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Background In epithelial ovarian cancer (EOC), 15-20% of the tumors do not respond to first-line chemotherapy (paclitaxel with platinum-based therapy), and in recurrences this number increases. Our aim is to determine the feasibility of cell proliferation assays of tumor cells isolated from malignant ascites to predict in vitro chemotherapy sensitivity, and to correlate these results with clinical outcome. Materials and methods Ascites was collected from twenty women with advanced EOC. Cell samples were enriched for tumor cells and EOC origin was confirmed by intracellular staining of CK7, surface staining of CA125 and EpCAM, and HE4 gene expression. In vitro sensitivity to chemotherapy was determined in cell proliferation assays using intracellular ATP content as an indirect measure of cell number. In vitro drug response was quantified by calculation of the drug concentration at which cell growth was inhibited with 50%. Clinical outcome was determined using post-treatment CA125 level. Results Cell samples of twenty patients were collected, of which three samples that failed to proliferate were excluded in the analysis (15%). Three other samples were excluded, because clinical outcome could not be determined correctly. In twelve of the fourteen remaining cases (86%) in vitro drug sensitivity and clinical outcome corresponded, while in two samples (14%) there was no correspondence. Conclusions Our study demonstrates the feasibility of drug sensitivity tests using tumor cells isolated from ascites of advanced EOC patients. Larger observational studies are required to confirm the correlation between the in vitro sensitivity and clinical outcome.

Arginase-1, which converts the amino acid L-arginine into L-ornithine and urea, is a promising new drug target for cancer immunotherapy, as it has a role in the regulation of T-cell immunity in the tumor microenvironment. To enable the discovery of small-molecule Arginase-1 inhibitors by high-throughput screening, we developed a novel homogeneous (mix-and-measure) fluorescence-based activity assay. The assay measures the conversion of L-arginine into L-ornithine by a decrease in fluorescent signal due to quenching of a fluorescent probe, Arginase Gold. This way, inhibition of Arginase-1 results in a gain of signal when compared with the uninhibited enzyme. Side-by-side profiling of reference inhibitors in the fluorescence-based assay and a colorimetric urea formation assay revealed similar potencies and the same potency rank order among the two assay formats. The fluorescence-based assay was successfully automated for high-throughput screening of a small-molecule library in 384-well format with a good Z'-factor and hit confirmation rate. Finally, we show that the assay can be used to study the binding kinetics of inhibitors.

Indoleamine 2,3-dioxygenase (IDO1) is a key regulator of immune suppression by catalyzing the oxidation of L-tryptophan. IDO1 expression has been related to poor prognosis in several cancers and to resistance to checkpoint immunotherapies. We describe the characterization of a novel small molecule IDO1 inhibitor, NTRC 3883-0, in a panel of biochemical and cell-based assays, and various cancer models. NTRC 3883-0 released the inhibitory effect of IDO1 on CD8-positive T cell proliferation in co-cultures of IDO1-overexpressing cells with healthy donor lymphocytes, demonstrating its immune modulatory activity. In a syngeneic mouse model using IDO1-overexpressing B16F10 melanoma cells, NTRC 3883-0 effectively counteracted the IDO1-induced modulation of L-tryptophan and L-kynurenine levels, demonstrating its in vivo target modulation. Finally, we studied the expression and activity of IDO1 in primary cell cultures established from the malignant ascites of ovarian cancer patients. In these cultures, IDO1 expression was induced upon stimulation with IFNγ, and its activity could be inhibited by NTRC 3883-0. Based on these results, we propose the use of ascites cell-based functional assays for future patient stratification. Our results are discussed in light of the recent discontinuation of clinical trials of more advanced IDO1 inhibitors and the reconsideration of IDO1 as a valid drug target.

Kinase inhibitors form the largest class of precision medicine. From 2013 to 2017, 17 have been approved, with 8 different mechanisms. We present a comprehensive profiling study of all 17 inhibitors on a biochemical assay panel of 280 kinases and proliferation assays of 108 cancer cell lines. Drug responses of the cell lines were related to the presence of frequently recurring point mutations, insertions, deletions, and amplifications in 15 well-known oncogenes and tumor-suppressor genes. In addition, drug responses were correlated with basal gene expression levels with a focus on 383 clinically actionable genes. Cell lines harboring actionable mutations defined in the FDA labels, such as mutant BRAF(V600E) for cobimetinib, or ALK gene translocation for ALK inhibitors, are generally 10 times more sensitive compared with wild-type cell lines. This sensitivity window is more narrow for markers that failed to meet endpoints in clinical trials, for instance CDKN2A loss for CDK4/6 inhibitors (2.7-fold) and KRAS mutation for cobimetinib (2.3-fold). Our data underscore the rationale of a number of recently opened clinical trials, such as ibrutinib in ERBB2- or ERBB4-expressing cancers. We propose and validate new response biomarkers, such as mutation in FBXW7 or SMAD4 for EGFR and HER2 inhibitors, ETV4 and ETV5 expression for MEK inhibitors, and JAK3 expression for ALK inhibitors. Potentially, these new markers could be combined to improve response rates. This comprehensive overview of biochemical and cellular selectivities of approved kinase inhibitor drugs provides a rich resource for drug repurposing, basket trial design, and basic cancer research.

Graphical abstract, Highlights • Side-by-side biochemical comparison of the inhibitors ABH, nor-NOHA and CB-1158. • Arginase-1 binding, inhibition and stabilization by ABH and CB-1158 is pH-dependent. • ABH and CB-1158 have slow association kinetics and a long target residence time. • At higher pH, the catalytic center adopts a more symmetrical coordination structure. • CB-1158 forms an additional hydrogen-bond network in the active site compared to ABH., Arginase-1 is a manganese-dependent metalloenzyme that catalyzes the hydrolysis of L-arginine into L-ornithine and urea. Arginase-1 is abundantly expressed by tumor-infiltrating myeloid cells that promote tumor immunosuppression, which is relieved by inhibition of Arginase-1. We have characterized the potencies of the Arginase-1 reference inhibitors (2S)-2-amino-6-boronohexanoic acid (ABH) and Nω-hydroxy-nor-L-arginine (nor-NOHA), and studied their pH-dependence and binding kinetics. To gain a better understanding of the structural changes underlying the high pH optimum of Arginase-1 and its pH-dependent inhibition, we determined the crystal structure of the human Arginase-1/ABH complex at pH 7.0 and 9.0. These structures revealed that at increased pH, the manganese cluster assumes a more symmetrical coordination structure, which presumably contributes to its increase in catalytic activity. Furthermore, we show that binding of ABH involves the presence of a sodium ion close to the manganese cluster. We also studied the investigational new drug CB-1158 (INCB001158). This inhibitor has a low-nanomolar potency at pH 7.4 and increases the thermal stability of Arginase-1 more than ABH and nor-NOHA. Moreover, CB-1158 displays slow association and dissociation kinetics at both pH 9.5 and 7.4, as indicated by surface plasmon resonance. The potent character of CB-1158 is presumably due to its increased rigidity compared to ABH as well as the formation of an additional hydrogen-bond network as observed by resolution of the Arginase-1/CB-1158 crystal structure.

Introduction: Indoleamine 2,3-dioxygenase (IDO1) is a heme-containing oxidoreductase enzyme that converts L-tryptophan (Trp) into N’-formylkynurenine (NFK). IDO1 is broadly expressed by tumor cells as well as immune cells in the tumor microenvironment of many cancers, which is often correlated with poor prognosis. Depletion of Trp and increased L-kynurenine (Kyn) levels induce immune tolerance by suppression of effector T-cell and natural killer cell functions, and activation of regulatory T-cells and myeloid-derived suppressor cells. Four small molecule inhibitors are currently investigated in phase III (linrodostat/BMS-986205), phase II (epacadostat/INCB024360) or phase I (MK7162 and LY3381916) clinical trials. Linrodostat, epacadostat and LY3381916 are reported as being selective for IDO1 over TDO, while details on MK7162 have not yet been disclosed. The compounds inhibit IDO1 with different mechanisms, with epacadostat binding to the heme-group in the catalytic center of IDO1, while linrodostat and LY3381916 bind to apo-IDO1 and compete with heme for the active site [1-3]. Experimental procedures Clinical and reference IDO1 inhibitors were characterized in biochemical assays for IDO1 and tryptophan 2,3-dioxygenase (TDO), a structurally different enzyme, which also catalyzes the conversion of Trp to NFK. Furthermore, the compounds were profiled in a panel of functional cell-based assays, including human cancer cell lines and assays based on IDO1- or TDO2-overexpressing HEK293 cells. A IDO1-expressing subline of the mouse B16F10 melanoma cell line was generated and used to develop a syngeneic mouse model at Charles River Laboratories (USA) to determine target modulation in vivo [4]. Stable gene expression was confirmed by qPCR and target modulation was examined by measurement of Trp and Kyn levels using LC-MS/MS. Results: Linrodostat has sub-nanomolar cellular potency, despite the absence of any biochemical activity on the timescale of our assays, which is consistent with its reported heme-competing mechanism of inhibition [1]. Linrodostat also inhibits different Cytochrome P450 enzymes with micromolar activity. In our biochemical assays, epacadostat was not selective for IDO1 over TDO, whereas in the IDO1- and TDO2-overexpressing HEK293 cell lines it was 2000 times selective for IDO1. High level expression of IDO1 in B16F10 cells did not result in enhanced tumor growth after grafting in syngeneic mice, which contrasts published data with a similar model [4]. Nonetheless, we observed strong modulation of Trp and Kyn levels in plasma and in tumors of the IDO1-overexpressing mouse model, compared to non-tumor bearing mice. Treatment of the B16F10-IDO1 model with epacadostat did not result in a reduction of tumor growth, though epacadostat did induce clear changes in Trp and Kyn in both plasma and tumor tissue. Conclusion: Our comparative study of the potencies and selectivities of IDO1 inhibitors, as well as our model for measuring in vivo target modulation, helps to identify strengths and weaknesses of current IDO1 inhibitors, and supports the development of new inhibitors. [1] Nelp et al. (2018) Proc. Natl. Acad. Sci. U.S.A. 115, 3249-3254; [2] Yue et al. (2017) ACS Med. Chem. Lett. 8, 486-491; [3] Dorsey et al. (2018) Proceedings: AACR Annual Meeting 2018, Abstract nr. 5245; [4] Holmgaard et al. (2015) Cell Rep. 13, 412-424. Citation Format: Yvonne Grobben, Joost C.M. Uitdehaag, Antoon M. van Doornmalen, Nicole Willemsen-Seegers, Diep Vu-Pham, Jos de Man, Rogier C. Buijsman, Guido J.R. Zaman. Side-by-side comparison of small molecule IDO1 inhibitors in biochemical and cell-based assays and development of a IDO1-expressing mouse model to evaluate target modulation [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B060. doi:10.1158/1535-7163.TARG-19-B060

Introduction: Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy. First-line therapy in advanced EOC is surgery in combination with platinum-based chemotherapy and paclitaxel. 15-20% of patients do not respond and in 80% of advanced cases, the disease recurs within three years. PARP inhibitors synergize with platinum therapy and have been approved for platinum sensitive EOC. Clinical trials with immunotherapies, such as PD-1/PD-L1 blockade, have so far not been successful. Currently, the only approved companion diagnostic is BRCA gene mutations for PARP inhibitors. More diagnostic assays to predict the clinical response to chemo- or immunotherapies are needed. We have developed a biomarker discovery platform using ascites of ovarian cancer patients. Experimental procedures Ascites was gathered from patients by punction or during debulking surgery. Cells were collected by centrifugation and characterized by flow cytometry using specific antibodies. Genomic DNA was sequenced using Illumina cancer gene panels. Gene expression was analyzed by quantitative PCR (qPCR). Cellular activity of the tryptophan metabolizing enzymes IDO1 and TDO was measured with NFK Green [1]. Levels of L-tryptophan and its metabolite L-kynurenine in ascites fluid and blood were determined with LC-MS/MS. In vitro data were related to tumor histopathology and clinical response data. Results: Low passage cell samples from twenty patients were profiled for sensitivity to various cytotoxic agents and targeted anti-cancer therapies in cell proliferation assays. In parallel the mutation status of fifty cancer genes including BRCA1 and 2 was assessed by DNA sequencing. The expression of genes implicated in resistance to chemotherapy (CCNE1, ABCB1) or immunotherapy (PD-L1, IDO1, TDO) was determined with qPCR. The immune status of ascites was analyzed by measuring the relative proportion of different immune cell populations, i.e., cytotoxic and regulatory T cells, monocytes, dendritic and natural killer cells. The expression of the immune suppressive markers PD-L1, IDO1 and TDO was related to the immune cell composition of the ascites, kynurenine-tryptophan ratio, and clinical response data. A tumor cell sample derived from a patient with low grade serous ovarian cancer (LGSOC) was heterozygous for an oncogenic NRAS mutation and was much more sensitive to MEK inhibitors than other samples not harboring the mutation. The cells also expressed IDO1 and high levels of PD-L1 at the cell surface. Two other samples derived from high grade serous ovarian cancer (HGSOC) expressed high PD-L1 and one also IDO1. Several HGSOC samples expressed TDO. One HGSOC sample showed high expression of the ABCB1 gene, encoding the multidrug transporter P-glycoprotein. The sample was relatively resistant to paclitaxel, a known substrate of P-glycoprotein Conclusion: Our study shows that in vitro drug sensitivity assays with primary patient samples can be used to confirm or identify predictive drug response biomarkers. In an ongoing study, in which hundred patients with HGSOC will be included, the in vitro drug response of tumor cells from ascites to first-line cytotoxic anti-cancer agents will be determined and compared to the clinical response of patients bearing specific genomic biomarkers. [1] Seegers et al. (2014) J Biomol Screen 19, 1266-1272 Citation Format: Guido J.R. Zaman, Judith E. den Ouden, Jelle Dylus, Antoon M. van Doornmalen, Winfried R. Mulder, Diep Vu-Pham, Suzanne J.C. van Gerwen, Joost C.M. Uitdehaag, Rogier C. Buijsman, Leon F. Massuger, Anne M. van Altena. A precision medicine platform to predict the clinical response to chemo- and immunotherapy for epithelial ovarian cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A044. doi:10.1158/1535-7163.TARG-19-A044

Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy. Standard treatment of advanced EOC is surgery in combination with chemotherapy, consisting of a platinum-based drug (carboplatin or cisplatin) and paclitaxel. Although most women show a good response to first line treatment, tumors do not respond in 15-20% of patients, whereas in 80% of cases of advanced EOC, the disease recurs within three years. Patients who have responded to first line treatment are referred to as ‘platinum sensitive’, and are treated with a PARP inhibitor in second line. Several other new targeted inhibitors are investigated in clinical trials, including PI3-kinase and bromodomain inhibitors. There is great need of new, more effective therapies with improved long-term treatment outcome, and diagnostic assays and biomarkers to predict chemotherapy response in the clinic. We have determined whether tumor cells isolated from ascites of patients with EOC can be used to develop in vitro cell proliferation assays. We also characterized the immune status of ascites by cytokine analysis and used flow cytometry to analyze the immune cell types in ascites. In a pilot study, ascites was gathered from 18 patients with advance stage EOC. Cells were isolated by centrifugation and tumor cells were separated from immune cells by adhering them to tissue culture plates. Sensitivity of tumor cells to platinum compounds, paclitaxel, PARP inhibitors, and other second line and investigational drugs, as well as drug combinations, was determined in cell proliferation assays, using intracellular ATP content as an indirect read-out of cell number [1]. The mutant status of a number of known oncogenes and tumor suppressor genes in the patient-derived cell samples was determined by DNA sequence analysis. Mutation status was related to histopathological data and in vitro drug response. We demonstrate that ascites from EOC patients contains many tumor cells and that these tumor cells can be used to perform drug sensitivity assays in vitro. In an ongoing study, in which ascites from hundred EOC patients will be included, the in vitro sensitivity to standard-of-care chemotherapy will be related to the immune status of the ascites and to the clinical outcome of the patient, which is defined by platinum sensitivity. An update of this study will be presented at the conference. [1] Uitdehaag et al. (2014) PLoS ONE 9(3): e92146 Citation Format: Guido Zaman, Judith E. den Ouden, Jelle Dylus, Antoon M. van Doornmalen, Rogier C. Buijsman, Astrid Eijkelenboom, Leon F. Massuger, Anne M. van Altena. Chemotherapy sensitivity of tumor cells from ascites of ovarian cancer patients: Relationship with immune status and clinical response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2221.

Target residence time (τ) has been suggested to be a better predictor of the biological activity of kinase inhibitors than inhibitory potency (IC50) in enzyme assays. Surface plasmon resonance binding assays for 46 human protein and lipid kinases were developed. The association and dissociation constants of 80 kinase inhibitor interactions were determined. τ and equilibrium affinity constants (KD) were calculated to determine kinetic selectivity. Comparison of τ and KD or IC50 values revealed a strikingly different view on the selectivity of several kinase inhibitors, including the multi-kinase inhibitor ponatinib, which was tested on 10 different kinases. In addition, known pan-Aurora inhibitors resided much longer on Aurora B than on Aurora A, despite having comparable affinity for Aurora A and B. Furthermore, the γ/δ-selective PI3K inhibitor duvelisib and the δ-selective drug idelalisib had similar 20-fold selectivity for δ- over γ-isoform but duvelisib resided much longer on both targets.

Arginase I (ArgI) is an important small molecule drug target in cancer immunotherapy. Arg1 converts L-arginine into L-ornithine and urea. Recruitment of ArgI-expressing myeloid-derived suppressor cells (MSDCs) at a tumor site results in the depletion of L-arginine, which causes reduced proliferation of T-cells and natural killer cells and inhibition of the antitumor immune response. In patient material, MSDC-induced T-cell suppression can be reverted by arginase inhibitors. ArgI inhibitors work synergistically with checkpoint inhibitor therapy in syngeneic mouse models. In order to find novel inhibitors for ArgI, we developed a set of tools to enable screening and hit validation. The first is an activity assay that enables the high-throughput screening (HTS) of compound libraries. Previously reported arginase assays are poorly compatible with HTS due to the requirement of multiple reactions steps, harsh assay conditions, or the use of low-turnover substrates other than L-arginine. We developed a novel ArgI activity assay, which has a homogenous format and requires only two addition steps before readout. The assay makes use of a fluorescence readout and has a high robustness (Z'-factor > 0.7). Progression of the assay can be followed in real time, allowing for kinetic experiments. To investigate the false positive hit rate, the assay was screened at the Pivot Park Screening Centre with a library consisting of 233 compounds with known interference in other assay formats. Using a simple background signal control, the number of false positives in this library was minimized to below 0.2%. The assay was subsequently used to accurately determine the dissociation constants and binding kinetics of the reference inhibitors ABH, NOHA and CB1158. In order to validate the binding of screening hits to ArgI, we developed a thermal shift assay. We observed a shift in the ArgI melting temperature of up to 3.8°C after addition of the most potent inhibitors. In addition, we developed a Surface Plasmon Resonance binding assay. This showed that ABH and CB1158 have long residence times on ArgI. To allow the development of novel inhibitors through rational drug design, we successfully crystallized human ArgI in a novel space group with higher symmetry (P63) compared to those previously reported (space group P3), and without the presence of hemihedral twinning. We determined a series of high resolution (< 1.7 Å) crystal structures at various pH values and with several ligands. These demonstrate that a series of peptide flips lies at the basis of the pH-dependent symmetry of ArgI and its unusually high pH optimum of 9.0 to 9.5. Finally, to assess the cellular activity of ArgI inhibitors, we examined 102 cancer cell lines for ArgI activity and correlated the results to public gene expression profiles. The novel assay portfolio will help to deliver a new generation of ArgI inhibitors. Citation Format: Yvonne Grobben, Joost C. Uitdehaag, Nicole Willemsen-Seegers, Werner W. Tabak, Martine B. Prinsen, Suzanne J. van Gerwen, Jan van Groningen, Johan Friesen, Helma Rutjes, Jos de Man, Rogier C. Buijsman, Guido J. Zaman. High-throughput fluorescence-based assay for screening of Arginase I inhibitors for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1944.

Introduction In epithelial ovarian cancer (EOC) patients, 15%–20% of the tumours do not respond to first-line chemotherapy treatment, i.e., paclitaxel combined with a platinum-based therapy. The aim of this pilot study was to validate a new approach to measure the sensitivity of ovarian cancer cells to chemotherapeutics. Furthermore, we aimed to relate the in vitro response of tumour cells from ascites to clinical response of ovarian cancer patients. Material and methods For this pilot study, we selected 18 patients with advanced stage EOC and collected ascites. Proliferation assays on tumour cells isolated from the ascites were performed using intracellular ATP content as an indirect measure of cell number. Clinical outcome was determined using status of remission after primary treatment, disease free survival (DFS), and the platinum-sensitivity. Complete remission was defined as a serum-CA125 Results and discussions The outcome of the proliferation assays, expressed by GI50 and using dose-response curves, corresponded to the clinical outcome in the patient in most cases. Of the 14 ascites samples eventually tested, seven of the in vitro responses were poor as well as the DFS of the patient. Four samples showed good in vitro response and the patient showed a good DFS as well. Only two of the samples tested did not show similarity in in vitro response and in clinical outcome of the patient. We also determined the in vitro response to second line therapies and new targeted therapies, including PARP- and bromodomain inhibitors. Conclusion This pilot study demonstrates the feasibility of using ascites tumour cells to perform drug sensitivity tests. These are promising results because it seems possible to predict accurately the response on first-line chemotherapy in advanced stages of EOC. However, larger observational studies are required to show a statistical significant relation, to determine the best clinical response measurements and to test second line therapies.

The journey towards a detailed mechanistic understanding of the anticoagulant action of heparin has resulted in synthetic mimetics with improved pharmacodynamic profiles. Inspired by the ternary complex formation of heparin with antithrombin III and thrombin, the active pentasaccharide fondaparinux has been succeeded by several clinical candidates, such as SR123781, that have tailor-made factor Xa and thrombin inhibitory activities combined with less aspecific binding (e.g. binding to platelet factor 4 involved in thrombocytopenia). Novel compounds with both antithrombin III-mediated inhibition of factor Xa and direct thrombin inhibition are emerging. Org42675 is one such compound, balancing dual inhibition of factor Xa and thrombin in one anticoagulant drug, with excellent pharmacokinetic properties and strong inhibitory activity toward clot-bound thrombin.

HTS has been executed with a luciferase reporter assay in CHO cells stably expressing an ERa/GCNF chimer. Confirmed hits could be optimized some 10 to 100 fold. Partial agonists identified during the hit optimization process behaved as antagonists in a competition based variant of the reporter assay. The latter assay format was also used to identify some full antagonists by HTS. GCNF binding of agonists and antagonists was confirmed by GCNF/cofactor interactions studies in solution.

Screening of new drugs on large cell panels is an important tool to study the biologic mechanism of drug response. From a combination of parallel in vitro tests and bio-informatics analysis, important conclusions can be drawn, for instance about drug selectivity in a cellular context, about resistance mechanisms, and about the patient population in which a drug is effective. This makes cell panel screening indispensable in modern drug discovery. We have set up a platform called Oncolines™ that comprises 102 cell lines from diverse tumor tissues. All cell lines are screened in parallel in high-throughput proliferation assays based on ATP-lite™. Compounds are tested with 9 point dose-response curves in duplicate. Curves are visually inspected. In the past, we have shown that this workflow leads to highly reproducible IC50s, which are necessary for genomic biomarker discovery [1]. For instance, the IC50s have been coupled to curated databases of somatic mutations and copy numbers (1). However, these changes only reflect a small percentage of oncogenic transformations. A more comprehensive view of oncogenic signaling inside a cell can be obtained from mRNA expression levels (2). Here, we describe a workflow to investigate drug response in the 102 cell line Oncolines™ panel based on basal gene expression levels. Correlations between gene expression levels and the log IC50s were calculated for more than 18,000 genes. To reduce the number of false-positive correlations, we considered only genes with a known biologic role in cancer or that were clinically actionable. Secondly, we filtered out genes that correlated indiscriminately with drug responses, by correcting for the average correlations seen in our profiling database of more than 150 inhibitors. This filtered gene list was structured graphically by combining it with information on protein-protein interactions (using the StringDB) or information on which genes are part of the same pathways (a method called Gene Set Analysis). Our method reveals that gene overexpression correlates with drug responses for a number of targets. For instance, EGFR, IGFR, or ALK kinase inhibitors have more potent effects in cell lines that overexpress EGFR, IGFR, or ALK, respectively, and the MDM2 antagonist nutlin is more active in MDM2-overexpressing cell lines. This is irrespective of the genomic alterations driving overexpression of these genes. The distinct response profiles of the spectrum-selective BTK/EGFR/ERBB2 inhibitor ibrutinib and the more selective BTK inhibitor alcalabrutinib could be distinguished as well. Finally, the analysis shows mechanisms of drug resistance. For instance, the response of vincristine strongly correlates to cellular ABCB1 expression, the P-glycoprotein/MDR1 drug transporter, but also to ABCC3, another ABC transporter. Combining basal gene expression levels with cell panel data therefore allows discovery of novel mechanisms of drug response and resistance. References: 1. Uitdehaag et al. Cell panel profiling reveals conserved therapeutic clusters and differentiates the mechanism of action of different PI3K/mTOR, Aurora kinase and EZH2 inhibitors. Mol Cancer Therap 2016;15:3097-3109. 2. Rees et al. Correlating chemical sensitivity and basal gene expression reveals mechanism of action. Nature Chem Biol 2016;12:109-16. Citation Format: Joost C.M. Uitdehaag, Jeffrey Kooijman, Jeroen A.D.M. de Roos, Martine B.W. Prinsen, Jelle Dylus, Suzanne J.C. van Gerwen, Rogier C. Buijsman, Guido J.R. Zaman. Combining cell panel screening with analysis of gene expression levels reveals features of drug response and resistance [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B155.

The protein kinase TTK is a critical component of the spindle assembly checkpoint (SAC), which regulates the proper attachment of sister chromatids during mitosis. TTK is essential for normal progression of the cell cycle and is upregulated in various aggressive cancers such as triple negative breast cancer (TNBC). Inhibition of TTK leads to an overriding of the SAC, premature progression of the cell cycle and mitotic catastrophe. TTK inhibition therefore contrasts with e.g. Aurora and PLK1 inhibition, which block the cell cycle [1]. Various studies have shown the clinical utility of TTK inhibition, particularly in combination therapy with paclitaxel [1,2]. In vivo, TTK inhibitor monotherapy has shown partial tumor growth inhibition without weight loss [1,2]. We have developed a novel class of TTK inhibitors based on a pyrimido-indolizine scaffold. These bind selectively and with high affinity to the ATP pocket of TTK and potently inhibit the proliferation of a variety of cell lines [1]. Previously we published a representative of this class, NTRC 0066-0, which has an IC50 of 0.6 nM in a biochemical assay for TTK activity and which is more than 200 times selective over a panel of 276 kinases [1]. NTRC 0066-0 has an average IC50 of 98 nM in proliferation assays in 66 different cell lines (Oncolines™ panel) [3]. To further improve the potency of the pyrimido-indolizine series, we determined the X-ray structures of a dozen of class representatives in complex with TTK. The results were compared to 3D complexes of other chemical scaffolds such as BAY-1161909, Mps-Bay2b, MPI-0479605, NMS-P715 and Mps1-IN1. The pyrimido-indolizine series uses an aromatic moiety to trap the catalytic lysine in the active site, enforcing a catalytically incompetent conformation of TTK. Thermal melting and surface plasmon resonance experiments demonstrate that this leads to a strong stabilization of the kinase domain and a slow dissociation rate for the compounds, which is one of the key determinants for potent cellular activity. We took advantage of these structure-activity relationships to develop analogs of NTRC 0066-0 with increased residence time and cellular potency. One such analog, NTRC 1501-0, inhibits the proliferation of 66 cancer cell lines with an average IC50 of 18 nM, while retaining selectivity over the kinome. It has good pharmacokinetic properties and shows no cross-reactivity with drug safety targets in vitro. In a xenograft model of the human TNBC cell line MDA-MB-231, it completely inhibited tumor growth at a low oral dose, without effect on body weight, indicating good tolerability. These data show, for the first time, that TTK inhibition as monotherapy can achieve complete inhibition of tumor growth. [1] Maia et al. (2015) Annals of Oncology 26, 2180-2192; [2] Wengner et al (2016) Mol. Canc. Therap. 15, 583-592; [3] Uitdehaag et al. (2016) Mol. Canc. Therap., in the press. Citation Format: Joost C. Uitdehaag, Jos de Man, Marion Libouban, Nicole Willemsen-Seegers, Jan Gerard Sterrenburg, Joeri J. de Wit, Jeroen A.D de Roos, Martine B. Prinsen, Rogier C. Buijsman, Guido J. Zaman. NTRC 1501-0, a TTK kinase inhibitor selected for its long target residence time, completely inhibits tumor growth in the MDA-MB-231 xenograft model for triple-negative breast cancer [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 4185. doi:10.1158/1538-7445.AM2017-4185

Indoleamine 2,3-dioxygenase (IDO1) and tryptophan 2,3-dioxygenase (TDO) are two structurally different enzymes that have a different tissue distribution and physiological roles, but both catalyze the conversion of tryptophan to N-formylkynurenine (NFK). IDO1 has been clinically validated as a small-molecule drug target for cancer, while preclinical studies indicate that TDO may be a target for cancer immunotherapy and neurodegenerative disease. We have developed a high-throughput screening assay for IDO1 and TDO based on a novel chemical probe, NFK Green, that reacts specifically with NFK to form a green fluorescent molecule with an excitation wavelength of 400 nm and an emission wavelength of 510 nm. We provide the first side-by-side comparison of a number of published inhibitors of IDO1 and TDO and reveal that the preclinical IDO1 inhibitor Compound 5l shows significant cross-reactivity with TDO, while the relative selectivity of other published inhibitors was confirmed. The suitability for high-throughput screening of the assays was demonstrated by screening a library of 87,000 chemical substances in 384- or 1536-well format. Finally, we demonstrate that the assay can also be used to measure the capacity of cells to metabolize tryptophan and to measure the cellular potency of IDO1 and TDO inhibitors.

A synthesis is presented of the cyclic trimeric d-oligonucleotide 3′-isopropylphosphate I, comprising one formacetal and two (3′ → 5′)-internucleosidic phosphodiester bonds. The ester linkages connect d-guanosine with the 3′ and 5′ ends of thymidine and 5-hydroxymethyl-2′-deoxyuridine-3′-isopropylphosphate (HMDUpiPr), respectively. The 5′-end of the thymidine unit is anchored via the formacetal bond to the allylic hydroxyl group of HMDUpiPr. The cyclic arrangement of the three d-nucleosides in I mimics, as based on molecular modeling, the key structural features of the conformationally constrained T7pG8pT9p-domain of the thrombin-binding DNA aptamer d(G1G2T3T4G5G6 T 7 G 8 T 9 G10G11T12T13G14G15). Biological evaluation showed that compound I did not exhibit anti-thrombin activity.

Trisubstrate UGT transition state analogue 2 is readily accessible by nucleophilic ring-opening of 1,2-anhydroglucose precursor 5 with diethylmalonate anion followed by reduction of the ethyl ester moieties ( 6→7 ). Subsequent C 6 oxidation ( 8→9 ), NIS/ cat . TfOH-mediated introduction of the androsterylmethylene unit ( 12→15 ) and phosphitylation with 5′-uridine phosphoramidite 16 furnished, after oxidation and deprotection, target derivative 2 , the two individual diastereomers of which ( 2a and 2b ) were separated by HPLC. Trisubstrate analogues 2a,b show a different inhibition pattern for several UGT isoforms, indicating isoenzyme selectivity. Moreover, C 7″ -epimers 2a and 2b exert a different inhibitory effect on UGT2B15.

Derivatization of the octadecathymidylate derivative 18 (T18 ODN) containing a free amine function with sulfo-SIAB® gave the corresponding iodoacetyl ODN 21. Conjugation of the latter with the thiol-containing pentasaccharide 17c gave pentasaccharide–ODN conjugate III, which exhibited anti-Xa and antithrombin activities of 173 U mg-1 and 5 U mg-1, respectively.

[purpose] An abnormal number of chromosomes, or ‘aneuploidy’, is a common feature of solid human tumors and a predictor of poor prognosis in breast, lung, brain and colorectal cancer. Aneuploidy is caused by malfunctioning of the Spindle Assembly Checkpoint (SAC), a surveillance mechanism that ensures the fidelity of chromosome segregation. The protein kinase TTK (commonly referred to as Mps1) is a component of the SAC. Inhibition of TTK gene expression by RNA interference and inhibition of TTK kinase activity by small molecule kinase inhibitors causes chromosome missegregation and cancer cell death. [experimental procedures] A novel class of compounds was identified that potently inhibits TTK enzyme activity and cancer cell line proliferation [1]. Its binding mode and that of reference inhibitors was characterized by protein crystallography. Binding kinetics and target residence time were determined by surface plasmon resonance using Biacore T200. Anti-proliferative activity was measured on a broad panel of cancer cell lines [2]. [results] The clinical candidate, NTRC 0066-0, inhibits TTK with subnanomolar potency (IC50) in a kinase enzyme assay and is more than 200 times selective over 276 kinases examined, including mitotic and cell cycle dependent kinases (CDKs). X-ray structures of the TTK kinase domain in complex with NTRC 0066-0 and analogs indicate that this class of compounds induces a large conformational shift in the glycine-rich loop, invoking an inactive kinase conformation. In surface plasmon resonance experiments, NTRC 0066-0 exhibited slow dissociation kinetics, resulting in a long target residence time. Parallel surface plasmon resonance experiments with mitotic kinases confirmed the exquisite selectivity of NTRC 0066-0 for TTK over Aurora and Polo-like kinases. NTRC 0066-0 potently inhibited the proliferation of a wide variety of human cancer cell lines with potencies in the same range as marketed cytotoxic agents. The crystal structure, binding kinetics and cellular potency of NTRC 0066-0 were compared to that of other TTK inhibitors such as Mps1-IN-2, AZD-3146, Mps-BAY2b and Bay 1161909 as well as analogs from the NTRC 0066-0 series. This suggest that the unique binding mode of NTRC 0066-0 results in long target residence time which contributes to its strong anti-tumor activity. In subsequent mouse xenograft models of human cancer cell lines, NTRC 0066-0 inhibited tumor growth as a single agent after oral administration at 20 mg per kg. [conclusions] NTRC 0066-0 is a novel TTK inhibitor with outstanding in vitro properties and potent anti-tumor activity in mouse xenograft models. Our data suggest that long target residence time corresponds with potent cellular activity for TTK inhibitors. References [1] Maia et al. (2015) Annals of Oncology 26, 2180-2192; [2] Uitdehaag et al. (2014) PLOS ONE 9(3) e92146. Citation Format: Jos de Man, Joost C.M. Uitdehaag, Nicole Willemsen-Seegers, Jan Gerard Sterrenburg, Joeri J.P. de Wit, Jeroen A.D.M. de Roos, Martine B.W. Prinsen, Rogier C. Buijsman, Guido J.R. Zaman. The unique binding mode of NTRC 0066-0, a novel inhibitor of the spindle assembly checkpoint kinase TTK (Mps1), leads to long target residence time and potent antitumor activity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2646.

[reaction--see text] Ionic liquid 1-butyl-3-methylimidazoliumhexafluorophosphate ([bmim]PF(6)) is described as an effective medium for ring-closing metathesis (RCM) using Grubbs catalysts. When [bmim]PF(6) was used as solvent, the RCM showed high conversions and a broad substrate tolerance. [bmim]PF(6) and the ruthenium catalyst were recycled after extraction of the product in the organic phase for at least three cycles.

Background: Pediatric T-cell acute lymphoblastic leukemia patients frequently harbor mutations in IL7Ra or downstream molecules encoded by JAK1, JAK3, N-RAS, K-RAS, NF1, AKT, and PTEN. These mutated signaling molecules can contribute to leukemia by disturbing a multitude of cellular processes such as the cell cycle, epigenetics, apoptosis, or affecting other important signal transduction pathways. Aims: We aimed to determine the overall incidence of mutations in IL7Ra and downstream signaling components in a large cohort of pediatric T-ALL patients. In order to find better treatment options for patients with these mutations, we analyzed the effect of selected IL7Ra-pathway inhibitors-individually and in combinations-on downstream signaling and cytotoxicity in Ba/F3 cells expressing each of the mutations. Methods: We sequenced 146 pediatric T-ALL patient samples for mutations in the FERM, pseudokinase and kinase domains of the Janus kinase gene family (JAK1, JAK2, JAK3, TYK2) and hotspot regions of N-RAS and K-RAS. We adapted the IL3-dependent Ba/F3 cell line to express mutant or wild type genes upon induction by doxycycline and assessed cell viability and signaling in the absence of IL3. Various IL7Ra-pathway inhibitors were tested using this system, and the synergy of combined inhibitors was determined by comparing the dose-response curve of different ratios of IC50-based inhibitor concentrations to the curves for each of the single inhibitors. The Combination Index was calculated using Calcusyn™ software. Results: IL7Ra, JAK, RAS, AKT and PTEN mutations are present in approximately 45% of patients and occur in a predominantly mutually exclusive fashion, suggesting they share aberrant activation of similar downstream targets. We found JAK1, JAK3 and RAS mutations as previously reported, but also identified new JAK1 mutations including V427M, L624YPILKV, E668Q, P815S, and T901G. A novel three-dimensional model of JAK1 reveals that mutations in JAK molecules affect important amino acids that are involved in the interaction between the pseudokinase and kinase domains, facilitating constitutive kinase activity. In our doxycycline-inducible IL3-dependent Ba/F3 system, expression of mutant genes-in contrast to the wild type genes-transforms Ba/F3 cells by supporting IL3-independent growth through activation of the RAS-MEK-ERK and PI3K-AKT pathways. We used this system to test the sensitivity to pharmacological inhibitors; IL7Ra and JAK mutant Ba/F3 cells are sensitive to JAK inhibition, so JAK inhibitors such as ruxolitinib may offer therapeutic potential for IL7Ra, JAK1 or most JAK3 mutated T-ALL patients. The RAS and AKT mutants respond to RAS-MEK and PI3K-AKT-mTOR inhibition, respectively, but are-as expected-insensitive to JAK inhibition. Remarkably, IL7Ra and JAK mutants are relatively resistant to downstream RAS-MEK-ERK or PI3K-AKT-mTOR inhibition, indicating that inhibiting just one of these downstream pathways is insufficient. We provide evidence of (cross-)activation of the alternate pathway when one of these pathways is inhibited. Combined inhibition of MEK and PI3K/AKT synergistically prevents proliferation of the IL7Ra- and JAK-mutants by efficiently blocking both downstream signaling pathways. Furthermore, this combined inhibition is cytotoxic in two out of five tested primary T-ALL specimens. Summary/Conclusion: We show that the combined inhibition of MEK and PI3K/AKT leads to strong and synergistic cytotoxic effects in the IL7Ra and JAK mutants and efficiently blocks signaling downstream of both pathways. This inhibitor combination is effective in two out of five primary T-ALL samples. Therefore, the cytotoxic effects of synergistic MEK and PI3K/AKT inhibition should be further explored as a therapeutic option for (relapsed) ALL patients. Disclosures Buijsman: Netherlands Translational Research Center B.V.: Equity Ownership, Other: founder and shareholder. Zaman:Netherlands Translational Research Center B.V.: Equity Ownership, Other: founder and shareholder.

The synthesis of ionic support 1 and its application in the preparation of a set of amides and sulfonamides is described. The potential of 1 is further exemplified by its use in a one-pot multistep ionic liquid phase assisted synthesis of tirofiban analogue 2.

The synthesis of three heparin analogues (i.e. compounds VI-VIII) having perphosphorylated thrombin binding domains (TBDs) is reported. These compounds were tested in vitro for their antithrombin III (ATIII)-mediated anti-Xa and antithrombin activities. Conjugates VI and VIII show a remarkable increase in antithrombin activity compared to the structurally related conjugates with persulfated TBDs (i.e. compounds IV and V), whereas compound VII displays a diminished activity.

The synthesis of a novel antithrombotic consisting of a heparin pentasaccharide conjugated to the active site inhibitor N-(2- naphtalenesulfonyl)-glycyl-( d )-4-aminophenyl-alanyl-piperidine (NAPAP) (i.e. compound I) is reported. This conjugate shows a unique pharmacological profile both in vitro and in vivo having direct anti-thrombin and ATIII-mediated anti-Xa activity. Furthermore, conjugate I has a prolonged in vivo half-life compared to NAPAP (1.5 h vs 9 min.).