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The molecular mechanisms contributing to immune suppression in ovarian cancer are not well understood, hampering the successful application of immunotherapy. Amino acid-metabolizing enzymes are known to contribute to the immune-hostile environment of various tumors through depletion of amino acids and production of immunosuppressive metabolites. We aimed to collectively evaluate the activity of these enzymes in high-grade serous ovarian cancer patients by performing targeted metabolomics on plasma and ascites samples. Whereas no indication was found for enhanced l-arginine or l-glutamine metabolism by immunosuppressive enzymes in ovarian cancer patients, metabolism of l-tryptophan by indoleamine 2,3-dioxygenase 1 (IDO1) was significantly elevated compared to healthy controls. Moreover, high levels of l-phenylalanine- and l-tyrosine-derived metabolites associated with interleukin 4 induced 1 (IL4I1) activity were found in ovarian cancer ascites samples. While l-tryptophan is a major substrate of both IDO1 and IL4I1, only its enhanced conversion into l-kynurenine by IDO1 could be detected, despite the observed activity of IL4I1 on its other substrates. In ascites of ovarian cancer patients, metabolite levels were higher compared to those in plasma, demonstrating the value of utilizing this fluid for biomarker identification. Finally, elevated metabolism of l-phenylalanine and l-tyrosine by IL4I1 correlated with disease stage, pointing towards a potential role for IL4I1 in ovarian cancer progression.

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.

Kinases are the major anticancer drug target class of the 21st century with nearly sixty small molecule kinase inhibitors approved for clinical use in the first two decades. While there are more than 500 kinases encoded by the human genome, currently approved inhibitors act primarily through approximately twenty different targets. Key to the success of kinase inhibitor therapy has been the simultaneous development of biomarker assays to enable the selection of patients most likely to respond. Predictive drug response biomarkers can be identified with cancer cell panel profiling, which is the parallel testing of compounds on a large panel of cancer cell lines. By correlating drug sensitivity with genomic information of the cell lines, strategies for patient stratification or drug repurposing have been developed [1-3]. In two earlier studies [2,3], we compared the kinase selectivity and the cellular inhibition profiles of all kinase inhibitors approved by the FDA until May 2018. Here, we will present the cancer cell panel profiling of all twenty small molecule kinase inhibitors approved since then. Several of these inhibitors act through well-known targets, such as ALK (lorlatinib), BRAF (encorafenib), EGFR (dacomitinib) and MEK1 (binimetinib, selumetinib). Others act through kinases for which no small molecule inhibitors have been approved before, such as CSF1R (pexidartinib), FGFR (erdafitinib, pemigatinib), c-MET (capmatinib), RET (selpercatinib, pralsetinib) and TRK (larotrectinib, entrectinib). All compounds were profiled on a panel of 102 cancer cell lines, known as Oncolines, representing a wide range of solid tumors and hematological malignancies, and harboring mutant and wild-type versions of many major cancer driver genes [3]. To determine similarities in the mechanisms underlying the anti-proliferative activity of the inhibitors, their IC50 fingerprint in the cell proliferation assays were compared by hierarchical clustering [4]. Compounds that act through the same primary kinase clustered together in this analysis. Exceptions were investigated further by profiling of additional cell lines, representing cancer gene alterations that were not present in the 102 cell line panel, such as FLT3 mutation and TRK-gene fusions, which occur in relatively small cancer patient populations. To compare the genomic targeting of kinase inhibitors acting on the same biochemical target, the cancer cell lines were classified as either “mutant” or “wild-type” for specific cancer gene alterations and were grouped per cancer gene. The relationship between drug sensitivity and cancer gene mutation status was examined. Detailed genomic biomarker analyses and comparative profiling results of novel BTK, EGFR, FGFR, FLT3 and TRK kinase inhibitors will be presented.[1] Mol Cancer Ther 2017;26:2609-17; [2] PLoS ONE 2014;9:e92146; [3] Mol Cancer Ther 2019;18:470-81; [4] Mol Cancer Ther 2016;15:3097-109 Citation Format: Jeffrey J. Kooijman, Wilhelmina E. van Riel, Martine B. Prinsen, Jelle Dylus, Jeroen A. de Roos, Yvonne Grobben, Nicole Willemsen-Seegers, Michelle Muller, Jos de Man, Yugo Narumi, Yusuke Kawase, Rogier C. Buijsman, Suzanne J. van Gerwen, Guido J. Zaman. Comparative cancer cell panel profiling of kinase inhibitors approved for clinical use from 2018 to 2020 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1480.

Site-specific incorporation of post-translationally modified amino acids into proteins, including histones, has been a subject of great interest for chemical and biochemical communities. Here, we describe a site-specific incorporation of structurally simplest trimethyllysine analogs into position 4 of the intact histone H3 protein. An efficient alkylation of cysteine 4 of the recombinantly expressed histone H3 provides a panel of trimethyllysine analogs that differ in charge, charge density, sterics, and chain length. We demonstrate that H3 histone that bears trimethyllysine analogs can be further assembled into the octameric histone complex that constitutes the nucleosome. Binding studies showed that H3 histone that possesses trimethyllysine analogs is well recognized by a PHD3 reader domain of human JARID1A. This work provides important (bio)chemical tools for fundamental biomolecular studies aimed at unravelling the molecular basis of the higher order nucleosome and chromatin assemblies.

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

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.