CD45+ CD11b+ F4/80+) localised near vessels within the central core. Although DMXAA induced haemorrhagic necrosis in subcutaneous B16-F10 tumors, antitumor activity overall was modest, and no significant survival benefit was observed compared to untreated tumor-bearing mice. Common at all sites was the presence of myeloid cells associated with tumor tissue, including resident and peripheral macrophages. DMXAA was recently shown to be an agonist for stimulator of interferon genes (STING) protein which triggers IFN-β signalling and innate immune gene expression in macrophages. DMXAA induces innate immunity against melanoma xenografts, where a similar presence of myeloid cells is observed before treatment. However, this activity was modest in aggressive B16-F10 melanomas. The precise reasons for this are unclear, but may involve immunosuppressive mechanisms. Our center is developing novel compounds to overcome immune-related resistance, such as inhibitors of indoleamine-2,3-dioxygenase, to be used with vaccines against melanoma. B16 metastatic melanoma is one challenging model that will be used to test these. Citation Format: Kimiora Henare, Raymond Yung, P. Rod Dunbar, Cristin G. Print, Lai-Ming Ching. Characterizing the tumor stroma of B16 melanoma at different sites. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A19. doi:10.1158/1538-7445.CHTME14-A19

Metabolites of omega-3 fatty acids can inhibit primary tumor growth, angiogenesis, and tumor metastasis. Effective treatment for brain tumors is an unmet need as many chemotherapies are unable to cross the blood-brain barrier efficiently. As fatty acids are capable of entering the brain, these agents may have potential for treating brain tumors. In this communication a reliable and sensitive LC-MS/MS method was developed and validated for quantitation of ethyl 16-{[(4-methylphenyl)carbamoyl]amino}hexadecanoate (CUT-EE), a urea-based analog of eicosapentaenoic acid. The assay has acceptable intra- and inter-assay accuracy and precision over the range 0.25-125ng/mL, and a lower limit of quantitation of 0.25ng/mL. Non-aqueous, mouse brain, and plasma solutions of CUT-EE were stable during short-term and long-term storage, and after three freeze-thaw cycles. This method was applied to determine concentrations of CUT-EE in brain and plasma following administration to mice at 50mg/kg. CUT-EE was cleared from the plasma within 24hr, whilst concentrations in the brain increased over 24hr and remained stable out to 48hr. The calculated area under the concentration curve in brain was over 100 times greater than that found in plasma, supporting further investigations of CUT-EE for its potential in treating brain cancers.

Background: Tryptophan catabolism along the kynurenine pathway is associated with a number of pathologies including cataract formation and cancer. Whilst the chemical reactions of kynurenine are well studied, less is known about the reactivity of its precursor N-formylkynurenine (NFK). We previously reported the generation of a strong fluorophore in an aqueous reaction of NFK with piperidine, and herein we describe its structure and mechanism of formation.Methods: Compounds were identified using NMR, mass and UV spectroscopic techniques. The products from the reaction of amines with amino acids were quantified using HPLC-MS.Results: The novel fluorophore was identified as a tetrahydroquinolone adduct (PIP-THQ), where piperidine is N-formylated and attached at its 2-position to the quinolone. NFK is initially deaminated to generate an unsaturated enone, which forms an adduct with piperidine and is subsequently converted into the fluorophore. Testing of a variety of other secondary amines showed that only cyclic amines unsubstituted at both positions adjacent to nitrogen could form fluorophores efficiently. The amino acids tryptophan and kynurenine, which lack the formamide group do not form such fluorophores.Conclusions: NFK forms fluorophores in a not previously published reaction with cyclic amines.General significance: Our study is the first to provide evidence for concurrent transamidation and substitution at the 2-position of a cyclic amine occurring under moderately-heated aqueous conditions with no added catalysts. The high reactivity of NFK demonstrated here could result in formation of biologically relevant metabolites yet to be characterised. (C) 2015 Elsevier B.V. All rights reserved.

Abstract As part of our studies to identify novel inhibitors for the immunosuppressive enzyme indoleamine 2,3-dioxygenase 1 (IDO1) for restoring tumor immunity, we investigated the ability of the enzyme's x-ray crystal structure to predict active molecules when used in molecular docking based virtual screening of fragment libraries. The 352 compound Zenobia Therapeutics fragment library was screened by differential scanning fluorimetry and 74 compounds were identified that altered the thermal stability of the protein. Of these, biochemical analysis showed that only 14 inhibited the enzyme activity by >50% at 1 mM, and hydrazine containing fragments were the most potent inhibitors of recombinant human IDO1 (rhIDO1), with the best being a benzothiazole compound retrieving an IC50 of 8 µM. We recently showed that phenylhydrazine is a more potent inhibitor (IC50 0.3 µM), which provided a dataset for testing various docking strategies to discover fragment inhibitors. The fragment library was docked into the IDO1 active site (PDB code 2D0T) using GOLD driven by the scoring functions ChemPLP, Goldscore, ASP or Chemscore. The three top scoring poses for each compound were kept then rescored by each scoring function. The combination of docking by Goldscore and rescoring by Chemscore to identify the top scoring pose gave the best outcome, an 8.3 fold enrichment of active compounds within the top 5% of the ranked library compared to random selection. Based on this docking and scoring strategy we performed a virtual screening campaign on a 14,000 compound library containing commercially available phenylhydrazine derivatives. Our characterization of phenylhydrazine binding indicated that the hydrazine moiety interacts with the heme iron. Based on this, a set of compounds was selected from the top 30% of poses kept from the docked library for biochemical testing that had predicted hydrazine to heme iron interactions. Of 21 compounds chosen, 10 were tested and 9 inhibited IDO1 with IC50 of < 10 µM. Also, compounds predicted to make a hydrogen bond acceptor to heme iron interaction were selected and an additional 9 compounds chosen of which 6 were tested and only 1 inhibited rhIDO1 with IC50 of < 10 µM. For these latter ones the hydrazine was not predicted to interact with the heme iron. This study indicates that the rhIDO1 structure would be useful for discovering inhibitors in a diverse fragment library, and an additional hydrazine to heme iron binding descriptor is useful for finding actives in a library of compounds containing the phenylhydrazine core. Citation Format: Simon Fung, Jack U. Flanagan, Christopher J. Squire, Brian D. Palmer, Lai-Ming Ching. A molecular docking strategy for identifying fragment inhibitors of indoleamine 2,3-dioxygenase 1 (IDO1). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5372. doi:10.1158/1538-7445.AM2014-5372

Glioblastomas are amongst the most highly vascularised tumours, and the pursuit of anti-angiogenic approaches such as bevacizumab has provided short-term benefits. The purpose of this study was to determine whether the vascular-disrupting agent, dimethylxanthenone-4-acetic acid (DMXAA), could provide longer-lasting therapeutic benefits in a murine model of glioblastoma.

It is 40years since cancer growth was correlated with neovascularisation. Anti-angiogenic drugs remain at the forefront of cancer investigations but progress has been disappointing and unexpected toxicities are emerging. Gap junction channels are implicated in lesion spread following injury, with channel blockers shown to improve healing; in particular preventing vascular disruption and/or restoring vascular integrity. Here we briefly review connexin roles in vascular leak and endothelial cell death that occurs following acute wounds and during chronic disease, and how connexin channel regulation has been used to ameliorate vascular disruption. We then review chronic inflammatory disorders and trauma in the eye, concluding that vascular disruption under these conditions mimics that seen in tumours, and can be prevented with connexin hemichannel modulation. We apply this knowledge to tumour vessel biology, proposing that contrary to current opinion, these data suggest a need to protect, maintain and/or restore cancer vasculature. This may lead to reduced tumour hypoxia, promote the survival of normal cells, and enable improved therapeutic delivery or more effective radiation therapy.