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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.

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: 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.

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.

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

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.

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.

[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.

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 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.).