TP53, NOTCH1 and DNMT3 to be mutated in 84.6%, 69.2% and 30.0% of sensitive cell lines, and in 62.5%, 0% and 0% of resistant cell lines, respectively. An immunohistochemistry study showed that p-ERK and RelB were associated as potential biomarkers of tipifarnib sensitivity and resistance, respectively. Data from RNA-seq show that tipifarnib at IC50 after 72 h downregulated a great variety of pathways, including those controlling cell cycle, metabolism, and ribosomal and mitochondrial activity. This study establishes tipifarnib as a potential therapeutic option in T-cell leukemia and TCL. The mutational state of NOTCH1, p-ERK and RelB could serve as potential biomarkers of tipifarnib sensitivity and resistance.Patients diagnosed with T-cell leukemias and T-cell lymphomas (TCLs) still have a poor prognosis and an inadequate response to current therapies, highlighting the need for targeted treatments. We have analyzed the potential therapeutic value of the farnesyltransferase inhibitor, tipifarnib, in 25 TCL cell lines through the identification of genomic and/or immunohistochemical markers of tipifarnib sensitivity. More than half of the cell lines (60%) were considered to be sensitive. Tipifarnib reduced cell viability in these T-cell leukemia and TCL cell lines, induced apoptosis and modified the cell cycle. A mutational study showed  T-cell lymphoproliferative disorders are a very heterogeneous group of lymphoid neoplasms characterized by the clonal expansion of mature T-lymphocytes in bone marrow, blood or other tissues. They include peripheral T- cell lymphoma (PTCL) and T-cell acute lymphoblastic leukemia (T-ALL).1. It comprises a set of disorders that includes peripheral T-cell lymphoma not otherwise specified (PTCL-NOS), angioimmunoblastic T-cell lymphoma (AITL), anaplastic lymphoma kinase (ALK-) positive and ALK-negative forms of anaplastic large cell lymphoma (ALCL), and enteropathy-associated TCL2. Despite the subtypes being very different at the molecular level, their treatment is basically similar, consisting of CHOP  and Etopoxide, or a similar regimen. Therapeutic responses to this treatment show a current general 5-year survival rate of only 10–30% and a rapid progression in some subtypes5.PTCL accounts for about 10% of all non-Hodgkin lymphomas (NHLs)6. These results highlight the urgent need for alternative treatment strategies focusing on molecular characterization of individual cases that could identify potential candidates for targeted therapy.T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy characterized by an accumulation of immature T-cell lymphoblasts in bone marrow and peripheral blood. In contrast with PTCL, clinical progress has clearly improved in T-ALL; the 5-year survival rate is currently 65–70%8. The relatively poor prognosis for PTCL4 is ultimately the consequence of our insufficient knowledge about the molecular pathogenesis of these tumors and the limited availability of therapeutic tools8.The classification of T-cell lymphomas/leukemias is complex and requires the integration of information about clinical, pathological and genetic factors, morphology, immunohistochemistry (IHC), flow cytometry, cytogenetics, and molecular biology13, and, for a given gene, the distributions of the mutations and their relative prevalence are quite variable.Deregulated survival pathways and mutated genes have been partially characterized in PTCL. Genetic alterations affecting TCR signaling operate as a common pathogenic mechanism in several TCL entities. Mutations in genes related to TCR co-stimulation and signaling were recently reported in a range of TCL entities, such as cutaneous T-cell lymphoma (CTCL), PTCL-NOS, AITL and ATLL (adult T-cell lymphoma/leukemia). Nonetheless, mutations in specific genes have a variable frequency in distinct entitiesJAK1, JAK3, PTEN, IL7R and NRAS signaling proteins15. Other common oncogenic lesions are the loss of the CDKN2A (p16) locus and aberrant NOTCH1 signaling18. Activating mutations in JAK1, JAK3 or IL7R lead to activation of the JAK/STAT pathway, resulting in the stimulation of proliferation and survival pathways in the leukemic cells and thereby the development of T-ALL20.The mutational landscape of T-ALL has recently been elucidated too. A variety of somatic mutations was described like, 21. Mutations in RAS family genes induce constitutive activation of RAS-mitogen-activated protein kinase (MAPK), which activates several downstream effectors that play a role regulating a variety of cell functions, including cell growth, survival and differentiation. In view of these findings and the molecular landscape of PTCL and T-ALL, we were prompted to investigate the Ras mutations and MAPK pathway activation further. Farnesyltransferase inhibitors (FTIs) were designed to disrupt Ras farnesylation and the membrane localization necessary for Ras function. This work and other studies have demonstrated activity in neoplasms lacking mutant Ras23, suggesting that it could inhibit farnesylation of multiple proteins, leading to the arrest of proliferation and the induction of apoptosis in a variety of preclinical models25.Aberrant activation of oncogenic Ras signal transduction is a very frequent finding in PTCL and T-ALL27 and leukemia22. A variety of phase II trial has shown that it improves early survival when administered as maintenance therapy in patients in remission28. Administered as a single-agent, tipifarnib can produce antitumor effect in pretreated patients29.Some phase I trials using tipifarnib have demonstrated its antineoplastic effects in solid tumors31 in a panel of 25 TCL and T-ALL cell lines, enabling us to determine the therapeutic value of tipifarnib in these cell lines, and to identify biomarkers that could predict the response to this drug and to measure the dynamic effects on cell viability, apoptosis, cell cycle and gene expression. These observations could facilitate the development of personalized therapy in patients with TCL and T-ALL. The selected panel of 25 cell lines includes cell lines derived from a wide array of T-cell lymphoproliferative disorders, including T-cell acute lymphoblastic leukemia (T-ALL), cutaneous T-cell lymphoma (CTCL), anaplastic large cell lymphoma (ALCL) and adult T-cell lymphoma/leukemia (ATLL).The present study evaluates the effect of inhibiting FTase with tipifarnib50 values. With this classification, we found 60% of cell lines were sensitive to tipifarnib after 96 h , TH2 (IL-4), Th17 (IL-17) and Treg (Foxp3) phenotypes by analyzing changes in the expression of their markers in eight cell lines: MOLT-14, RPMI-8402, SU-DHL-1, L-82, HPB-ALL, HUT-78, JURKAT and HH. Those cytokines that allow the characterization of inflammatory, non-inflammatory and regulatory subpopulations were measured . No phenotypic changes were observed when cells were cultured in the presence of tipifarnib at IC50 and 2x IC50 , the mutational state of NOTCH1, ERK activation , sex and cell subtype. In this case, the magnitude of the change represents a coefficient that increases with the IC50 value  of <0.25 was used33 to generate testable hypotheses. The results suggested that the TCR pathway, G protein-coupled receptor activity and G2/M checkpoints were correlated with highly sensitive cases, while NFkB activation by the TNFα, IFNγ and IFNα response, ribosome and p53 pathway were correlated with resistant cell lines   and RAS-GTPase activating protein (GAP) genes, respectively . The mutational state of the RAS, RAS-GEF and RAS-GAP genes was not associated with drug sensitivity .These results prompted us to focus our analysis on the RAS/MAPK pathway and other T-cell lymphoma pathways, such as TCR, JAK/STAT, NFAT and NFκB. We selected only cell lines with genomic data available from COSMIC or CCLE or generated by our group (Supplementary Table 05) Fig. . 52% andty Figs.  and 4e, To elucidate the consequences of farnesyltransferase inhibition in TCL and T-ALL cell lines, we performed RNA-seq in three highly sensitive cell lines . Differential gene expression was analyzed with and without tipifarnib. We detected 24 differentially expressed (DE) genes common to all three cell lines Fig. .Figure 5. Several of the downregulated genes are involved in the cell cycle: UNG prevents mutagenesis; UHRF1 regulates chromatin structure and gene expression; SSRP1 affects nucleosome disassembly and transcription elongation; MYBL2 influences cell-cycle progression; MCM2 and MCM3 are involved in replication. Other deregulated genes are closely related to enzymatic and mitochondrial activity . The heat shock response genes, HSPD1, HSPA9 and HSP90AB1, are other downregulated genes.Two of these upregulated genes are related to damage response and the prevention of infection by retroviruses 35, are shown in Fig. GSEA was performed under more stringent conditions  at clinically achievable concentrations .Here we report the effectiveness of the farnesyltransferase inhibitor tipifarnib in a panel of 25 TCL and T-ALL cell lines, integrating TP53, NOTCH1 and DNMT3A to be mutated in 84.6%, 69.2% and 30.0% of cells in sensitive cell lines, respectively, in contrast to resistant cell lines, in which the mutational frequencies were much lower   Figs. . The assNOTCH1 and the activation of ERK could serve as potential biomarkers of sensitivity to tipifarnib, and the positivity of RelB (by IHC) could be a biomarker of resistance to the drug. Other studied markers, such as CD30, showed no such significant associations  and three resistant (37.5%) cell lines harbored mutations in RAS genes (not significant (n.s.)); eight sensitive (61%) and three resistant (37.5%) cell lines harbored mutations in RAS-guanine nucleotide exchange factors (GEFs) (n.s.) and five sensitive (38%) and two resistant (25%) cell lines harbored mutations in RAS-GTPase-activating protein (GAP) genes (n.s.). The mutational state of the RAS, RAS-GEF and RAS-GAP genes was not associated with tipifarnib sensitivity. The mutational state of all the RAS-GEF genes was associated with ERK activation and RelB negativity .Given the association between ERK activation and tipifarnib sensitivity, we focused our attention on the RAS/MAPK pathway and other T-cell leukemia/lymphoma-related pathways, such as TCR, JAK/STAT, NFAT and NFκB, which are known to be involved in T-cell leukemia/lymphomaUnder basal conditions, RNAseq analysis of 25 cell lines indicated that the TCR pathway, G protein-coupled receptor activity and G2/M checkpoints were correlated with greater sensitivity to tipifarnib, while NFkB activation by TNFα, IFNγ and IFNα response, ribosome and p53 pathway were correlated with resistance. These results replicate and confirm our previous observations that associated mutational status and pathway activation with tipifarnib sensitivity.To improve our understanding of the biological mechanism of action of tipifarnib, we performed RNA-seq in three of the most sensitive cell lines . We detected more than 300 differentially expressed genes , which is a farnesylated small GTPase, a key protein in the Rheb-Notch-Rheb loop, that positively regulates mTOR signaling49.The mammalian target of rapamycin (mTOR) is another commonly activated pathway in TCL and T-ALLNOTCH1 and the activation of ERK could serve as potential biomarkers of tipifarnib sensitivity, and a high level of RelB expression could be a biomarker of resistance to the drug. These results require validation in clinical samples or PDXs.This study suggests that tipifarnib is a potential therapeutic option in T-cell lymphomas and T-cell acute lymphoblastic leukemia. The results of our study suggest that the mutational state of 8). All cell lines were purchased or authenticated before use and were tested for mycoplasma .We included 25 TCL and T-ALL cell lines representing tumor types such as T-cell acute lymphoblastic leukemia/lymphoma , anaplastic large cell lymphoma , cutaneous T-cell lymphoma , and adult T-cell lymphoblastic leukemia   and cultured with Roswell Park Memorial Institute (RPMI) 1640 Medium (Thermo Fisher Scientific) containing 10% fetal bovine serum (Gibco), except for the MJ cell line, which was cultured with Gibco Iscove’s Modified Dulbecco’s Media (IMDM) medium (Thermo Fisher Scientific) containing 20% fetal bovine serum.BE-13, DND-41, HD-MAR-2, HPB-ALL, HSB-2, JURKAT, KE-37, L-82, MHH-TALL-2, MOLT-14, MOLT-16, P12-ICHIKAWA, PF-382, RPMI-8402, SR-786 and TALL-1 were purchased from the DSMZ German Collection of Microorganisms and Cell Cultures. Except for HSB-2, they were cultured with Roswell Park Memorial Institute (RPMI) 1640 Medium containing different proportions of fetal bovine serum: 10% for DND-41, HD-MAR-2, HPB-ALL, JURKAT, KE-37, L-82, MOLT-14, MOLT-16, P12-ICHIKAWA, PF-382 and RPMI-8402; 20% for BE-13 and MHH-TALL-2; 15% for SR-786 and TALL-1. HSB-2 was cultured with Gibco Iscove’s Modified Dulbecco’s Media (IMDM) medium containing 10% fetal bovine serum.MyLa was purchased from the European Collection of Authenticated Cell Cultures (ECACC) and was cultured with Roswell Park Memorial Institute (RPMI) 1640 Medium containing 10% fetal bovine serum.2 incubators at 37 °C in an atmosphere containing 5% CO2.All cells were cultured in CO50  values were calculated. Tipifarnib was provided by Kura Oncology . Growth curves were derived and IC50 values determined using the Cell Titer-Glo Luminescent Cell Viability Assay from Promega  at 0, 24, 72 and 96 h. IC50 values were calculated with Graphpad Prism v5 .All cell lines were tested for tipifarnib sensitivity in two steps, for which purpose they were cultured in appropriate medium in T96 multi-well plates . Individual optimal cell line seeding densities were determined before deriving growth curves with increasing concentrations of tipifarnib  using DMSO as the vehicle for 96 h. Six replicates of all experiments were carried out, and IC2. Cells were maintained in standard conditions for 24 h, after which they were exposed to IC50 and 2x IC50 values of tipifarnib and DMSO as a vehicle for 96 h. The positive and negative controls for the next analysis were included. To identify dead and apoptotic cells, 100 μL of Annexin Red Working Solution were added to each 100 μL of cell suspension and samples were incubated for 15 min at 37 °C. Samples were washed and resuspended in Assay Buffer HSC and then 5 μL of 7-ADD were added. Samples were incubated at room temperature (RT) for 5 minutes in the dark before analysis by Fluorescence-Activated Cell Sorting (FACS). Double-positive cells (Annexin-V and 7-ADD) were considered in the late apoptotic stage or necrotic cells, whereas cells that were positive for Annexin-V but negative for 7-ADD were those in early-stage apoptosis; in this study, we were interested in these early apoptotic cells. To assess the effects on DNA synthesis, cells were grown in each appropriate medium and, after treatment with tipifarnib, incubated for a further 2 h with EdU (5-ethynyl-2′-deoxyuridine), which is a nucleoside analog of thymidine that is incorporated into the DNA during active DNA synthesis . Immediately afterwards, cells were prepared for cytometry following the manufacturer’s recommendations. To calculate the percentage blockade of tipifarnib in the G0 phase and to prevent these cell lines from entering the G2 phase of replication, the percentage inhibition of tipifarnib with respect to DMSO was measured.Three of the most sensitive cell lines were subjected to apoptosis and cell-cycle analysis. Flow cytometry assays were used for both analyses, using a Flow Cellect Annexin Red Kit  and a Click-iT Plus EdU Alexa Fluor 647 Flow Cytometry Assay Kit , following the manufacturers’ instructions. Cells were seeded in 12-well plates  and incubated for 24 h at 37 °C in a humidified atmosphere of 5% CO8, we obtained genomic data from genomic repositories from T-cell lymphoma/leukemia cell lines. Specifically, we combined genomic data from CCLE , COSMIC Cell Lines Project , EGAS00001000268 , exomes produced previously by our group . Variants were annotated using the Variant Effect Predictor . Known SNPs with an allelic frequency greater than 1% in public databases  were filtered out.As previously described by our groupet al.8, total RNA was extracted using TRIzol reagent (Invitrogen) according to the manufacturer’s protocol. Total RNA was assayed for quantity and quality using the Qubit RNA BR or HS Assay Kit on the Qubit 2.0 Fluorometer (Life Technologies), and the RNA 6000 Nano Assay on a Bioanalyzer 2100 (Agilent). The RNASeq libraries were prepared using TruSeq Stranded mRNA LT Sample Preparation Kit . Briefly, total RNA (500 ng) was enriched for the mRNA fraction and fragmented by divalent metal cations at high temperature . To achieve directionality, the second-strand cDNA synthesis was performed in the presence of dUTP. Mondejar et al.8 described how the blunt-ended double-stranded cDNA was 3′adenylated and Illumina indexed adapters were ligated. The ligation product was enriched over 15 PCR cycles and the final library was validated on an Agilent 2100 Bioanalyzer with the DNA 7500 assay (Agilent). Libraries were sequenced on HiSeq2000  in paired-end mode with a read length of 2 × 76 bp using a TruSeq SBS Kit v4 in a fraction of a sequencing v4 flow cell lane, following the manufacturer’s protocol. Image analysis, base calling and quality scoring of the run were processed using the manufacturer’s Real Time Analysis (RTA 1.18.66.3) software, followed by generation of FASTQ sequence files.As previously described by Mondejar 51 with ENCODE parameters for long RNA. Annotated genes (GENCODE version 27) were quantified using RSEM version 1.3.052 with default parameters. Differential expression was analyzed with limma-voom53 adjusted for sex, cell subtype and 18 other hidden factors detected by surrogate variable analysis (sva). The IC50 was considered as a continuous variable, and only genes with at least 1 cpm in 10 samples or more were selected for the analysis.RNA-seq was performed in 25 cell lines under basal culture conditions. RNA-seq paired-end reads were mapped against the human reference genome (GRCh38) using STAR version 2.5.3a50 and DMSO in three biological replicates. RNA-seq reads were mapped and genes were annotated as described above. Principal component analysis of the ‘rlog’-transformed expression data was done using the top 500 most variable genes with the ‘prcomp’ R function and ‘ggplot2’ R library. Two outlier samples belonging to DMSO_SUDHL1 and TIPI_SUDHL1 were removed. Differential expression was analyzed with DESeq2 version 1.1054 for each cell line separately. Heatmaps with the top 50 differentially expressed genes were generated with the ‘pheatmap’ R package. Gene set enrichment analysis (GSEA) was performed from a background of > 5000 gene sets of the Hallmark collection of the Molecular Signatures Database (MSigDB), curated  and Gene Ontology (GO) gene sets (C5 collection). Expression data have been deposited in the Sequence Read Archive (SRA) under BioProject accession reference PRJNA603759.To further elucidate the consequences of farnesyltransferase inhibition in TCL and T-ALL cell lines, RNA-seq was performed in three cell lines  treated with tipifarnib for 72 h at IC50 of tipifarnib and DMSO in plates of T12 wells . All experiments were done in triplicate.The expression of the markers IL-2, IL-4, IFN-g, IL-17, RORγT and Foxp3 was tested by FACS in eight cell lines , enabling possible changes in the TH1/TH2/Treg/Th17 phenotypes to be studied. Cell lines were incubated for 96 h at 1x and 2x the ICThe chi-square or Fisher exact test were used to determine the significance of associations between the presence or absence of markers and sensitivity. Estimates were considered statistically significant for two-tailed values of p < 0.05. All analyses were carried out using IBM SPSS for Windows version 20 .Supplementary Table Legends.Supplementary Tables.


Restoration of blood flow to ischaemic heart inflicts ischaemia/reperfusion (I/R) injury, which manifests in metabolic and morphological disorders. Klotho is a protein with antioxidative and antiapoptotic activity, and is involved in the regulation of inflammation and fibrosis. The aim of the current research was to determine the role of Klotho in the heart subjected to I/R injury, as well as to study Klotho as a potential cardioprotective agent. Human cardiomyocytes and Wistar rat hearts perfused using Langendorff method subjected to I/R have been used. Hemodynamic parameters of heart function, markers of I/R injury, and gene and protein expression of Klotho were measured. Human cardiomyocytes were also incubated in the presence of recombinant Klotho protein, and the viability of cells was measured. There was a higher expression of Klotho gene and protein synthesis in the cardiomyocytes subjected to I/R injury. The compensatory production and release of Klotho protein from cardiac tissue during I/R were also shown. The treatment of cardiomyocytes subjected to I/R with Klotho protein resulted in increased viability and metabolic activity of cells. Thus, Klotho contributes to compensatory mechanism during I/R, and could be used as a marker of injury and as a potential cardiopreventive/cardioprotective agent. The expression of Klotho gene is observed mainly in the kidneys and brain.The aim of this study was to investigate the role of Klotho protein in the heart tissue subjected to I/R injury, as well as to study Klotho as a potential cardioprotective agent.2In this study, animal tissues obtained from our other project were used. All animal procedures conformed to the Guide to the Care and Use of Experimental Animals published by the Polish Ministry of Science and Higher Education. This investigation was approved by the Ethics Committee for Experiments on Animals at the Ludwik Hirszfeld Institute of Immunology and Experimental Therapy Polish Academy of Sciences, Wroclaw, Poland (Resolution 14/2016 of 20 April 2016).2.12 atmosphere in Dulbecco's Modified Eagle's Medium (Sigma‐Aldrich) containing Cardiac Myocyte Growth Supplement (ScienCell Research Laboratories), 5% foetal bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin . Cells were passaged at 90% confluence using 0.25% trypsin‐EDTA (Sigma‐Aldrich).The primary human cardiac myocytes (HCM) were purchased from ScienCell Research Laboratories. The cells were cultured at 37°C in a water‐saturated, 5% CO2.22, 5 mmol/L KCl, 2.1 mmol/L MgCl2, 5.5 mmol/L glucose and 10 mmol/L taurine) containing additional 55 μmol/L CaCl2 and 0.75 mg/mL BSA. In an in vitro chemical ischaemia group, cells were incubated in HEPES buffer containing 4.4 mmol/L 2‐deoxyglucose (to inhibit glycolysis) and 4.0 mmol/L sodium cyanide (an inhibitor of cellular respiration).g, the buffer was removed and cells were incubated in aerobic conditions with HEPES buffer for 20 minutes at RT (reperfusion). After reperfusion, the buffer was removed by centrifugation at 1500 g for 5 minutes, and the cell pellet was homogenized. The cells from aerobic control group were incubated aerobically for 50 minutes in HEPES buffer at RT. In the Klotho experimental groups , the cells underwent experimental protocol in the presence of Klotho protein  during whole procedure. Myocytes from aerobic group subjected to Klotho protein were tested to check the cytotoxicity of Klotho.Cardiac myocytes in culture underwent in vitro chemical I/R in accordance with the guidelines for experimental models of myocardial ischaemia and infarction.2.3g for 5 minutes at 4°C and then were transferred into a fresh tube. The homogenates were stored at −80°C for further experiments.Cells underwent three cycles of freezing (in liquid nitrogen) and thawing (at 37°C) in the homogenization buffer . Then, myocytes were homogenized mechanically on ice (three times for 10 seconds) with a hand‐held homogenizer. Supernatants were obtained by centrifugation at 10 000 2.4−ΔCt. 2−ΔCt was equal to the relative transcriptional mRNA level of Klotho gene in cells that were exposed to aerobic conditions and cells subjected to I/R.Total RNA from HCM was isolated with TRIZOL reagent (Thermo Fisher Scientific) according to the manufacturer's instruction. Microultraviolet (UV) spectrophotometer  was used to evaluate the concentration and purity of RNA. Reverse transcription of pure RNA samples (200 ng) was conducted in order to prepare cDNA with iScript cDNA Synthesis Kit (Bio‐Rad) according to the instructions provided. Briefly, the reverse transcription was carried out at 42°C for 30 minutes and inactivated at 85°C for 5 minutes. The expression level of glucose‐6‐phosphate dehydrogenase (G6PD) was used as internal reference for Klotho. To analyse expression of Klotho gene in a ratio to G6PD gene, real‐time quantitative PCR (RT‐qPCR) and CFX96 Real‐Time System (Bio‐Rad) were used. Briefly, the final volume of the reaction mix was 30 μL and included iTag Universal Sybr Green Supermix with ROX (Bio‐Rad), forward and reverse primers , water and cDNA (40 ng). The primers for Klotho were designed by us and synthesized by TIB Molbiol . The sequences of primers 5′–3′ are as follows: Human Klotho F: AGGGTCCTAGGCTGGAATGT and Human Klotho R: CCTCAGGGACACAGGGTTTA. The amount of particular mRNAs relative to G6PD was calculated as 22.5Klotho protein concentration in cell homogenates was measured quantitatively using Sandwich Human Klotho ELISA Kit from Biorbyt (Biorbyt Ltd.), according to manufacturer's instruction. After resetting the blank well, the optical density (OD) (at 450 nm) of each well was measured by Spark multimode microplate reader (Tecan Trading AG). Klotho protein concentration was normalized to total protein concentration in cell homogenates and expressed in pg/μg protein. The concentration of Klotho was compared in cells that were exposed to aerobic conditions and cells subjected to I/R.2.65 cells/well. When the cell confluence reached approximately 90%, myocytes underwent in vitro chemical I/R injury in the cell culture plate  for 15 minutes in the dark and rinsed with PBS. ZOE Fluorescent Cell Imager (Bio‐Rad) was used to estimate the bright green fluorescence for Klotho and blue fluorescence for DAPI. Image J 1.52a software (NIH) was used to analyse the area of fluorescence of each image. The number of cells was assessed by measuring the fluorescence of cells’ nuclei stained by DAPI (blue fluorescence). The cell surface expression of Klotho was assessed by measuring the green fluorescence of fixed cells stained with anti‐Klotho antibodies and normalized to the number of cells (blue fluorescence). Data were collected from three independent images trials for each experiment and shown as AU. The cell surface expression of Klotho was compared in cells that were exposed to aerobic conditions and cells subjected to I/R.The cells were cultured in 24‐well cell culture plate (Greiner Bio‐One GmbH) at a density of 1 × 102.74 cells/well for 24 hours and then subjected to in vitro chemical I/R injury in the presence and absence of 1 µg/mL Klotho protein  was performed.2.84 cells/well for 24 hours and then subjected to in vitro chemical I/R injury in the presence and absence of 1 µg/mL Klotho protein  and the vital exclusion dye DAPI.2.92PO4, 1.2 mmol/L MgSO4, 3.0 mmol/L CaCl2, 25 mmol/L NaHCO3, 11 mmol/L glucose and 0.5 mmol/L EDTA, pH 7.4. Spontaneously beating isolated hearts were cannulated by the aorta on a Langendorf system (EMKA Technologies) and perfused at a constant pressure of 60 mm Hg with Krebs‐Henseleit Buffer at pH 7.4, at 37°C and gassed continuously with 5% CO2/95% O2. After stabilization, the hearts were subjected to protocol of global I/R injury. As haemodynamic end‐points of cardioprotection,To determine the expression of Klotho protein at the tissue level, isolated rat heart model was used. Ten‐ to 11‐week‐old male Wistar rats (250‐350 g) were treated with buprenorfin  and anaesthetized with sodium pentobarbital (0.5 mL/kg i.p.). The hearts were rapidly excised from animals and rinsed by immersion in ice‐cold Krebs‐Henseleit Buffer containing 118 mmol/L NaCl, 4.7 mmol/L KCl, 1.2 mmol/L KH2.10Hearts in I/R group were subjected to aerobic perfusion for 25 minutes. After oxygenation, the heart perfusion was stopped (by cessation of the buffer flow) and hearts underwent global no‐flow ischaemia for 22 minutes.2.11g at 4°C for 15 minutes, and the supernatant was collected and stored at − 80°C for further experiments.Hearts previously frozen at −80°C were crushed using a mortar and pestle in liquid nitrogen and then homogenized in ice‐cold homogenization buffer containing: 50 mmol/L Tris‐HCl (pH 7.4), 3.1 mmol/L sucrose, 1 mmol/L dithiothreitol, 10 mg/mL, leupeptin, 10 mg/mL soybean trypsin inhibitor, 2 mg/mL aprotinin and 0.1% Triton X‐100. The homogenate was centrifuged at 10 0002.12The expression of Klotho protein in the rat heart tissue was determined qualitatively by immunoblot and quantitatively by ELISA test in aerobic control and I/R groups. The content of Klotho protein in coronary effluents was determined by immunoblot. In the Western blot analysis, an aliquot of 20 μg of total proteins from heart homogenates and an aliquot of 40 μL of coronary effluents were separated on 10% SDS‐PAGE and then transferred onto nitrocellulose membrane (Bio‐Rad), which were then blocked with 5% BSA. Klotho protein was detected with rabbit anti‐Klotho polyclonal antibody 1:1000  and secondary goat anti‐rabbit IgG horseradish peroxidase conjugate antibody 1:5000 . The membranes were developed with the ClarityTM Western ECL substrate (Bio‐Rad) using the ChemiDocTM MP System. Target protein bands in coronary effluents were quantified using Quantity One Software (Bio‐Rad). 120‐130 kDa Recombinant Human Klotho  and 65‐70 kDa Recombinant Human Klotho  were used as positive controls. The content of Klotho protein in coronary effluents was expressed as AU and normalized to CF. For quantitative evaluation of Klotho protein in the heart tissue, Rat Klotho ELISA Kit (Cusabio Technology LLC) was used according to manufacturer's instruction. Each sample was assayed in duplicate. Klotho protein concentration was normalized to total protein concentration in tissue homogenates and expressed in pg/μg protein. The level of Klotho was compared in hearts that were exposed to aerobic conditions and hearts subjected to I/R.2.13To determine the activity of LDH in coronary effluents, Lactate Dehydrogenase Activity Assay Kit (Sigma‐Aldrich) according to manufacturer's instruction was used. LDH is a stable cytosolic enzyme that is released upon membrane damage/permeability or cell lysis and serves as a marker of cell damage. LDH level was normalized to CF and compared in coronary effluents of hearts that were exposed to aerobic conditions and hearts subjected to I/R.2.14To assess an influence of I/R injury on isolated rat hearts, the number of death cells using the CytoTox‐Glo™ Cytotoxicity Assay (Promega) according to manufacturer's instruction was tested. The measurement of extracellular activity of dead‐cell protease was performed in coronary effluents and normalized to CF. The number of death cells was compared in hearts that were exposed to aerobic conditions and hearts subjected to I/R.2.15Bradford method was used to determine protein concentration in the cardiac tissue and cell homogenates. BSA  served as the protein standard. For measuring total protein concentration, Bio‐Rad Protein Assay Dye Reagent (Bio‐Rad) and Spark multimode microplate reader (Tecan Trading AG) were used.2.16t test or Mann‐Whitney U test was used for comparison between two groups of measurement data. ANOVA or non‐parametric test with post hoc tests for multiple groups was used. Correlations were assessed using Pearson's or Spearman's test, as appropriate. Results were expressed as mean ± SEM, and a value of P < .05 was regarded as statistically significant.Experimental data were analysed using GraphPad Prism 6 software (GraphPad Software). Shapiro‐Wilk normality test or Kolmogorov‐Smirnov test was used to assess normality of variances changes. The Student's 33.1The analysis showed significantly higher expression of Klotho mRNA in myocytes subjected to I/R in comparison with aerobic control group Figure . Then, w3.2Cardiac mechanical function was decreased in hearts subjected to I/R in comparison with aerobically perfused hearts, showing increased heart injury Table . The rel3.3P < .05, r = −.7) and positively correlated with LDH level   Figure .3.4To check the potential cardioprotective effect of Klotho protein, HCM were subjected to I/R in the presence and absence of Klotho protein. The treatment of cardiomyocytes with recombinant human Klotho (1 µg/mL) resulted in increased viability of cells during I/R. To check the toxicity of Klotho protein on cardiomyocytes, cells from aerobic control group were incubated with Klotho. Supplementation of recombinant Klotho protein decreased the number of dead cells in I/R group in comparison with I/R group without Klotho addition and did not show toxicity in aerobic control group Figure .3.5To explore an impact of Klotho on the metabolic activity of cardiomyocytes, the vital inclusion dye FDA and the vital exclusion dye DAPI were used. Cells that stained with FDA and excluded DAPI were considered viable, whereas cells that excluded FDA and stained with DAPI were considered necrotic. I/R injury led to decreased metabolic activity of the cardiomyocytes. The treatment with Klotho protein (1 µg/mL) significantly increased metabolic activity of the cardiomyocytes that underwent I/R in comparison with Klotho untreated I/R group Figure .4As the discovery of Klotho, a significant amount of efforts has been devoted to characterize its protective role in oxidative stress, inflammation, fibrosis and apoptosis.There are 3 types of Klotho protein: membrane‐bound (135 kDa), shed and secreted. It is known that membrane proteases cut membrane‐bound Klotho to generate shed types of Klotho: full‐length 130 kDa molecule (the major product of shedding) and a fragment of molecular mass of about 68 kDa. The alternative transcriptional termination of Klotho gene leads to production of secreted Klotho (about 65 kDa). Soluble types of Klotho protein (shed and secreted) are found in the blood, urine and cerebrospinal fluid.A large number of studies over last decade have greatly enriched our knowledge of correlation between Klotho deficiency and risk of CVD.In our study, we showed that Klotho protein was intensively released from cardiac tissue subjected to ischaemic damage. Thus, we propose that enhanced expression of Klotho, its further release from cardiomyocytes into extracellular space and increased level in the body fluids may be useful as a marker of ischaemic heart injury. This hypothesis can be supported by the evidence of increased serum Klotho level in patients after heart failure.Research from the last few years showed protective effect of Klotho overexpression or administration during ischaemic injury of the brain and kidneys.5Our research showed that: (a) there is an increased compensative production of Klotho protein in the cardiomyocytes during I/R to protect ischaemic heart from further injury; (b) an enhanced production of Klotho during I/R and its release to extracellular space can be used as a marker of heart damage; and (c) Klotho administration protects the cardiomyocytes against I/R injury and improves their metabolism after ischaemic damage.In conclusion, this study confirmed the potential cardioprotective role of Klotho in the development of I/R injury. We propose that administration of Klotho protein into hearts may prevent the cardiomyocytes from the damage or reduce the area of injury. We strongly suggest that Klotho protein can be valuable both as a marker of injury and as a potential cardioprotective agent and contributes to compensatory mechanism which mitigates the initial damage under cardiac stress.We are also aware that further studies on the role of exogenous Klotho on cardioprotection on in vivo model are needed. So the next goal of our study is to check whether administration of Klotho before I/R or during I/R will be cardioprotective, and how this cardioprotection is induced.The authors confirm that there are no conflicts of interest.All authors participated in the design, interpretation of the studies, analysis of the data and review of the manuscript; AO, AKZ and IBL designed the research study; AO, AKZ, MB and IBL performed the research; AO and IBL analysed the data; AO wrote the paper; IBL made critical revision of the manuscript.


EZH2 and other members of the polycomb repressive complex and EZH2 inhibition in RMC tumor spheroids resulted in decreased viability. High throughput drug screening of both cell lines revealed several additional candidate compounds, including bortezomib that had both in vitro and in vivo antitumor activity. The activity of bortezomib was shown to be partially dependent on increased oxidative stress as addition of the N‐acetyl cysteine antioxidant reduced the effect on cell proliferation. Combining bortezomib and cisplatin further decreased cell viability both in vitro and in vivo that single agent bortezomib treatment. The UOK353 and UOK360 cell lines represent novel preclinical models for the development of effective forms of therapy for RMC patients.Renal medullary carcinoma (RMC) is a rare, aggressive disease that predominantly afflicts individuals of African or Mediterranean descent with sickle cell trait. RMC comprises 1% of all renal cell carcinoma diagnoses with a median overall survival of 13 months. Patients are typically young (median age—22) and male  and tumors are characterized by complete loss of expression of the SMARCB1 tumor suppressor protein. Due to the low incidence of RMC and the disease's aggressiveness, treatment decisions are often based on case reports. Thus, it is critical to develop preclinical models of RMC to better understand the pathogenesis of this disease and to identify effective forms of therapy. Two novel cell line models, UOK353 and UOK360, were derived from primary RMCs that both demonstrated the characteristic SMARCB1 loss. Both cell lines overexpressed  RCC consists of several distinct subtypes that vary in incidence and demonstrate associations with specific histologic, genetic, and clinical features. Renal medullary carcinoma (RMC) is a rare and aggressive subtype of RCC that predominantly afflicts individuals of African or Mediterranean decent with sickle cell trait.SMARCB1 in chromosome band 22q11.23, is a core subunit of the SWI/SNF chromatin remodeling complex. Loss destabilizes the complex and disrupts its equilibrium with the polycomb repressive complex 2 (PRC2) resulting in dysregulation of transcription activity.SMARCB1 gene.SMARCB1 mutant MRT mouse xenograft models have shown complete responses.A central feature of RMC is loss of expression of the SWI/SNF‐related matrix‐associated actin‐dependent regulator of chromatin subfamily B member 1 (SMARCB1) protein, also known as integrase interactor 1 (INI1), BRG1‐associated factor 47 (BAF47), or sucrose non‐fermenting 5 (SNF5).SMARCB1 loss and (2) high throughput drug screening. Both cell lines produced tumor xenografts in nude mice, with UOK360 more rapidly, providing models for both in vitro and in vivo analysis of potential therapeutic targets.We endeavored to establish new RMC cell lines in hopes of elucidating their biology and defining effective treatments. Herein we report the development of two patient tumor‐derived cell lines, UOK353 and UOK360, that resulted from spontaneous transformation and represent models for RMC. These cell lines allow the adoption of a two‐pronged approach to identification of potential therapeutics using (1) a targeted approach focused on exploiting 22.1Patients were evaluated and managed at the Hatfield Clinical Research Center, National Institutes of Health (NIH). Tumor tissues were obtained from biopsy or surgical resection and used for cell line production. Peripheral blood and tumor samples were obtained for DNA extraction. All patients signed the Urologic Oncology Branch protocol, NCI‐97‐C‐0147 that has been approved by the Institutional Review Board of the National Cancer Institute and that includes the production and use of cell line models. Informed consent was acquired only after the patient had fully understood what might be done with samples obtained from them and the implications of our research.2.2d‐glucose) DMEM medium supplemented with 10% fetal calf serum and 2 mM l‐glutamine. Short tandem repeat (STR)‐based DNA‐profiling was performed on the cell lines and the matched peripheral blood DNA to confirm the origins of both cell lines.Surgically resected tumor tissue from one patient and a tumor biopsy from a second patient were utilized to establish two cell lines, UOK353 and UOK360, in accordance with protocols and techniques previously described by the Urologic Oncology Branch.2.3FISH and SKY were performed on both UOK353 and UOK360, on interphase and metaphase spreads, respectively. Interphase cells were hybridized with a SMARCB1 break apart probe . Images were imaged on ×63 objective using a Leica Thunder Imager . Metaphase spreads (n = 25) were scored for chromosomal copy number and for structural aberrations as previously described.2.4SMARCB1 and TP53 genes identified by the OncoVar v.4 assay were validated by an orthogonal sequencing methodology to validate the accuracy of the mutation calling. Sanger sequencing was performed on PCR products generated using a Qiagen Taq PCR Core Kit  that were bidirectionally sequenced using the BigDye Terminator v.1.1 Cycle Sequencing Kit , in accordance with manufacturer's protocols. Sequence reactions were cleaned with Performa DTR Plates  and capillary electrophoresis was performed on an ABI 3730/ABI 3130xl Genetic Analyzer automated sequencing machine (Applied Biosystems). Sanger Sequencing was conducted at the CCR Genomics Core, NCI. Forward and reverse sequences were evaluated using Sequencher 5.4.6 .DNA was extracted from cell pellets, tumors, and peripheral blood leucocytes using a Promega Maxwell 16 Cell, Tissue, or Blood DNA Purification Kit . The UOK353 and UOK360 cell line DNAs were assessed using the OncoVar v.4 assay provided by Genetics Branch, National Cancer Institute (NCI). The assay performs hybrid capture sequencing analysis for genomic variants on a panel of cancer‐related genes, including the known kidney cancer associated genes.2.5SMARCB1 (Hs00992521_m1), SMARCA4 (Hs00946396_m1), and PBRM1 (Hs00217778_m1), components of the PRC2 complex—EZH2 (Hs01016789_m1), SUZ12 (Hs00248742_m1), and EED (Hs00537777_m1), and downstream expression markers of activation of the NRF2‐antioxidant response element (ARE) signaling pathway—NQO1 (Hs00168547_m1), HMOX1 (Hs01110250_m1), SQSTM1 (p62) (Hs01061917_g1), GCLC (Hs00155249_m1), and GCLM (Hs00978072_m1).Total RNA was extracted from cell lines grown in 10 cm dishes using Direct‐zol Miniprep , following a standard protocol. Cell lines were grown to a confluency of approximately 80%‐90%, washed with 5 mL of sterile PBS and lysed using 1 mL of Trizol reagent. Following the standard RNA extraction protocol, the RNA was resuspended in 50 μL of RNase‐free water and the RNA concentration was measured using a NanoDrop 2000 UV‐Vis Spectrophotometer (Thermo Fisher Scientific). For each cell line, cDNA was synthesized from 1 μg of total mRNA using the SuperScript VILO cDNA Synthesis Kit (Thermo Fisher Scientific) in a 20 μL volume. The cDNAs were diluted 10‐fold with 180 μL of RNase‐free water and 2 μL was used in 10 μL reaction volume for RT‐PCR amplification using an ABI ViiA7 real‐time PCR system (Thermo Fisher Scientific). Expression levels were normalized to the control 18S housekeeping gene (Hs99999901_s1) and calculated using the ViiA7 software as comparative CT (ΔΔCT) values. The non‐immortalized normal kidney cell line HRCE was designated to represent the normal expression level with a value of 1. TaqMan Gene Expression Assays (Thermo Fisher Scientific) were used to assess the expression levels of several genes, including components of the SWI/SNF complex—2.62PO4, 10 mM Tris pH 8, 0.1% mercaptoethanol). Protein concentration was quantified by the Bio‐Rad protein assay . Proteins (20 μg) were separated by electrophoresis on NuPage 4%‐20% Bis‐Tris gels  and transferred onto Immobilon‐P membranes  according to the manufacturers' directions. Western blot analyses of proteins were carried out by using anti‐Ku80 (C48E7) , anti‐BAF180 (ABE70) (Millipore Sigma), anti‐BRG1/SMARCA4 (SC‐17796) , anti‐BAF57/SMARCE1 (A300‐810A) , anti‐SNF5/ SMARCB1 (612111) , anti‐EZH2 (ab3748) , anti‐SUZ12 (ab126577) (Abcam), and anti‐EED (ab4469) (Abcam). The primary antibodies were detected using fluorescently labeled anti‐mouse and anti‐rabbit secondary antibodies at a dilution of 1:10 000 and obtained from Li‐Cor and were visualized by scanning the blots on a Li‐Cor Odyssey (Thermo Fisher Scientific).Sub‐confluent cells were trypsinized and washed once in PBS. Total proteins were extracted by solubilizing the cell pellets in urea buffer  at 48 hours posttreatment. The Cell Titer Glo assay was conducted according to the manufacturer's instructions.2.82 until formation was confirmed by eye under a microscope. UOK353 did not produce usably spheroids, only loose aggregates, and where not analyzed. However, UOK360 cells produced tight spheroids and these were evaluated with the positive control SMARCB1‐deficient MRT cell line G401. After spheroids had been determined to have formed, drugs were added at ×2 concentration to reach ×1. All drugs were incubated for 48 hours and evaluated using Cell Titer Glo3D. For GSK126, every 3‐4 days 100 μL of the 200 μL of media in each well was removed and replaced with 100 μL fresh media containing ×2 the desired drug concentration. Cell Titer Glo 3D assay was conducted according to the manufacturer's instructions.Cell lines were evaluated using several EZH2 inhibitors, GSK126, EPZ6438, and JQEZ5, but these agents require an extended time period to be effectively evaluated that is not compatible with 2D in vitro assays in these fast‐growing cell lines, so 3D spheroid viability assays were utilized.2.9Samples were treated with equimolar concentration of the indicated compounds and viability as determined by Cell Titer Glo was utilized as the endpoint. The combination index calculation was performed using CompuSyn software .2.106 UOK353 or 1‐3 × 106 UOK360 cells were suspended in a 0.2 mL mixture of 50% PBS and 50% Matrigel Matrix  and subcutaneously injected into 10 NCI Athymic NCr‐nu/nu mice (obtained from Charles River Frederick Research Model Facility) to evaluate the tumorigenic potential of these cell lines. All animal care protocols used had been approved by the Institutional Animal Care and Use Committee (IACUC) and were in accordance with National Cancer Institute guidelines.Approximately 5 × 102.11Quantitative high throughput screening (qHTS) was conducted with the assistance of the National Center for Advancing Translational Sciences, NIH. Five hundred UOK353 or UOK360 cells were plated into white, solid‐bottom, 1536‐well tissue culture treated plates using a Multidrop Combi cassette dispenser. The cells were then treated with the compounds of the MIPE library. Viability was assessed using Cell Titer‐Glo assay (Promega) 48 hours posttreatment and luminescence was measured on a ViewLux plate reader .2.12Invasion assays were performed using the xCELLigence system as previously described2.13CellROX® green reagent is a cell‐permeant dye used for measuring oxidative stress in live cells (Thermo Fisher Scientific Inc.). The probe is weakly fluorescent while in a reduced state and exhibits bright green photostable fluorescence upon oxidation by ROS. Cells were evaluated at 48 hours posttreatment.33.1UOK353 was derived from a 42‐year‐old female, African‐American patient with sickle‐cell trait who initially presented with flank pain and was found to have a 4.1 cm right kidney mass and bulky retroperitoneal lymphadenopathy Figure . The patProcured tissue from the surgery‐resected primary renal tumor of the first patient and biopsy tissue from the second patient was used to derive two new, spontaneously immortal cell lines (grown for more than 20 passages), UOK353 and UOK360 respectively.3.2SMARCB1 gene (22q11.23) and FISH analysis using SMARCB1 break apart probes demonstrated that the break occurred within the SMARCB1 gene resulting in loss of activity  immunohistochemistry staining was observed in both patient tumors. The mechanisms of this loss were investigated with spectral karyotypic (SKY) and genomic analysis. SKY analysis of UOK353 revealed that the modal number of chromosomes was hypodiploid, with consistent loss of one copy of chromosomes 4, 9, and 15 Figure . Transloy Figure . The FISSMARCB1   at a known cancer mutation hotspot  Figure . In addit Figure .3.3SMARCB1 gene mRNA expression, relative to primary renal proximal tubule epithelial cells (RPTEC), and no detectable levels of SMARCB1/SNF5 protein by western blot analysis  have shown that the loss of SWI/SNF complex subunits, such as SMARCB1/SNF5, result in an up‐regulation and dependency upon an opposing chromatin remodeling complex, the polycomb repressive complex 2 (PRC2).s Figure . Howevers Figure ,B. Conves Figure . Due to 3.4In collaboration with the National Center for Advancing Translational Science (NCATS), a high throughput drug screen utilizing a library of FDA approved drugs and compounds currently undergoing clinical trials was performed on UOK353 and UOK360 cells in 2D culture. These screens identified several candidate drugs that were effective in both cell lines, including the toll‐like receptor 7/8/9 antagonist CPG‐52364, the neurokinin 3 receptor antagonist osanetant, and the histone deacetylase (HDAC) inhibitor panobinostat that were selected for confirmation Figure . Further3.5NQO1, HMOX1, SQSTM1, GCLC, and GCLM. Notably, both cell lines demonstrated extremely low basal expression levels for NQO1 and reduced expression of HMOX1, while other members of the NRF2‐ARE signaling pathway demonstrated expression comparable to RPTEC cells  when the tumor sizes were compared to vehicle after 21 days of treatment  with an improved suppression of the tumor growth rate compared to single agent treatment, but tumors were not eliminated  injection twice weekly with either 0.9% saline vehicle (n = 8), 1 mg/kg bortezomib (n = 7), 5 mg/kg cisplatin (n = 7), or a combination of bortezomib and cisplatin (n = 7) and followed for 21 days. Single agent treatment with bortezomib demonstrated little effect and with cisplatin slowed the rate of xenograft proliferation that reached statistical significance , having sickle cell trait, and presenting with metastatic disease disseminating from RMC tumors that demonstrated loss of SMARCB1 (SNF5/INI‐1) staining.These lines therefore provide the opportunity to identify new potential treatments for RMC by investigating potential targeted therapies or utilizing high throughput drug screens. Previous studies of malignant rhabdoid tumors (MRTs) with SMARCB1/SNF5 loss had shown that those tumors upregulate expression the PRC2 complex, containing the essential subunit EZH2, and develop a significant sensitivity EZH2 inhibitors such as EPZ‐6438 (tazemetostat).While the efficacy of EZH2 inhibitors in RMC is still unknown, several small‐scale studies have demonstrated a response with bortezomib or platinum‐based combination chemotherapies, such as MVAC .This study also identified a response in these cell line models to panobinostat, an HDAC inhibitor, that could affect the gene expression dysregulation resulting from loss of SMARCB1/SNF5. Panobinostat inhibited proliferation and may represent a potential component of a combination therapy rather than a single agent treatment but highlights the potential for using preclinical models for drug discovery.In summary, UOK353 and UOK360 represent two novel human cell line models for RMC and the first in vivo mouse xenograft models of RMC. These models will provide an invaluable tool for research and preclinical drug testing. Initial analysis of these models confirmed the potential for combination therapy of bortezomib and cisplatin in RMC and highlighted other potential therapeutic options for patients with advanced renal medullary carcinoma.The authors declare no conflicts of interest.Data S1: Supporting InformationClick here for additional data file.


Introduction: mTOR inhibitors are anticancer agents affecting mTOR/AKT/PI3K pathway that is one of the most important in human cancer cells. Hyperactivation of mTOR/AKT/PI3K and overexpression of this pathway members are frequently reported in uterine sarcoma and carcinosarcoma.in vitro models.Present study is aimed to assess the activity of the two mTOR inhibitors (rapamycin - RAP and sapanisertib - MLN) as a single agent and combined with gemcitabine  in uterine sarcoma and carcinosarcoma Material and methods: SK-UT-1 and SK-UT1-B (uterine carcinosarcoma), MES-SA (leiomyosarcoma) and ESS-1  cell lines were used. An MTT assay was performed to examine the cytotoxicity of RAP, MLN and mixtures: RAP+MLN, RAP+GEM, MLN+GEM against these cells. The interactions between tested compounds were assessed in isobolographic analysis.Results and conclusions: Carcinosarcoma cell lines (both SK-UT-1 and SK-UT-1B) do not respond to RAP and respond relatively weakly to MLN treatment. Additive and supraadditive effects were noted for combined treatment with GEM and MLN. Endometrial stromal sarcoma cell line (ESS-1) occured to be sensitive to both RAP and MLN, but the response was stronger for MLN. Additive effect of all tested drug combinations was observed for ESS-1. Leiomyosarcoma cell line (MES-SA) was found sensitive to both mTOR inhibitors. Additive effects in combinations of GEM, RAP and MLN were observed, what makes them promising for future preclinical and clinical trials. Additivity with slight tendency towards antagonism between GEM and MLN observed in MES-SA cell line is unexpected finding and might prompt the mechanistic research aimed to explain this phenomenon. It integrates many cell functions including growth, proliferation, translation and transcription Such properties made it one of the most actively investigated molecular target in oncology. Many substances affecting mTOR have been identified so far.The mTOR protein executes its metabolic function by composing complexes with raptor and mLST8/GβLTORC1) and with rictor and mSIN,GβL TORC2)  and with  and witTORC1 regulates mostly protein synthesis by phosphorylation of translation factors, TORC2 controls cell survival and cytoskeleton functions, both complexes are linked by AKT Streptomyces hygroscopicus bacteria. It is widely used as immunosupresant in prevention of transplanted organ rejection and restenosis of coronary vessels (stents coated with rapamycin). Rapamycin binds and deactivates mTOR molecule. It was proved to efficiently inhibit activity of TORC1 complex The most 'classic' mTOR inhibitor is rapamycin (sirolimus), from which the names of protein and pathway were created, it is natural compound isolated from culture of MLN  is novel allosteric and ATP-competitive mTOR inhibitor. Unlike rapamycin, this compound inhibits both TORC1 and TORC2 complexes. The mechanism of overcoming rapamycin resistance by MLN was revealed by Rodik-Outmezguine et al. Gemcitabine is commonly using chemotherapeutic, according to recent recommendations is one of agents to be used in standard systemic treatment of uterine sarcomas Hyperactivation of mTOR/AKT/PI3K and overexpression of this pathway members are frequently reported in uterine sarcoma and carcinosarcoma The majority of studies that investigated the effectiveness of various mTOR inhibitors , in sarcomas, were designed to assess single agent activity There is limited number of studies already finished and ongoing assessing the performance of MLN added to conventional chemotherapy but these do not recruit patients with neither uterine sarcomas nor carcinosarcomas Presently, there is a gap in knowledge about interactions between tested compounds in uterine sarcoma models. In present study we propose to evaluate the activity of both mentioned mTOR inhibitors in combination with gemcitabine as a standard therapeutic agent, that may indicate the directions for future therapeutic regimens.The study is aimed to assess the activity of RAP and MLN as a single agent, in combination and combined with gemcitabine in uterine sarcomas and carcinosarcomas in vitro models.Four cell lines were selected to experiments:- SK-UT-1 and SK-UT1-B- derived from uterine carcinosarcoma - MES-SA- derived from leiomyosarcoma- ESS-1- derived from low grade endometrial stromal sarcoma.Cell lines were obtained from the Laboratorio de Investigación Traslacional, MD Anderson Cancer Centre in Madrid, Spain. The SK-UT-1, SK-UT-1B and MES-SA cells were cultured according to ATCC recommendations 20The methodology of cell cultures and detailed description of used cell lines was described in details previously Cell viability was assessed by performing MTT test. Cells were plated in microplates and incubated in presence of various concentrations of RAP , MLN , GEM  and following 1:1 mixtures: RAP+MLN, RAP+GEM, MLN+GEM for 96 h. Afterwards MTT  -2,5-diphenyltetrazolium bromide) solution was added for 3 h of incubation. Living cells metabolize MTT into purple formazan crystals that are solubilised. The solution was examined optically in light 570 nm length with ELX-800 plate reader  and analyzed with Gen5 software . All experiments were repeated three times and the median value was used for calculations.The IC50 values were calculated by computer-assisted log-probit analysis. Differences in cells viability between control and particular concentrations of tested compounds were evaluated with one-way ANOVA Tukey's post-hoc testing. P<0.05 was considered to indicate a statistically significant difference. The analysis was performed using GraphPad Prism 5.0 .In order to analyze pharmacodynamic interactions between tested substances isoblography was used. The methodology of this analysis was described in our previous paper Detailed results of single agent activity of RAP and MLN measured in MTT test in SK-UT-1, SK-UT-1B, ESS-1 and MES-SA cell lines are presented on Figures 50 values obtained for each agent in monotherapy in all tested cell lines are summarized in table 50 values were not determined.ICThe results of MTT test performed to assess the activity of combinations of tested drugs in SK-UT-1, SK-UT-1B, ESS-1 and MES-SA were presented in Figure The results of isobolographic analysis indicating type of interactions between examined substances are presented in Figures Resistance of carcinosarcoma cell lines  to rapamycin and relatively weaker response to MLN than observed in other investigated cell lines was noticed. Such observation were not reported yet in available literature. The resistance to rapamycin might be explained by feedback activation of AKT and as consequence activation of mTOR pathway. The results of genetic analysis by Jones et al. revealed that the activity of mTOR pathway in carcinosarcomas is less altered, comparing to type I endometrial cancer 50 cell viability was decreased to about 50% in SK-UT-1 and about 30% in SK-UT-1B such results have to be considered as promising and this combination might occur to be useful in clinical practice. No data regarding activity of mTOR inhibitors in combination with chemotherapy in uterine carcinosacoma models or in clinical practice are available in literature up to date. Bae‐Jump et al. revealed synergistic effect of rapamycin and cisplatin combination in endometrial cancer cells, however the cells that were used were derived from type I endometrial cancer Interestingly, we noticed additive and supra-additive effects of combined GEM and MLN in SK-UT-1 and SK-UT-1B. Taking into consideration that we achieved relatively deep inhibition of cell viability in presence of relatively low concentrations of tested compounds. When both substances were mixed in 1/10 of their ICAdditive effect of all tested combinations was observed in endometrial stromal sarcoma cell line (ESS-1), though it responded to treatment with rapamycin moderately. Endometrial stromal sarcoma tumours are, in most cases, hormone-sensitive. In systemic treatment of these tumours anti-oestrogen agents are widely using in combinations with cytostatics and radiotherapy - DNA synthesis alteration by GEM- mTOR pathway inhibition- Oestrogen receptor deactivation.The importance of mTOR signalling in leiomyosarcoma tumours biology is known in vitro as well as in vivo in patients suffering from such conditions as i.a. breast cancers, gliomas There are two main limitations of mTOR inhibitors usefulness in clinical practice. One is acquisition of resistance by cancer cells, second one is toxicity to patients. Acquiring the resistance to mTOR inhibitors (most commonly rapamycin and its derivates) was detected in various tumours' models Multidrug regimens have a chance to overcome both of them. Obviously, these may allow decreased dosing by exploiting additivity and synergy in drugs combinations. Hence, lead to reduce dose-depending toxicity, but targeting multiple cellular pathways at the same time gives, also a chance to avoid formation of cells resistance to anti-neoplastic agent. When many pathways are altered simultaneously then single mutation resulting with resistance to one of agents is not associated with survival handicap and risk of resistance is much lower comparing to monotherapy.The mTOR pathway is considered as one of regulating mechanisms responsible for cell sensitivity to GEM. Such relation was already proved in various cancers including breast and pancreatic Interestingly, the combination of GEM and mTOR inhibitors is already in early phases clinical trials. So far results of the studies are inconsistent. Combination of temsirolimus  and GEM showed promising activity in pancreatic cancer in vivo. Secondly, the reported project do not include any mechanistic research, partially the activity mechanisms of used compounds were previously described The present study is not free of limitations. The most important is using only 2D models of uterine sarcomas and carcinosarcoma, such models are easily reproducible and allow to perform isobolographic analysis of relations between tested substances, but on the other hand do not fully reflect behaviour of tumours 1. Carcinosarcoma cell lines (both SK-UT-1 and SK-UT-1B) do not respond to RAP and respond relatively weak to MLN treatment. Although, additive and supraadditive effects were noted in combined treatment with GEM and MLN.2. Additive effect in all tested combinations was observed in endometrial stromal sarcoma cell line (ESS-1). This suggests that combinations of mTOR inhibitors and GEM might be effective combination in treatment of such tumours, especially in cases of hormone-insensivity.3. Leiomyosarcoma cell line (MES-SA) appeared as the most sensitive to both mTOR inhibitors among all of tested cell lines. Additive effects observed in combinations of GEM, RAP and MLN make them promising for future preclinical and clinical trials.4. Tendency to antagonism between GEM and MLN observed in MES-SA cell line is unexpected finding and might prompt mechanistic research aimed to explain this phenomenon


IL3RA  is a cell membrane protein frequently expressed in acute myeloid leukemia (AML) and Hodgkin lymphoma; therefore, it is a promising therapeutic target for cancer treatment. Here, we introduce BAY-943, a novel IL3RA-targeting antibody–drug conjugate that shows potent and selective efficacy in IL3RA-positive AML and Hodgkin lymphoma cell lines. In IL3RA-positive AML mouse models, BAY-943 improved survival and reduced tumor burden. Impressively, treatment with BAY-943 induced complete tumor remission in 12 out of 13 mice in an IL3RA-positive HL model. BAY-943 showed a favorable safety profile without any signs of toxicity in rats and monkeys. Overall, these preclinical results support the further development of BAY-943 for the treatment of IL3RA-positive hematologic malignancies.IL3RA (CD123) is the alpha subunit of the interleukin 3 (IL-3) receptor, which regulates the proliferation, survival, and differentiation of hematopoietic cells. IL3RA is frequently expressed in acute myeloid leukemia (AML) and classical Hodgkin lymphoma (HL), presenting an opportunity to treat AML and HL with an IL3RA-directed antibody–drug conjugate (ADC). Here, we describe BAY-943 (IL3RA-ADC), a novel IL3RA-targeting ADC consisting of a humanized anti-IL3RA antibody conjugated to a potent proprietary kinesin spindle protein inhibitor (KSPi). In vitro, IL3RA-ADC showed potent and selective antiproliferative efficacy in a panel of IL3RA-expressing AML and HL cell lines. In vivo, IL3RA-ADC improved survival and reduced tumor burden in IL3RA-positive human AML cell line-derived (MOLM-13 and MV-4-11) as well as in patient-derived xenograft (PDX) models (AM7577 and AML11655) in mice. Furthermore, IL3RA-ADC induced complete tumor remission in 12 out of 13 mice in an IL3RA-positive HL cell line-derived xenograft model (HDLM-2). IL3RA-ADC was well-tolerated and showed no signs of thrombocytopenia, neutropenia, or liver toxicity in rats, or in cynomolgus monkeys when dosed up to 20 mg/kg. Overall, the preclinical results support the further development of BAY-943 as an innovative approach for the treatment of IL3RA-positive hematologic malignancies. Interleukin 3 receptor subunit alpha  is the α subunit of the heterodimeric IL-3 receptor. Together with the β subunit, it forms a functional high-affinity receptor for IL-3 . IL3RA i+CD38− AML blasts as compared to expression in normal cells. Furthermore, these IL3RA-overexpressing cells have been shown to be able to initiate and maintain the leukemic process in immuno-deficient mice and thus act as leukemic stem cells [Several studies have indicated that IL-3 and its receptor play important roles in the progression of AML , and indem cells . Consequem cells ,21. Takeem cells .Here, we exploited a novel pyrrole subclass payload that potently inhibits the kinesin spindle protein (KSP/KIF11/Eg5) in biochemical and cellular assays to develop an ADC to target IL3RA on cancer cells ,23,24,25KSP is active in all proliferating cells and therefore, KSP inhibitors (KSPi) representing various structural classes have resulted in neutropenia, mucositis, and stomatitis in clinical trials ,30,31,32To generate the IL3RA-ADC BAY-943, a non-cell-permeable KSPi was conjugated randomly to the lysine residues of a humanized derivative of the anti-IL3RA antibody 7G3 , TPP-947The binding affinity of the IL3RA-targeting antibody TPP-9476 (IL3RA-Ab) to human and cynomolgus monkey IL3RA was assessed by surface plasmon resonance (SPR) and flow cytometry. IL3RA-Ab showed high affinity to both the human and the cynomolgus monkey IL3RA protein with dissociation constants (KD) of 11 nmol/L and 16 nmol/L, respectively, as determined by SPR. No binding to murine IL3RA was observed. Furthermore, IL3RA-Ab bound specifically to the IL3RA-expressing human hematologic cancer cell lines MOLM-13, MV-4-11, and KG-1 as determined by flow cytometry A.As the prerequisite for ADC activity is to effectively deliver the cytotoxic payload into the cells, we next studied the ability of IL3RA-Ab to internalize upon target binding. The fluorescently labeled IL3RA-Ab showed highly specific, target-dependent internalization in the IL3RA-positive MOLM-13 and MV-4-11 cell lines with >3.5-fold enhancement as compared with the non-specific internalization of the isotype control antibody B,C. In f50 18–21 nmol/L in MOLM-13 cells; Since IL3RA-Ab demonstrated the essential properties of an effective ADC antibody, we conjugated a non-cell permeable KSPi to the lysine residues of the IL3RA-Ab TPP-9476 via a novel legumain-cleavable peptide linker  to produ50) values at the subnanomolar to nanomolar range in the IL3RA-positive AML  and HL  derived cell lines, whereas little activity was observed in the tumor cell lines with low levels of or negative for IL3RA membrane expression . Moreover, in IL3RA-positive AML and HL cell lines, a 10 to 100-fold higher sensitivity to IL3RA-ADC compared to the non-targeted isotype control ADC was observed  cell line-derived xenografts: MOLM-13 human AML and MV-4-11 human biphenotypic leukemia models in mice. Both the MOLM-13 and MV-4-11 cell lines harbor s in AML . The medIn the vehicle and the isotype control ADC groups, nearly all mice had symptoms of leukemia, i.e., splenomegaly and paralysis of hind limbs. In addition, the mean body weights decreased in these treatment groups, indicating that the mice suffered from leukemia . HoweverThe antitumor efficacy of IL3RA-ADC was also tested in the subcutaneous MOLM-13 and MV-4-11 xenograft models in mice. Repetitive dosing with IL3RA-ADC resulted in a significant suppression of tumor growth in both models compared to the isotype control ADC, while the standard-of-care cytarabine showed no activity in these models . FurtherNMP1, FLT3-ITD, IDH1, IDH2, and DNMT3A  reduced tumor burden compared to the vehicle or isotype control ADC, as indicated by a decreased number of human CD45 (hCD45)/human IL3RA (hIL3RA)-positive cells in blood on day 56 (both p < 0.001). In addition, treatment with IL3RA-ADC using either the preventive or therapeutic setting resulted in prolonged MSTs of 107 or 108 days, respectively  or a therapeutic (treatment started on day 34) setting. IL3RA-ADC administered at 10 mg/kg inhibited the growth of IL3RA-positive AML cells, as indicated by temporarily reduced numbers of hCD45-positive cells in blood compared to vehicle or isotype control ADC in both settings .The safety, including possible changes in the hematologic cell populations, of IL3RA-ADC was evaluated in the cynomolgus monkey in two range-finding studies with single or repeated dosing. IL3RA-ADC was well-tolerated without adverse events typically observed with ADCs containing other payload classes, such as thrombocytopenia, neutropenia, or signs of liver toxicity. In addition, mucositis, a dose-limiting toxicity for small molecule KSP inhibitors in clinical studies , was notThe effect of the toxophore metabolite BAY-716 was also analyzed after a single dose of 0.25 mg/kg in rats (data not shown). No laboratory or histopathology findings were observed, indicating that this non-cell-permeable toxophore metabolite does not induce toxic effects thereby underlining the good safety profile of IL3RA-ADC.Despite the recent progress in the treatment of AML, clinical outcomes have improved only minimally over the past three decades. Therefore, novel therapeutic agents with a larger therapeutic window and a favorable tolerability profile are urgently needed to improve the therapeutic outcome for AML patients. Increasing evidence indicates that IL3RA is highly expressed in leukemic stem cells but not in normal hematopoietic stem cells, and it associates in AML with treatment response, minimal residual disease detection, and prognosis ,17. ConsHere, we explored a novel concept to improve the therapeutic window and safety of KSP inhibition by targeting a non-cell-permeable KSP inhibitor as ADC to AML cells, and thereby, sparing fast-dividing healthy cells from KSP inhibition. This provides a payload with a novel mode of action and would be a new therapeutic option for the treatment of IL3RA-positive malignancies. The investigated IL3RA-targeting ADC  consists of a humanized anti-IL3RA antibody conjugated with a stable lysine linkage to a potent proprietary KSPi via a protease-cleavable linker, producing a non-cell-permeable payload metabolite.KSP is an ATP-dependent plus-end directed motor protein, which generates force and moves along microtubules, and it is involved in the separation of the centrosomes, the generation of the bipolar spindle, and thereby plays an important role in mitosis . The inhAntibody–drug conjugates (ADCs) are one solution that has been proposed to mitigate the toxic side effects of anti-mitotic therapies and to broaden their therapeutic window. In fact, currently more than 60 ADCs against multiple targets in solid and hematologic tumors are in clinical trials ,46, and The IL3RA-ADC BAY-943 demonstrated the capability of delivering a novel cytotoxic payload to IL3RA-positive cells. The IL3RA antibody TPP-9476 to which the KSP inhibitor payload is linked via a legumain-cleavable peptide linker showed high binding affinity and specificity to IL3RA and bound specifically to IL3RA-expressing human AML and HL cell lines. The IL3RA antibody internalized into the lysosomes of IL3RA-positive MOLM-13 and MV-4-11 AML cell lines, and IL3RA-ADC demonstrated high cytotoxic potency in IL3RA-positive MV-4-11 and MOLM-13 AML and HDLM-2 and L-428 HL derived cell lines. Furthermore, in the IL3RA-positive cell lines tested, IL3RA-ADC showed 10 to 1000-fold cytotoxicity compared with the isotype control ADC, indicating high target selectivity. The less prominent selectivity observed in MOLM-13 and THP-1 cells may be explained by a non-specific uptake of the isotype control ADC to AML cells differentiated along the macrophage lineage.In an in vivo setting, IL3RA-ADC administered at 10 mg/kg increased survival in both the IL3RA-positive MOLM-13 and MV-4-11 cell line-derived and IL3RA-positive AM7577 and AML11655 patient-derived AML xenograft models harboring molecular alterations associated with poor prognosis in AML. The increased survival was accompanied by a reduction in the growth of IL3RA-positive AML cells and tumor size in the systemic and subcutaneous models, respectively. In the IL3RA-positive HDLM-2 subcutaneous Hodgkin lymphoma model, IL3RA-ADC treatment also resulted in significant antitumor efficacy with most animals being tumor-free at the end of the study.The body weights of the animals decreased over the course of the study, indicating that they suffered from leukemia. However, no IL3RA-ADC treatment-related body weight losses were observed, suggesting good tolerability , particu+Lin− cell population, and transient decreases in basophils and IL3RA-positive basophils. Importantly, liver toxicity, thrombocytopenia, and neutropenia, which are frequently observed with ADCs in the clinic and in cynomolgus monkey preclinical studies [The good tolerability of the IL3RA-ADC was confirmed by repeated dose safety and immunotoxicity assessments in the cynomolgus monkey with no changes in the portion of the CD34 studies ,49, wereCell lines were acquired from DSMZ  unless otherwise noted and cultured according to the provider’s instructions. Human MDA-MB-231 breast cancer, NCI-H292 non-small cell lung cancer, MV-4-11 and THP-1 acute monocytic leukemia, KG-1 acute myelogenous leukemia, Rec-1 mantle cell lymphoma, and Ramos Burkitt’s lymphoma cells were obtained from ATCC . Human OVCAR-8 ovarian cancer cells were acquired from the NCI-60 Human Tumor Cell Line Panel . The human diffuse B cell lymphoma cell line HBL-1 was obtained from Dr. Georg Lenz  and cultivated in RPMI 1640 supplemented with 10% fetal calf serum (FCS). The human Hodgkin lymphoma cell line L-428 (source not known) was cultivated in RPMI 1640 supplemented with 10% FCS. Cancer cell lines were obtained between 2002 and 2012, authenticated using short tandem repeat DNA fingerprinting at DSMZ , and subwww.syncom.eu; Groningen, the Netherlands), cytarabine (HT0476) from Accord Healthcare GmbH , and staurosporine (#S4400) from Sigma-Aldrich .The anti-IL3RA antibodies TPP-9476 and TPP-8988 (recognizes a different epitope in the extracellular domain of IL3RA than TPP-9476) and the isotype control antibody TPP-754, the IL3RA-ADC BAY-943, the isotype control ADC BAY-229 (with TPP-754), the non-cell-permeable toxophore metabolite BAY-716 (active toxophore metabolite of IL3RA-ADC), and the cell-permeable small molecule KSPi BAY-331 were manufactured at Bayer AG. Ispinesib (SYNT1009) was purchased from Syncom  was generated by humanization of the murine anti-IL3RA antibody 7G3  as descrThe generation and characterization of the IL3RA-Ab is described in the ® FlowSight® or the Guava easyCyteTM flow cytometers  and analyzed using the IDEAS® software or the guavaSoft 2.6 software .Internalization and colocalization experiments were performed in MOLM-13 and MV-4-11 AML cell lines using flow cytometry-based imaging. The IL3RA-specific antibody TPP-9476, IL3RA-ADC BAY-943, corresponding isotype control antibody TPP-754, and isotype control ADC BAY-229 were lysine-conjugated with a ten-fold molar excess of CypHer 5E mono NHS ester  at pH 8.3. The reaction mixture was purified by chromatography , followed by centrifugation . Alexa 488  was utilized as a constitutive dye. The fluorescence was measured using the Amnis4/well) were incubated with the labeled antibodies (10 µg/mL) at 37 °C, 5% CO2 for 0, 1, 2, and 6 h. The fluorescence was measured using the Amnis® FlowSight® or the Guava easyCyteTM flow cytometers and analyzed using the IDEAS® or the guavaSoft 2.6 software. The kinetics of the internalization were determined based on the analysis of the median fluorescence intensity (MFI) over time.For the internalization assay, the tumor cells (5 × 104/well) were incubated with the labeled antibodies (20 µg/mL) at 37 °C, 5% CO2 for 0 h, 0.5 h, 2 h, and 6 h. The lysosomal compartment marker CytoPainter LysoGreen  was added 30 min before the end of the incubation period. After incubation, the cells were washed and resuspended in ice-cold FACS (fluorescence-activated cell sorting) buffer consisting of phosphate-buffered saline (PBS) and 3% FCS. The lysosomal colocalization was analyzed with FACS image analysis using the IDEAS® software.For the colocalization studies, MOLM-13 and MV-4-11 tumor cells . Cells (2000–5000 cells/well) were incubated at 37 °C, 5% CO2 for 24 h and the compounds were added at concentrations of 3 × 10−11–3 × 10−7 M  in triplicates. Cell viability was determined at the beginning (day 0) and after 72 h incubation in the presence or absence of ADCs. The IC50 of the growth inhibition was calculated in comparison to day 0. The IL3RA antigen density was determined with the QIFI  quantitative flow cytometry assay using the murine anti-IL3RA antibody clone 7G3 .The antiproliferative activity of IL3RA-ADC was determined in a panel of human tumor cell lines using the CellTiter-GloAll animal experiments were conducted in accordance with the German Animal Welfare Law and approved by Berlin authorities . When a body weight loss of >10% was observed, treatment was ceased until recovery. In the systemic models, mice were sacrificed individually when signs of leukemia were observed . The molecular alterations of the tested in vivo models are described in 6 or 5 × 106 cancer cells in 0.9% NaCl, respectively. The mice were treated with i.p. injection of IL3RA-ADC at 2.5 or 10 mg/kg once weekly (Q7D), every two weeks (Q14D), or every three weeks (Q21D). In the MOLM-13 model, treatments were started on day 10, and the study was terminated on day 124. In the MV-4-11 model, treatments were started on day 3, and the study was terminated on day 174 after tumor cell injection.For the systemic MOLM-13 and MV-4-11 models, female CB17-SCID  or NOD SCID  mice were injected intravenously (i.v.) with 200 µL of 7.5 × 106 cancer cells in PBS at CrownBio . The development of AML was monitored by flow cytometric analysis of the percentage of hCD45 cells in blood. On day 38 after tumor cell injection, when approximately 4% of hCD45-positive cells were present, the mice were randomized, and treatments were started. The mice were treated with i.v. injections of IL3RA-ADC at 2.5 or 10 mg/kg, Q7D or the isotype control ADC at 10 mg/kg, Q7D. In the first IL3RA-ADC treatment group, the initial dose of 2.5 mg/kg, Q7D was increased to 10 mg/kg, Q14D from the second administration onwards (indicated as 2.5 + 10 mg/kg). The study was terminated on day 104 after tumor cell injection.For the systemic AM7577 PDX model, female NOD/SCID mice  were injected i.v. with 100 µL of 1.4 × 10®  were injected i.v. with 400 µL of 1 × 107 cancer cells in PBS at EPO Berlin-Buch GmbH . The development of AML was monitored by the percentage of hCD45-positive cells in blood as determined by flow cytometry. Treatments were initiated on day 5 after tumor cell injection (preventive setting) or day 34 after tumor cell injection (therapeutic setting) when approximately 46% of hCD45-positive cells were detected in blood. The mice were treated with i.v. injections of IL3RA-ADC at 10 mg/kg, Q7D or the isotype control ADC at 10 mg/kg, Q7D. The study was terminated on day 118 after tumor cell injection.For the systemic AML11655 PDX model, female CIEA NOG mice7 cancer cells suspended in 30% Matrigel/70% medium. Tumor volume (0.5 × length × width2) was determined based on twice weekly measurement of tumor area by a caliper (length and width). Treatments with IL3RA-ADC  or ispinesib  were started on day 49 when the tumors had reached a mean size of 100 mm3. The study was terminated on day 84 after tumor cell injection.For the HDLM-2 Hodgkin lymphoma model, female CB17-SCID mice  were injected subcutaneously (s.c.) with 100 µL of 1 × 10The subcutaneous MOLM-13 and MV-4-11 models as well as safety studies are described in the p values < 0.05 were considered significant.Statistical analyses were performed using R  . Flow cyThe novel IL3RA-ADC with a differentiated mode-of action demonstrates selective binding and internalization to IL3RA-positive cells, which translates into selective and efficacious antitumor activity in IL3RA-positive AML and Hodgkin lymphoma models. By employing a KSP inhibitor, a stable lysine linkage between the payload and the antibody, and a legumain-cleavable linker resulting in a non-cell-permeable payload metabolite, IL3RA-ADC presents a new alternative for the treatment of IL3RA-positive malignancies. Using the KSPi as a payload in an ADC is expected to result in manageable toxicity and a broader therapeutic window compared to that reported for the systemic application of KSPi in clinical trials. Our data support further development of the IL3RA-ADC BAY-943 as an innovative approach for the treatment of patients with IL3RA-positive AML.


PAC in dogs is infrequent, difficult to differentiate from TCC and usually characterized by poor prognosis, enhancing the value of the few available cell lines. However, as cell lines adapt to culturing conditions, a thorough characterization, ideally compared to original tissue, is indispensable. Herein, six canine PAC cell lines and three TCC cell lines were profiled by immunophenotype in comparison to respective original tumor tissues. Three of the six PAC cell lines were derived from primary tumor and metastases of the same patient. Further, two of the three TCC cell lines were derived from TCCs invading into or originating from the prostate. Cell biologic parameters as doubling times and chemoresistances to commonly used drugs in cancer treatment  were assessed. All cell lines were immunohistochemically close to the respective original tissue. Compared to primary tumor cell lines, metastasis-derived cell lines were more chemoresistant to doxorubicin, but equally susceptive to carboplatin treatment. Two cell lines were multiresistant. COX-2 enzyme activity was demonstrated in all cell lines. However, meloxicam inhibited prostaglandin E2 production in only seven of nine cell lines and did neither influence metabolic activity, nor proliferation. The characterized nine cell lines represent excellent tools to investigate PAC as well as TCC in prostate and urinary bladder of the dog. Furthermore, the profiled paired cell lines from PAC primary tumor and metastasis provide the unique opportunity to investigate metastasis-associated changes PAC cells undergo in tumor progression. The combination of nine differently chemoresistant PAC and TCC cell lines resembles the heterogeneity of canine lower urinary tract cancer.Canine prostate adenocarcinoma (PAC) and transitional cell carcinoma (TCC) of prostate and urinary bladder are highly invasive and metastatic tumors of closely neighbored organs. Cell lines are valuable tools to investigate tumor mechanisms and therapeutic approaches  For resIn general, cell lines are established from tumor-burdened individuals. Ideally, investigated features of the primary tumor are representative for the tumor type or subtype and stay preserved in the derived cell line . Howeverp53 have also been detected and correlated with poor survival , n0 the cell count after 24 h, t the time [h] and r the specific growth rate of each cell line. Doubling times (DT) [h] were calculated using the formula DT [h] = ln(2) / ln(r).Cell lines were seeded in 6-well plates 2*102 supply achieved constant cultivation conditions as described above.Cell lines were seeded in 96-well plates 7,500 cells/well. Morphology and growth behavior were surveyed with a live cell Imaging microscope  over 72 h in 100x magnification. Pictures of the same position were taken every 15 minutes with the program LAS AF 2.6.0. A heating unit, a humidifier and regulated CODoxorubicin  was diluted to 100 μM in PBS and carboplatin  was diluted to 10 mM in cell culture medium. Therefore, solvent controls in cell culture medium used for controls in doxorubicin and carboplatin inhibition were not necessary. Meloxicam was purchased from Selleck Chemicals  and solved in dimethyl sulfoxide (DMSO) to a concentration of 75 mM. Solutions were sterile filtered and aliquots were stored at -20 °C.® AQueous One Solution Cell Proliferation Assay, Promega, Mannheim, Germany). All cell lines were seeded in 96-well plates 7,500 cells/well. The next day, medium was exchanged, and cells were exposed in quadruplets to increasing concentrations of doxorubicin  or carboplatin . Dilutions were chosen based on doxorubicin serum concentrations in patient dogs [® AQueous One Solution Cell Proliferation Assay, Promega) was conducted according to manufacturer’s protocol. The absorbance was recorded at 490 nM using a Synergy2 plate reader . Blanks were subtracted.The inhibitory effect of doxorubicin and carboplatin on metabolic activity was ascertained by MTS assay  were calculated using GraphPad Prism Software 3.0 .5 cells/well and exposed to the respective concentrations of doxorubicin or carboplatin used in MTS assay the next day. For carboplatin, 0.5 and 1000 μM were omitted again, as these concentrations showed little differences to 0 and 2000 μM in MTS assays. Controls were supplied with fresh medium. After 72 h of exposure, medium including floating cells and debris was collected. Adherent cells were detached with 1 ml TrypLE™Express (Life Technologies GmbH), pooled with the respective medium and centrifuged for 10 min at 160 g. The obtained pellets were stained with Annexin V-FITC Detection Kit plus  according to manufacturer’s protocol and fluorescence signals were measured by flow cytometry using a MACSQuant® Analyzer 10 . Data analysis was performed with FlowJo 10.3 software. Debris was excluded and proportions of vital, apoptotic and late apoptotic cells were determined.Additionally, proportions of vital, apoptotic and late apoptotic cells were assessed after doxorubicin and carboplatin exposure. Cells were seeded in 6-well plates 1.5*104 cells/well and in 96-well plates 7,500 cells/well. The next day, medium was exchanged and cells were exposed to 1μM, which resembles the therapeutic steady state plasma concentration in dogs [For meloxicam response regarding total cell number and metabolic activity, cells were seeded in 24-well plates 4*10 in dogs , and 10 4 cells/well in 12-well plates. The next day, medium was changed and cells were treated in duplicates with 1 and 10 μM meloxicam. Controls were incubated with 0.13% (v/v) DMSO. After 24 h of exposure, medium was collected, centrifuged for 10 min at 160 g for removal of cells and debris and stored at -80 °C. PGE2 concentrations in medium samples were measured in duplicates using a competitive ELISA  according to manufacturer’s protocol.The functionality of COX-2 enzyme was ascertained by production of prostaglandin E2 (PGE2). Each cell line was seeded in a density of 7.5*10Statistical analysis of data was performed with SAS software 7.1 . Means and standard deviations are displayed. For comparison to untreated controls in metabolic activity, cell count and proportions of vital, apoptotic and late apoptotic cells, Dunnett’s test for multiple comparisons was applied. Additionally, Student’s t-tests were performed for comparison between PGE2 concentrations at 1 and 10 μM meloxicam. The confidence levels were set to 95% , 99%  and 99.9% . All experiments were conducted three times independently, except from cell count and apoptosis rate of doxorubicin and carboplatin response, which were conducted four times and from PGE2 concentrations, which were measured once.FFPE prostate tissue samples P1, P2, P3 and P4.1 were histopathologically classified as infiltrative PAC . All fouIn both metastasis-derived cell lines Metadcarc1511.2 and Metadcarc1511.3, cells were distinctly larger than cells of the primary tumor cell line Adcarc1511.1 and showed numerous cytoplasmic vacuoles, which were also observed in Adcarc1508 and TCC0840 cells  between 0.9151 and 0.9999.Doubling times of cell lines varied between 18.2 and 38.5 h . The lonTCC0840, Adcarc0846, TCC1506, Adcarc1508, Adcarc1511.1, Metadcarc1511.2 and Metadcarc1511.3 grew in monolayers in cobblestones-like patterns. Adcarc0846 and Adcarc1508 tended to form piles when reaching confluence. Adcarc1258 and TCC1509 had low anchoring potential and did not establish uniform monolayers in culture. Instead, cells formed prominent protrusions when attached to the well bottom and tended to form aggregates with increasing cell density. Videos of growth behavior are provided as After 72 h of exposure to doxorubicin, all cell lines showed decreased metabolic activity at 200 nM and higher Figs  and 4. RCell count IC50 values for doxorubicin varied between 3.6 nM in Metadcarc1511.3 and 95.7 nM in Adcarc1258. At 2000 nM doxorubicin, total cell numbers in all PAC and TCC derived cell lines were reduced below seven percent, total cell numbers of cell lines originating from metastases were reduced below 16%.Carboplatin metabolic activity IC50 values varied between 33.6 and 129.3 μM in all cell lines with Metadcarc1511.2 and Metadcarc1511.3 ranging in the lower half . ReductiCell count IC50 values for carboplatin ranged from 3.8 μM in TCC1509 and Adcarc1511.1 to 10.9 μM in Metadcarc1511.2 . ReductiDoxorubicin and carboplatin induced dose-dependent increases of apoptotic cell proportions and decreases of vital cells in nearly all cell lines Figs  and 8. NAfter 72 h exposure to carboplatin, Adcarc1511.1 was the cell line reacting first with increased apoptosis rates at 20 μM. At 200 and 500 μM carboplatin, all cell lines except from TCC0840 showed increased apoptotic rates.Metabolic activities and total cell numbers did not decrease in any cell line treated with meloxicam in both concentrations .Basal PGE2 concentrations in medium ranged beyond 1000 pg/ml in all cell lines except from Adcarc1258, Adcarc0846 and TCC1509 . MeloxicThe presented study provides nine thoroughly characterized cell lines of canine lower urinary tract cancer. Therefore, combined in a panel, these cell lines offer a great opportunity to evaluate therapeutic strategies in any case of lower urinary tract cancer in dogs, including TCC located in the prostate and PAC metastasis.When comparing initial characterizations of different human ,45,67,68in vitro models are required for repeatable results in further experiments, cell lines were herein characterized in later passages.Some of the previously mentioned human and canine PAC and TCC cell lines were characterized in early and some in later passages. However, subculturing-induced gene expression changes were reported to happen within the first ten passages , particuMetastasis research is an important part in human and veterinary cancer research. Various studies were carried out using metastasis-derived prostate cancer cell lines like LNCaP and PC-3  or metasThe broad range of the herein analyzed cell lines’ IC50 values for doxorubicin are comparable with human prostate and bladder cancer cells  and withIn most cell lines, cell counts decreased before apoptotic proportions of cells increased, indicating that doxorubicin and carboplatin first inhibit proliferation and induce apoptosis at higher concentrations. However, in Adcarc1258 after exposure to doxorubicin and in TCC1509 after exposure to both drugs, apoptosis began at lower concentrations compared to the other cell lines, which makes these cell lines more sensitive to apoptosis inducing stimuli.Exposed to high concentrations of doxorubicin, still a number of cells survived in both metastasis-derived cell lines and in TCC0840. Especially in those cell lines, apoptotic proportions of cells did not increase until 2000 nM, indicating that doxorubicin inhibits proliferation, but does not induce apoptosis. Apoptotic proportions of TCC0840 were relatively high in controls and in all treated samples. It remains uncertain if the detachment of cells or the staining procedure induced apoptosis in this cell line or if a certain proportion of this cell line constantly underwent apoptosis. Changes of vital and apoptotic proportions of cells in cell lines Adcarc1511.1 and Metadcarc1511.3 that were observed at 200 and 100 μM doxorubicin but not at higher concentrations might be of artificial origin. Prominently in both metastasis-derived cell lines after treatment with doxorubicin and in Adcarc1258, TCC0840, Adcarc0846 and Adcarc1511.1 after carboplatin exposure, total cell numbers decreased much faster than metabolic activities. Various transporters and other avoidance mechanisms are involved in doxorubicin and carboplatin resistance ,80. Assuin vitro, which was also demonstrated for human prostate cancer cell lines [in vitro studies [Daily administered COX-2 inhibitors increase survival times in dogs with prostate and bladder tumors ,10,81. Hll lines  and canill lines –85. Consll lines , supportll lines ,85. In A studies ,87 mightp53 mutation cannot be excluded. Specific mutations affecting the binding site of the herein used antibody might prevent it from binding to p53 [Due to a short half-life of its wild type, p53 is almost exclusively detectable in accumulated and mutated forms by immunohistochemistry ,14. MutaEpithelial to mesenchymal transition is a common pathological process in canine metastatic prostate cancer ,91. It iAll tumors apart from P1 possess basal cell features by expressing pan-CK and luminal cell characteristics by staining positive for CK7 and CK8/18, which supports the involvement of basal and luminal cells in canine prostate cancerogenesis ,95. UPIILike in the human prostate cancer cell line LNCaP , Adcarc1Transcriptomic analysis of canine PAC tissues referred to non-cancerous controls will be the next step to identify upregulated cancer-specific targets for promising inhibitors . Furtherin vitro tools that are immunohistochemically close to the respective tumor tissue. Adcarc1511.1, Metadcarc1511.2 and Metadcarc1511.3 provide the unique opportunity to investigate functional cellular features associated with canine PAC metastasis, as they were derived from primary tumor and metastasis of the same patient. TCC0840 and TCC1509 represent the uncertain percentage of TCC with invasion or emergence in prostate tissue. Adcarc1258 and TCC0840 represent multi-resistant canine PAC and TCC, while TCC1506 and TCC1509 are chemosensitive. Both metastasis derived cell lines Metadcarc1511.2 and Metadcarc1511.3 are resistant to apoptotic effects induced by doxorubicin, but susceptive to carboplatin treatment.Summarized, this thorough characterization enriches canine lower urinary tract cancer research with nine valuable S1 Tablem = male; f = female; n = neutered; original tissue ; cell line names are explained as institution (Tiho = University of Veterinary Medicine Hannover); species (D = dog); tissue origin ); diagnosis ; abbreviations of cell lines written in bold; none = cell lines have not been published yet; n.a. = tissue of patient no. 5 is missing, as the patient owners declined surgery and necropsy; *diagnosis by cytology of cells obtained by fine needle aspiration biopsy; **in total remission after a combination protocol of vincristine, asparaginase, cyclophosphamide, doxorubicin, prednisone and lomustin.(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S1 Fig(TIFF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S1 Video(MP4)Click here for additional data file.S2 Video(MP4)Click here for additional data file.S3 Video(MP4)Click here for additional data file.S4 Video(MP4)Click here for additional data file.S5 Video(MP4)Click here for additional data file.S6 Video(MP4)Click here for additional data file.S7 Video(MP4)Click here for additional data file.S8 Video(MP4)Click here for additional data file.S9 Video(MP4)Click here for additional data file.