We analyzed 4467 breast cancers and identified GABRP as top expressed gene in TNBC with low expression in most normal tissues. GABRP protein was localized to cell membrane with broad range of receptors/cell  and expressed by nearly half of breast cancers tissues. GABRP gene knockdown inhibited TNBC cell growth and colony formation in vitro and growth of MDA-MB-468 xenografts in nude mice. Commercially available anti-GABRP antibody (5–100 μg/ml) or de novo generated Fabs (20 μg/ml) inhibited TNBC cell growth in vitro. The same antibody conjugated to mertansine (DM1) also showed significant anticancer activity at nanomolar concentrations. Our results indicate that GABRP is a potential novel therapeutic target for ADC development.The goal of this study was to identify a novel target for antibody-drug conjugate (ADC) development in triple negative breast cancer (TNBC), which has limited treatment options, using gene expression datasets and  Antibody drug conjugates (ADCs) allow targeted delivery of highly toxic agents to cancer cells that express a specific antigen without extensive adverse effects on normal tissues4. These drugs are emerging as a potential new strategy to treat TNBC. Preliminary results from clinical trials with IMMU-132 , CDX-011 , and SGN-LIV1A (that targets a zinc transporter SLC39A6) show around 30% objective tumor response rates in patients with metastatic TNBC that progressed on multiple prior lines of chemotherapies . Our goal was to identify new targets for ADC development in TNBC. We compared mRNA expression profiles of TNBC with other breast cancer subtypes to identify genes that are overexpressed on TNBC surface, and identified gamma-amino-butyric-acid receptor π subunit (GABRP) as the most promising candidate gene for further functional experiments.Triple negative breast cancers (TNBC) lack amplification of HER2 and expression of estrogen and progesterone receptors, and represent 15% of all breast cancers. Currently the only approved treatment options for newly diagnosed TNBC patients are chemotherapy agents7. GABAA receptors in the central nervous system are most frequently made up of two α, two β and γ subunits7 and function as inhibitors of synaptic transmission. The function of the π subunit is not clear; it is not detectable in neuronal tissues and it is predominantly expressed at low levels in reproductive organs such as the uterus, ovary, prostate and breast9. GABRP levels change during the reproductive cycle and gestation and are upregulated during the window of implantation in the human endometrium, suggesting a role in these processes12. We have previously identified GABRP as a highly expressed gene in TNBC13. In this paper, we investigate the function of GABRP in TNBC growth in vitro and in vivo and assess its potential as a candidate for ADC development.The GABRP gene encodes the π subunit of the gamma-aminobutyric acid (GABA) A receptor. The GABAA receptor is a heteropentameric, ligand-gated chloride channel composed of multiple subunits including α1–6, β1–3, γ1–3, δ, ε, θ, π and ρ1–3. Each subunit consists of a conserved extracellular domain, 4 transmembrane domains (TM1–4) and intracellular loops). GABRP was also overexpressed in the basal subtype of TCGA breast cancer cohort  < 0.0001 observed in two independent datasets. GABRP displayed the highest fold-change (8.18×) in the MDACC cohort Fig.  and in t1Cort Fig. . All expTM) and gpNMB   and non-TNBC (SKBR3 and BT-474) cells express comparable GABRP protein levels Fig. . This ob21. However, clinical response to ADC is determined by multiple factors including receptor turnover rate, inflammatory and immune response, and by-stander effects of the toxins released, in addition to surface receptor density.We quantified GABRP density on cell surface using QuantiBRITE PE flowcytometry assay Fig. . ReceptoWe next examined GABRP protein with IF Fig. , and chrin vitro results, we observed significantly reduced tumor growth and tumor formation in the knockdown cells compared to shRNA control or the parent MDA-MB-468 cells in female athymic Nude-Foxn1 mice  were 3.80 and 4.10 respectively.We commissioned ADCs from Levena Biopharma and requested conjugation of DM1 using a stable linker to (i) commercially available ECD bindingpolyclonal GABRP-antibody (GABRP-Ab) and (ii) polyclonal isotype control IgG. Excess DM1 was removed after conjugation and free DM1 was limited to <5% in each ADC batch by centrifugal ultrafiltration using 50 kDa molecular weight cut-off (MWCO) Amicon columns (7x rounds) and size exclusion chromatography (SEC). Unconjugated antibody was 5% for each ADC, as determined by hydrophobic interaction chromatography (HIC)-HPLC. Drug to antibody ratios (DAR) calculated using ultraviolet-visible spectrophotometry 22. We therefore, tested the naked (unconjugated) commercially available ECD-binding GABRP-Ab that suppressed the growth of breast cancer cell lines, particularly MDA-MB-468, in a dose-dependent manner starting at 20 μg/ml. The ICD-binding GABRP-Ab had no effect in concentrations up to 100 μg/ml  in the same cell lines. These effects are specific as CRISPR KO cell lines are more refractory to anti-GABRP Fab induced growth inhibition . These effects of anti-GABRP-DM1 were significant but modest indicating that the antibody and the conjugation technique will require further optimization23.The anti-GABRP antibody-DM1 conjugate (GABRP Ab-DM1) showed significantly higher growth inhibitory effect at nanomolar concentrations (10–500) than control (IgG)-DM1 Fig. . InteresFig. . These eOur results suggest that GABRP is a potential novel therapeutic target in TNBC. Its membrane localization, high expression in breast cancers, low expression in most normal tissues and important role to sustain cell growth makes it a candidate for ADC development. We also provide proof-of-concept experiments demonstrating anticancer activity of a commercial naked anti-GABRP antibody, anti-GABRP Fab, and a DM1 conjugated ADC version of the same commercial antibody.19; 1000–10,000 for CD33, the target of gemtuzumab ozogamicin21; 1000–9000 for CD54, the target of the therapeutic antibody BI-505, on the surface of myeloma cells19.The distribution of GABRP mRNA expression across many cancer types suggests that an anti-GABRP-ADC may have therapeutic activity in subsets of several currently difficult-to-treat cancers including gastric, colorectal and lung. The generally low expression in normal tissues is reassuring, but we noted relatively high mRNA expression in the upper airways and lungs, although expression levels were still lower than seen in breast cancer  and synthetically generated Fabs induced greater growth inhibition than the commercially available polyclonal antibodies.Antitumor activity of naked antibody is not a prerequisite for successful ADC development, as naked antibodies against targets such as CD30, CD138, SLC39A6, gpNMB alone do not show significant antitumor activity, despite substantial clinical activity of the conjugated antibodies. However, several successful ADCs’ targets are important for cell survival and therefore the naked antibody also has clinical activity (e.g. trastuzumab). We demonstrated in siRNA and CRISPR knockout experiments that GABRP expression is important for TNBC cell growth, both in vitro. We observed enhanced inhibitory activity compared to the isotype-matched control in GABRP expressing cells and not in GABRP KO isogenic background. The relatively low activity of our ADCs may be due to less than optimal internalization of the IgG and non-uniformity of DM1 conjugation. Using our method of conjugation, DM1 can bind to any of ~30 available IgG lysine sites and therefore the resulting ADCs are a mixture composed of variable number of toxic cargo on each antibody. Higher than 3/4 DM1 molecules conjugated to an antibody can reduce the antigen-binding potency, solubility and stability of an ADC26. The observed cytotoxicity of the isotype control-DM1 conjugates at higher concentrations is likely due to non-specific antibody internalization or free DM1 in the medium. These results support the target function of GABRP but also highlight the need for further antibody and conjugation optimization23.To further credential GABRP as a potential therapeutic target, we used a commercially available anti-GABRP ECD-antibody for ADC generation and tested its effect on cell growth Our systematic evaluation for target identification and functional validation followed by generation of specific GABRP Fabs and ADCs helped building a robust preclinical package for drug development. These steps provide a framework that can be extended to the discovery of other novel ADC targets, and in other cancer types.28 were used as the discovery cohort to define candidate TNBC-genes. A previously described gene expression dataset (Affymetrix U133A or U133Plus2.0 arrays) of 4467 breast cancer patients compiled from 40 publicly available data sets30 was used for validation. Additional details are in the Supplementary Methods.Affymetrix U133A gene expression profiles from 294 fine-needle aspiration (FNA) biopsies of stage I-III breast cancers at MD Anderson Cancer Center (MDACC)TNBC  and non-TNBC HER2-amplified (SKBR-3 and BT-474) cell lines were purchased from the American Type Culture Collection  where all cell lines were authenticated by short tandem repeat profiling, karyotyping, morphology and cytochrome C oxidase I testing. Cell lines were obtained between 2011 and 2016, and used at passages 3–9, and cultured less than 3 months after resuscitation. Additional details are in the Supplementary Methods.6cells/100 mm plate and grown to subconfluency, followed by extraction by RIPA buffer containing proteinase and phosphatase inhibitors for whole cell lysates. Membrane and cytoplasmic protein fractions were extracted using Subcellular Protein Fractionation Kit (Thermo Scientific). Lysate samples were electrophoresed in 4–12% NuPAGE SDS-polyacrylamide midigels (Life Technologies Corporation) and transblotted onto PVDF membrane as described previously31. For details, see Supplementary Methods.Cells were plated at 1 × 10Number of GABRP receptors on cell surface was quantified by BD QuantiBRITE PE (BD BioSciences) flow-cytometric analysis. GABRP ECD-binding specific monoclonal antibody-fragments (Fabs) were used in GABRP detection on cell surface in Fig. Cells grown to subconfluency in 24-well culture plates were fixed with 4% formaldehyde, permeabilized with 0.5% triton-X, blocked by 1% BSA and incubated overnight with primary antibody against GABRP (ICD binding), followed by staining with rabbit secondary antibody-tagged with AlexaFlor488 fluorochrome (green). Cell nuclei were counter-stained with Hoechst (blue) dye. Fluorescence was visualized under confocal microscope and 20X images were captured using imaging software (Perkin Elmer).GABRP IHC was performed on 5-μm whole tissue-sections of formalin-fixed paraffin embedded (FFPE) breast cancer tissue microarray  This TMA containing 100 invasive breast cancer tissues with annotated clinical stage, pathology grade, IHC markers  status, was stained using GABRP antibody (Santa Cruz Biotech). For details, see Supplementary Methods.Supplementary Methods. Knockdown of GABRP was also achieved by ~60% efficiency of GABRP CRISPR knockout (KO) in cell lines (polyclones); GABRP knockout (KO-1 and KO-2) MDA-MB-468 and HCC1143 cell lines were generated according to protocol described previously32. Surveyor T7 endonuclease assays were conducted as described previously33. Primer sequences and protocol details are in Supplementary Methods.Human GABRP siRNA oligonucleotides were purchased from Ambion . For stable knockdown, we produced GABRP shRNA encoding pRETRO-SUPER-GABRP plasmid. For details, see Soft agar plates were photographed and colonies were enumerated using ImageJ software version 1.46r.Female athymic Nude-Foxn1 nude mice (Nu/Nu) (Harlan) were housed under specific pathogen-free conditions, at The University of Texas MDACC approved by the American Association for Accreditation of Laboratory Animal Care in accordance with the current regulations and standards of the U.S. Department of Agriculture, Department of Health and Human Services, and National Institutes of Health. Experimental details are in the Supplementary Methods.Cell lines were plated at 2000 cells/well (3 wells/group) in clear bottom opaque-walled 96-well culture plates (Thermo Scientific) in media containing 5% FBS, and exposed to respective experimental treatments and counted in a hemocytometer after staining by 0.4% trypan blue or luminescence measured using the ATP-based CellTiter-Glo® luminescent cell-viability assay (Promega).To generate a novel ADC against GABRP, commercially available ECD binding GABRP antibody (Abcam) was conjugated to mertansine (DM1), a potent thiol-containing maytansinoid toxin, by ‘Immunogen Conjugation’ method to achieve drug to antibody ratio (DAR) of 3–4 . Further details are in the Supplementary Methods.Supplementary InformationSupplementary Table 1Supplementary Table 3


Targeted cancer therapy is based on exploiting selective dependencies of tumor cells. By leveraging recent functional screening data of cancer cell lines we identify Werner syndrome helicase (WRN) as a novel specific vulnerability of microsatellite instability-high (MSI-H) cancer cells. MSI, caused by defective mismatch repair (MMR), occurs frequently in colorectal, endometrial and gastric cancers. We demonstrate that WRN inactivation selectively impairs the viability of MSI-H but not microsatellite stable (MSS) colorectal and endometrial cancer cell lines. In MSI-H cells, WRN loss results in severe genome integrity defects. ATP-binding deficient variants of WRN fail to rescue the viability phenotype of WRN-depleted MSI-H cancer cells. Reconstitution and depletion studies indicate that WRN dependence is not attributable to acute loss of MMR gene function but might arise during sustained MMR-deficiency. Our study suggests that pharmacological inhibition of WRN helicase function represents an opportunity to develop a novel targeted therapy for MSI-H cancers. BRCA1/2 mutations or impaired DNA mismatch repair (MMR), are a common characteristic of tumor cells, accelerating the accumulation of DNA mutations or chromosomal aberrations that are required for neoplastic growth and transformation  is a member of the RecQ DNA helicase subfamily . RecQ heIn contrast to the previously described tumor-suppressive role of WRN, we demonstrate in this study that WRN possesses a context-dependent critical pro-survival function for cancer cells. By leveraging a recently defined map of cancer cell specific vulnerabilities  and a cohttps://oncologynibr.shinyapps.io/drive/; redundant siRNA activity) sensitivity scores ≤−3, indicative of cell essentiality  .In order to explore this hypothesis we developed a Random Forest model using an MSI feature list defined by Boland and Goel . This mo−8, Fisher’s exact test). Of the 18 cell lines classified as MSI-H, 15 cell lines (83%) were sensitive to WRN depletion using an RSA value of ≤−3 to define WRN dependency -mediated knock-down of WRN in a panel of three MSS  and three MSI-H  CRC cell lines. In agreement with the results from Project DRIVE, WRN depletion using a mixture of four siRNA duplexes (Pool) or an individual siRNA (#1) targeting WRN profoundly affected viability in MSI-H, but not in MSS CRC models . In contTP53-wild-type MSI-H CRC line HCT 116 would reverse the sensitivity to WRN knock-down. However, WRN/p53 co-depletion exacerbated the reduction in viability compared to WRN knock-down alone  did not display sensitivity to knock-down of WRN . It is nwn alone . TP53-wiepletion .To determine whether reconstitution of MLH1 and MSH3 in the MSI-H CRC line HCT 116 would revert WRN dependence, we utilized variants of HCT 116 harboring copies of human chromosome 2 , chromosome 3  or chromosome 3 and 5  derived from normal fibroblasts . In bothWe carried out CRISPR-Cas9 depletion assays in MSS and MSI-H CRC models to independently confirm the selective WRN dependencies observed in shRNA/siRNA studies. Cell lines were stably transduced with Cas9 followed by transduction of lentiviral particles co-expressing GFP and single guide RNAs (sgRNAs) targeting WRN or the essential replication factor RPA3 . To inveTo further dissect the relevance of WRN exonuclease and helicase function in WRN-dependent cell models, we generated FLAG-tagged, siRNA-resistant WRN (WRNr) expression constructs harboring loss-of-function mutations within the exonuclease-  and helicase-domain , or both domains   Figure . Wild-tyIn order to investigate the cellular basis for the viability reduction of MSI-H cancer cells upon WRN depletion, we monitored the consequences of WRN loss-of-function using immunofluorescence analysis of the nuclear membrane protein LAP2β and Hoechst DNA staining in MSS and MSI-H CRC cell lines. SW480 did not display any phenotypic differences upon transfection with NTC and WRN-targeting siRNAs . StrikinThe observed nuclear integrity and mitotic defects caused by WRN depletion in MSI-H cells could be the consequence of preceding genome maintenance aberrations. This hypothesis is reinforced by the important role of RECQ family helicases, including WRN, in genome integrity . To inteTo test whether signs of DNA damage can already be detected in WRN-depleted interphase cells, we analyzed the intensity of nuclear γ-H2AX signals. We observed a strong induction of γ-H2AX upon siRNA-mediated depletion of WRN in MSI-H, but not MSS, CRC cancer cell lines . StrikinOur analyses suggest that WRN helicase is essential for maintaining genome integrity in MSI-H cells by preventing chromosome breaks and erroneous chromosome fusions. The observed mitotic chromosome aberrations in WRN-depleted MSI-H cells can also explain the aforementioned nuclear morphology and mitotic defects, including micronuclei, lagging chromosomes and chromatin bridges. The detection of DNA damage markers in interphase nuclei of WRN-depleted MSI-H cells suggests that the aforementioned mitotic and post-mitotic aberrations originate from genome integrity defects in interphase. The correlation of chromosome aberrations and nuclear abnormalities with MSI-H status following loss of WRN function indicate that genome integrity defects are responsible for the profound reduction in viability of MSI-H cancer cells.Treatment paradigms for MSI-H tumors have recently shifted with the approval of the immune checkpoint agents pembrolizumab, nivolumab and ipilimumab, targeting programmed cell death 1 (PD-1) and cytotoxic T-lymphocyte-associated Protein 4 (CTLA-4) in this patient segment . PembrolThe highest prevalence of MMR-deficiency/MSI-H status is observed among CRC, gastric and endometrial cancers . Recent Our data suggest that similar to the tumor agnostic activity of checkpoint blockade, MMR deficiency and MSI-H status represent a genetic determinant for WRN dependency regardless of tumor type. As indicated by the MLH1/MSH3 reconstitution and depletion studies, the genetic interaction of WRN and MMR genes cannot readily be recapitulated using isogenic cell models and thus seems to be distinct from acute and hard-wired synthetic lethal interactions, such as described in cancer cells for the alternate BAF complex ATPases SMARCA2/SMARCA4 or the cohesin subunits STAG1/STAG2 . In the Upon WRN loss-of-function we observe a strong and rapid decrease in the viability of MSI-H cell models that is accompanied by nuclear abnormalities and cell division defects. In particular, we find that WRN-depleted MSI-H cancer cells display chromosome breaks, chromatin bridges and micronuclei indicative of genome instability that is highlighted during cell division. The strong induction of γ-H2AX in WRN-depleted MSI-H interphase cells suggest that pervasive DNA damage, e. g. DNA double strand breaks, accumulate already in interphase prior to entry into mitosis. The occurrence of these defects and phenomena in MSI-H but not MSS cells upon WRN inactivation suggests that these aberrations might be causally linked to the selective reduction in viability in MSI-H cells. While rescue studies using WRN variants clearly indicate WRN helicase function as the critical enzymatic activity in MSI-H cell models, CRISPR domain scanning indicates a potential structural requirement of the exonuclease domain and the HTH loop of WRN.WRN is a member of the RecQ helicase family which fulfils pleiotropic functions in DNA repair . MMR actThe chromosome breaks and radial chromosomes observed in MSI-H cells upon WRN depletion indicate the generation and/or persistence of DNA double strand breaks. Future research is required to dissect the molecular basis for this effect. It is conceivable that WRN is required to process and resolve DNA repair or replication intermediates that arise in MMR-deficient cells or that MMR is required to cope with intermediates emerging upon compromised WRN function. The identification of the molecular basis of the WRN-MSI-H relationship will also help to understand why some rare outlier MSI-H cell lines see  do not rWrn-null mice display no overt phenotype and do not recapitulate the premature ageing or cancer predisposition conditions of Werner syndrome, unless crossed into a Terc-null background  involved in MMR  and ii) genetic target genes of MSI in CRC . We nextGenomic DNA was isolated using QIAampDNA mini kit . Per reaction 2 ng of genomic DNA was used for fluorescent PCR-based analysis of the mononucleotide microsatellite marker length using the Promega MSI Analysis System, Version 1.2 kit. Microsatellite fragment length of a standard panel of markers  according to the ‘Revised Bethesda Guidelines for Hereditary Nonpolyposis Colorectal Cancer (Lynch Syndrome) and Microsatellite Instability’  was analHCT 116 cells were cultured in McCoy’s 5A medium  with glutamax supplemented with 10% fetal calf serum (FCS), hTERT RPE-1 cells were cultured in DMEM:F12  supplemented with 10% FCS and 0.01 mg/ml Hygromycin B. RKO, SW480, CaCo-2 and SK-CO-1 cells were cultured in EMEM  with glutamax supplemented with 10% FCS and Na-Pyruvate. SNU-C4 cells were cultured in RPMI1640 medium  with glutamax, supplemented with 10% FCS, 25 mM HEPES and 25 mM NaHCO3. LS1034 cells were cultured in RPMI-1640  supplemented with 10% FCS. MFE-280 cells were cultured in 40% RPMI 1640 , 40% DMEM  supplemented with 20% FCS and 1X insulin-transferrin-sodium selenite . HEC-265 cells were cultured in EMEM  with glutamax supplemented with 15% FCS. ISHIKAWA cells were cultured in EMEM  with glutamax medium supplemented with 5% FCS and NEAA. HEC-6 cells were cultured in EMEM  with glutamax supplemented with 15% FCS, NEAA and Na-Pyruvate. HT-29_CRISPR-Cas9 cells were cultured in McCoy’s 5A  with glutamax supplemented with 10% FCS and 10 µg/ml Blasticidin . HCT 116_CRISPR-Cas9 cells were cultured like the parental cell line but supplemented with 2 µg/ml Puromycin. All supplements were obtained from GIBCO, FCS (SH30071.03) from GE Healthcare Life Sciences and Puromycin from SIGMA (P9620). Lentiviral particles were produced using the Lenti-X Single Shot system . Following lentiviral infection, stably transduced pools were generated using Puromycin selection  or Blasticidin (HT-29: 10 µg/ml). Sources, MSI status and authentication information  of cell lines used in this study are provided in For knock-down experiments, cells were transfected with ON-TARGETplus SMARTpool siRNA duplexes  targeting WRN (L-010378–00), MLH1 (L-003906–00) or MSH3 (L-019665–00) using Lipofectamine RNAiMAX reagent according to the manufacturer’s instructions . For WRN knock-down, additionally an individual siRNA was used (J-010378–05). Chromosome spreads, immunoblotting, immunofluorescence and live cell imaging experiments were performed using a final siRNA concentration of 20 nM. Cell viability assays were performed using 10 nM siRNA in 96-well plates in a total volume of 100 µl per well. Viability was determined using CellTiter-Glo . Seven days post-transfection, 100 µl CellTiter-Glo solution was added directly to the cell medium, mixed and incubated for 10 min prior to determination of the luminescence signal. Crystal violet staining was performed in 24-well format using a total volume of 1000 µl per well. Seven days post-transfection, cells were fixed with 4% formaldehyde in PBS and stained twice for 30 min with crystal violet . For co-depletion of p53 and WRN 10 nM of the respective siRNA duplexes each were used for immunoblot and viability assay.Cell pellets were resuspended in extraction buffer .The following antibodies were used: WRN (8H3) mouse mAb , mouse anti-GAPDH , mouse anti-FLAG , mouse anti-LAP2ß , mouse anti-p53 , rabbit anti-Actin  dilution), mouse anti-MLH1 , rabbit anti phospho-Histone H2A.X (Ser139)  a rabbit anti-MSH3  and secondary rabbit , mouse anti-IgG-HRP  and mouse Alexa Fluor 488 .RNA was isolated 472 hr post-transfection and reversely transcribed using SuperScript VILO kit (Thermo Scientific). All qPCR analyses were performed with the QuantiTect Multiplex PCR kit  on a StepOne Real-Time PCR Sytem (Applied Biosytems) with a total of 45 cycles. Constitutive maintenance gene 18S rRNA  and human WRN , human MLH1  and human MSH3  TaqMan probes were used. WRN expression was normalized to 18S rRNA expression levels and is indicated relative to the NTC control.To introduce mutations in WRN in SW480, the following sgRNA sequences were cloned into pSpCas9 BB-2A-GFP pX458 : GGCCACCATTATACAATAGA (EN-domain_01), GCAGTTAAAAAGGCAGGTGT (EN-domain_02) and GTCTTGCCGATCAATATCGC (RQ-domain_01). Cells were transfected with X-tremeGENE 9 DNA Transfection Reagent , sorted after 48 hr for GFP positive cells and diluted to obtain single cell clones. WRN knock-out was monitored by immunoblotting.Stable Cas9 expressing cell lines, using either Blasticidin or Puromycin as selection markers, were transduced with vectors encoding GFP and sgRNAs targeting different domains of WRN . On day pLVX-WRN-3xFLAG-IRES-Puro, pLVX-WRN-3xFLAG-IRES-Puro K577M, pLVX-WRN-3xFLAG-IRES-Puro E84A and pLVX-WRN-3xFLAG-IRES-Puro E84A _K577M for siRNA-resistant transgene expression were generated by gene synthesis  based on the WRN cDNA sequence NCBI NM_000553.5 followed by cloning into the parental pLVX vector . Codon optimization changes were introduced into WRN coding sequences to render the transgenes siRNA-resistant.For immunofluorescence, 72 hr post siRNA transfection cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.2% Triton X-100 in PBS for 10 min and blocked with 3% BSA in PBS containing 0.01% Triton X-100. Cells were incubated with primary LAP2ß or phospho-Histone H2A.X (Ser139) and secondary antibody , DNA was counterstained with Hoechst 33342 . Coverslips and chambers were mounted with ProLong Gold . Images were taken with an Axio Plan2/AxioCam microscope and processed with MrC5/Axiovision software . For quantification of γ-H2AX immunofluorescence, nuclei were identified based on Hoechst staining using segmentation in ImageJ 1.52a and corresponding γ-H2AX mean intensities of nuclei were determined.2 atmosphere.For chromosome spread analysis, Nocodazole  and 2 mM caffeine was added to the medium for 6 hr. Cells were harvested and hypotonically swollen in 40% medium/60% tap water for 5 min at room temperature. Cells were fixed with freshly made Carnoy’s solution , and the fixative was changed three times. For spreading, cells in Carnoy’s solution were dropped onto glass slides and dried. Slides were stained with 5% Giemsa (Merck) for 4 min, washed briefly in tap water and air dried. For chromosome spread analysis two independent slides were scored blindly for each condition. Live-cell imaging was performed using Spinning Disk Confocal UltraView Vox Axio Observer equipped with Plan apochromat 20x/0.8 objective (Zeiss) and an electron-multiplying charge-coupled device 9100–13 camera (Hamamatsu Photonics). The microscope was controlled using Volocity software (Perkin-Elmer). DNA was counterstained with 100 nM SiR-Hoechst 3 hr before the start of imaging. At 24 hr post siRNA transfection cell nuclei were imaged in 2 z slice sections spaced 6 μm every 6 min for 48 hr. For the imaging, cells were seeded into glass bottom 24 well SensoPlate (Greiner) with imaging medium . During live cell imaging, cells were maintained at 37°C in a humidified 5% CO In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, including Wolf-Dietrich Heyer as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Jeffrey Settleman as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Ray Monnat (Reviewer #2); Petr Cejka (Reviewer #3).Thank you for submitting your article "Werner syndrome helicase is a selective vulnerability of microsatellite instability-high tumor cells" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:This intriguing new report identifies the WRN RecQ helicase as a new and potentially useful vulnerability in microsatellite-unstable ('MSI-high or MSI-H') cancer cell lines. The results are worth reporting after some additional work on the manuscript.The authors identified a dependency of MMR-deficient cells on the RecQ-like helicase WRN, which constitutes a significant novel finding with far-reaching implications for improving treatment of MMR-deficient tumors. The selective dependency was identified in a large screen of 398 cancer cell models covering ~8,000 genes as published in 2017 and the correlation of MMR status and WRN dependency is highly significant . The observation is validated in multiple cell line models for MMR+ and MMR- colorectal cancer and endometrial cancer cell lines  and with WRN KO in 2 independent MMR+ and MMR- colorectal cancer cell lines  using viability and colony forming assays. Back-complementation activity depended on the WRN ATPase/helicase activity but not the nuclease activity in HCT 116 cells . The results with another MMR- cell line, RKO, are less clear , suggesting the involvement of additional variables. Figure 5, Figure 6 and Figure 7 demonstrate that WRN knockdown in MMR-deficient cells leads to hallmarks of genomic instability including bridges and micronuclei , which is confirmed by life imaging  and endpoint analysis of mitotic spreads . The claims of the manuscript are largely well supported by the data with one exception . Overall, the authors consistently use multiple cell lines and approaches to corroborate the dependence of MMR-deficient cells on WRN. The manuscript is very well written, and the results are displayed very clearly in the Figures and Tables.Essential revisions:1) Figure 4—figure supplement 1 and subsection “WRN dependency in MSI-H CRC is linked to its helicase function”: There is a significant difference between the back-complementation results in HCT 116  and RKO  and the description in the text does not reflect the results of Figure 4—figure supplement 1. Figure 4 shows that the back-complementation with wildtype and nuclease-deficient WRN is very effective, whereas the ATPase/helicase-deficient mutant as well as the ATPase/helicase nuclease double mutant cannot complement the WRN siRNA knockdown cells. For RKO , the difference between nuclease and helicase mutants appears statistically insignificant as the error bars overlap. The text should be changed to more accurately reflect these results.2) The authors meticulously show the dependence of the effect on WRN status, but the dependence on MMR status is only correlative. A key concern is that all experiments were carried upon depletion of WRN in MMR-deficient cell lines. These cell lines have a high mutator phenotype and accumulate mutations in a number of other genes, in particular in those containing microsatellites. Therefore, to confirm the key conclusion of this manuscript that the lethality is a result of simultaneous MMR and WRN deficiency, the experiment should also done the other way round, i.e. to deplete key MMR factors (MLH1 would be the best) in WRN-deficient cells, or to perform co-depletion experiments. Additionally, WRN depletion should also be carried out in HCT 116 +ch3 cell line, in which the expression of MLH1 was corrected.HCT 116 and RKO are both MLH1 deficient. What is known about additional mutations in these cell lines that may contribute to this difference? This above approach might also address the difference between both cell lines. For RKO a complementation system was described in Yan et al., 2003.3) WRN inhibitors need to be referenced and discussed more fully. In addition to the Rosenthal et al., 2010 reference, at least two WRN inhibitors have been around for 5+ years. They are highly and directly relevant to the topic of this manuscript, and are being pursued in other clinical contexts  that may provide additional insight. These inhibitors are readily available, and the authors' results make a straight-ahead prediction of what a dose-response outcome should be in their CRC cell line panels to provide a bit of pre-clinical data. The authors should acquire the WRN inhibitors and do dose-response curves on their MSI-H and MSS cell lines, and show that their WRN KO lines are insensitive (or largely insensitive) as a demonstration of target specificity.eLife. Your revised article has been favorably evaluated by Jeffrey Settleman (Senior Editor), a Reviewing Editor, and two reviewers.Thank you for resubmitting your work entitled "Werner syndrome helicase is a selective vulnerability of microsatellite instability-high tumor cells" for further consideration at The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:The Abstract should be updated to reflect the significant insight that the MHL1 WRN synthetic lethality is context-dependent and not absolute.Reviewer #1:In this revision the authors meticulously addressed the comments made in the initial review. They conducted significant new experimentation that strengthens the conclusion about the role of the nuclease activity of WRN .The authors devoted significant efforts to address essential revision 2, conducting 3 lines of experimentation that clearly demonstrate that the MLH1 WRN synthetic lethality is not hard wired but is context dependent. This significantly affects the overall conclusion of the study, and it is very important that these new data were included in the revision and discussed. What I am missing is an update of the abstract to reflect this important point.The authors also nicely addressed essential revision 3 testing 2 WRN inhibitors and 2 controls in their cell models. The results provide important insights about the inhibitors but do not contribute much mechanistic insight regarding the MLH1 WRN synthetic lethality. While the authors briefly refer to WRN inhibitors in the discussion, they elected not to include the data in the manuscript. I agree with their choice, as the inhibitors show little promise in terms of target specificity.Point for final acceptance:Update the Abstract to reflect the major conclusion that the MLH1 WRN synthetic lethality is context dependent and not absolute.Reviewer #3:The authors carried out experiments to address issues raised during the initial revision.I now support publication of this paper. Although the effect seems "indirect" as far as MMR status is concerned, it is still very significant with regards to the therapeutic potential. Also, this is now a very competitive topic. Essential revisions:1) Figure 4—figure supplement 1 and subsection “WRN dependency in MSI-H CRC is linked to its helicase function”: There is a significant difference between the back-complementation results in HCT 116  and RKO  and the description in the text does not reflect the results of Figure 4—figure supplement 1. Figure 4 shows that the back-complementation with wildtype and nuclease-deficient WRN is very effective, whereas the ATPase/helicase-deficient mutant as well as the ATPase/helicase nuclease double mutant cannot complement the WRN siRNA knockdown cells. For RKO , the difference between nuclease and helicase mutants appears statistically insignificant as the error bars overlap. The text should be changed to more accurately reflect these results.We agree with the reviewer’s analysis that in RKO cells the back-complementation studies do not clearly indicate the differential relevance of helicase and nuclease function in the context of WRN dependence. We have revised the text in the manuscript accordingly:“In RKO cells expressing WRNr variants, we observed a slightly a stronger dependency on WRN helicase ATP-binding function compared to exonuclease activity upon knock-down of endogenous WRN”.Since the determination of the relevant enzymatic function of WRN in MSI-H cancer cells is critical for both the mechanistic understanding and from a drug development perspective we have performed WRN back-complementation experiments in an additional WRN-dependent MSI-H cell model, the endometrial carcinoma cell line HEC-265. We generated monoclonal lines with stable expression of FLAG-tagged, siRNA-resistant WRN (WRNr) expression constructs harboring loss-of-function mutations within the exonuclease-  and helicase-domain , or both domains . Similar to the findings in HCT 116 cell we observed that exogenous expression of the nuclease-dead form of WRNr, but not the ATP-binding deficient and double-mutant forms rescued the effect of endogenous WRN depletion. WRNr expression levels were similar or higher for the mutant WRNr variants compared to WRNr wild-type.This data, which we have included in the revised version of the manuscript as Figure 4—figure supplement 1C, is supportive for a predominant function of WRN helicase in MSI cancer cell lines.2) The authors meticulously show the dependence of the effect on WRN status, but the dependence on MMR status is only correlative. A key concern is that all experiments were carried upon depletion of WRN in MMR-deficient cell lines. These cell lines have a high mutator phenotype and accumulate mutations in a number of other genes, in particular in those containing microsatellites. Therefore, to confirm the key conclusion of this manuscript that the lethality is a result of simultaneous MMR and WRN deficiency, the experiment should also done the other way round, i.e. to deplete key MMR factors (MLH1 would be the best) in WRN-deficient cells, or to perform co-depletion experiments. Additionally, WRN depletion should also be carried out in HC T116 +ch3 cell line, in which the expression of MLH1 was corrected.HCT 116 and RKO are both MLH1 deficient. What is known about additional mutations in these cell lines that may contribute to this difference? This above approach might also address the difference between both cell lines. For RKO a complementation system was described in Yan et al., 2003.We agree that demonstrating whether WRN dependency of MSI-H cancer cell lines is attributable to an acute and context-independent synthetic lethal interaction or an acquired vulnerability related to the MMR-deficient status of these cell lines would substantially strengthen the relevance of this study. In line with the reviewers’ suggestions we have therefore addressed a potential co-dependence of WRN and MMR gene function with several approaches:1) Test of differential WRN dependency in wild-type vs. MLH1-knock-out models of SW480 and CaCo-2.2) Test of differential WRN dependency in parental vs. MLH1 and MLH1/MSH3 reconstituted HCT 116 cell lines .3) Test of differential MLH1/MSH3 dependency in parental vs. WRN-knock-out models of SW480.1) Test of differential WRN dependency in wild-type vs. MLH1-knock-out models of SW480 and CaCo-2.To monitor whether loss of MLH1 would induce WRN dependency we generated MLH1-deficient clones in two MSS CRC cell lines (SW480 and CaCo-2) using CRISPR-Cas9 mediated gene knock-out and applied siRNA-mediated knock-down of WRN.For both SW480- and CaCo-2-derived MLH1 knock-out clones we did not observe increased sensitivity to WRN knock-down compared to non-edited, wild-type clones. This indicates that at least in the context of established MSS CRC cancer cell lines MLH1 loss-of-function alone does not induce WRN dependency. Of note, WRN dependency was also not observed at later passages and MLH1 knock-out did not induce MSI in both models at >9 months cultivation post MLH1 knock-out.2) Test of differential WRN dependency in parental vs. MLH1 and MLH1/MSH3 reconstituted HCT 116 cell lines We thank the reviewer for pointing out the HCT 116 +ch3 cell line as a semi-isogenic, MLH1-proficient model to the parental MMR-deficient HCT 116 cell line used in our study. We have analyzed the effect of WRN knock-down in the HCT 116 +ch3 cells along with the MMR-deficient control line HCT 116 +ch2 and the HCT 116 +ch3 +ch5 line . We find that neither back-complementation of MLH1 alone or in combination with MSH3 reverses the strong WRN dependency of HCT 116 cells – although we observe a slightly increased viability upon WRN knock-down in the HCT 116 +ch3 and HCT 116 +ch3 +ch5 models compared to HCT 116 parental cells and the +ch2 reconstituted control model . siRNA-mediated depletion of WRN mRNA was equally efficient in all cell models .3) Test of differential MLH1/ MSH3 dependency in parental vs. WRN-knock-out models of SW480.To monitor whether loss of WRN would induce dependency on MLH1 and/or MSH3, we analyzed the effect of individual or combined knock-down of MLH1 and MSH3 on the viability of wild-type (WRN-proficient) and two WRN-knock-out monoclonal SW480 cell lines. Neither single nor concomitant knock-down of MHL1 and MSH3 affected cell viability of the WRN-deficient SW480 cell lines  despite efficacious knock-down of both genes . This data is in line with the studies outlined above and argue against an acute and direct synthetic lethal interaction of WRN and MLH1/MSH3 genes.In summary, the results outlined above suggest that the WRN dependence of MSI-H/MMR-deficient cancer cell lines might not be attributable to a hard-wired, context independent synthetic lethality such as described for the paralog genes STAG1/STAG2  or SMARCA2/SMARCA4 . Instead, our data argue for an acquired dependency on WRN function as a consequence of persistent MSI-H status and/or MMR-deficiency.MLH3 reconstituted HCT 116 lines (2.) and the SW480 WRN-knock-out models (3.) in the revised version of this manuscript as Figure 2—figure supplement 2 and Figure 2—figure supplement 3. This is of particular relevance since our results are in partial discrepancy to a recent preprint manuscript reporting the full reversion of WRN dependency in the MLH1/MSH3 reconstituted HCT 116 +ch3 +ch5 model . In our experiments, we only detect a minor improvement in viability upon WRN depletion in HCT 116 +ch3 +ch5 model. We have also revised the Discussion section accordingly.To incorporate this relevant finding we have included the studies using the MLH1/It is plausible that WRN dependency is a consequence of alterations in genetic targets of MSI (i.e. genes harboring repetitive DNA sequences that are particularly vulnerable to the loss of MMR activity). To this end, we analyzed the effect of siRNA-mediated knock-down of genes commonly mutated in MSI-H cancer cell lines on viability of parental vs. WRN-knock-out SW480 cell lines. We did not observe an increased dependency on any of the genes tested the context of concomitant WRN loss. This argues against an acute synthetic lethal interaction of WRN and MSI target genes. However, we cannot rule out synthetic lethal interactions based on combined loss of MSI target genes and have addresses this accordingly in the Discussion section of the revised manuscript.3) WRN inhibitors need to be referenced and discussed more fully. In addition to the Rosenthal et al., 2010 reference, at least two WRN inhibitors have been around for 5+ years. They are highly and directly relevant to the topic of this manuscript, and are being pursued in other clinical contexts  that may provide additional insight. These inhibitors are readily available, and the authors' results make a straight-ahead prediction of what a dose-response outcome should be in their CRC cell line panels to provide a bit of pre-clinical data. The authors should acquire the WRN inhibitors and do dose-response curves on their MSI-H and MSS cell lines, and show that their WRN KO lines are insensitive (or largely insensitive) as a demonstration of target specificity.We thank the reviewers for pointing out the use of published WRN inhibitors in the context of this study and we agree that it is highly relevant to assess the potential clinical value of our findings with the available chemical matter.50 values below 10 µM in the majority of the tested cell lines (see Author response table 1). However, MSI-H CRC and endometrial carcinoma cell lines did not show increased sensitivity to treatment with the two WRN inhibitors compared to MSS cell models. The BLM inhibitor ML-216 was largely ineffective in the majority of cell lines at concentrations up to 10 µM. As a positive control, we included the pan-HDAC inhibitor panobinostat which showed potent inhibition of cell viability in the nM range across all cell models.We have tested two structurally related WRN inhibitors, NSC 19630  and NSC 617145  and a chemical probe for the RecQ helicase BLM  across a panel of MSS and MSI cell models. Both NSC 19630 and NSC 617145 affected cell viability in 7-day dose-response proliferation assays with ICWe have included two SW480 monoclonal cell lines with an engineered deletion of WRN in order to assess the on-target activity of the WRN inhibitors. Both SW480 WRN knock-out lines display sensitivities lower or similar to the parental cell line or a WRN-proficient SW480 monoclonal cell line. In the absence of the protein target this indicates that the observed toxicity of the WRN inhibitors might be at least in part attributable to non-WRN related off-target effects.While these results do not preclude on-target activities of the tested WRN inhibitors in line with the literature data, we suspect that the inhibitors lack the potency and selectivity required for the cellular assessment of WRN function in the context of this study. Since the potential off-target activities pose a challenge in interpreting the cellular effects compared to the genetic inactivation of WRN, we have not included this data in the revised version of this manuscript. We have included the references to the WRN inhibitors NSC 19630 and NSC 23867477 in the revised Discussion section of the manuscript.Reviewer #1:[…]Point for final acceptance:Update the Abstract to reflect the major conclusion that the MLH1 WRN synthetic lethality is context dependent and not absolute.The Abstract has been updated to reflect the new experimentation. We have included a statement that WRN dependence is not acutely linked to loss of MMR gene function, but rather a consequence of sustained MMR-deficiency. The Abstract has further been slightly modified to adhere to the 150 word limitation despite addition of the new information.


Uveal melanoma (UM) is the most common primary intraocular tumor that arises from neoplastic melanocytes in the choroid, iris, and ciliary body. Electrochemotherapy (ECT) has been successfully established for the treatment of skin and soft tissue metastatic lesions, deep-seated tumors of the liver, bone metastases, and unresectable pancreas lesions. The aim of this study was to evaluate the effect of ECT in vitro in 3D spheroid culture systems in primary and metastatic UM cell lines. We also investigated the chick embryo chorioallantoic membrane (CAM) as an in vivo model system for the growth and treatment of UM tumors using ECT. The cytotoxic effect of ECT in 3D spheroids was analyzed seven days following treatment by assessment of the size and MTT  assay. The cytotoxicity of ECT after intratumoral or intraarterial administration was evaluated histologically. In vitro and in vivo ECT caused a significant reduction in tumor size and viability compared to electroporation or chemotherapy in both sections of our study. The current results underline the effectiveness of ECT in the treatment of UM and prepare the way for further investigation of its potential application in UM. Uveal melanoma (UM) is the most common primary intraocular tumor in adults, with an incidence of 5.1 cases per million annually in the USA and between 4 and 7 per million per year in Europe . A higheGNAQ) and quinine nucleotide binding protein subunit alpha 11 (GNA11) [CYSLTR2, and PLCB4, while those involved in UM progression/metastasis include BAP1, SF3B1, EIF1AX, and SRSF2. There are some mutations detected at a low frequency , where the clinical significance is currently unknown [The development of UM is associated with the presence of related ocular or cutaneous melanocytic lesions, fair complexion and a light iris . The mol (GNA11) . Other g unknown ,7.The first-line therapeutic options for UM include radiotherapy (brachytherapy and proton beam radiotherapy), phototherapy , and surgery  . The selElectrochemotherapy (ECT) and its effectiveness have been established in the treatment of skin and superficial soft tissue metastatic lesions, and recently have been described for the application in deep-seated tumors of the liver, bone metastases, and unresectable pancreas cancer ,10,11. IElectroporation (EP) increases the permeability of the cell membrane after the application of short electric pulses on both sides of the membrane, resulting in the formation of transient pores, allowing for the delivery of large hydrophilic molecules to the cytosol ,13. The In vitro 3D culture systems have been developed to incorporate in vivo growth conditions of tumor masses, and to better preserve the biological characteristics and microenvironment of tumors than conventional monolayer cultures . Tumor-dThe aim of this study was to evaluate the effect of bleomycin in five primary and metastatic UM spheroid culture systems, when combined with EP or used alone. Subsequently, we tested the CAM as an in vivo assay to develop and treat UM nodular masses.The spheroid growth of different UM cell lines of primary and metastatic origin was calculated in the cross-sectional area of the spheroids after seven days of treatment. In four of five cell lines, the most significant reduction of size after ECT compared to untreated spheroids (control group) was detected in primary tumor cell lines and metastatic cell lines after treatment with 750 V/cm after application of 2.5 µg/mL bleomycin (ECT) a,b.Chemotherapy alone and EP alone showed a nonsignificant reduction of spheroid growth. The cytotoxic effect of all tested therapeutic modalities was higher in metastatic than in primary tumor cell lines. The mean difference compared to the control group was 56.2% and 19.4%, respectively. Morphological changes of spheroids at day seven following ECT were more prominent in the metastatic cell lines and resulted in a loss of sphericity and loose cells around the spheroid . The aveThe UM cell lines formed not spherical but oblate spheroids, with a loose association of cells. To validate the microscopic observations of spheroid size, a viability assay was performed using MTT assay seven days following treatment c,d. ChemThe 92.1 cell line is a highly pigmented UM cell line, making it suitable for an in vivo CAM assay, since the cell‒Matrigel grafts can be easily observed for a long period of time without artificial labeling of the cells. We utilized the ‘in ovo’ CAM assay to analyze in vivo the cytotoxic effect of ECT on UM cells based on cell line 92.1. The survival rate of the embryos was 52% on embryonic day 16 (E16).Different histochemical and immunohistochemical stainings were performed to characterize the human UM cells on the CAM layer. PAS staining was used to distinguish the CAM layer and the Matrigel grafts, including human 92.1 cells a. The MaIn successfully cultivated CAMs, the cell‒Matrigel grafts were predominantly flat at E16 a. ApproxWe compared the cytotoxic effects of ECT on 3D tumor organoids at day 5 after intraarterial and intratumoral injection of bleomycin (E16). The control group consisted of cell‒Matrigel grafts without as well as after treatment with EP or chemotherapy alone. ECT with intraarterial injection of bleomycin mostly resulted in necrosis and apoptosis of tumor cells, especially in the peripheral Matrigel graft region near the chicken tissue or near blood vessels . FurtherThe intratumoral ECT led to large intratumoral areas of necrosis in the center of the Matrigel grafts . In the Compared to ECT, in the control group samples without treatment or chemotherapy alone, no significant effect on the viability of tumor cells in Matrigel grafts could be detected .This novel study investigated the effect of ECT in vitro and in vivo, setting a milestone for further experiments and clinical application. We demonstrate the first results following ECT using lower doses than the peak plasma concentration (0.5–5.0 µg/mL) of bleomycin. Bleomycin can be administered intratumorally or intravenously ,21,22,23The response of the different UM cell lines varied following treatment. The metastatic cell lines OMM1 and MM28 were more sensitive to ECT with bleomycin. It is postulated that the cell size, shape, and membrane characteristics may affect EP ,28. An aHamburger and Hamilton classified in 1951 the stages of development of the chick embryo and enabled in vivo biomedical research . The chiThe references in the literature to the possible use of ECT in the treatment of ocular melanoma are limited. The Collaborative Ocular Melanoma Study suggested that invasive therapeutic modalities are not associated with a lower mortality rate in comparison with eye-conserving therapies for patients with medium-sized melanomas . ECT couwww.loorg.org). All three cell lines have been described as having disomy 3 and nuclear expression of the BAP1 protein. The authentication of cell lines was performed. Two further cell lines MM28 (ATCC CRL-3295) and MP46 (ATCC CRL-3298) were purchased from LGC standards in 2019. These cell lines are characterized by monosomy 3 or BAP1 mutation [3 cells in round bottom 96 well ultra-low attachment plates  containing 200 µL complete medium. For the experiments three days tumor spheroids were used.UM cell lines 92.1, Mel270, and OMM1 were kindly provided by Prof. Sarah Coupland and Dr. Helen Kalirai of the Liverpool Ocular Oncology Research Group , or with high-voltage electrical pulses electroporation (750V/8 pulses) in the absence of bleomycin  or in the presence of 2.5 µg/mL bleomycin  using a voltage pulse generator . Details of the EP protocol are as follows: two parallel aluminum electrodes 4 mm apart, eight pulses, 100 µs pulse duration, 5 Hz repetition frequency, and 750 V/cm pulse strength. As a negative control, a further sample remained untreated (control). Twenty-four hours after treatment, the spheroids were washed three times with PBS and incubated in a fresh complete medium. Spheroids were harvested seven days following treatment. The analysis was performed in three to eight independent biological replicates on different dates.The growth of spheroids was analyzed seven days after treatment using bright-field microscopy by calculating the cross-sectional area of the spheroids using the image processing software program ImageJ Fiji  . The relSpheroids of UM cell lines are flat-faced spheres. Thus, MTT assay can be used as an indirect method to analyze the viability of the spheroids. MTT assay  was performed seven days after treatment according to the manufacturer’s instructions. In short, the medium was removed from spheroids, and 110 µL fresh medium including 10 µL AB solution was added. After four hours, the formacan was resuspended using 100 µL isopropanol. Then 200 µL spheroid solution were transferred to a 96-well plate and spectrophotometrically analyzed using Tecan Infinity M Plex .6 cells) were mixed 1:1 with Matrigel (Corning B.V.) in a total volume of 40 µL. Cell‒Matrigel grafts were placed on top of the CAM near to chicken vessels. The window was resealed with adhesive tape and eggs were incubated at 37.7 °C until E11. At E11, the samples were treated by ECT. In detail, 50 µL bleomycin  were injected into a chicken artery using a 30 gauge needle  or into a tumor-formed cell‒Matrigel graft  ((A)750 V/cm pulse strength or(B)1000 V/cm pulse strength.Fertilized chicken eggs were obtained from Ovovac GmbH, Thallwitz, Germany. Eggs were incubated in an incubator  at 37.7 °C with 60% humidity. At embryonic day three (E3), approximately 7‒8 mL of albumin were removed from the apical side of the egg using a 20 gauge needle and syringe without detaching the embryo or the yolk. At E4 a small window was cut in the top surface a. UM celjection) b,c. Subsjection) d. The foAs a control, untreated samples (control), as well as samples with bleomycin-only treatment (chemotherapy) and samples with only EP treatment, were included in the analyses. Eggs were returned to the incubator until E16. Matrigel grafts with surrounding CAM tissue were harvested from each embryo and fixed in 4% paraformaldehyde for 24 h and embedded in paraffin at E16 e–g. Seri2O2 for 10 min. Antigen detection, including blocking of background staining, was performed using a ZytoChem Plus HRP Polymer Kit . Tissue sections were incubated with primary antibodies for 30 min at 37 °C , MelanA monoclonal antibody 1:50 , and antihuman melanosome 1:100 , followed by DAB/HRP detection. Sections were counterstained with Mayer’s hematoxylin solution for 30 s.Paraffin sections were incubated at 60 °C for 2 h and afterwards dehydrated with xylene and decreasing ethanol concentration, followed by distilled water. Antigen retrieval was performed using 10 mM sodium citrate solution (pH6) for 30 min in a water bath at 95 °C. The endogenous peroxidase activity of the sections was blocked by using 3% Ht-test was used to determine the statistical significance of spheroid viability and growth. Results were determined to be significantly different if p-values were ≤ 0.05.All analysis was performed using IBM SPSS statistics 25  for Windows software. Student’s This study presents the positive results of ECT with bleomycin in metastatic as well as primary UM 3D spheroids with or without monosomy 3. Moreover, we discussed the use of the chick embryo model in UM and its treatment with ECT. The application of ECT may result in globe-sparing therapy for large UM. Additionally, the use of ECT could be considered in localized metastatic UM. Further investigations in vivo and in vivo are required in order to establish this new modality as another therapeutic option in the treatment of UM.


Protein kinase C iota (PKCι) is an enzyme highly expressed in NSCLC, regulating PAK1 signaling. In the present study we explored whether the PKCι-PAK1 signaling pathway approach can be an efficient target in different types of NSCLC cell and mouse models.The effect of IPA-3 (PAK1 inhibitor) plus auranofin (PKCι inhibitor) combination was evaluated by cell viability assay, colony formation and western blotting assay, using three types of NSCLC cell lines: EGFR or KRAS mutant adenocarcinoma and squamous cell carcinoma with PAK1 amplification. In addition, for clinical availability, screening for new PAK1 inhibitors was carried out and the compound OTSSP167 was evaluated in combination with auranofin in cell and mice models.The combination of IPA-3 or OTSSP167 plus auranofin showed high synergism for inhibiting cell viability and colony formation in three cell lines. Mechanistic characterization revealed that this drug combination abrogated expression and activation of membrane receptors and downstream signaling proteins crucial in lung cancer: EGFR, MET, PAK1, PKCι, ERK1/2, AKT, YAP1 and mTOR. A nude mouse xenograft assay demonstrated that this drug combination strongly suppressed tumor volume compared with single drug treatment.Combination of IPA-3 or OTSSP167 and auranofin was highly synergistic in EGFR or KRAS mutant adenocarcinoma and squamous cell carcinoma cell lines and decreased tumor volume in mice models. It is of interest to further test the targeting of PKCι-PAK1 signaling pathways in EGFR mutant, KRAS mutant and squamous NSCLC patients. Non-small cell lung cancer (NSCLC) is the leading cause of cancer related deaths and comprises several histological subtypes: lung adenocarcinoma (LUAD), squamous cell carcinoma (SCC) and large cell carcinoma. Despite the identification of targeted druggable driver mutations and rearrangements, most cases have poor survival , 2. ReceThis study aimed to estimate the therapeutic effect of PKCι-PAK1 signaling pathways in different types of NSCLC.Detail of standard methodologies for cell culture, reagents, cell viability assay, colony formation assay, and western blotting analysis are described in Supplementary materials and methods.The measurement of PAK1 kinase was performed on a Caliper LabChip EZ Reader II equipped with a 12-sipper chip in Profiler Pro separation buffer supplemented with CR-8 and analyzed using EZ Reader software . The test compounds were incubated with PAK1 kinase and fluorescence-labeled substrate Peptide 14 (Sequence RRRLSFAEPG) in the kinase assay buffer at 30 °C for 10 min, followed by adding 9.5 μM ATP to initiate the reaction. After incubation for 1 h at 30 °C, the phosphorylated and unphosphorylated substrates were separated and detected by EZ Reader II device. IPA-3 was used as a positive control. The separation of peptide 14 was performed under the following optimized conditions: upstream voltage = − 500 V, downstream voltage = − 2400 V and pressure = − 1.4 psi using a marker dye consisting of unphosphorylated peptide 14. After PAK1 inhibitors were screened from the Target Mol-Inhibitory-Library, the anti-tumor effects of candidates were estimated by MTT assay using A549 cell lines.6 cells suspended in phosphate-buffered saline mixed 1:1 with Corning Matrigel  via right flank. Tumor size was measured in two orthogonal directions using calipers every 2 days and weights were determined every 2 days. After tumors became palpable (~ 100–300 mm3), mice were randomized into a vehicle group and treated groups with IPA-3 alone, auranofin alone, OTSSP167 alone, IPA-3 plus auranofin, or OTSSP167 plus auranofin. Each reagent was suspended in 1% [weight/volume (w/v)] Kolliphor HS15 and administered once daily by intraperitoneal administration (IPA-3 and auranofin) or oral gavage (OTSSP167) of 10 mg/kg. Mice in the untreated group were given the same volumes of 1% Kolliphor HS15. The tumor volume (mm3) was estimated using the equation length × (width)2 × 0.5.Four to five-week-old female nude mice were kept in individually ventilated cages (5 per cage) with access to food and water, at 20 °C and 50 ± 20% relative humidity under a 12:12 h light-dark cycles and pathogen free conditions. Three cell line xenograft tumors were established by subcutaneously injecting 4 × 10p value of less than 0.05 was regarded as significant.In MTT and colony forming assays, the strength of interaction between reagents was determined by calculating the combination index (CI) according to method of isobologram-combination index . The CIEGFR mutated), H23 (KRAS mutated) and H520 (PAK1 overexpression). We evaluated cell viability and colony formation inhibition. Results indicated that the IPA-3 plus osimertinib combination showed synergism in H23 cell lines in cell viability assays with a CI of 0.73. IPA-3 plus trametinib was also synergistic in the H23 cell line (CI was 0.74)  combination with several compounds used in cancer treatment, such as EGFR inhibitors (osimertinib and afatinib), PKCι inhibitor (auranofin), MEK inhibitor (trametinib), and a Flk3/Syk, and potential multiple PKC inhibitor (midostaurin), several lung cancer cell lines possessing different molecular characteristics were tested, such as HCC827  , H23 (KRAS-mutant LUAD) and H520 (PAK1 overexpression). Also, downregulation of protein expression of EGFR, MEK, AXL, Src and STAT3 was observed.  or combination of IPA-3 or OTSSP167 plus auranofin or vehicle were inoculated via intraperitoneal administration (auranofin and IPA-3) or oral gavage (OTSSP167) every 2 days for 2 or 4 weeks. Combination therapy showed stronger anti-tumor effect compared to mono therapy in 3 mice model cell lines. IPA-3 plus auranofin inhibited tumor growth, both in volume and weight, compared to IPA-3 alone or auranofin alone. The combination of OTSSP167 plus auranofin showed significant inhibition of tumor growth in the 3 mice model cell lines compared to single OTSSP167 or auranofin treatment  relieves inhibition by disrupting the PAK1 homodimer . PAK1 isOTSSP167 is a maternal embryonic leucine-zipper kinase (MELK) inhibitor with anti-cancer effect reported in several tumors, as well as in chronic lymphocytic leukemia . The antThe phosphorylated residue Ser144 of PAK1 was inhibited in three cell lines. The phosphosites, Ser21 or Thr423, also contribute towards PAK1 activation, although, Ser144 is the most critical for PAK kinase activity . PhosphoOur model showed that some PAK1 phosphosites were inhibited by, not only IPA-3, but also auranofin alone. IPA-3 did not inhibit PKCι expression. This suggests that auranofin inhibits PAK1 from the upper stream of PKCι-PAK1 signaling and stronger inhibition of PKCι-PAK1 signaling resulted in stronger synergism. Although osimertinib monotherapy in HCC827 cell lines or trametinib monotherapy in H23 cell lines also inhibited some phosphorylated PAK1, the combination of auranofin plus these reagents was not synergistic and showed less abrogation of RTK and non-RTK. In addition, midostaurin, a FLT3 inhibitor, and a potential inhibitor of all PKC isoforms  was not In the mice xenograft model, a significant decrease in tumor volume was confirmed by the OTSSP167 plus auranofin combination. Although IPA-3 plus auranofin showed anti-tumor effect, it did not reach a significant difference. OTSSP167 suggested stronger PAK1 inhibition compared to IPA-3 in western blotting; phosphorylated PAK1 at Thr423/Ser144/Thr204 in HCC827 cell lines and Thr423 in H23 cell lines were inhibited by OTSSP167 alone, but not by IPA-3 alone. These differences between IPA-3 and OTSSP167 might explain the difference in results of the mice xenograft model. Although mice body weight was significantly decreased in OTSSP167 alone, or OTSSP167 plus auranofin in all 3 cell lines, there was no significant difference between OTSSP167 alone, and OTSSP167 plus auranofin. Chung et al. showed OTSSP167 was tolerable in the mice model without adverse body weight loss . Adverse. Although OTSSP167 inhibited phosphorylated PAK1, we cannot rule out that other activators of PAK1 can intervene in lung cancer, like MLK3 and TNFα.Our research has centered on targeting PKCι-PAK1 signaling and was effective with auranofin plus OTSSP167 in 3 lung cancer models in vitro and in vivoEGFR mutant, KRAS mutant, and squamous NSCLC patients.Auranofin has been investigated in a clinical trial for NSCLC and small cell lung cancer (NCT01737502). A Phase I study of OTSSP167 for solid tumors (NCT01910545) and a feasibility evaluation for healthy volunteers (NCT02768519) have been completed. Other clinical trials of OTSSP167 are ongoing in breast cancer (NCT02926690) and leukemia (NCT02795520). Targeting PKCι-PAK1 signaling pathways is of interest to be further tested clinically in The Combination of PKCι-PAK1 inhibitors was highly synergistic in EGFR and KRAS mutant adenocarcinoma and squamous cell carcinoma of the lung, in both in vitro and in vivo mice models. It is warranted to further test the therapeutic strategy of targeting PKCι-PAK1 signaling pathways in EGFR mutant, KRAS mutant and squamous NSCLC patients.Additional file 1: Table S1. Profiles of cell lines and IC50 (nM) of each reagent. Table S2. The profiles of tested potential PAK1 inhibitors. Figure S1. IPA-3 and auranofin potentiates colony formation inhibition in H23 and H520 cell lines. Figure S2. Western blotting by treatment using single reagent or combination of IPA-3 plus auranofin in H23 and H520 cell lines. Figure S3. Optimization and validation of the kinase assay for PAK1 inhibitor screening. Figure S4. Summary of cell growth inhibition using potential PAK1 inhibitors. Figure S5. OTSSP167 plus auranofin potentiates colony formation inhibition in H23 and H520 cell lines. Figure S6. Anti-tumor effects of single reagent or combination treatment using IPA-3, auranofin, and OTSSP167 in nude xenograft model in H23 and H520 cell lines.


Aconitum, Delphinium, and Garrya (Ranunculaceae) and classified according to their chemical structures as C18-, C19- or C20-diterpenoid alkaloids. The extreme toxicity of certain compounds, e.g., aconitine, has prompted a thorough investigation of how structural features affect their bioactivities. Therefore, natural diterpenoid alkaloids and semi-synthetic alkaloid derivatives were evaluated for cytotoxic effects against human tumor cells . Among the tested alkaloids, C19-diterpenoid  and C20-diterpenoid  alkaloids exhibited significant cytotoxic activity and, thus, provide promising new leads for further development as antitumor agents. Notably, several diterpenoid alkaloids were more potent against MDR subline KB-VIN cells than the parental drug-sensitive KB cells.Diterpenoid alkaloids are isolated from plants of the genera  Cancer therapy mainly involves surgery, chemotherapy, radiation therapy, immunotherapy, monoclonal antibody therapy, and hormone therapy. Chemotherapy generally refers to the use of cytotoxic drugs to treat cancer. Plant alkaloids are one major class of chemotherapeutic drugs ,6,7,8,9.Aconitum, Delphinium, and Garrya (Family Ranunculaceae) species and are classified according to their chemical structures as C18-, C19- or C20-diterpenoid alkaloids , lactone , 7,17-seco, and rearranged ones .,11.Aconiss toxic . 19-diterpenoid alkaloids have been studied extensively and reviewed [Aconitum extracts as poisons in hunting and warfare [Aconitum plants have been used as “bushi”, an herbal drug in some prescriptions of traditional Japanese medicine for the treatment of hypometabolism, dysuria, cardiac weakness, chills, neuralgia, gout, and certain rheumatic diseases [The pharmacological properties of the Creviewed . Aconitireviewed . This ex warfare , althougdiseases . Howeverdiseases ,16,17. S19- and C20-diterpenoid alkaloids on the growth of the A172 human malignant glioma cell line [19- and C20-diterpenoid alkaloids were also investigated [20-diterpenoid derivatives showed significant suppressive effects against the Raji non-Hodgkin’s lymphoma cell line [20-diterpenoid alkaloids on the growth of the A549 human lung cancer cell line were examined and subsequent structure-activity relationships for the antiproliferative effects against A549 cells were considered [19-diterpenoid  and C20-diterpenoid  alkaloids have shown significant antiproliferative activity, as well as provided promising new leads for further development as antitumor agents.Our previous study demonstrated the effects of various naturally occurring and semi-synthetic Cell line . Antitumnsidered . Since 219-diterpenoid alkaloids included 21 natural alkaloids, aconitine (1), deoxyaconitine (2), jesaconitine (3), deoxyjesaconitine (4), aljesaconitine A (5), secojesaconitine (6), mesaconitine (8), hypaconitine (9), hokbusine A (10), 14-anisoyllasianine (12), N-deethylaljesaconitine A (13), aconine (14), lipomesaconitine (15), lipoaconitine (16), lipojesaconitine (17), neolinine (18), neoline (19), 14-benzoylneoline (20), isotalatizidine (21), karacoline (22), and 3-hydroxykaracoline (23), isolated from the rhizoma of Aconitum japonicum THUNB. subsp. subcuneatum (NAKAI) KADOTA [19-diterpenoid alkaloids, 3,15-diacetyljesaconitine (7) [11) [6) and mesaconitine (8), respectively  KADOTA ,26,27,28(7) [11)  preparedectively , were al19-diterpenoid alkaloids, both natural alkaloids  and synthetic analogs (7 and 11), were inactive (IC50 > 20 or 40 μM) [15~17) exhibited cytotoxic activity against five human tumor cell lines  (17) showed significant cytotoxicity against four tested cell lines with IC50 values of 6.0 to 7.3 μM, but weak cytotoxicity against KB-VIN (IC50 = 18.6 μM) [15) showed moderate cytotoxicity against the KB cell line (IC50 = 9.9 μM), but weak cytotoxicity against the other four human tumor cell lines (IC50 =17.2 ~ 21.5 μM) [16) was weakly cytotoxic (IC50 = 13.7 ~ 20.3 μM) against all five human tumor cell lines [17 may be important to the cytotoxic activity of aconitine-type C19-diterpenoid alkaloids.Eighteen of the 23 tested aconitine-type Cr 40 μM) ,28,30 (T KB-VIN) . Lipojes21.5 μM) . Lipoaco19-diterpenoid alkaloid group included 12 natural alkaloids, namely nevadensine (24), N-deethylnevadensine (25), and virescenine (27), purified from rhizoma of Aconitum japonicum subsp. subcuneatum [26), delphinifoline (28), delcosine (34), 14-acetyldelcosine (34–43), and 14-acetylbrowniine (35), purified from root of Aconitum yesoense var. macroyesoense (NAKAI) TAMURA [30), pacifiline (31), pacifinine (32), and pacifidine (33), purified from seeds of Delphinium elatum cv. Pacific Giant [19-diterpenoid alkaloids from this subtype were synthetic alkaloids, N-deethyldelsoline (29) [34-1) [34-2) [34-24) [34-42) [34-45) [34-46) [34-3~34-23, 34-25~34-41, 34-44, and 34-47) [34) or delsoline (34-42)  [19-diterpenoid alkaloids, both natural alkaloids  and a synthetic alkaloid (29), were inactive (IC50 > 20 or 40 μM). All tested delcosine derivatives that contain an acetyl or methoxy group, both natural alkaloids  and synthetic analogs , were inactive (IC50 > 20 μM). However, acylation, except with an acetyl group, of the C-1 and/or C-14 hydroxy group of 34 led to various degrees of antiproliferative activity. Among the C-1 esterified alkaloids, the synthetic derivatives 34-6, 34-8, 34-10, and 34-18 exhibited significant potency against all cell lines . Also, alkaloids 34-3, 34-16, 34-17, 34-21, 34-25, 34-27, 34-31, 34-32, 34-38, and 34-40 showed moderate potency toward all cell lines (average IC50 12.7−20.7 µM). While alkaloid 34-32 displayed good antiproliferative activity (IC50 8.7 µM) against KB cells, it was much less potent against A549, MDA-MB-231, and KB-VIN cells. Alkaloids 34-5, 34-13, 34-15, 34-29, 34-35, 34-37, and 34-41 exhibited only weak potency against all cell lines (average IC50 22.0−26.5 µM). Finally, alkaloids 34-24, 34-30, and 34-34 were inactive against all five human tumor cell lines, while 34-12, 34-33, and 34-39 showed limited potency.The tested lycoctonine-type  Cescenine , purifie) TAMURA ,32,33,34rowniine , purifieine (29) , 1-acety) [34-1) , 1,14-di) [34-2) , 1-(4-tr [34-42) , 1,14-did 34-47) , prepare (34-42) . These 4 KB-VIN)  . Alkaloid 34-9 (average IC50 11.9 µM) showed significant antiproliferative activity against MDA-MB- 231 and KB cells  comparable with 34-19 and 34-20, but was less potent against MCF-7 and A549  and inactive against KB-VIN. Alkaloid 34-23 exhibited only weak potency toward all cell lines (average IC50 23.7 µM). Alkaloids 34-4, 34-7, 34-11, 34-14, 34-26, 34-36, 34-45, 34-46, and 34-47 were inactive against all five human tumor cell lines, while 34-22 and 34-28 showed limited potency.Among the derivatives esterified at both C-1 and -14, alkaloids 34-3, 34-6, 34-8, 34-10, 34-13, 34-21, 34-25, 34-27, and 34-35) were significantly more potent compared with corresponding C-1,14 diacylated delcosine derivatives . Thus, a C-1 acyloxy group and C-14 hydroxy group are crucial for enhanced antiproliferative activity of 1-derivatives. Regarding alkaloids 34-18 , 34-19 (pentafluorobenzoate at C-1 and C-14), and 34-20 , all three alkaloids were essentially equipotent against three of the five tumor cell lines, while 34-18 was somewhat less potent than the diacylated alkaloids against MCF-7 and KB-VIN cells.Particularly, C-1 monoacylated delcosine derivatives  generally ranked somewhat below those of the most potent alkaloids, except against the MCF-7 cell line, where they were even less active.Striking observations from the data in 34-19 and 34-20 with one or two pentafluorinated benzoyl esters, respectively, showed significant potency against all five tested cell lines. Alkaloid 34-9 with two 3-nitro-4-chlorobenzoyl groups showed good potency against certain cell lines. Similarly, the 1-monoacylated alkaloids with the highest potency against the five tumor cell lines contained 3-nitro-4-chloro- (34-8) and pentafluoro- (34-18) as well as 4-dichloro-methyl- (34-10) benzoyl esters. The chlorinated alkaloids 34-8 and 34-10 as well as 34-6, which has 3,5-dichloro substitution on the benzoate ester, were more potent than 34-5 with only a single chloro group or 34-13 with chloro and fluoro groups. Similarly, alkaloid 34-18 showed increased antiproliferative activity against the five tumor cell lines compared with other fluorinated alkaloids 34-13~34-17, 34-21~34-27. Moreover, with some exceptions against certain cell lines, alkaloids with bromo (34-3 and 34-4), dimethylamino (34-12), dimethoxy (34-29), trimethoxy (34-30), diethoxy (34-31), benzyloxy (34-32), cyano (34-33), methylenedioxy (34-34 and 34-35), nitro (34-45 and 34-46), and ethoxy (34-47) substituted benzoate esters or phenylacetyl (34-37), cinnamoyl (34-38 and 34-39), 1-naphthoyl (34-40), and anthraquinone-2-carbonyl (34-41) esters were less potent or inactive. The identity of the substituent(s) on the acyl group affected the antiproliferative potency. Notably, among the 1,14-diacyl and 1-acyl-14-acetyl derivatives, only alkaloids Interestingly, the active alkaloids were generally effective against P-gp overexpressing MDR subline KB-VIN, while alkaloids such as vincristine and paclitaxel are ineffective due to excretion from the MDR cells by P-gp. These results suggest that these diterpenoids are not substrates for P-gp.19-diterpenoid alkaloids included 19 natural alkaloids, delcorine (36), delpheline (37), pacinine (38), yunnadelphinine (39), melpheline (40), bonvalotidine C (41), N-deethyl-N-formylpaciline (42), N-deethyl-N-formylpacinine (43), isodel-pheline (44), pacidine (45), eladine (46), N-formyl-4,19-secopacinine (47), N-formyl-4,19-secoyunna-delphinine (48), iminoisodelpheline (49), iminodelpheline (50), laxicyminine (51), N-deethyl-19-oxo-isodelpheline (52), N-deethyl-19-oxodelpheline (53), and 19-oxoisodelpheline (54), purified from seeds of Delphinium elatum cv. Pacific Giant [19-diterpenoids were synthetic derivatives (37-1~37-22) [37  Clphinine , melphellpheline , bonvalotidine C , N-deethpaciline , N-deethpacinine , isodel--22) [37 . 19-diterpenoid alkaloids were evaluated for antiproliferative activity against human tumor cell lines [19-diterpenoid alkaloids, both the natural alkaloids (36~54) and synthetic analogs that did not contain a C-6 ester group (37-20 and 37-22), were inactive (IC50 > 20 or 40 μM). Among the C-6 esterified alkaloids, 37-1, 37-17, and 37-18 exhibited the highest average potency toward four tested cell lines . Alkaloids 37-3, 37-5~37-7, 37-9, 37-10, 37-12, 37-13, 37-16, and 37-19 showed moderate potency against all tested cell lines (average IC50 13.9−20.8 µM). However, alkaloid 37-13 showed significantly increased cytotoxic activity (IC50 10.2 μM) against A549 cells compared with 37-1, 37-17, and 37-18, but was generally less potent against DU145 and KB cells. While alkaloids 37-12, 37-13, 37-16, and 37-19 displayed good antiproliferative activity  against KB-VIN cells, they were much less potent against A549, DU145, and KB cells. Alkaloids 37-4 and 37-21 were inactive against all tested cancer cell lines, while 37-2, 37-8, 37-11, and 37-14 exhibited only weak potency toward all cell lines (average IC50 23.0−29.2 µM).All tested lycoctonine-type  Cll lines ,41,42,4337-17~37-19 , followed by alkaloids 37-16, 37-12, 37-1, 37-13, and 37-9 . Generally, all active alkaloids showed the highest potency against the KB-VIN cell line compared with the other three tested cancer cell lines. Moreover, alkaloids 37-12, 37-16, 37-13, and 37-19 showed over two-fold selectivity between the two cell lines . Alkaloids 37-2, 37-5, and 37-17 displayed weak selectivity between the KB and KB-VIN cell lines . Finally, alkaloids 37-1, 37-3, 37-6~37-9, 37-11, 37-14, 37-15, and 37-18 displayed similar potency against the KB and KB-VIN cell lines .The most noticeable observations from the data in 37-1), fluoro , trifluoromethyl , ethoxy (37-16), or benzyloxy (37-17) substituents on the acyl group. Against the KB-VIN cell line, alkaloids 37-18 and 37-19 with both fluoro and trifluoromethyl/methyl groups were more potent than 37-9 with only a single trifluoromethyl group and even more potent than 37-2 with a single fluoro group. Similarly, alkaloid 37-13 showed increased cytotoxic activity against most cell lines compared with the related fluorinated alkaloids 37-14 and 37-15. Moreover, alkaloids with nitro, methoxy, phenyl, trifluoromethoxy, trifluoromethythio, and methyl carboxylate groups on a C-6 benzoate ester were generally less potent.The identity of the substituent on the C-6 acyl group affected the cytotoxic potency. For instance, the alkaloids with the highest potency against the KB-VIN cell line contained chloro , isolated from rhizoma of Aconitum japonicum subsp. subcuneatum [20-diterpenoid alkaloids, lucidusculine (57), flavadine (58), 12-acetyllucidusculine (59), 1-acetyl-luciculine (60), dehydrolucidusculine (61), dehydroluciculine (62), and 12-acetyldehydroluciduscu-line (63), purified from roots of Aconitum yesoense var. macroyesoense [20-diterpenoid alkaloid derivatives (56-1~56-7) [56)  and synthetic analogs , were inactive (IC50 > 20 or 40 μM). Among the synthetic alkaloids, alkaloid 56-5 exhibited only weak potency toward the tested cell lines . Because the related alkaloids 57, 60, 56-2~56-4, 56-6, and 56-7 were inactive against all tested cancer cell lines, a C-1 hydroxy group, C-12 acyloxy group, and C-15 acetoxy group found in 56-5 could be needed for antiproliferative activity of luciculine derivatives.One natural actaline-type Calkaloid , aconicacuneatum ,  ,32,45 we-7) [56)  and testll lines ,30 , 9-hydroxynominine (65), and torokonine (66), isolated from rhizoma of Aconitum japonicum subsp. subcuneatum [67), purified from roots of Aconitum yesoense var. macroyesoense [67-1~67-19) [67 were tested also.Tested hetisine-type  Ccuneatum ,28  ,30,46,4720-diterpenoid alkaloids were evaluated for antiproliferative activity against four human tumor cell lines [64~67) and synthetic , were inactive (IC50 > 20 or 40 μM). Kobusine derivatives 67-5, 67-7, 67-10, 67-18, and 67-19 exhibited the highest average potency over the four tested cell lines , and alkaloids 67-8, 67-13, and 67-14 showed moderate potency . However, while alkaloid 67-14 showed good cytotoxic activity (IC50 9.6 μM) against DU145 cells, it was much less potent against A549, KB, and KB-VIN cells. All tested hetisine-type  Cll lines ,28,30  or even converted an inactive to an active alkaloid . Consequently, all of the most potent analogs  of 67 were esterified at both C-11 and C-15. Among these analogs of 67-5, 67-7, 67-10, 67-18, and 67-19) were quite potent (IC50 < 10 µM) against KB-VIN. Indeed, alkaloid 67-19 exhibited a significantly low IC50 value of 3.1 µM. The ratios of KB to KB-VIN (IC50 KB/IC50 KB-VIN) were greater than 0.73 for all active alkaloids, with many alkaloids displaying comparable potency against the two cell lines, in contrast with paclitaxel (ratio of 0.0067). Alkaloid 67-19 showed over 1.3-fold selectivity with the greatest cytotoxic activity against KB-VIN (IC50 KB/IC50 KB-VIN: 1.32).Striking observations from the data in 20-diterpenoid alkaloids pseudokobusine (68) and 15-veratroylpseudokobusine (68-11) were purified from the roots of Aconitum yesoense var. macroyesoense [68-1~68-10, 68-12~68-37) [68.The two tested natural hetisine-type  Cyesoense ,32  ,47,48,4968 analogs) C20-diterpenoid alkaloids were evaluated for antiproliferative activity against four human tumor cell lines [68 and 68-11) and synthetic analogs , were inactive (IC50 > 20 µM). The pseudokobusine derivatives 68-5, 68-15, 68-19, 68-20, 68-24, and 68-32 exhibited the highest average potency over the tested cell lines . Alkaloids 68-7, 68-10, 68-12, 68-13, and 68-22 showed moderate potency over all tested cell lines (average IC50 13.5-16.8 µM). However, although alkaloid 68-10 showed good cytotoxic activity (IC50 8.0 µM) against A549 cells, it was much less potent against DU145, KB, and KB-VIN cells.All tested hetisine-type  and two C-11,15 di-esterified alkaloids (68-5 and 68-19) exhibited average IC50 values of less than 10 μM. Certain C-11 , C-6,11 (68-4 and 68-12) and C-6,15 (68-13) esterified alkaloids were generally less potent, while all C-6  and C-15  mono-substituted alkaloids, as well as the tri-substituted analog (68-18), were inactive. Thus, all more active (IC50 < 10 µM) C20-diterpenoid alkaloids in this classification had an ester or ether group on the C-11 hydroxy and were 11-monoester/11,15-diester analogs of 68 (OH at C-6).Among the analogs of 68-5, 68-15, 68-19, 68-20, 68-24, and 68-32) were quite potent (IC50 < 10 µM) against KB-VIN. Indeed, alkaloid 68-32 exhibited a low IC50 value of 5.2 µM. The ratios of KB to KB-VIN (IC50 KB/IC50 KB-VIN) were greater than 0.70 for all active alkaloids, with many alkaloids displaying comparable potency against the two cell lines, in contrast with paclitaxel (ratio of 0.0067). Alkaloids 68-12, 68-13, and 68-20 showed over 1.3-fold selectivity with their greatest cytotoxic activity against KB-VIN .The data in 20-diterpenoid alkaloid derivatives 68-7 and 68-22 showed important suppressive effects against Raji cells. Further study indicated that 68-22 inhibited extracellular signal-regulated kinase phosphorylation but induced enhanced phosphoinositide 3 kinase phosphorylation, leading to accumulation of Raji cells in the G1 or sub G1 phase [In mechanism of action studies on selected diterpenoid alkaloids, the hetisine-type C19- and C20-diterpenoid alkaloids. Totally, 199 natural alkaloids and their derivatives were evaluated against four to five human tumor cell lines. Among all alkaloids, 128 alkaloids were non-toxic (IC50 > 20 or 40 µM) and 51 alkaloids showed moderate antiproliferative effects (average IC50 = 10–40 µM). General summaries are described briefly below, and the most active compounds are shown in We have synthesized acylated derivatives of various C19-diterpenoid alkaloids, the fatty acid ester at C-8 and the anisoyl group at C-14 found in 17 may be important to the cytotoxic activity. Compounds without the fatty acid ester at C-8 were inactive, and compounds with an unsubstituted benzoyl group at C-14 were less potent.Among the aconitine-type C19-diterpenoid alkaloids, the most active alkaloids were lycoctonine-type C19-diterpenoid alkaloids with two different substitution patterns, C-1 (delcosine derivatives) and C-6 (delpheline derivatives). Delcosine derivatives 34-6, 34-8, 34-10, and 34-18, which are acylated at the C-1 hydroxy, as well as delpheline derivatives 37-1, 37-17, and 37-18, which are acylated at the C-6 hydroxy, exhibited the greatest potency over all tested cell lines, including MDR KB-VIN. Among the C19-diterpenoid alkaloids, a C-1 acyloxy group and C-14 hydroxy group were important for improved antiproliferative activity. The C-1,14 diacylated delcosine derivatives were generally less potent than corresponding C-1 monoacylated delcosine derivatives. The 1-monoacylated alkaloids with the highest potency (IC50 4−6 µM) against five tested cell lines contained 3-nitro-4-chloro- (34-8) and pentafluoro- (34-18) as well as 4-dichloromethyl- (34-10) benzoyl esters. Two or one pentafluorinated benzoyl esters were also found in the two most consistently potent alkaloids (34-19 and 34-20) among the 1,14-diacyl and 1-acyl-14-acetyl derivatives.Among the lycoctonine-type  C19-diterpenoid alkaloids, none of the tested compound reached the potency levels of the most active 7,8-diol compounds. However, three 6-acylated delpheline derivatives 37-17~37-19 did show significant potency against the KB-VIN cell line . Interestingly, the two latter compounds contained fluorinated benzoyl esters. In addition, among 19 tested delpheline derivatives, four compounds  showed over two-fold selectivity between the MDR and parental cell lines . Among the lycoctonine-type  C20-diterpenoid alkaloids showed significant antiproliferative potency. Only 12-benzoyllucidsuculine (56-5) with C-1 hydroxy, C-12 acyloxy, and C-15 acetoxy groups showed even weak potency.None of the 15 tested actaline- and napelline-type C20-diterpenoid alkaloids, the most active alkaloids were hetisine-type C20-diterpenoid alkaloids with two different substitution patterns, C-11,15 (kobusine) and C-6,11,15 (pseudo-kobusine). Hetisine-type C20-diterpenoid alkaloids 67-5, 67-7, 67-10, 67-18, 67-19, 68-5, 68-15, 68-19, 68-20, 68-25, and 68-32, which are acylated or tritylated at the C-11 hydroxyl, exhibited the greatest potency over all tested cell lines, including MDR KB-VIN. All five most active kobusine derivatives  are acylated at both C-11 and C-15. All tested derivatives with a hydroxy group at either C-11 or C-15 were inactive or much less active. All six most active pseudo-kobusine derivatives  contain a free hydroxy group at C-6. The substituent at C-11 is either a benzoyl/cinnamoyl ester  or a trityl ether (68-32). Finally, the moiety at C-15 is a hydroxy group  or benzoyl ester . Among C19- and C20-diterpenoid alkaloids were also investigated [20-diterpenoid derivatives (68-7 and 68-20) showed significant suppressive effects against the Raji non-Hodgkin’s lymphoma cell line [Furthermore, previously our study, Antitumor properties and radiation-sensitizing effects of various types of novel derivatives prepared from C19- and C20-diterpenoid alkaloids. All alkaloids and their derivatives were screened against four to five human tumor cell lines. Alkaloids 37-2, 37-9, 37-17, 37-18, 56-5, 67-7, 67-14, 67-19, 68-4, 68-12, 68-20, 68-22, 68-24, and 68-32 showed comparable potency against KB and KB-VIN cancer cell lines, and some alkaloids showed tumor- selective activity. Alkaloids 37-12, 37-13, 37-16, and 37-19 exhibited greater inhibitory activity against drug-resistant KB-VIN cells (2.15~2.57-fold) than the parental KB cells. These results demonstrate that modified lycoctonine-type C19-diterpenoid alkaloids and hetisine-type C20-diterpenoid alkaloids are not substrates of P-gp and could be effective against P-gp overexpressing MDR tumors. These promising new lead alkaloids merit continued studies to evaluate their potential as antitumor agents, particularly with enhanced resistant tumor selectivity. In addition, our results from modification-based antitumor activity studies can be used for further development of anticancer drugs overcoming an MDR phenotype.We have synthesized acylated derivatives of various C