New insights into the pathophysiology of GVHD garnered from our understanding of the immunological pathways within animal models have been pivotal in driving new therapeutic paradigms in the clinic. Successful clinical translations include histocompatibility matching, GVHD prophylaxis using cyclosporine and methotrexate, posttransplant cyclophosphamide, and the use of broad kinase inhibitors that inhibit cytokine signaling (e.g. ruxolitinib). New approaches focus on naïve T cell depletion, targeted cytokine modulation and the inhibition of co-stimulation. This review highlights the use of animal transplantation models to guide new therapeutic principles.Allogeneic hematopoietic cell transplantation (HCT) is a curative treatment for hematologic malignancies, bone marrow failure syndromes, and inherited immunodeficiencies and metabolic diseases. Graft- In 1963, Mathé and colleagues reported the first case of a human allogeneic bone marrow transplantation (BMT) recipient that survived beyond a year. This patient had complete engraftment and the development of a lethal “secondary disease” was described  and administered allogeneic splenocytes survived for a shorter time than those transplanted with syngeneic splenocytes . The recescribed . These pescribed .Although many investigators lost their enthusiasm for BMT, several groups increasingly utilized animal models to gain a better understanding of the allogeneic barrier with regard to both GVHD and graft rejection. Studies of allogeneic BMT in Seattle using dog models in the 1980s provided the scientific groundwork for the field leading to the concepts of histocompatibility matching, conditioning regimens and pharmacological GVHD prophylaxis . These fFor a detailed discussion of the various mouse models of GVHD currently available and the penetrance of disease therein, we refer the reader to some of the excellent reviews on this subject –17.versus CD8) responsible for the induction of GVHD in each model  effects. In 1956, Barnes et al. reported that leukemia-bearing mice receiving allogeneic cells eventually died of GVHD without evidence of leukemia  defined by the transcription factor FoxP3 are pivotal for the maintenance of self-tolerance and the induction of tolerance after allogeneic hematopoietic stem cell transplantation (HCT). Depletion of CD25 in mice –46. Moga in mice . Pretran in mice . Followi in mice –54 but d in mice . Promisi in mice . Additio in mice .GVHD prophylaxis using calcineurin inhibitors (cyclosporin or tacrolimus) reduce the expansion of effector T cells (Teff) by blocking IL-2 and prevent acute GVHD, but fail to reduce chronic GVHD –60. CalcIn the setting of BMT, donor T cells are infused into recipients that have potentially experienced tissue injury by prior treatments of the underlying malignancy, infections, and more immediately, pre-transplant conditioning. The inflammatory environment invoked by these therapies predispose to a state of enhanced alloantigen-presentation. Johnson and Truitt demonstrated that delayed infusion of donor T cells induced less severe GVHD . The Fer+ or CD11b+ APCs do not prevent GVHD  were responsible for donor T cell activation and the induction of acute GVHD . They suent GVHD , 87.γ to mediate these effects by IEC  and a differentiation signal in the form of cytokine as defined above. The recognition of the importance of cognate costimulatory signals has led to the investigation of the relative ability of inhibitory antibodies that block these pathways in preclinical models. Thus CTLA-4-Ig that inhibits CD80 and CD86, anti-CD28 and anti-CD40 have all been shown to attenuate GVHD in preclinical models –97. The γ, GM-CSF, IL-17) can in turn invoke target tissue apoptosis and secondary myeloid cell migration to amplify GVHD [reviewed in  are generated in response to chemoradiotherapy during conditioning and promote the activation of APCs. Other cytokines act in a costimulatory role to promote pathogenic T cell differentiation . Finally pivotal T cell derived cytokines . Some of these limitations can be overcome by more thorough study of these variables in mice  and/or the use of non-human primates or dog models . Additional limitations include the widespread use of cell lines to study graft-versus-leukemia effects and the lack of relevant models to study pathogen-specific immunity in the context of new therapies, at least until recently. Nevertheless, to date almost all effective therapeutic paradigms that are now established in humans have their genesis in animal models, suggesting that these systems will continue to provide valuable insights and therapeutic advances to the field. Importantly, it would seem critical that well-established preclinical systems are utilized to analyze the effects of various therapeutic interventions before they are translated into early phase clinical trials.Mouse models of GVHD faithfully recapitulate the pathological lesions seen in clinical disease and allow the dissection of pathogenic TT and GH, wrote the manuscript. All authors contributed to the article and approved the submitted version.TT is supported by JSPS KAKENHI . GRH is supported by NIH R01 HL148164. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.TT: Grants from Kyowa Kirin, Chugai, Sanofi, Astellas, TEIJIN PHARMA, Fuji Pharma, NIPPON SHINYAKU, Personal Fees from Novartis, Merck, Kyowa Kirin, Takeda, Pfizer, Bristol-Myers Squibb, Non-Financial Support from Janssen, Novartis.GRH has consulted for Generon Corporation, NapaJen Pharma, Neoleukin Therapeutics, iTeos Therapeutics and has received research funding from Roche Pharmaceuticals, Compass Therapeutics, Syndax Pharmaceuticals, Applied Molecular Transport and iTeos Therapeutics.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Streptomyces (S.) griseus isolate KJ623766, a recovered soil bacterium previously screened in our lab that showed promising cytotoxic activities against various cancer cell lines. Methods: Production of cytotoxic metabolites from S. griseus isolate KJ623766 was carried out in a 14L laboratory fermenter under specified optimum conditions. Using a 3--2,5-diphenyl tetrazolium-bromide assay, the cytotoxic activity of the ethyl acetate extract against Caco2 and Hela cancer cell lines was determined. Bioassay-guided fractionation of the ethyl acetate extract using different chromatographic techniques was used for cytotoxic metabolite purification. Chemical structures of the purified metabolites were identified using mass, 1D, and 2D NMR spectroscopic analysis. Results: Bioassay-guided fractionation of the ethyl acetate extract led to the purification of two cytotoxic metabolites, R1 and R2, of reproducible amounts of 5 and 1.5 mg/L, respectively. The structures of R1 and R2 metabolites were identified as β- and γ-rhodomycinone with CD50 of 6.3, 9.45, 64.8 and 9.11, 9.35, 67.3 µg/mL against Caco2, Hela and Vero cell lines, respectively. Values were comparable to those of the positive control doxorubicin. Conclusions: This is the first report about the production of β- and γ-rhodomycinone, two important scaffolds for synthesis of anticancer drugs, from S. griseus.Background: This study aimed to produce, purify, structurally elucidate, and explore the biological activities of metabolites produced by  Worldwide cancer incidence and mortality have always been a concern to the community. The cancer mortality rate has generally declined over the years; however, there is still an increased mortality rate in poorer countries that receives considerable attention from healthcare professionals . This suMicrobial metabolites can be utilized as novel anticancer agents; with fewer side effects . StreptoStreptomyces have made imperative contributions to human health with their capabilities to produce various important active metabolites, including anticancer agents [Streptomyces peucetius bacterium. It has widespread use as a chemotherapeutic agent since the 1960s [2 [Various members of r agents . Many drr agents . Doxorubhe 1960s , Unfortu1960s [2 . Doxorub1960s [2 .Streptomyces spp. and used in the treatment of various types of cancers, including leukemias, lymphomas, breast, stomach, uterine, ovarian, bladder, and lung cancers [Streptomyces spp. [S. griseus strains including two new benzoxazines, the chandrananimycin E and dandamycin, [S. griseus, and biosynthetic genes of S. griseus have been heterologously cloned, expressed, and had their biological activities evaluated in vivo [S. griseus isolate KJ623766, a soil bacterial isolate previously screened in our lab that showed promising cytotoxic activities against various cancer cell lines.Anthracyclines are drugs extracted from  cancers . Regardl cancers . The mos cancers . Daunoru cancers . Anthracces spp. . These aces spp. . Severaldamycin,  chromomydamycin, . In addi in vivo . A recen in vivo . TherefoCaco2 (CD50 14 µg/mL) than Hela cell line (CD50 20 µg/mL), which indicates the presence of metabolites with higher selectivity to human colorectal adenocarcinoma  were obtained. All the fractions were subjected to 3--2,5-diphenyl tetrazolium-bromide (MTT) assay to measure the cytotoxic activity of each. The fraction SG-3 (70% Hexane) showed potential cytotoxic activity against ell line .m/z 385.05 (M − H)− corresponding to the molecular formula C20H18O8 (The metabolite R1 (5mg) was isolated as a reddish powder, ESI-MS revealed pseudomolecular ion peak at C20H18O8 . As showC20H18O8 .This is confirmed by COSY  correlatm/z 369.09 (M−H)− corresponding to the molecular formula C20H18O7 (The metabolite R2 (1.5 mg), a red colored metabolite, showed an identical 1H NMR spectrum to R1 except in the replacement of H-7 at δH 5.25  in R1 with the methylene protons δH 2.98  and 2.90 , corresponding to H-7a and H-7b in R2. A comparison of 1H NMR spectrum of R2  with thaC20H18O7 . TherefoC20H18O7 .S. griseus isolate KJ623766, a recovered soil isolate previously screened in our lab that exhibited promising cytotoxic activity against various cancer cell lines [Natural products are likely to provide many of the lead structures, and these will be used as templates for the construction of novel compounds with enhanced biological properties, so natural products have been the mainstay of cancer chemotherapy for the past 30 years . These nll lines . The proll lines  in orderCaco2 and Hela cell lines, the CD50 were 6.3 and 9.45 µg/mL, respectively, while lower cytotoxic activity was shown against the Vero cell line (CD50 = 64.8 µg/mL). Being cancer cell lines, Caco2 and Hela cell lines have lost their normal regulation of growth or cell death and were more sensitive to the effect of cytotoxic agents than the Vero cell line. The lower cytotoxic effect of certain metabolites on the Vero cell lines usually reflects safety and potential of these metabolites to be used in future treatment of cancer. In addition, the Vero cell line is a continuous non-tumourigenic one when a cell passage is not prolonged [For, metabolite R1, it showed potent cytotoxic activity against rolonged . Thereforolonged . TherefoCaco2 and Hela cell lines, the CD50 were 7.11 µg/mL and 9.35 µg/mL, respectively. While lower cytotoxic activity was shown against Vero cell line [CD50 = 67.3 µg/mL). Accordingly, both β- (R1) and γ-rhodomycinone (R2) showed potent cytotoxic activities against Caco2 and Hela cell lines as compared to their activities on the Vero cell line, confirming their potential applicability to be used as potential anticancer agents in humans. These results matched with those of Tsuji et al. [Caco2 cell line matched with results obtained by Tan et al. [Streptomyces sp. MUM256 crude extract against Colon Cancer Cell HCT116. Results revealed that the ethyl acetate fraction of MUM256 extract was shown to exhibit the highest cytotoxicity towards HCT116 among the fractions tested, followed by the hexane fraction and the aqueous fraction as the least toxic against HCT116 cells. Lesser toxicity was observed when the extract was evaluated against a normal colon cell line CCD-18Co at all the concentrations tested in the study [Hela cell line matched with results obtained by Kim et al. [Streptomyces bulli GJA1, against ovarian cancer cell lines, OV90 and ES2. Results revealed potent antiproliferative activities against both cell lines [Comparison of the 1H NMR spectrum of R2 with that of known anthracyclins in the literature showed strong similarity to the known metabolite γ-rhodomycinone . Like β-i et al.  which shn et al.  who examhe study . On the m et al.  who evalll lines . MoreoveStreptomyces spp. used in the treatment of various types of cancers [Streptomyces cultures that exhibited potent cytotoxic activities [Streptomyces broth yielded twelve new anthracycline antibiotics including three new ε-rhodomycinone derivatives [Streptomyces strains producing aglycones and nucleotide deoxysugars to yield more potent cytotoxic derivatives with high potential to be used in the treatment of various cancers in humans [Streptomyces venezuelae-based combinatorial biosynthetic systems have previously been developed for the production of glycosylated derivatives of doxorubicin and its biosynthetic intermediates [Both β- and γ-rhodomycinone belong to the antharcycline family of drugs extracted from various  cancers . They artivities ,28. Purin humans . Anthracmediates . This symediates .S. griseus isolate KJ623766. To the best of our knowledge, this the first report of β- and γ-rhodomycinone production from S. griseus that can be used as precursors for the construction of new derivatives with more potent cytotoxic activities. Further studies should be conducted to explore the biosynthetic pathways of the respective metabolites, as well as to produce new derivatives of the anthracycline family with higher antitumor activities and lower side effects. The latter can be achieved by structural modifications or microbial transformation of the recovered metabolites.Bacteria and bacterially produced bioactive compounds have recently emerged as a promising alternative for cancer therapeutics . BacteriStreptomyces strain from Egyptian soil sample, S. griseus KJ623766, was identified using 16S ribosomal RNA gene sequences  [http://www.wfcc.info/ccinfo/collection/col_by_country/e/20/, accessed on 14 September 2018) belonging to the world data center of microorganisms (WDCM) . This isolate was preserved onto starch nitrate agar slants  and sub-cultured every month, while slant-medium (50:50)  was used for long term preservation.Locally isolated J623766) . It was Vero cell line, ATCC No.CCL-81), colorectal adenocarcinoma derived from the human colon (Caco2 cell line), and epithelioid carcinoma derived from the human cervix (Hela cell line) were obtained from VACSERA, Egypt. The Caco2 and Hela cell lines were used to study the cytotoxic activities of the crude extract and isolated metabolites; stock cultures of these cell lines were grown in T-75 tissue culture flasks containing 20 mL of RPMI-1640 medium with 1% antibiotic antimycotic solution and 10% fetal bovine serum. The medium was changed at 48 h intervals and cells dissociated with trypsin solution . Vero cell line is a continuous non-tumourigenic when a cell passage was not prolonged and was used to analyze the cytotoxic activities of isolated metabolites [Three cell lines, including kidney and epithelial cells derived from the African green monkey (abolites . It was abolites .3 1 g per 1 L of distilled water in 100 mL flask) and incubated at 28 °C with shaking 200 RPM for 72 h in a C5KC shaking incubator . The culture obtained was used to inoculate the main culture.This was carried out according to Peng et al.  with somv/v) in subsequent manner [Caco2 and Hela cancer cell lines using MTT assay [50 value is ≤30 μg/mL [1 L of CFCS was extracted with ethyl acetate at the level of 1:1  on Kromasil ODS preparative column (10 mm × 250 mm) at flow rates of 4 mL/min and UV detection at 254, yielding metabolites R1 and R2  consisting of a mixing HPLC quaternary pump, a PDA-UV-Vis detector 190–840 nm, a fraction collector, and a sample loading module. For system controlling and process monitoring, Interchim Software 5.0 was used. 360 mg sample was dissolved in 50 mL of methanol then introduced into the column via dry load using 4 gm silica. Elution started with hexane:ethyl acetate (100%:0%) followed by ethyl acetate: methanol (100%:0%) gradient to yield 117 fractions. Elution was monitored using TLC  with UV-detection and spraying with vanillin sulphuric acid spray reagent to yield 12 major fractions (SG1-SG12); these fractions were subjected to MTT assay to measure the cytotoxic activity of each against 1 and R2 . MTT assv/v) under aseptic conditions, the run started under the previously stated conditions. The fermentation process was left for 72 h, during which foam was suppressed using silicon oil. The culture obtained was centrifuged at 6000 RPM for 10 min using an EBA20 Centrifuge , and the cell free culture supernatant (CFCS) obtained was collected.This was done according to Radwan et al.  with som®xevo-tqd®  and NMR spectroscopic analysis, which was recorded on a Bruker AVANCE HD III 400 MHz spectrometer . NMR samples were dissolved in methanol-d4 or chloroform-d3  and transferred to 3 mm NMR tubes .Structures of the isolated metabolites were determined based on LC-ESI-MS analysis performed using waters50 for each. Control wells contained two aliquots of 100 μL of ethyl acetate extract of soybean meal medium (1 mg dissolved in 5% DMSO and tissue culture medium) and 100 μL of tissue culture medium, followed by two-fold serial dilutions. Values were compared to those of the positive control doxorubicin. After 24 h incubation period at 37 °C in a CO2 incubator, the wells were washed with PBS, followed by incubation with 100 μL MTT solution (1 mg/mL) per well for 1 h at 37 °C. Supernatants were obtained by decantation and the cells were mixed with 100 μL DMSO per well to dissolve formazan particles. Elutes of the 12 wells of each tested metabolite were obtained and measured spectroscopically at 540 nm using differential wavelengths of 630 nm in a Plato R496 Microplate reader AMD diagnostics, Graz, Austria. Control wells were similarly handled, and the percentage cytotoxicity was calculated by the following formula [An MTT assay was carried out as previously reported  with som formula .CytotoxS. griseus isolate KJ623766 in a 14L fermenter and studied the different recovery methods for downstream processing. The structures of isolated metabolites were determined based on the mass, 1D, 2D NMR, COSY, and HSQC BMR spectroscopic analysis and identified as β- and γ-rhodomycinone, respectively. The respective metabolites were biologically analyzed using various cancer cell lines for their cytotoxic actives and exhibiting promising cytotoxic activities. Accordingly, S. griseus isolate KJ623766 could be used as a potential industrial strain for the commercial production of β- and γ-rhodomycinone to be used for production of different derivatives of antitumor antibiotics of the anthracycline family.This study targeted large scale production of cytotoxic metabolites by


The expression of 84 key genes associated with unfolded protein response (UPR) was assessed in four OS cells  by RT2 Profiler PCR Arrays. Based on results, we performed both siRNA and inhibitor assays focusing on IRE1α-XBP1 and PERK pathways. All OS cell lines showed resistance to PERK inhibitors. Furthermore, ATF4 and EIF2A inhibition by siRNA did not affect the survival of OS cell lines. On the other hand, IRE1α-XBP1 inhibition by toyocamycin suppressed OS cell growth (IC50: < 0.075 μM) and cell viability was suppressed in all OS cell lines by silencing XBP1 expression. The expression of XBP1s and XBP1u in OS cell lines and OS surgical samples were confirmed using qPCR. In MG63 and U2OS, toyocamycin decreased the expression level of XBP1s induced by tunicamycin. On the other hand, in 143B and KHOS, stimulation by toyocamycin did not clearly change the expression level of XBP1s induced by tunicamycin. However, morphological apoptotic changes and caspase activation were observed in these two cell lines. Inhibition of the IRE1α-XBP1s pathway is expected to be a promising new target for OS.Osteosarcoma (OS) is the most common primary malignant bone tumor. However, the therapeutic results of the advanced cases at the first visit were still extremely poor. Therefore, more effective therapeutic options based on molecular profiling of OS are needed. In this study, we investigated the functions of endoplasmic reticulum (ER) stress activities in OS and elucidated whether ER stress inhibitors could exert antitumor effectsThe online version contains supplementary material available at 10.1007/s12672-021-00453-2. Osteosarcoma (OS) is the most common primary malignant bone tumor; it peaks during childhood/adolescence and after the age of 50 years. The standard protocol for the treatment of patients with OS was established more than 30 years ago , and limited therapeutic progress has been made since then . The theRecently, studies have explored the therapeutic effects of targeting endoplasmic reticulum (ER) stress and unfolded protein response (UPR) using these inhibitors in several tumors , 3. Our The 143B and MG63 cell lines were obtained from the American Type Culture Collection (ATCC). The KHOS and U2OS cell lines were provided by Dr. Melinda Merchant . All cell lines were grown in Dulbecco’s modified Eagle's medium (DMEM) supplemented with 10% FBS and 1% penicillin/streptomycin.-∆∆Ct method.The overall expression of 84 key genes associated with the UPR was determined with the RT2 Profiler PCR Arrays , using an RT2 SYBR Green ROX qPCR Master Mix (Qiagen). Arrays were analyzed using mRNA from four OS cell lines. Thermal cycling was performed using ABI-7500Fast  with initial denaturation at 95 °C for 10 min, 40 cycles at 95 °C for 15 s, and 60 °C for 1 min. The signal was acquired at 60 °C for each cycle. The cycle threshold (Ct) values obtained in quantification were used to calculate fold changes in mRNA abundance using the 2EIF2A (Assay ID Hs00230684_m1), ATF4 (Assay ID Hs00909569_g1), and GAPDH (Assay ID Hs02758991_g1). Custom qPCR sets were designed for XBP1s, XBP1u, and TATA-box binding protein (TBP) for separate quantifications. These primer and probe sequences were as follows: XBP1s , XBP1u , TBP . Plasmids for standard curves were generated by cloning cDNA fragments of XBP1s, XBP1u, and TBP into the pCRII TOPO vector (Invitrogen). The amounts of XBP1s and XBP1u relative to the housekeeping gene, TBP, were determined using the standard curve method. The amounts of other genes relative to the housekeeping gene, GAPDH, were determined using the comparative Ct method.RNA was extracted using RNeasy Plus Mini kit . All quantitative real-time PCR (qPCR) was performed with TaqMan Fast Advanced Master Mix (Applied Biosystems) on an Applied Biosystems Step One Plus Real Time PCR System in accordance with standard protocols. qPCR was performed using predeveloped TaqMan assays  for XBP1 siRNA knockdown was also performed using pre-designed XBP1 siRNA , EIF2A siRNA , and ATF4 siRNA  using Lipofectamine™ RNAiMAX reagent (Thermo Fisher Scientific). After 72 h, RNA from each cell line was isolated, and its expression was validated using quantitative real-time PCR.For the knockdown expression studies, we used four cell lines . XBP1, EIF2A, and ATF4, 2000 to 5000 OS cells were plated into 96-well plates on day 1. Transfection was performed on the same day with 25–50 nM of the siRNA reagents, as described above. After 72 h, the cell proliferation ability of OS cell lines was assessed using a Cell Counting Kit-8  and a microplate reader .For knockdown proliferation studies with respect to XBP1 pathway inhibitor. GSK2606414  and ISRIB  were used as PERK pathway inhibitors. OS cells were seeded into 96-well plates at a density of 3000–10000 cells/well. The next day, different concentrations of inhibitors or DMSO (as a vehicle control) were added to each well. After 72 h, the inhibitory effect of these inhibitors on the growth of OS cell lines was assessed using a Cell Counting Kit-8  and a microplate reader . The IC50 was calculated using GraphPad Prism software version 9.2.0 .Toyocamycin  was used as an IRE1α-–9 μM: minimum dose) for 12 h, and after DMSO (as a vehicle control) exposure for 18 h.Because two OS cell lines (143B and KHOS) kept unexpectedly high-level expression of XBP1s even after Toyocamycin treatment while showing morphological apoptotic change, apoptotic assays were performed for these cell lines. These cells were plated into 96-well plates at a density of 5000 cells/well, and the next day, tunicamycin (TM) or DMSO (as a vehicle control) were added to each well. After 3 h and 6 h, apoptosis (caspase-3/7 activity) was measured using the Apo-ONE Homogeneous Caspase-3/7 Assay kit . Furthermore, apoptosis assay (caspase-3/7 activities) was performed at the following time points: after TM stimulation for 18 h, after TM stimulation for 6 h and subsequent toyocamycin stimulation . ATF4 and EIF2A showed the highest and second highest expressions, respectively, among ER stress genes across all OS cell lines. In addition, XBP1 showed the third highest expression in three cell lines , and the fifth highest expression in KHOS. Furthermore, stimulation with tunicamycin led to enhanced expression of PERK pathway genes, including ATF4, EIF2A, DDIT3, PPP1R15A, and DNAJC3 . Furthermore, HSPA5, an upstream gene of the main UPR pathway, was also upregulated by tunicamycin stimulation. These findings indicated that tunicamycin stimulation enhanced the upstream UPR pathway gene and stimulated the PERK pathway among the three ER stress pathways. Based on these findings, we focused on the PERK and IRE1α pathways for further analysis.Three major signaling pathways in the ER stress response are inositol-requiring enzyme 1α (IRE1α), PKR-like ER kinase (PERK), and activating transcription factor 6 (ATF6), all of which are involved in tumorigenesis –5. We peEIF2AK3 of the PERK pathway, while ISRIB is an inhibitor of EIF2A phosphorylation of the PERK pathway. The IC50 of GSK2606414 was shown to be 1.7 µM in ARPE-19  treated with GSK2606414 for 72 h [Recently, two PERK inhibitors have been developed: GSK2606414 and ISRIB. GSK2606414 is an inhibitor of ATF4 and EIF2A was performed via siRNA-mediated knockdown of ATF4 and EIF2A in the four OS cell lines. qPCR confirmed a significant decrease in ATF4 and EIF2A mRNA levels in all OS cell lines . In the cell proliferation assays, by silencing the expression of ATF4, cell viability was not significantly suppressed, except for U2OS . Additionally, silencing EIF2A expression did not significantly suppress cell viability in any of the OS cell lines .To investigate the association between the PERK pathway and viability of OS cells, the inhibition of XBP1 inhibition on OS cell lines. We had previously reported that toyocamycin showed the highest anti-tumor effect on Ewing’s sarcoma cells [Next, we examined the effect of IRE1α-ma cells . In Ewinma cells . ToyocamXBP1 pathway and malignant behaviors in Ewing sarcoma cells [XBP1s and XBP1u expressions were analyzed in the four OS cell lines and eight clinical surgical materials. All OS cell lines showed the mRNA expression of XBP1s and XBP1u .Our previous proteomic analyses demonstrated critical associations between the IRE1α-ma cells . XBP1s aP1u Fig. A. MG63 cXBP1 pathway and the survival of OS cell lines, inhibition of XBP1 by siRNA was performed in four OS cell lines. The knockdown of both XBP1s and XBP1u was confirmed using qPCR. In the cell proliferation assays, we also confirmed that cell viability was suppressed in all OS cell lines due to the silencing of XBP1 expression  induced the expression of XBP1s in a time-dependent manner and suppressed the expression of XBP1u in all OS cell lines . On the other hand, in 143B and KHOS, stimulation by toyocamycin did not clearly change the expression level of XBP1s induced by TM. However, morphological apoptotic changes were observed in these two cell lines .Toyocamycin is a selective IRE1α inhibitor that shows antitumor effects and induces apoptosis in cancer cells. Tunicamycin (TM) generally induces ER stress and enables the processing of nes Fig. . We nextTo verify the different effects of toyocamycin after TM stimulation on the two OS cell lines (143B and KHOS), we evaluated apoptotic activity using the caspase-3/7 assay. After TM stimulation for up to 6 h, caspase-3/7 activity was not evident in the OS cells, and morphological apoptotic change was not evident . Toyocamycin treatment at a low dose after TM stimulation elevated caspase-3/7 activity in two OS cell lines (143B and KHOS), and morphological apoptosis changes were evident . On the other hand, in MG63 and U2OS cells, morphological apoptosis changes were not evident  after TM stimulation for 6 h and toyocamycin treatment at a low dose after TM stimulation. These findings were consistent with the morphological changes observed in the OS cell lines following stimulation with toyocamycin.The endoplasmic reticulum (ER) is a major intracellular compartment involved in protein folding and maintenance of cell homeostasis , 8. To m-EIF2A-ATF4 activation contributes to tumor progression and metastasis, and is ultimately associated with drug resistance [Tumor cells escape from ER stress by UPR, making the adjacent environment suitable for tumor survival and tumor growth , 10. IREsistance , whereassistance . In thisXPB1 is a downstream transcriptional factor of the IRE1α pathway and plays an important role in cancer progression. It has been shown that the loss of XBP1 induces a terminal UPR that blocks proliferation and differentiation during mammary gland development [XBP1 strongly inhibited cell proliferation in all OS cell lines, which is consistent with a previous study showing the antitumor effect of XBP1 knockdown in two OS cell lines [XBP1 mRNA splicing [Several studies have revealed an association between the IRE1α pathway and malignant tumors, including apoptosis, cell differentiation, invasion, metastasis, and drug resistance . XPB1 iselopment . In thisll lines . Functioll lines , 16, 17.splicing . These fXBP1 expression during TM/toyocamycin treatment, we found that TM stimulation induced XBP1s expression in all OS cell lines. Furthermore, we confirmed that XBP1s expression was decreased and XBP1u was increased after treatment with toyocamycin in two OS cell lines (U2OS and MG63). However, this switching of XBP1 expression after toyocamycin treatment was not clear in the other two OS cell lines (KHOS and 143B), and XBP1s expression remained at a high level. Interestingly, these two OS cell lines were not examined in a previous study showing anti-tumor effects on XBP1 blocking in OS [XBP1s overexpression and apoptosis, it has been known that activation of JNK (MAPK8) cooperates with p38 and induces apoptosis [MAPK8 expression was not enhanced . Furthermore, it has been reported that sustained activation of XBP1 splicing induces apoptosis in normal tissues [XBP1 splicing induces apoptosis in tumor cells, we observed caspase activation in 143B and KHOS cells, after toyocamycin treatment at a low dose after TM stimulation, and morphological apoptotic changes were evident. Interestingly, TM treatment for 6 h followed by toyocamycin treatment for 12 h induced morphological apoptotic changes in 143B and KHOS with caspase activation, while high levels of XBP1s expression were preserved in these two cells as well as under TM stimulation. The reason for this paradoxical change in OS cells was unclear because high levels of XBP1s were preserved while morphological apoptotic changes occurred.Regarding ng in OS . Notablypoptosis , 13. How tissues , 19. Alt, we investigated the functions and malignant activities of ER stress response in OS, and further elucidated whether inhibitors of ER stress response had antitumor effects. Our findings demonstrated critical associations between ER stress response and malignant behavior in OS. Furthermore, we found that IRE1α inhibitors exerted antitumor activity in OS. As XBP1s expression was consistently observed in OS clinical samples and XBP1s expression was not related to the chemotherapeutic state, inhibition of this pathway is expected to be a new promising target for OS patients.In ConclusionAdditional file 1: Table S1. List of the 84 key genes associated with the UPR (unfolded protein response) on RT2 Profiler PCR Arrays. (XLSX 15 KB)Additional file 2: Table S2. Clinicopathological information of eight OS clinical samples. (XLSX 9 KB)Additional file 3: Figure S1A-1. Gene expression data of the 84 key genes associated with the UPR without any stimulation (143B and MG63). Green: PERK pathway; Yellow: IRE1α pathway; Blue: ATF6 pathway. Figure S1A-2. Gene expression data of the 84 key genes associated with the UPR without any stimulation (KHOS and U2OS). Green: PERK pathway; Yellow: IRE1α pathway; Blue: ATF6 pathway. Figure S1B: ER stress response analysis using the RT2 Profiler PCR Arrays after TM stimulation shows a high expression of DDIT3 (CHOP) followed by EIL2A and ATF4. XBP1s and XBP1u were not included in this array , and were, therefore, separately examined using qPCR. Figure S2: Cell viability by ATF4 knockdown in OS cell lines. ATF4 siRNA knockdown in four OS cell lines  was performed to examine the associations between ATF4 expression and cell viability. A Suppression of ATF4 expression evaluated using qPCR. The expression of ATF4 in each OS cell line is suppressed by all ATF4 siRNAs. B The cell viability in OS cell lines is not affected by ATF4 siRNA knockdown. Figure S3 Cell viability following EIF2A knockdown in OS cell lines. EIF2A siRNA knockdown in four OS cell lines  was performed to examine the association between EIF2A expression and cell viability. A Suppression of EIF2A expression is evaluated using qPCR. The expression of EIF2A in each OS cell line is suppressed by all EIF2A siRNAs. B The cell viability in OS cell lines is not affected by EIF2A siRNA knockdown. Figure S4. Caspase-3/7 assay in OS cell liens. Caspase-3/7 activity is not evident in OS cells after TM stimulation for up to 6 h. However, it is activated by prolonged stimulation (18 h) in KHOS cell. Caspase-3/7 activity in 143B and KHOS cells is elevated by toyocamycin treatment after TM stimulation. Figure S5. Morphological apoptotic changes in OS cell liens. In MG63 and U2OS cells, morphological apoptosis changes are not evident after TM stimulation for up to 6 h and toyocamycin treatment after TM stimulation. On the other hand, in 143B and KHOS cells, morphological apoptosis changes are evident after low dose toyocamycin treatment after TM stimulation. (PPTX 4759 KB)


Epirubicin (EPI) is one of the potent breast cancer (BC) chemotherapeutic agents, but its adverse effects limit its efficacy. Herein, EPI was selected to be loaded in liposomal carrier, which has been targeted by a monoclonal antibody, Herceptin. The preparation process of liposomes was a modified ethanol injection method followed by Herceptin conjugation. The in vitro cell toxicity and cellular uptake of optimum formulation against HER2+ and HER2− cancer cell lines were evaluated. The results showed that the drug loading (DL%) and encapsulation efficiency (EE%) of liposome preparation method yielded 30.62% ± 0.49% and 62.39% ± 8.75%, respectively. The average size of naked liposomes (EPI‐Lipo) and immunoliposomes (EPI‐Lipo‐mAb) was 234 ± 9.86 and 257.26 ± 6.25 nm, with a relatively monodisperse distribution, which was confirmed by SEM micrographs. The release kinetic followed Higuchi model for both naked and immunoliposomes. In vitro cytotoxicity study on three different BC cell lines including BT‐20, MDA‐MB‐453 and MCF‐7 demonstrated higher toxicity of EPI in the Herceptin conjugated form (EPI‐Lipo‐mAb) in comparison with the free EPI and EPI‐Lipo in HER2 overexpressing cell line. In addition, the cellular uptake study showed a higher uptake of immunoliposomes by MCF‐7 cells in comparison with naked liposomes. In conclusion, these data show that the targeted delivery of EPI to breast cancer cells can be achieved by EPI‐Lipo‐mAb in vitro, and this strategy could be used for breast cancer therapy with further studies. Despite different therapeutic strategies, BC has the highest mortality rate after lung cancer . More thAnthracyclines exist in many cytotoxic regimens for the treatment of several malignancies such as sarcoma, BC and lymphoma as they diminish disease relapse and improve survival rate . EpirubiHerein, EPI was encapsulated in the novel immunoliposomes consisting of 1,2‐dioleoyl‐sn‐glycero‐3‐phosphoethanolamine (DOPE) and cholesterol (Chol), and was targeted for HER2 receptors by Herceptin functionalization. Liposomes were prepared by ethanol injection method followed by extrusion and mAb conjugation. Immunoliposomes were optimized according to physicochemical properties, cellular uptake was assessed and, finally, cytotoxicity was studied for the optimized formulation in three different BC cell lines to investigate the in vitro tumour cell cytotoxicity.22.1Mw 145.5 kDa) was supplied by LuyePharma. Epirubicin was purchased from Sigma‐Aldrich Co.DOPE was purchased from Lipoid AG. Chol, N‐hydroxycarbidiimide (NHS), 3‐‐2,5‐diphenyltetrazolium bromide (MTT), 1‐ethyl‐3‐(3‐dimethyl aminopropyl) carbodiimide (EDC), trypsin‐EDTA, were obtained from Merck. Potassium dihydrogen phosphate, dimethyl sulfoxide (DMSO), analytical grade ethanol and acetone were purchased from Sigma‐Aldrich Co. Trastuzumab  was obtained and stored at 4°C for further analysis  [Amide bond formation is the basis of trastuzumab conjugation. So, for the preparation of functionalized nanoliposomes, the mAb should be activated with EDC and NHS. NHS ester creates reactive acylating agents with nucleophiles such as primary amine and releases its leaving group to form a stable amide bond. The addition of aminated antibody to prepared liposomes was performed under NGermany) .Figure 2.2.3The Bradford protein assay is a spectroscopic analytical procedure used to measure the concentration of protein in a solution. For this aim, 180 μl of Coomassie dye plus reagent was added to an ambient amount of free dispersion of mAb, and after 10 min of incubation, the absorbance was measured at 595 nm using a UV‐Visible instrument (Schimadzu). The results were compared to a standard curve of mAb solution in the concentration range of 15–60 μg/ml [2.3Wa, Ws, and WDOPE,Ch were the weight of the added drug, the analysed weight of the drug in the dialysis medium, and the weight of DOPE and cholesterol added to prepare liposomes, respectively.The percentage of incorporated EPI was calculated indirectly and has been reported as drug loading (DL)  and enca2.42.4.1The samples (naked liposomes and immunoliposomes) were diluted with distilled water, and after drying for 12 h, they were placed on an aluminium pan and coated with a gold thin layer. The scan was conducted at an accelerating voltage of 10 kV by capability of point‐to‐point scanning by XL30 microscope  for scanning electron microscopy (SEM) images .The particle size, poly dispersity index (PDI) and zeta potential of the naked liposomes and immunoliposomes were measured by dynamic light scattering (DLS) . The vesicles were diluted with distilled water (ratio of 1:3) and measurements were conducted at room temperature.2.4.2−1.Fourier transform‐infrared spectroscopy (FT‐IR) spectra of samples were studied by Schimadzu IR‐prestige 21 FTIR spectrometer to find the sample interactions. Therefore, the evaluated samples were naked and immunoliposomes, free mAb, Chol, DOPE and EPI. Each sample tablet was prepared by mixing 2 mg of sample with 10 mg KBr followed by compression. Transition mode was applied for sample evaluation and the spectral region was 450–4000 cm2.4.3Thermal characteristics of the samples including naked and functionalized liposomes, free mAb, cholesterol, DOPE, EPI and physical mixture were characterized by differential scanning calorimeter  at the temperature range of 20–375°C and heating rate of 10°C/min. Reference was An empty aluminium pan was used as a reference, and dry nitrogen was used as effluent gas during the experiment .2.5Franz diffusion cell was selected to evaluate the in vitro drug release profile of naked liposomes and immunoliposomes . The relR2) of the models given in Table Different kinetic models were evaluated to find the best model fitted to in vitro release data. The analysis was done according to correlation coefficient R of the m2.64 cells/well followed by 24‐h incubation for sufficient adhesion of cells. The different formulations containing different concentrations of free EPI, phospholipid, naked EPI‐loaded liposomes and EPI‐loaded immunoliposomes were added to wells and incubated for 24 h; each sample was repeated six times. Then the samples were replaced with 200 μl fresh Dolbecco's modified eagle's medium followed by 20‐min incubation. The medium was removed, then 200 μl RPMI medium and 20 μl of MTT dye solution (10% in phosphate buffer pH = 7.4) were added to each well and incubated for 2.5 h (at 37°C and 5% CO2). Finally, the medium was removed, and formazan crystals were solubilized by 200 μl of DMSO; the reacted dye was dissolved and the absorbance was measured at 570 nm by a microplate reader .The cytotoxicity assay was studied in three different cell lines containing MCF‐7, MDA‐MB‐453, and BT‐20; the method was based on MTT assay, which shows the viable cells. For this purpose, the cells were cultured in 96‐well plates ; the cell density was 1.5 × 10A)test/(A)control × 100, where (A)test is the absorbance of each test sample and (A)control is the absorbance of control sample. The culture medium without cells was considered as control, and the IC50 of samples (the concentration which inhibits the growth of 50% of cells) was calculated by Sigma plot software [The cell viability (%) was calculated by . According to the results of Table One of the important factors for drug release is particle size by the way that smaller particles lead to higher surface area so the drug exposure for dissolution will increase and higher amount of drug will be dissolved in the medium . Figure 3.550 of each formulation was evaluated; the results are depicted in Figure MTT assay was selected for in vitro cytotoxicity investigation of formulations. MCF‐7, MDA‐MB‐453 and BT‐20 cell lines were exposed to different concentration of formulations. The viable cells were counted and ICIt can be concluded that the phospholipid (DOPE) did not show any cytotoxicity effect on none of the cell lines in the treated concentrations.50 of EPI in three formulations , as tabulated in Table 50 of EPI in MCF‐7 cells is higher for immunoliposomes compared to naked liposomes. As this cell line is HER2−, the immunoliposomes could not attach to cell surface as much as to cause lower IC50 and so higher potency. On the contrary, the IC50 of EPI in BT‐20 cell line is much lower for immunoliposomes than naked liposomes. The IC50 of EPI in MDA‐MB‐453 is much lower even than the difference of BT‐20 for immunoliposomes in comparison with naked liposomes. The reason can be the overexpression of HER2 for MDA‐MB‐453 cells that leads to higher efficacy of loaded EPI in mAb coupled liposomes in this cell line.Evaluation of the average IC50, which can be related to the ER+ and PR+ characteristic of MCF‐7 cell line. The overexpression of ER and PR on the cell surface can lead to higher affinity of immunoliposomes [50 has been reduced to about a third of the free drug.Comparison of the effect of immunoliposomes in three cell lines shows that EPI is more effective in MCF‐7 and BT‐20 than MDA‐MB‐453 and confirms by lower ICiposomes . The resCellular uptake of naked and immunoliposomes to MCF‐7 cells was studied by fluorescence microscopy. There was no need for addition of a fluorescent dye, due to EPI fluorescence property. The amount of cellular uptake of EPI increased along with higher exposure time of formulation to cells but the fluorescence intensity was high enough to be detected even after four hrs of exposure. As shown in Figure 4In the described experiments, EPI‐loaded liposomes and HER2‐targeted liposomes were prepared, and after the physicochemical characteristic evaluations, these formulations were assessed by in vitro cytotoxicity and cellular uptake study. As the liposomes were targeted for HER2 receptors by Herceptin, the drug efficacy was higher than free EPI in HER2 overexpressing cells. Therefore, this kind of EPI‐loaded drug delivery system can be a potent carrier for HER2+ BCs.Higher efficacy of immunoliposomes can be due to cytostasis/cytolysis which is caused by the carrier binding to HER2 receptors as was elucidated by Vaidya et al. They prepared dual functionalized immunoliposomes by trastuzumab (anti‐HER2) and OKT‐3 (anti‐CD3) antibodies to study the doxorubicin efficacy. The results showed high cytotoxicity for the functionalized formulations, especially when effector cells exist. They demonstrated that the mechanism can be cytostasis/cytolysis in addition to slow drug release from functionalized liposomes .The mechanism of EPI uptake in HER2+ cell lines can be through receptor‐mediated endocytosis as reported by Amin et al. in the study of mAb conjugated liposomes containing idarubicin in HER2+ BC cell lines. It has been observed that trastuzumab conjugated liposomes cause more efficient attachment of drug delivery to overexpressed HER2 cell lines and so higher cellular uptake will happen .Yan Wu et al. studied the EPI pharmacokinetic parameters in animal model after encapsulating in thermosensitive liposomes. The results showed that liposomes can increase the circulation time of EPI, and higher drug exposure will lead to higher antitumour activity .Rodallec et al. studied the efficacy and tumour cell uptake of targeted liposomes containing Docetaxel for HER2+ BC model in vitro and in vivo. They found that immunoliposomes caused higher efficacy than free drug and antibody. They related this effect to better drug delivery because despite the higher tumour internalization, the localization of immunoliposomes was not higher for immunoliposomes, even in HER2+ cells. The results show that the EPR effect is as potent as antibody conjugation in drug targeting .5Further in vivo analysis should be conducted to confirm the results.In the present study, Herceptin conjugated EPI‐loaded liposomes were prepared and the physicochemical properties were evaluated. EPI was successfully encapsulated which was confirmed by high EE%. The negative surface charge of particles leads to high stability of formulation and a good choice for intravenous injection. The Higuchi release kinetic shows that drug will be released continuously during 24 h. Different formulations based on various aqueous‐to‐organic phase ratio and components were prepared. The optimum formulation according to size and size distribution was selected and in vitro cellular uptake and cytotoxicity studies showed that Herceptin conjugated liposomes have higher cell toxicity in HER2+ cell lines compared to naked liposomes. Therefore, Herceptin decorated liposomes can improve EPI efficacy in HER2+ BCs. The authors declare no conflict of interest.


Salmonella enterica serovars are frequently associated with disease outbreaks in low-moisture foods (LMF) due to their ability to adapt efficiently to desiccation stress. These serovars are often persistent during food processing. Disruption of these resistance responses was accomplished previously using the membrane-active lipopeptide, paenibacterin. This study was initiated to determine how desiccation resistance mechanisms are overcome when Salmonella Tennessee, a known resistant serovar, is treated with the membrane-active food additives carvacrol and thymol. Knowing that the minimum inhibitory concentrations (MICs) of carvacrol and thymol against Salmonella Tennessee are 200 and 100 µg/mL, the concentrations tested were 100–400 and 50–200 µg/mL, respectively. Results show that desiccation-adapted Salmonella Tennessee, prepared by air drying at 40% relative humidity and 22–25 °C for 24 h, was not inactivated when exposed for 4.0 h to less than 2xMIC of the two additives. Additionally, treatment of desiccation-adapted Salmonella Tennessee for 120 min with carvacrol and thymol at the MIC-level sensitized the cells (1.4–1.5 log CFU/mL reduction) to further desiccation stress. Treating desiccation-adapted Salmonella Tennessee with carvacrol and thymol induced leakage of intracellular potassium ions, reduced the biosynthesis of the osmoprotectant trehalose, reduced respiratory activity, decreased ATP production, and caused leakage of intracellular proteins and nucleic acids. Carvacrol, at 200–400 µg/mL, significantly downregulated the transcription of desiccation-related genes  as determined by the reverse-transcription quantitative PCR. The current study revealed some of the mechanisms by which carvacrol and thymol combat desiccation-resistant Salmonella Tennessee, raising the feasibility of using these additives to control desiccation-adapted S. enterica in LMF.Some  Salmonella enterica serovars are highly adaptable to dry conditions, and this adaptation allows the pathogen to survive for extended periods in low-moisture foods (LMF), and cause frequent disease outbreaks [Salmonella Tennessee, Agona, Bredeney, Mbandaka, Montevideo, and Enteritidis, which caused salmonellosis linked to consumption of products such as peanut butter, breakfast cereal, tahini, and almonds. Serovars having this phenotype invoke several mechanisms upon exposure to desiccation conditions; these mechanisms include accumulation of potassium ions, biosynthesis of the osmoprotectant trehalose, increased fatty acid metabolism, and production of cellulose and fimbriae [Salmonella serovars to dry conditions was found to involve collateral adaptive responses, such as the ability to form biofilm and induction of viable but nonculturable state [Some nontyphoidal utbreaks . Dry-tolfimbriae . Adaptable state ,4. Collele state .Salmonella serovars on dry products such pistachios, nuts, or in-shell pecans; these methods include heat-spray, irradiation, and hot water treatment [S. enterica serovars; the lipopeptide rendered the pathogen sensitive to dry conditions [Salmonella, in similar fashion to paenibacterin, were investigated [Salmonella serovars.Researchers explored the suitability of different methods to combat desiccation-resistant reatment ,7,8. In nditions . Howevernditions . Alternastigated . Of thesSalmonella, in view of their activity against bacterial cytoplasmic membranes. In a previous study, the two additives sensitized desiccation-adapted Salmonella to heating at 55 °C for 15 min [Salmonella effect of carvacrol was apparent during spray- or freeze-drying of liquid milk, and this effect was noticeable during storage of the dried milk at room temperature [Salmonella. Unveiling such mechanisms will help in confirming the ability of the two compounds to overcome desiccation adaptation in Salmonella. This knowledge could facilitate developing membrane-active agents that de-adapt desiccation-resistant Salmonella, with the goal of improving the safety of LMF. Therefore, the present study was conducted to investigate the mode of action of membrane-active carvacrol and thymol against the desiccation resistance mechanisms employed by S. enterica.Carvacrol and thymol are monoterpene phenolic compounds that have been studied extensively for their antimicrobial, antitumor, and anti-inflammatory activities ,14,15,16r 15 min . Furtherperature . These fCarvacrol and thymol  were dissolved in sterile water containing dimethyl sulfoxide  at 5% level. Control experiments were completed using sterile aqueous DMSO (5%).Salmonella enterica serovar Tennessee E2007000304, a known desiccation-resistant strain [Salmonella Tennessee demonstrated stronger resistance to desiccation stress when compared to Salmonella Typhimurium [t strain , was obtt strain , and to himurium , and a gSalmonella Tennessee cells were prepared as described previously [S. enterica cells were grown overnight in Tryptic Soy broth  at 37 °C to a cell density of 109 CFU/mL, as determined by plating on Tryptic Soy agar . Cells of the incubated cultures were harvested by centrifugation at 4 °C and 8000× g for 5 min, and finally resuspended in fresh TSB. Aliquots  from cell suspensions were placed in empty plastic Petri dishes  with a final population of 109 CFU per plate, and air dried in a biosafety cabinet for 24 h at 22–25 °C under ca. 40% relative humidity; survivors are considered desiccation-adapted cells. The adapted cells were collected by dispensing 1 mL of the resuspension medium onto the dried cells. The resuspension medium was saline (0.85% NaCl), TSB, or M9 minimal medium supplemented with glucose, depending on the medium required in a given experiment, to obtain desiccation-adapted cell suspension  containing 5 mM glucose . Portions of these cell suspensions (90 µL) were added to wells of a black 96-well microplate (Corning) followed by the addition of the PBFI probe at a final concentration of 2 µM. Then, 10 µL of carvacrol or thymol was added to each well to achieve final concentrations of 80 to 800 µg/mL of each compound, and polymyxin (10 µg/mL) served as a positive control. Concentrations of released potassium ions were measured as changes in fluorescence using a microplate reader  at excitation and emission wavelengths of 346 and 505 nm, respectively. Fluorescence measurements were captured every 100 s and normalized by subtracting the background noise. The potassium-sensitive benzofuran isophthalate probe  was used to determine the release of potassium ions from desiccation-adapted eviously ,11. BrieSalmonella Tennessee desiccation-adapted cells in saline  at 280 and 260 nm, respectively, and results are expressed as µg/mL. n saline  were treSalmonella Tennessee desiccation-adapted cells in TSB , an electron acceptor that indicates dehydrogenase activity, was used as described previously [To measure respiratory activity in eviously . BrieflySalmonella Tennessee cells in saline  from additive-treated and untreated cells were distributed into 96-well, clear-bottom, black microtiter plates (Corning), and the ATP levels were determined using an assay kit  according to the manufacturer’s instructions. The ATP levels were determined by measurement of changes in luminescence using a microplate reader (PerkinElmer). The decrease in ATP levels in treated cells was calculated in reference to the untreated control.Desiccation-adapted n saline  were treSalmonella Tennessee desiccation-adapted cells in TSB  and the reference gene, gapA, which encodes glyceraldehyde-3-phosphate dehydrogenase [Salmonella Tennessee cells and compared to the untreated control cells using the 2−ΔΔCt method [s in TSB  were treus study  using rerogenase , were met method . Primer p value of <0.05.All experiments were performed in triplicates and each experiment was repeated twice, independently. Each data point was represented as mean ± SD of the two independent repeats. The statistical analysis was performed using a statistical software . Analysis of variance (ANOVA) with Tukey pairwise comparisons were used to determine significant differences between treatment groups or comparing pairs of treatment. Statistical significance was considered at Salmonella Tennessee was performed as reported in previous studies [The preparation of desiccation-adapted dry cells of  studies ,11, descSalmonella Tennessee E2007000304 were determined in a previous study to be 200 and 100 µg/mL, respectively [Salmonella Tennessee, suspended in saline  than those of the untreated control , when compared to the untreated control (p > 0.05) Salmonella populations when compared to the untreated control during the 4.0-h exposure period. These findings suggest that 2× MIC, at least, of carvacrol or thymol is required to inactivate desiccation-adapted Salmonella Tennessee. The minimum inhibitory concentrations (MICs) of the food additives carvacrol and thymol against non-desiccation-adapted cell suspensions of ectively . Based on saline , was tre control A. Simila control B. HoweveSalmonella Tennessee cells were treated with carvacrol or thymol at 1× MIC level for 120 min and then subjected to a second round of desiccation, the populations behaved differently, compared to the desiccation-adapted additive-untreated control , respectively, compared to the untreated populations, which decreased only by 0.3 log CFU/mL after the second cycle of desiccation. This indicates ability of carvacrol or thymol to sensitize Salmonella Tennessee to desiccation stress. When desiccation-adapted  control . Results+) is required for S. enterica to adapt to desiccation conditions [+ leakage could counteract desiccation resistance. Carvacrol or thymol at levels ranging from sublethal to lethal concentrations caused rapid leakage (p < 0.05) of potassium ions (after 100 s of exposure to the additive) from desiccation-adapted Salmonella Tennessee cells, compared to the untreated desiccation-adapted control (p < 0.05) when the concentration of carvacrol or thymol was >100 or >80 µg/mL, respectively.Accumulation of potassium ions  as shown in p < 0.05; p > 0.05) when the two additives were compared for their ability to induce leakage of the intracellular nucleic acids.The ability of carvacrol and thymol to induce potassium leakage prompted us to investigate the capability of these additives to invoke the leakage of intracellular macromolecules, such as proteins and nucleic acids. Results showed that carvacrol caused leakage of intracellular proteins in a concentration-dependent manner . Thymol caused similar effect at all tested concentrations but the decrease in trehalose levels was significant only when 100 and 200 µg/mL of the compound were tested (p < 0.05). The capability of carvacrol and thymol to inhibit trehalose biosynthesis in desiccation-adapted iccation . Figure e tested B. GeneraSalmonella Tennessee desiccation-adapted cells, suspended in M9 broth medium that was supplemented with glucose  among the various concentrations of each food additive or even between the inhibitory effects of the two additives.  glucose , were me glucose . SalmoneSalmonella cells, we explored their ability to affect the production of ATP, which is produced through cytoplasmic membrane- and respiration-related processes [Salmonella to adapt to desiccation [Salmonella cells suspended in saline (p < 0.05).Because carvacrol and thymol are membrane-active compounds, and both decreased the respiratory activity in rocesses . Producticcation ,19. Desin saline  were expn saline A, with tn saline B, but thproV, kdpA, otsB, and STM1494) are necessary for desiccation adaptation and are mainly involved in proline accumulation, K+ transport, trehalose synthesis, and osmoprotection, respectively [proV and STM1494, whereas a concentration of 400 µg/mL  was needed to significantly downregulate (>2-fold decrease) the expression of proV, STM1494, and kdpA (The genes investigated in this study (ectively ,26. Exprand kdpA . In contS. enterica in LMF, the bacterium continues to cause frequent outbreaks linked to these foods [S. enterica in LMF, such as almonds, pecans, pistachios, and walnuts, among others [Salmonella expresses thermal resistance [Despite the efforts of processors to control se foods ,28. The se foods  and causg others . Howeversistance , which csistance ,32. Salmonella and to sensitize the pathogen to drying and mild thermal processing. One of these strategies is utilizing cytoplasmic membrane-active agents to de-adapt desiccation-resistant Salmonella into a phenotype that is sensitive to drying and thermal processing. The incorporation of membrane-active agents, such as carvacrol and thymol, in liquid foods before drying could be an effective approach to sensitize Salmonella to the mild food processing [Several antimicrobials, including oxidizing agents, such as peroxyacetic acid, hydrogen peroxide, and propylene oxide, were used for nonthermal decontamination of LMF. Use of these agents may cause oxidation of food components, which could alter product quality . Althougocessing .Salmonella Tennessee and Eimsbuettel in response to desiccation [Salmonella Tennessee E2007000304. Carvacrol and thymol are membrane-active food additives that were found capable of sensitizing desiccation-adapted S. enterica to mild heat treatment, and spray- or freeze-drying [Salmonella Tennessee showed tolerance to 1× MIC carvacrol or thymol; these additives were needed at 2× MIC to cause significant reduction in Salmonella populations (S. enterica serovars in response to treatment with the membrane-active paenibacterin or other chemical antimicrobials [In a previous study, the microbial-produced lipopeptide paenibacterin disrupted the adaptation mechanisms employed by iccation . In the e-drying . In the ulations . Similarcrobials ,35.Salmonella Tennessee to accumulate K+, synthesize trehalose and ATP, retain respiratory activity, and maintain the intracellular contents, is indispensable for desiccation adaptation [+ in a concentration-dependent manner at levels ranging from sublethal to lethal concentrations (+ may be attributed to the increased cytoplasmic membrane permeability, which can be facilitated by carvacrol or thymol [Salmonella’s early responses to hyperosmotic shock or desiccation conditions [+ leakage results, the additives also caused the release of intracellular proteins and nucleic acid (The ability of aptation . Thus, ttrations ; this efr thymol . Potassinditions . Similareic acid ; this coSalmonella, since the sugar protects the bacterium against cellular collapse during water loss by forming hydrogen bonding with the membrane phospholipids [Salmonella Tennessee (Salmonella to desiccation. Evidence for the role of trehalose in desiccation resistance was demonstrated in the finding that desiccation-sensitive Salmonella Typhimurium LT2 accumulates inadequate amounts of trehalose [The biosynthesis of the trehalose is important for desiccation adaptation in holipids . Carvacrennessee . This efrehalose .Salmonella Tennessee by more than 70% (Based on the results of the current study, carvacrol and thymol reduced the respiratory activity in desiccated than 70% . A plausthan 70% , may be proV, STM1494, and kdpA (Salmonella. In contrast, the anti-desiccation effect of thymol could not be attributed to ability of this compound to alter the expression of desiccation-related genes (Salmonella Enteritidis with carvacrol decreased expression of a panel of cellular proteins that include few transporter proteins [Origanum vulgare, which include carvacrol and thymol, were capable of crossing the cytoplasmic membrane of Salmonella Enteritidis and acting directly on the bacterial DNA [It was observed that carvacrol at 200–400 µg/mL downregulated the expression of and kdpA ; these ged genes . The dirSalmonella is presented schematically in Salmonella Tennessee permeabilizes the cells, causing leakage of the intracellular small ions, such as K+, whose intracellular accumulation is needed as a primary event in resistance to desiccation. With the increase in cell-membrane permeability, efflux of other intracellular molecules, such as DNA and proteins, starts. Compromising the cytoplasmic membrane permeability could also alter the proton motive force and reduce respiration. Additionally, the phenol group of carvacrol or thymol may interact with ATPase, causing its inactivation. Several other direct or indirect cascades of events could affect the transcription of genes necessary for desiccation adaption; however, this inference needs future research.The proposed mechanism of action of carvacrol or thymol against desiccation-resistant S. enterica in low–moisture foods. The ability of carvacrol and thymol to act on the cytoplasmic membrane of Salmonella Tennessee impaired the desiccation resistance in this pathogen via altering the membrane permeability and inducing the leakage of intracellular osmoprotectants. Considering that Salmonella Tennessee has been linked to salmonellosis outbreaks in several low-moisture foods, the current findings imply that carvacrol, thymol, or similar membrane-active agents can protect these foods against this pathogen and potentially against other desiccation-resistant serovars. However, further research is needed to confirm the efficacy of these additives under different desiccation conditions and in various low-moisture foods.This study provided molecular evidence that the additives carvacrol and thymol are feasibly usable in mitigating desiccation-resistant


Transcriptional and phosphoproteomic profiling reveals that the combination converges to downregulate the MAPK-ERK signaling pathway, a result not predicted by single-agent analysis alone. Correspondingly, the addition of a MEK inhibitor synergizes with either dasatinib or cabozantinib to increase its efficacy. This study, by using approved, clinically relevant drugs, provides the rationale for the design of effective combination treatments in NCCRCC that can be rapidly translated to the clinic.The lack of effective treatment options for advanced non-clear cell renal cell carcinoma (NCCRCC) is a critical unmet clinical need. Applying a high-throughput drug screen to multiple human kidney cancer cells, we identify the combination of the VEGFR-MET inhibitor cabozantinib and the SRC inhibitor dasatinib acts synergistically in cells to markedly reduce cell viability. Importantly, the combination is well tolerated and causes tumor regression   Non-clear cell renal cell carcinomas have poor prognoses and limited treatment optionsDrug screens identify cabozantinib and dasatinib combination as synergistic in NCCRCCCombining cabozantinib with dasatinib induces regression of NCCRCC tumors in miceIntegrated multi-omic analyses links therapeutic efficacy to suppression of MAPK activity One important and challenging question in kidney cancer is how do we treat treat non-clear cell kidney cancers? Lue et al. tackle this question by performing drug screens in non-clear kidney cancer cells and mouse models and identify the cabozantinib-dasatinib combination as a promising clinically relevant therapeutic option. VHL tumor suppressor gene. Consequently, clear cell RCC (CCRCC) is responsive to drugs that directly or indirectly inhibit angiogenesis.VHL gene, and are broadly classified as non-clear cell RCC (NCCRCC) or variant histology RCC. NCCRCC show less responsiveness to antiangiogenics and have no effective treatment options.Renal cell carcinoma (RCC) accounts for >400,000 new cancer cases and 175,000 deaths per year worldwide.,We and others identified SRC, an intracytoplasmic tyrosine kinase, as a novel therapeutic target in RCC.VHL intact (wild type [WT]) and null, resulting in single-agent and dasatinib combination responses. These studies revealed cabozantinib as a promising drug combination with dasatinib. Cabozantinib inhibits several tyrosine kinases that are biologically relevant in RCC, including VEGFRs, MET, and AXL,,in vivo testing in representative VHL intact RCC models. Strikingly, while single-agent dasatinib and cabozantinib recapitulated the clinical responses to restrain tumor growth, the combination caused marked tumor regression. Comprehensive integration of transcriptome and phosphoproteomic analysis of the combination therapy revealed rewiring of the kinome, with inhibition of mitogen-activated protein kinase (MAPK) signaling required for cytotoxic synergy. Our studies have identified promising drug combinations that transcend lineage and genetic landscape to induce cytotoxicity, suggesting broad utility across different kidney cancer subgroups.Here, we took a systematic approach toward identifying co-targeting strategies for dasatinib by performing a combination drug screen using a chemogenomic library of mechanistically annotated, clinically relevant approved and investigational drugs that inhibit pathways involved in growth, metabolism, and apoptosis in human RCC cell lines that were VHL WT: ACHN, SN12C, TK10, UO31, CAKI-1, and VHL Null: 786-0, 769-P, A498; 50 (the drug concentration necessary to inhibit growth by 50% compared to the untreated condition), the minimum viability, and the AUC (area under the dose-response curve) between drug alone and drug+dasatinib. Subsequently, we determined the leads for secondary screening with the following rationale: a drug should have an effect in multiple cell lines, but an effect across all cell lines is not necessary. Therefore, for a specific measurement such as GI50 or AUC, we considered drugs that were in the top 50% of the measurement for all drugs in more than half of the cell lines  and drugs that were in the top 25% of the measurement for all drugs in >1 cell line . We applied these criteria to 3 calculations  and noted the drugs that passed the criteria for multiple measurements , generating a further 6,720 dose-response signatures. The growth inhibition values from this secondary screen across different drug doses and combinations were analyzed for synergy using the Bliss independence model.25  of dasatinib and observed a leftward shift with corresponding decreases in cell viability  to the 5 human VHL WT RCCs in our panel, cabozantinib remained as one of the top combination partners for dasatinib . In agreement with the CellTiter-Glo data, combined treatment with dasatinib and cabozantinib increased apoptosis and decreased proliferation in RCC cells in the panel compared to single-agent treatment B and S4CThese experiments suggested several potential therapeutic partners for dasatinib. We selected cabozantinib for further evaluation because it has been approved for both first- and second-line treatment in CCRCC,in vivo in 2 xenograft tumor models. While dasatinib and cabozantinib exhibited antitumor effects on ACHN and CAKI-1 human RCC xenografts, the combination potently inhibited tumor growth and caused tumor regression  of ACHN human NCCRCC cells treated with dasatinib, cabozantinib, and the combination for 24 h. Based on prior work and reports, robust transcriptional signatures emerge ∼24 h after treatment with drugs that affect cell viability such as cell-cycle- and cell death-related signals. These can be captured by RNA-seq profiling, in addition to more specific treatment-related changes, and inform on mechanisms of action.To home in on the genes and phosphopeptides changes specifically induced by the combination treatment, we developed a statistical and network approach to compare across these treatments B. We ana,,Within the transcriptomic data, 324 genes were significantly better explained after the inclusion of an interaction term, indicating that these genes are preferentially activated after combination treatment (likelihood ratio test: q < 0.01). To identify molecular nodes that may mediate the response to the combination treatment, we performed a master regulator (MR) analysis to identify transcription factors or signaling molecules that serve as signal integration hubs. Specifically, we used the MARINa  and VIPER  algorithms and a gene regulatory network derived from The Cancer Genome Atlas cervical kidney renal papillary cell carcinoma (TCGA KIRP) cohort to infer MR activity from genes ranked by the effect of interaction of expressionWe used a similar analytical approach to analyze the proteomic data and identified 959 proteins that could be explained by an interaction between dasatinib and cabozantinib. Kinase activity driving the effect of drug interaction was inferred from the phosphoproteomic data using KSEA (q < 0.01), and demonstrated the concordant downregulation of cell-cycle-associated kinases CDK1/CCD2. Notably, KSEA also suggested the downregulation of multiple nodes of the MAPK pathway  with the dasatinib-cabozantinib combinationTo integrate across transcriptional and phosphoproteomic datasets, we leveraged the TieDIE algorithm with the Multinet interaction network to combine inferred transcriptional MRs, inferred kinases, and kinases into an integrated network associated with response to the combination drug treatment.To better define the effect of MAPK pathway inhibition, we examined 9 drugs with known MEK inhibitor activity  in combination with dasatinib against the 5 VHL WT RCC cells  in our screen. We generated drug dose-response curves for these MEK inhibitors alone and with dasatinib to examine the drug effect. All 9 structurally unrelated MEK inhibitors have limited single-agent activity, but they were more potent in combination with dasatinib, as seen by the decreased cell viability and leftward shift in dose-response curves F and 7G.−20). Conversely, antimetabolite chemotherapy agents were significantly de-enriched by the same analysis—for example, gemcitabine (p = 4.52 ×10−22)  resource, L1000FWD, which provides interactive visualization of 16,000 drug and small-molecule-induced gene expression signatures in 68 cell lines. ×10−22) B. These MET oncogene, chromophobe tumors exhibit alterations in mitochondrial DNA, RCCs that occur due to TFE translocation) and some that are not. For advanced NCCRCC, the only treatment options have been largely extrapolated from agents studied in CCRCC, including anti-angiogenics and mTOR inhibitors. In general, studies have demonstrated that patients with NCCRCC have a worse prognosis, with lower response rates to these therapies.11,,,,NCCRCC (or variant histology RCC) accounts for ∼25% of kidney cancer, with papillary, chromophobe, clear cell papillary, collecting duct, medullary, and sarcomatoid variants accounting for the majority of subtypes.The lack of treatment options for NCCRCC patients represents a critical unmet clinical need. Here, we sought to identify preclinical synergistic drug combinations that would transcend lineage and genetic landscape to induce cytotoxicity in NCCRCC and ultimately lead to deep and durable responses in patients. Accordingly, we present a model to predict and develop novel combination therapies through joint analysis of high-throughput datasets that interrogate multiple aspects of the kidney cancer cell . We posit that our phosphoproteomics signaling output likely reflects the direct results of treatment , whereas the transcriptional signatures likely represent the consequences that come from signaling pathway modulation . Consequently, integrating the phosphoproteome and transcriptome is a strength because we gained insight into several previously undescribed combinations in NCCRCC—for example, dasatinib with cabozantinib, dasatinib with MEK inhibitors, and cabozantinib with MEK.in vivo, confirming the robustness of our experimental screening approach. We observed that multiple NCCRCC cells appear to respond similarly to the combination, suggesting broad utility across this histologically diverse group of cancers. Mechanistically, the dasatinib-cabozantinib combination converges to suppress MAPK signaling to induce cytotoxicity. Critically, this suggests that combining dasatinib or cabozantinib with MEK inhibitors could be an alternate combination. In support of this, we observed decreased cell viability with these previously untested combinations. Taken together, our preclinical findings provide rationale for novel combination clinical trial designs.We demonstrate that the dasatinib-cabozantinib combination has potent synergy in 2-dimensional (2D) cell culture models. Importantly, this combination induces tumor regression ,What then is the most straightforward path to the clinic? We speculate that these findings have direct clinical implications for cabozantinib, providing rationale for further clinical study of cabozantinib in NCCRCC and supporting future combination treatments. Retrospective studies suggest that cabozantinib has activity in patients with NCCRCCin vitro, we and others,in vivo. This suggests that the cabozantinib may not directly induce apoptosis or may stimulate other types of programmed cell death mechanisms in vitro.The caveats here are limitations of cell culture and mouse xenograft studies and how these can be extrapolated to clinical trials. While we noted that increasing doses of cabozantinib alone suppressed proliferation in human RCC cells Although the dasatinib-cabozantinib combination was well tolerated in mice, the potential clinical toxicities in the setting of patients who have exhausted multiple lines of therapies and with comorbidities are part of the real-world study considerations. Accordingly, appropriate dose-finding studies will be needed as a first step to explore safety and feasibility, either stand-alone or incorporated into larger efficacy trials.thomasge@ohsu.edu)Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, George V. Thomas  supplemented with 10% fetal bovine serum at 37°C in a 5% COWe did screening using a library of 292 small molecules  GI50 fold change: Drugs that have at least 5-fold change in GI50 ratio between drug alone and drug+dasatinib (ratio GI50 drug/ GI50 combination) in 50% of cell lines (n = 38). 2) AUC difference: Drugs that have AUC difference of more than 0.9 (average is 0.9) between drug alone and drug+dasatinib in at least 50% of cell lines (n = 31). 3) AUC % change: Drugs that have AUC % change of more than 25% (average is 25%) between drug alone and drug+dasatinib in 66% (4 of 6) cell lines (n = 31). 4) GI50 of combination: Combinations that are in top 50%  in 50% (3 of 6) cell lines and pass through the minimum viability cutoff of 15% across cell lines that  (n = 18). 5) AUC of combination: Combinations that are in the top 50%  and in 50% (3 of 6) cell lines and pass through the minimum viability cutoff of 15% across cell lines that  (n = 13). We shortlisted the drugs based on the above criteria and picked 28 drugs that were identified by at least 4 of the 5 parameters. These 28 drugs were further subjected to the secondary screening, which involved a dose-matrix of 6X8 (6 doses of dasatinib and 8 doses of the drug). The growth inhibition values from the secondary screening were subjected to the estimation of synergy using Calcusyn. Calcusyn calculates Combination Index (CI) for drug combinations: CI < 1 is synergistic; CI = 1 additive and CI > 1 is antagonistic effects.6 cells were diluted in 50 μl of PBS and 50 μl of Matrigel (BD Biosciences) and were injected subcutaneously into the right and left flank of each mouse (8 mice per treatment arm). Tumors were monitored until they reached an average size of 50-80mm3 (approximately 2 weeks), at which point treatments were begun. dasatinib  was administered by oral gavage 5 days/week. Cabozantinib  were administered by oral gavage 3 days/week. The in vivo dosing for dasatinib and cabozantinib were chosen based on prior work from our group and others: Dasatinib has been administered as a single agent in the 25-50mg/kg range,,,in vivo, we examined our in vitro 6x8 dose matrix dasatinib-cabozantinib combination results, which demonstrated higher doses of cabozantinib were required to decrease ACHN cell viability and so we chose 25mg/kg dasatinib+30mg/kg cabozantinib in vivo dose. Conversely, the in vitro dasatinib-cabozantinib combination indicated that lower doses of cabozantinib when combined with higher doses of dasatinib elicited decreased CAKI-1 cell viability. Dasatinib and cabozantinib were dissolved in NMP/PEG. Tumors and mouse weights were measured twice weekly. At least 6-8 mice per treatment group were included in the analyses , and the investigators were not blinded to the identity of the study groups. All mice were euthanized using CO2 inhalation followed by cervical dislocation per institutional guidelines at Oregon Health and Science University.All animal experiments were approved by the Institutional Animal Care and Use Committee at OHSU (IACUC protocol: TR02_IP00000022). The studies are compliant with all relevant ethical regulations regarding animal research. All mice were housed in the OHSU animal facility approved by AAALAC in accordance with NIH guidelines. The animal facility provides care for a variety of animals and provides services of feeding and watering, cage cleaning and room sanitation, plus observation and health assessment of laboratory animals. All nude mice were housed in sterile conditions and handled in a sterile laminar airflow room by trained staff, supervised by several veterinarians. Six-week old female mice  were utilized for human renal cell carcinoma xenografts. For both ACHN and CAKI-1 cell lines 2x10,Cell viability assays were performed by plating cells/well in 96-well plates in triplicate and treating the following day with the indicated agent: dasatinib (dose range of 0–400 nM) and cabozantinib (dose range of 0–10 μM). These doses were based on prior work from our group and others.Cells were plated in 6 well dishes and treated the following day with the indicated agents. Treatments were for 24 hours, after which cells were washed with ice cold PBS and lysed with RIPA buffer (Sigma). Phosphatase inhibitor cocktail set II and protease inhibitor cocktail set III (EMD Millipore) were added at the time of lysis. Lysates were centrifuged at 15,000 g x 10 min at 4°C. Protein concentrations were calculated based on a BCA assay (Thermo Scientific) generated standard curve. Proteins were resolved using the NuPAGE Novex Mini Gel system on 4% to 12% Bis-Tris Gels (Invitrogen). For western blotting, equal amounts of cell lysates (15-20 μg of protein) were resolved with SDS-PAGE, and transferred to membranes. The membrane was probed with primary antibodies, washed, and then incubated with corresponding fluorescent secondary antibodies and washed. The fluorescent signal was captured using LI-COR  Odyssey Imaging System, and fluorescent intensity was quantified using the Odyssey software where indicated. The following antibodies were used for western blots: p-SRC (Y416), SRC, p-MET (Y1234/1235), p-p44/42 MAPK (Erk1/2) (T202/Y204), ERK and β-actin (AC15). Ki67 and cleaved caspase 3 were used for immunohistochemistry. Dasatinib, cabozantinib and cobimetinib were purchased from Selleck chemicals.Immunostaining was performed following deparaffinization and rehydration of slides. Antigen retrieval was performed in a pressure cooker using citrate buffer (pH 6.0) for 4 min. Nonspecific binding was blocked using Vector mouse IgG blocking serum 30 min at room temperature. Samples were incubated at room temperature with rabbit monoclonal antibodies cleaved caspase 3, and Ki67. Slides were developed with Vector Immpress rabbit IgG and Vector Immpress mouse IgG (Vector Laboratories) for 30 min at room temperature. Chromogenic detection was performed using Vector Immpact DAB (Vector Laboratories) for 3 min. Slides were counterstained with hematoxylin. A 3DHistech MIDI Scanner (Perkin Elmer) was used to capture whole slide digital images with a 20x objective. Images were converted to into MRXS files and computer graphic analysis was completed using inForm 1.4.0 Advanced Image Analysis Software (Perkin Elmer).H&E slides of formalin fixed, paraffin embedded tissue was used to assess morphological integrity of tumor samples. Once integrity was confirmed, immunofluorescent analysis was performed for p-SRC (Y416), p-MET (Y1234/1235). Four μ sections were cut, de-paraffinized and rehydrated. Antigen retrieval was performed using citrate for 4 min in a pressure cooker. Slides were blocked using 2.5% normal goat serum for 30 min then incubated in primary antibody for 1hr followed by secondary antibody mouse anti-rabbit Alexa 488  for 30 min. Slides were rinsed in PBS, air-dried, and coverslipped using Dako mounting media with Dapi.RNA was extracted from triplicate 24-hour treated cell lines using the RNeasy extraction kit (QIAGEN), and the quality if the extracted RNA was verified using an Agilent 2100 Bioanalyzer. Unstranded, poly-A-selected RNA-seq libraries were prepared using a TruSeq RNA Library Prep kit (Illumina). Read depth and splice junction details are in Differentially expressed genes in drug-treated samples were identified with DESeq2 (v1.18.1)qij is proportional to the predicted true concentration of transcripts in sample j, xjr are the design matrix components for sample j, and βir are the coefficients (log2 fold changes). The full modelxjdβid), the cabozantinib effect (xjcβic), and an interaction effect (xjdcβidc). The reduced modelxjdβid) and the cabozantinib effect (xjcβic).Significance of the cabozantinib-dasatinib interaction effect on gene expression was assessed using a likelihood ratio test to compare two generalized linear models. Both models feature logarithmic link functionsWe considered genes to be affected by the interaction of the drugs if the full model explained the observed effects significantly better than the reduced model .Enrichment of specific pathways and ontological terms in the gene most affected by the drug interaction was assessed using the Broad Gene Set Enrichment Analysis (GSEA) (v3.0) Java applicationβidc), using a network generated from gene expression profiles of the TCGA kidney renal papillary cell carcinoma cohort.Master Regulator analysis was used to infer transcription factors responsible for the observed drug interaction-induced changes in gene expression. The R package VIPER (v1.12)Interaction effects in the phosphoproteomic data were analyzed in R using the limma (3.36.3)βidc. A heat diffusion algorithm was then run to model diffusion of “heat” from the input nodes to nearby nodes in the network, and a subnetwork of agreement between the data types was identified by identifying nodes that receive heat from both data sources.An integrated network of inferred drug interaction effects on transcription and phosphoprotein levels was created using TieDie (v1.0)Similar and opposite drug-induced expression signatures were identified from lists of differentially expressed genes using the L1000 Fireworks Display (L1000FWD) web service. Differentially expressed genes were identified by selecting genes significantly better explained using a full model containing an interaction term, compared to a reduced model containing only terms for single agents (as described above in https://www.uniprot.org).Phosphopeptides were enriched and analyzed by mass spectrometry as previously described in detail.Quantitative phosphopeptide data were log10 transformed and missing data were imputed before applying quantile normalization as previously described.,Kinase substrate enrichment analysis (KSEA) was performed as previously described.P value less than 0.05 was considered statistically significant. Further statistical details, including values of n and definitions of what n represents, can be found in the figure legends. Additional statistical analysis specific to phosphoproteomics and RNA Seq are listed above. Immunohistochemistry: P-values were calculated using one-way ANOVA, with Bonferroni’s multiple comparison test. ∗ denotes p < 0.05, ∗∗ denotes p < 0.01, and ∗∗∗ denotes p < 0.001. The plots here were created using R version 3.6.1. Packages needed include ggplot2 and dplyr.Statistical significance between two groups was assessed by unpaired Student’s t test. Mouse tumor size was analyzed by 2-way ANOVA with time and drug as factors, using GraphPad Prism. Mouse weight during treatment was analyzed by repeated-measures 2-way ANOVA, with time and drug as factors. All cell culture experiments were per-formed at least two times. Data are presented as mean ± s.e.m., and a


Allogenic hematopoietic stem cell transplantation  represents a potent and potentially curative treatment for many hematopoietic malignancies and hematologic disorders in adults and children. The donor-derived immunity, elicited by the stem cell transplant, can prevent disease relapse but is also responsible for the induction of graft-versus-host disease (GVHD). The pathophysiology of acute GVHD is not completely understood yet. In general, acute GVHD is driven by the inflammatory and cytotoxic effect of alloreactive donor T cells. Since several experimental approaches indicate that CD4 T cells play an important role in initiation and progression of acute GVHD, the contribution of the different CD4 T helper (Th) cell subtypes in the pathomechanism and regulation of the disease is a central point of current research. Th lineages derive from naïve CD4 T cell progenitors and lineage commitment is initiated by the surrounding cytokine milieu and subsequent changes in the transcription factor (TF) profile. Each T cell subtype has its own effector characteristics, immunologic function, and lineage specific cytokine profile, leading to the association with different immune responses and diseases. Acute GVHD is thought to be mainly driven by the Th1/Th17 axis, whereas Treg cells are attributed to attenuate GVHD effects. As the differentiation of each Th subset highly depends on the specific composition of activating and repressing TFs, these present a potent target to alter the Th cell landscape towards a GVHD-ameliorating direction, e.g. by inhibiting Th1 and Th17 differentiation. The finding, that targeting of Th1 and Th17 differentiation appears more effective for GVHD-prevention than a strategy to inhibit Th1 and Th17 cytokines supports this concept. In this review, we shed light on the current advances of potent TF inhibitors to alter Th cell differentiation and consecutively attenuate GVHD. We will focus especially on preclinical studies and outcomes of TF inhibition in murine GVHD models. Finally, we will point out the possible impact of a Th cell subset-specific immune modulation in context of GVHD. Allogenic hematopoietic stem cell transplantation  represents a potent and potentially curative treatment for many hematopoietic malignancies and hematologic disorders in adults and children. Its success is based on a complete replacement of the patients’ immune system by a myeloablative conditioning regimen and reconstitution from a healthy donor stem cell graft. The donor derived immunity can prevent disease relapse but is also responsible for the main complication of allo-HSCT, the graft-versus-host disease (GVHD).Acute GVHD pathophysiology is not completely understood yet. In general, acute GVHD is driven by the inflammatory effect of donor T cells upon antigen-recognition of allo-antigens presented by host antigen-presenting cells (APCs). The subsequent alloreactive cytotoxicity of activated T cells effects the GVHD target organs  and leads to an amplification loop of inflammation there.Since several experimental approaches indicate that CD4 T cells play a key role in initiation and progression of acute GVHD, the contribution of the different CD4 T helper (Th) cell subtypes in the pathomechanism and regulation of the disease is a central point of current research. Acute GVHD is thought to be driven by a Th1/Th17/Th22 axis whereas Treg cells are attributed to attenuate GVHD effects. As the differentiation of each Th subset highly depends on the specific composition of activating and repressing transcription factors (TFs), these present a potent target to alter the Th cell landscape towards a GVHD-ameliorating direction by the inhibition of Th1 and Th17 differentiation. In this review, we discuss the current advances of potent of potent TF inhibitors in order to alter Th cell differentiation and attenuate GVHD in murine models.To date, eight different T helper cell types are known: Th1, Th2, Th9, Th10, Th17, Th22, follicular T helper cells (Tfh) and regulatory T cells (Treg). Th cell lineages derive from naïve CD4 T cell progenitors and lineage commitment is initiated by the surrounding cytokine milieu and subsequent changes in the TF profile. Each T cell subtype has its own effector characteristics, immunologic function, and lineage specific cytokine profile, leading to the association with different immune responses Figure 1In 1986, Mosmann and colleagues identified two distinct classes of CD4 helper T cells, which exhibited a different cytokine profile. The differentiation in these two classes, later called Th1 and Th2, was found to be stable and deterministic . In contIn the early 2000s, Szabo et al. discovered that the underlying mechanisms of the Th1/Th2 paradigm was the initiation or repression of distinct genetic programs upon activation, directed by Th lineage specific master transcription factors . With thFurther studies on the mechanisms, how T-bet and GATA3 mediate Th1 and Th2 differentiation respectively, revealed the mutual inhibition of the two master TFs and the involvement of many more interacting molecules and relevant signaling cascades –17. T−beTh17 cells were first described as an independent and distinct Th subset from Th1 and Th2 cells, producing IL-17a, IL-17f, IL-22 and IL-21 as effector cytokines in the early 2000s , 27. FirRorc) was identified to be uniquely expressed in mouse Th17 cells and necessary for Th17 differentiation γt  are characterized by their immunosuppressive capacity and are essential mediators of self-tolerance. Already in the 1960’s it was found that a thymus-derived cell population was mediating immunologic tolerance. Later on, Sakaguchi and colleagues characterized these cells further as CD4 T cells expressing the IL-2 receptor alpha chain (CD25) . HoweverIn contrast to other effector T helper cells, regulatory T cells differentiate in the thymus [thymus-derived Tregs (tTregs)], dependent on high affinity interaction with complexes of MHC-II and tissue-restricted self-antigens and IL-2 receptor signaling . HoweverFoxp3 locus  of the p3 locus . CNS1, rp3 locus , 59–62, p3 locus . CNS2, wp3 locus , 64–69. p3 locus , 58, 62.p3 locus , while Bp3 locus .in vitro  treatment was shown to modulate Th1/Th2 differentiation by inhibiting the production of the isoprenoid derivates farnesly-pyrophosphate (PP) and geranylgranyl-PP, of the mevalonate pathway. Inhibition of these isoprenoid derivates combined by AT or individually by a farnesyltransferase inhibitor (FTI) or a geranylgeranyltransferase inhibitor (GGTI) respectively, resulted in an upregulation of GATA3, and in case of AT and FTI treatment also an downregulation of T-bet expression in antigen-primed T cells . GGTI anathology . STAT5, athology .in vitro induced Tregs (iTregs) as a GVHD therapeutic revealed effective protection in the early phase after transplantation but unstable FoxP3 expression over time led to aggravation of GVHD, making this approach less promising as initially thought  like Cyclosporine A (CyA) of tacrolimus (FK506) for example block TCR-proximal signaling by inhibition of NFAT. Even though CNIs remain standard of care for GVHD prevention, they also interfere with the valuable GVL-effect by impairing donor immunity and disruption of Treg function and survival –165. ComThe following section will provide more detailed information on various new therapeutic agents, divided by substance classes, which have been successfully evaluated in GVHD alone or in combination with standard of care therapeutics in the recent years 2. ImporEpigenetic modulation of transcription is a promising approach to indirectly inhibit TF expression. The acetylation of histones, regulated by histone acetyl transferases (HATs) and histone deacetylases (HDACs), is an epigenetic mark, which influences chromatin structure and ultimately gene expression. The use of HDACs and HDAC-inhibitors (HDACi) can modulate this balance and subsequently alter gene expression.Valproic acid (VPA), a HDACi of the short-chain fatty acid category, was shown to indirectly decrease STAT5 phosphorylation and dampen T-bet expression in NK cells . In a movia the CNS1 regulatory element  inhibitor. Sirt-1 represses AP-1, Smad3 and FOXO-transcription factors which regulate pTreg differentiation  element –209 and  element . Pre-cli effects . Stabili effects , 211. Anon model . Howevervia decreased STAT3 phosphorylation and RORγt expression downstream of IL-6R signaling. Administration of Givinostat inhibited experimental colitis development by skewing the Th17/Treg balance in the lamina propria , a HDACi of the hydroxamic acid category, was also reported to suppress Th17 polarization and enhance FoxP3 expression and hence Treg differentiation  propria  and redu propria . Virinos propria . Additio propria . Hence, ntiation .via induction of suppressor of cytokine signaling 1 (SOCS1) (Romidepsin (Istodax), a cyclic peptide class HDACi, was shown to effectively suppress allo-responses in a mixed lymphocyte reaction (MLR) . Recentl (SOCS1) . Howevervia TF modulation. The success of HDACi in preventing GVHD is also displayed by multiple clinical studies validating their beneficial effect often in combinatory therapy in patients  but also the predisposition for newly acquired infections due to major immune suppression of especially Th1 T cells but also other immune cell populations required for viral clearance. First clinical trials with the HDACi Vorinostat and Panobinostat in GVHD patients did not show an augmentation for risk of infections while Romidepsin treated patients with T cell lymphoma more often experiences infections –232. StuTogether, given the promising results of some TF-modulators in clinical studies, we expect a fundamental contribution of TF-inhibitors to improve GVHD therapy in the future.JC and EU wrote the manuscript and approved the submitted version.The laboratory of EU has been supported by the Frankfurt Cancer Institute FCI/DKTK (to EU), by the German Research Foundation DFG , by the German Cancer Aid, the “Alfred & Angelika Gutermuth-Stiftung” and by “Menschen für Kinder e.V.”The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.