Background: Microsatellite and chromosomal instability have been investigated in Hodgkin lymphoma (HL). Materials and Methods: We studied seven HL cell lines (five Nodular Sclerosis (NS) and two Mixed Cellularity (MC)) and patient peripheral blood lymphocytes (100 NS-HL and 23 MC-HL). Microsatellite instability (MSI) was assessed by PCR. Chromosomal instability and telomere dysfunction were investigated by FISH. DNA repair mechanisms were studied by transcriptomic and molecular approaches. Results: In the cell lines, we observed high MSI in L428 (4/5), KMH2, and HDLM2 (3/5), low MSI in L540, L591, and SUP-HD1, and none in L1236. NS-HL cell lines showed telomere shortening, associated with alterations of nuclear shape. Small cells were characterized by telomere loss and deletion, leading to chromosomal fusion, large nucleoplasmic bridges, and breakage/fusion/bridge (B/F/B) cycles, leading to chromosomal instability. The MC-HL cell lines showed substantial heterogeneity of telomere length. Intrachromosmal double strand breaks induced dicentric chromosome formation, high levels of micronucleus formation, and small nucleoplasmic bridges. B/F/B cycles induced complex chromosomal rearrangements. We observed a similar pattern in circulating lymphocytes of NS-HL and MC-HL patients. Transcriptome analysis confirmed the differences in the DNA repair pathways between the NS and MC cell lines. In addition, the NS-HL cell lines were radiosensitive and the MC-cell lines resistant to apoptosis after radiation exposure. Conclusions: In mononuclear NS-HL cells, loss of telomere integrity may present the first step in the ongoing process of chromosomal instability. Here, we identified, MSI as an additional mechanism for genomic instability in HL. TP53 is still a matter of debate [Hodgkin lymphoma (HL) is a unique disease in which the neoplastic Hodgkin and Reed–Sternberg (HRS) cells constitute only 1–2% of the tumor tissue, the remaining tumor microenvironment being composed of various inflammatory cells ,4 and thf debate . Howeverf debate ,8,9. TheTwo important models have been implicated in the genomic instability observed in cancer cells: microsatellite instability (MSI) and chromosomal instability, of which the latter inversely correlates with MSI in hereditary non-polyposis colorectal cancer  and deteOnly a single study has previously investigated the implication of MSI in HL, and concluded that MSI is not involved in the oncogenic process . NeverthThe aim of this study was to investigate mechanisms underlying genomic instability in HL through combined cytogenetic and molecular approaches. We demonstrate, for the first time, the involvement of MSI in HL cell lines. The loss of the protective function of telomeres in NS-HL cell lines induced chromosomal fusions (dicentric chromosome formation) and “breakage-fusion-bridges” (B/F/B) cycles, resulting in a series of chromosomal breaks and duplications, which can lead to chromosome imbalances, gene amplification, non-reciprocal translocation, and altered gene expression. In MC-HL cell lines, the high level of spontaneous double strand breaks (DSB) within and outside telomeres, detected using 53BP1 foci, induced the formation of dicentric chromosomes and the lagging of acentric chromosomes (micronuclei with only telomere sequences) and B/F/B cycles. Transcriptome analysis demonstrated the difference in DNA repair mechanisms between the NS-HL and MC-HL cell lines. Finally, a NS-HL cell line exhibited high radiation sensitivity compared to MC-HL cell line. In addition, we validated our findings in a large cohort of NS-HL and MC-HL patients.TP53 and the clonal homogeneity of these cell lines  showed the presence of 100% red colonies generated from three of the cell lines , indicating the nonfunctional status of ll lines . Sequencangement . We observed telomere shortening (less than 6 kb) in three cell lines  A. Only KThe use of Q-FISH permits the analysis of telomere aberrations: telomere loss, telomere deletions, and interstitial telomeres in metaphases. We observed a high rate of telomere loss and deletion in the HL cell lines and a lymphoblastoid cell line (REMB) with short telomeres C. NevertAll HL cell lines demonstrated numerical chromosomal aberrations A. In theThe second common characteristic was the presence of clonal and sporadic dicentric chromosomes B. Only tJAK2-specific FISH revealed the presence of several gene copies  and found the subtelomeric sequences of 15q to be in the breakpoint of the dicentric chromosome A,B, showWe used the same approach to study the MC-HL cell lines, and lack of sub-telomeric sequences in dicentric chromosome breakpoints was observed also in L1236 and KHM2 cells . NeverthClonal dicentric chromosomes in KMH2 cells were characterized by a specific configuration with the pericentrometric breakpoints and the two centromeres in close proximity . In addiThe clonal structural chromosomal aberrations identified in each cell line are summarized in Numerical chromosomal aberrations were present in all HL cell lines, with a higher frequency of gains than losses. We observed the loss of chromosome Y in four cell lines derived from men. Chromosome 13 was lost from five cell lines and chromosomes 15 and 8 from two. There were gains for chromosomes 19 (6/7), 9(6/7), 5 (5/7), 11 (5/7), 18 (5/7), and 17 (4/7) .MYC, in all the NS -HL cell lines (JAK2 in 6/7 cell lines. HDLM2 exhibited a particularly high copy number for JAK2 (eight copies)  , includiTP53 mutation nor MSI instability and telomere shortening in the HL cell lines. Nevertheless, the NS HL cell lines with high MSI showed a high frequency of CCRs and B/F/B aberrations. In addition, KMH2, a MC HL cell line, showed a high frequency of CCRs relative to L1236 cells. There was no correlation between chromosomal instability and p < 10−16; p < 10−16; p < 10−9, respectively) and (2) telomere length, telomere loss, and telomere deletions in NS-HL and MC-HL patients  (p < 10−5). These characteristics reflect our observations in the HL cell lines and the long telomeres observed in KMH2 cells.We examined telomere dysfunction in circulating lymphocytes from 100 NS-HL and 23 MC-HL patients, as previously published ,22, to vctively) . InteresWe restricted the transcriptome analysis in this study to the comparison of NS HL with MC HL cell lines. We found 3,755 transcripts and 2,415 unique genes to be differentially expressed between the NS-HL  and MC-HL (L1236 and KHM2) cell lines . The affp = 0.005)  .MRE11 by immunofluorescence.DNA repair ontology and the affected pathways are part of a Reactome pathway . DNA repThe presence of two different mechanisms underlying genomic and chromosomal instability in HL cells suggests that the existence of a different pathway in DNA repair may be an important biological event that influences treatment resistance in HL. Therefore, we explored the radiation sensitivity of the HL cell lines. The HL cell lines and two lymphoblastoid B cell lines (NAD and REMB) were exposed to ionizing radiation and two approaches were used to assess radiation sensitivity: (a) apoptosis, including cell cycle arrest after 6Gy of irradiation by FACs analysis, and (b) mRNA expression of PUMA, P21, ZMAT3, Sesterin1 (SESN1), and CCNG1 at two and 24 h after 2Gy of irradiation . These gThe mechanisms underlying genomic instability and the primary transforming events of HL are still obscure ,9. In thWe report, for the first time, that MSI is involved in HL. We confirmed the results obtained for L1236 concerning the absence of MSI ,26,27.TP53 status of HL has been the subject of debate and remains controversial [TP53 mutations in lymph nodes contrasts with the levels of its expression which are often high [TP53 in three cell lines and DNA sequencing revealed the same mutations previously described in L1236, HDLM2, and L428 [TP53 status in HL using a sensitive technique reinforces the concept that TP53 mutation may be involved in the pathology of some cases of HL [TP53 status in lymph nodes derived from relapsing or refractory HL patients. The correlation between TP53 status and high-grade progression in B-cell lymphoma has been previously established [TP53 mutation were characterized by high MSI. For MC-HL, we did not detect MSI in the L1236 cell line, which carries mutated TP53, and conversely, we detected a high frequency of MSI in the KMH2 cell line, without a TP53 mutation.The oversial . The lacten high . The disten high . In thisand L428 . This rees of HL  and perhes of HL  or HL asablished ,32, as wChromosomal instability, a second mechanism of genomic instability, has been defined as an accumulation of point mutations, translocations, chromosomal gains and losses, telomere shortening, and defects in the nuclear architecture, which may cause genome instability .Here, we demonstrate the involvement of telomere dysfunction in chromosomal instability in HL. We first confirmed previous published data using seven HL cell lines and a large cohort of HL patients ,15 and vSecond, we demonstrate, for the first time, a significant difference between telomere length in NS-HL and MC-HL patients and cell lines. MC-HL patients showed high heterogeneity and a subset of patients showed long telomeres, as well as the KMH2 cell line. All NS-HL patients and cell lines presented telomere shortening associated with a high frequency of telomere aberrations (loss of one or two telomeres). Mononuclear cells presented longer telomeres than RS cells, but a higher frequency of telomere loss. We speculated that the loss of telomere integrity of mononuclear cells in NS-HL cell lines may represent the first step in the ongoing process of chromosomal instability. Such telomere dysfunction induced the formation of dicentric chromosomes, which have been observed in oncogenic processes, particularly hematological disorders .Jak2 in the HDLM2 cell line. The findings reported here suggest that chromosomal instability in NS-HL cell lines can arise as a consequence of telomere dysfunction in the early stages of HL tumorigenesis. HRS cells, the end of the proliferative stage, were characterized by telomere shortening, the accumulation of complex chromosomal rearrangements, and fewer telomere aberrations, resulting in a stable stage. Of note TP53 mutation, detected in L428 and HDLM2 cell lines, play a role in telomere shortening as well as the accumulation of chromosomal rearrangements and chromosomal instability [In the HDLM2 and L428 cell lines, we identified the subtelomeric sequences at the breakpoint of clonal dicentric chromosomes, in addition to the presence of interstitial telomere sequences. These dicentric chromosomes developed into large chromatin bridges connecting the daughter cells. The chromatin bridges did not appear to give rise to micronuclei , but contability ,38,39.TP53 mutations plays a major role in telomere dysfunction [Nevertheless, the mechanisms of dicentric formation in MC-HL cell lines are not related exclusively to telomere dysfunction. The lack of sub-telomere sequences at the dicentric breakpoints which were usually localized to the pericentromeric region, discards the possibility that telomere dysfunction exclusively induced the formation of dicentric chromosomes in the MC HL cell lines. We propose that spontaneous DNA DSBs detected by IF-FISH and associated with a higher frequency of 53BP1 foci, were produced in pericentromeric regions due to cellular metabolism or a genetic program, and induced the formation of dicentric chromosomes with specific configurations. Nucleoplasmic bridges without signals associated with the presence of micronuclei with only telomere sequences  may lend support to this hypothesis. These dicentric chromosomes contributed to the subsequent accumulation of CCRs and alterations related to B/F/B cycles. This profile was found in KMH2 cells. However, in L1236, the function  and the Dicentric chromosomes that have been observed in oncogenic processes, in particular in hematological disorders ,41,42,43TP53 status and MSI and thus the type of HL cell line. We demonstrate, for the first time, the radiation sensitivity of NS-HL cell lines and confirm the relevance of radiation therapy in HL treatment [The response to ionizing radiation is associated with the reatment . The L54reatment . Neverthreatment .Transcriptome analysis confirmed this difference between the NS and MC HL cell lines with respect to DNA repair processes, telomere maintenance, and cell cycle and apoptotic process.The human HL-derived cell lines L428 , L591 5, SUP-HD1Peripheral blood lymphocytes were obtained from 100 patients with NS HL and 100 healthy donors following informed consent . The col2 in RPMI 1640 medium  supplemented with 10% fetal bovine serum  and 1% antibiotic-antimycotic (Gibco).The HL cell lines and in vitro Epstein-Barr virus (EBV) infected cell lines NAD, REMB, G36, and RV10 were cultured at 37 °C in an atmosphere containing 5% CO2, in a humidified atmosphere to block them in metaphase. After harvesting the cells and centrifugation for 7 min at 1400 RPM at room temperature, the supernatant was removed and the cells were re-suspended in a solution of warm (37 °C) 0.075 M potassium chloride (KCl)  and incubated for 20 min in a 37 °C water bath (hypotonic shock). For pre-fixation of the cells, approximately five drops of fixative (3:1 ethanol/acetic acid) was added to each tube under permanent agitation and the tubes centrifuged for 7 min at 1400 RPM at room temperature. The supernatant was removed and the cells suspended in fixative solution and centrifuged using the same parameters. After two additional rounds of these fixative steps, the cells were stored in fixative solution at 4 °C overnight and metaphases were spread on cold, wet slides the next day. Slides spread with metaphases were dried overnight at room temperature and stored at −20 °C until further use.Cells were exposed to colcemid (0.1 µg/mL)  for 2 h at 37 °C, 5% COImmuno-phenotyping of Hodgkin lymphoma cell lines was performed on a LSRII flow cytometer (BD) using FITC- and APC-conjugated mouse anti-human specific monoclonal antibodies for CD30 (Ber-H83) and CD15 (HI98), respectively. Both antibodies were obtained from BD Biosciences . Staining for CD15 and CD30 was performed according to the manufacturer’s instructions. After the addition of 1 μg/mL Hoechst  to discriminate dead cells, the cells were analyzed by FACS as previously described.Cell-cycle fractions were determined by Hoechst nuclear staining. Briefly, cells were harvested, washed in PBS, and incubated in Hoechst solution for 30 min at room temperature. Data were collected using a FACSCalibur flow cytometer (BD Biosciences) and analyzed using FlowJo Version 7.5.5 . The results represent the mean value of three independent experiments.+ cells, leading to the generation of white colonies. Thus, colonies containing wild-type TP53 were white (Ade2+) and those containing the TP53 mutation red. After identification of non-functional TP53 samples, red colonies were cloned and sequenced by the Sanger method to characterize the TP53 mutation. Finally, we classified the mutations using the IARC p53 database according to their impact on the protein.To study the functionality of the TP53 gene, a functional assay was performed in accordance with the method described by Flaman et al. . The assThe Revised Bethesda Guidelines were used to determine the MSI status. Analysis of MSI was performed by five-plex polymerase chain reaction (PCR) using a panel of five quasimonomorphic mononucleotide repeat markers  that map to intron 15, intron 16, the 5′ untranslated region (UTR), 3′ UTR, and 3′ UTR of MSH2, c-kit, SLC7A8, transmembrane protein precursor B5, and ZNF2 genes. A single 5-plex polymerase chain reaction (PCR) allowed co-amplification of all five markers, which were subsequently analyzed using a genetic analyzer  and computerized fragment analysis for each of the five primer pairs used in the PCR.The denatured PCR products were separated by capillary electrophoresis using the genetic analyzer and were further analyzed using commercially available software . HL cell lines with instability of three markers were defined as MSI-H, those with < 3 as MSI-L, or MSS if instability was not identified .l-lysine-coated glass slides at 700 rpm for 4 min, fixed with 10% formalin for 10 min, and treated with 0.25% Triton X-100 solution for 10 min. After blocking with 5% bovine serum albumin , the cells were incubated overnight in 4 °C with primary antibody. After washing in PBS, cells were treated with Cyanine 3 anti-mouse IgG  or FITC anti rabbit  secondary antibody at 37 °C for 45 min. cells were mounted with p-phenylene diamine after counterstaining with 4,6-diamidino-2-phenylindole (Sigma-Aldrich). As a negative control, staining was carried out in the absence of primary antibody.Cells were cytospun onto poly-g after 5 h of colchicine (0.09 mg/mL) treatment at 37 °C in a humidified atmosphere of 5% CO2. The pellet was washed in 1× PBS at 37 °C and re-centrifuged. The cells were subjected to hypertonic shock by resuspension in 34 mM citrate at 37 °C to obtain a suspension of cells at a concentration of 60,000 cells/mL and incubated for 1 h at 37 °C. The suspension (200 μL) was subsequently applied to polylysine slides by cytospin. Following fixation  3%, sucrose 2%), cells were immunostained as described. Prior to telomere hybridization with the PNA probe (CCCTAA)3-FITC, cells were successively fixed , washed in PBS, and dehydrated (50/70/100 ethanol).IF-FISH was performed using a protocol similar to one described previously ,64. CellTelomere quantification was performed using the Q-FISH technique with a Cy-3-labelled PNA probe specific for (TTAGGG) . Two approaches were developed. The first approach consisted of the quantification of telomere length in interphase cells, permitting the investigation of intercellular variation in a large number of scored cells. Quantitative image acquisition and analysis were performed using Metacyte software . The mean fluorescence intensity (FI) of telomeres was automatically quantified in 10,000 nuclei on each slide. Settings for exposure and gain remained constant between captures. The experiment was performed in triplicate. The second approach consisted of quantifying telomere length in metaphases using automated acquisition module Autocapt software  and a ZEISS Plan-Apochromat 63×/1.40 oil and CoolCube 1 Digital High Resolution CCD Camera with constant settings for exposure and gain. The mean telomere length was measured and telomere loss and telomere doublet scoring were performed. This approach allows the study of intra-cellular variation of telomere length. The experiment was performed in triplicate. Telomere length, measured as mean fluorescence intensity (FI), strongly correlated with telomere length measured by Southern blot analysis using the telomeric restriction fragment (TRF). The mean telomere length is expressed in kb.Cytogenetic slides were subjected to telomere and centromere  staining followed by M-FISH  technique. The protocol was described previously ,52.Micronucleus assay was performed in the absence of cytochalasineB. The protocol was previously described . In addiTRF1, TRF2 and Ku 86 expressions were assessed by western blotting on lysates of HL cell lines. Cells were sonicated in 500 µL of a buffer containing 8 M urea, 150 mM β-mercaptoethanol, 50 mM Tris-HCl (pH 7.2) and centrifuged for 30 min at 4 °C to remove cellular debris. Samples were subjected to electrophoresis on 12% (p53 and Ku) or 6% (DNA-PKcs) SDS–polyacrylamide gels, blotted onto nitrocellulose membranes, and developed using the ECL system . To verify that equivalent amounts of each sample were loaded, the filters were additionally probed with anti-actin antibody . Densitometry was performed to evaluate the intensity of Ku70, p53, DNA-PKcs, and actin bands.6 cells were irradiated at 6Gy and harvested 24-h after irradiation. The protocol was previously published [To evaluate the sensitivity of cell lines to apoptosis, 10ublished .Cell-cycle fractions were determined by Hoechst nuclear staining. Briefly, cells were harvested, washed in PBS, and incubated in Hoechst solution for 30 min at room temperature. Data were collected using a FACSCalibur flow cytometer (BD Biosciences) and analyzed using FlowJo Version 7.5.5. The results represent the mean value of three independent experiments.Basal and post-irradiation expression of five radiation-responsive genes  was determined by quantitative real-time PCR in HL cell lines at 2 h and 24 h after 2Gy irradiation. The presented data show the rapport between basal gene expression and that after exposure. The protocol was previously published .Total RNA was extracted from frozen HL cells and controls (REMB lymphoblastoide cell line) using the AllPrep DNA/RNA/protein Mini kit , quality-controlled using a 2100 BioAnalyzer , and quantified using a Nanodrop 2000c spectrophotometer . The WT Expression Kit  was used to prepare cDNA from 10 μg purified cRNA, originally synthesised and purified from 0.25 μg of total RNA, following the manufacturer’s instructions.Micronucleus (MN) scoring was performed after telomere and centromere staining. Automated scoring of mononuleated cells was performed using Metafer 4 image analyser software  and a Zeiss Axioplan 2 imager . Ten thousand mononucleated cells per culture  were processed in the image analyzer. Following telomere and centromere staining, MN were classified based on the detection of centromeric regions and telomeric sequences. We detected MN with telomeric and centromeric sequences , MN with only telomeric sequences (acentric chromosome or chromatid fragments), and MN without any sequences . In addition, nucleoplasmic bridges (NPBs) were assessed following telomere and centromere staining (dicentric chromosomes caused by telomere end fusion) or without (dicentric chromosomes resulting from misrepair of DNA strand breaks). Nuclear buds (NBUDs) were also assessed to study the process of elimination of amplified DNA possibly generated via BFB cycles (with telomere sequences or without) or the process of elimination of excess chromosomes may occur in polyploidy cells to facilitate aneuploidy rescue. The scoring of chromosomal aberrations was performed after telomere and centromere staining to score dicentric chromosomes and various types of acentric fragments with four telomeres (resulting from a fusion event), two telomeres, representing terminal deletions, and acentric fragments without any telomere sequence, representing interstitial deletions. Karyotype analysis was then performed using the M-FISH technique on the same slide. The introduction of telomere and centromere staining in the establishment of complex karyotypes allows easy detection and robust classification of chromosomal abnormalities. Complex chromosomal rearrangements were defined as those chromosomes involved in three or more chromosomal exchanges.Similarly BFB chromosomes were defined as those chromosomes that were involved in multiple cycles of repetitive fusions and breakage following the loss of a telomere .Metaphase images were acquired using automated acquisition module Autocapt software  and a ZEISS Plan-Apochromat 63×/1.40 oil and CoolCube 1 Digital High Resolution CCD Camera. The analysis was carried out using Isis software . Automatic scoring of MN following TC staining was performed using MN-score software . Spectral karyotype analysis was performed using Isis software .Linear regression, supported by the least squares method and the Fisher test, was performed to describe the relationship between the chosen factors. The normality of signal distribution was verified with the Shapiro-Wilk test. Because of the significant departure from normality, the non-parametric tests: Wilcoxon rank sum test and exact Wilcoxon-Mann-Whitney test were used to compare telomere length, telomere loss and telomere deletion in NS-HL and MC-HL patients versus healthy donors.For transcriptome analysis, the KMH2 and L1236 cell lines (MC) were considered as one group (6 samples) and L428, L591, SUP, HDLM2 cell lines as another . The Shapiro-Wilk test was applied to check on distribution normality. The hypotheses on the equality of median values between groups were tested by nonparametric exact Wilcoxon-Mann-Whitney test due to non-normal signal distributions. Benjamini-Hochberg procedure was used to control False Discovery Rate caused by multiple testing. For each transcript, the effect size (ES) translated to the Spearman’s r correlation coefficient was calculated using a formula dedicated for non-normal distribution and the U-Mann-Whitney test . AccordiHere, we provide the first evidence for the involvement of MSI to the genomic instability observed in HL. The second main finding in this study is the existence of two different mechanisms that effect chromosomal instability in HL, reflecting the clinical differences observed in terms of remission and survival of NS and MC-HL patients . This stIn the MC HL cell lines, we demonstrate that chromosomal instability is not only related to telomere dysfunction, but that dicentric formation was induced through a specific non-homologous end joining (NHEJ) pathway of DSB repair, essentially in the pericentromeric region. These processes involve the formation of specific dicentric chromosomes with both centromeres in close proximity. This may result from difference in radiation sensitivity. In line with HL cell lines findings, we report telomere dysfunction in circulating lymphocytes from NS-HL patients and high telomere heterogeneity in MC-HL patients. Altogether, our results suggest that telomere dysfunction in SN-HL induces chromosomal instability necessary for HL initiation.


Muehrcke's lines are a type of apparent leukonychia that are common in patients receiving chemotherapeutic agents. They are self‐limited, and there is no need to more workup. Muehrcke's lines are a type of apparent leukonychia that are common in patients receiving chemotherapeutic agents. They are self‐limited, and there is no need to more workup. There was no significant point in the past medical history. The laboratory data including renal function test, liver function test, and serum albumin levels were normal. Treatments with acitretin 25 mg daily were initiated. After 5 months of treatments, paired transverse white lines were noted involving all the fingernails and made patient anxious. On each finger, there were two pale lines 1 mm in width, parallel to the lunula with regular border. The lines were disappeared with pressing on the nail plate, and they did not move distally during nail growth (Figure After 2 months of discontinuation of acitretin, patient represented with white lines faded Figure B.3Firstly, Muehrcke described a nail condition as two parallel lines of pallor in the nail bed in the patients with low serum albumin,Muehrcke’s lines are double white band parallel to the lunulaThe most important differential diagnosis is Mee’s line in which there is disturbance in nail plate but not in nail bed.Muehrcke's lines could be present in the absence of hypoalbuminemia.This condition is asymptomatic and resolves after drug withdrawal.The authors declare that they have no competing interests.FM: designed and collected the data and wrote, read, and approved the final manuscript. FD: collected the data and wrote, read, and approved the final manuscript.This study does not require any approval from the ethics committee.The authors have consent for publication.


The aggressive B-cell non-Hodgkin lymphomas diffuse large B-cell lymphoma (DLBCL) and mantle cell lymphoma (MCL) are characterised by a high proliferation rate. The anaphase-promoting complex/cyclosome (APC/C) and its co-activators Cdc20 and Cdh1 represent an important checkpoint in mitosis. Here, the role of the APC/C and its co-activators is examined in DLBCL and MCL.The expression and prognostic value of Cdc20 and Cdh1 was investigated using GEP data and immunohistochemistry. Moreover, the therapeutic potential of APC/C targeting was evaluated using the small-molecule inhibitor proTAME and the underlying mechanisms of action were investigated by western blot.We demonstrated that Cdc20 is highly expressed in DLBCL and aggressive MCL, correlating with a poor prognosis in DLBCL. ProTAME induced a prolonged metaphase, resulting in accumulation of the APC/C-Cdc20 substrate cyclin B1, inactivation/degradation of Bcl-2 and Bcl-xL and caspase-dependent apoptosis. In addition, proTAME strongly enhanced the anti-lymphoma effect of the clinically relevant agents doxorubicin and venetoclax.We identified for the first time APC/C as a new, promising target in DLBCL and MCL. Moreover, we provide evidence that Cdc20 might be a novel, independent prognostic factor in DLBCL and MCL. Although the standard-of-care regimen R-CHOP  has significantly improved the survival rates, MCL remains incurable and up to one-third of the DLBCL patients are or become refractory.4 However, these agents induce severe toxicity and are associated with multidrug resistance development.5 Clinical studies are currently ongoing to find more selective cell cycle targets by blocking either the interphase (e.g. Cdk inhibitors) or mitotic entry .6 In B-cell NHL, the anti-lymphoma effects of these agents were either disappointing or the trials were suspended early due to toxicity issues.9 Recent preclinical studies show that targeting molecules involved in the mitotic exit, such as the anaphase-promoting complex/cyclosome (APC/C), is a better strategy since it provokes a more permanent mitotic arrest, thereby reducing the chance of mitotic slippage and thus enhancing mitotic cell death.10 The activation of the APC/C, an E3-ubiquitin ligase, depends on the interaction with its co-activators Cdc20 or Cdh1. The spindle assembly checkpoint (SAC) prevents APC/C activation in early mitosis. When bi-orientation of the chromosomes is achieved in the metaphase, the SAC pathway is inactivated and APC/C–Cdc20 binding occurs. This interaction leads to proteasomal degradation of the substrates cyclin B1 and securin, subsequently allowing the onset of the anaphase.11 Cdc20 is thereafter replaced by Cdh1 and the APC/C–Cdh1 targets Cdc20, aurora kinases and Plk-1 during mitotic exit. In early G1 phase, Skp2 and mitotic cyclins are degraded, resulting in increased p21, p27 and cyclin D levels, which is necessary to maintain the G1 phase.12The clinical use of cell cycle targeting agents, such as the microtubule-targeting agent vincristine, has proven that this is an interesting approach in the treatment of high-grade B-cell NHL.13 Wang et al. were the first to demonstrate APC/C mutations in human cancer cells, showing that a disruption of some of the APC/C subunits contributes to tumorigenesis by deregulation of key cell cycle regulators.14 Increased Cdc20 expression is correlated with poor prognosis in several human cancers,21 and depletion of Cdc20 in various cancer cell lines resulted in tumour metaphase arrest and induction of apoptosis.23 Moreover, inhibition of Cdh1 has been associated with tumorigenesis and decreased Cdh1 expression is observed in breast and colon cancer.25 The small molecule TAME (tosyl-L-arginine methyl ester) was discovered as a specific APC/C inhibitor in the past decade. It mimics the IR-tail of the co-activators and blocks the interaction between APC/C and Cdc20 or Cdh1.26 Previously, we and others demonstrated that proTAME (permeable variant of TAME) treatment results in a reduced viability, a growth arrest and apoptosis of malignant plasma cells.27Several studies have already suggested the involvement of the APC/C and its co-activators in tumorigenesis of different cancers and their potential as a new therapeutic target.Currently, however, little is known about the therapeutic potential of targeting the APC/C and its co-activators in DLBCL and MCL. The aim of this study is to investigate the expression of the APC/C co-activators Cdc20 and Cdh1 and the therapeutic potential of APC/C targeting in DLBCL and MCL.29 , GSE5631530 , GSE1645531 (containing gene expression data of 7 indolent and 15 aggressive MCL patients) and GSE3613332 (containing gene expression data of 13 human DLBCL cell lines and 5 MCL cell lines) were used. Raw CEL files were obtained from the Gene Expression Omnibus (GEO) and gcrma-normalisation was performed in R using bioconductor. Survival analysis of GEP microarray data was done using Genomicscape (http://genomicscape.com). The following probe sets were used: 202870-s-at (Cdc20) and 209416-s-at (Cdh1).The publicly available GEP datasets GSE1084633 All samples were stained with Cdc20 antibody  using an automated immunostainer Benchmark XT . As a positive control, tonsils with secondary follicles were used. Two hundred tumour cells were counted and the percentage of tumour cells with a staining of any intensity was determined at a magnification ×400 on a Leica DM2000 microscope. The counting was performed in hot-spot areas by means of an ocular grid. Based on the R-IPI of the DLBCL patients, they were divided into good and poor prognosis groups. Cells were scored MYC and Bcl-2 positive as we described previously.33Patient samples were collected at the Department of Pathology in Brussels  and the Department of Biopathology in Montpellier . Paraffin-embedded samples available from seven DLBCL patients were selected in Brussels and 3-μm-thick sections from tissue microarrays containing three representative 0.6-mm cores of routinely processed tissues from 27 DLBCL patients were included from Montpellier.2atmosphere. All cell lines were obtained from ATCC and regularly tested for mycoplasma contamination. They were authenticated by STR profiling.All the MCL  and ABC–DLBCL cell lines (U2932 and RI-1) were maintained in the RPMI-1640 medium  supplemented with 10% foetal calf serum (FCS)  and 2 mM glutamine . The GCB–DLBCL cell lines  were maintained in the IMDM medium (Life Technologies) supplemented with 10% FCS and 2 mM glutamine. Cells were cultured at 37 °C in a humidified 5% COThe APC/C inhibitor proTAME was obtained from R&D Systems . Venetoclax and rituximab were obtained from Selleckchem , doxorubicin hydrochlorate was purchased from Sigma-Aldrich  and apcin was kindly provided by Dr. R.W. King .p-value of p < 0.05 was considered significant) was determined by a Mann–Whitney U test (to compare two groups) and a one-way ANOVA with Bonferonni correction for multiple testing.Prognostic significance of Cdc20 and Cdh1 gene expression was calculated using the MaxStat R package. Statistical differences in overall survival were calculated by a log-rank test and survival curves were plotted using Kaplan–Meier method. Multivariate analysis was performed using the Cox proportional hazards model. Graphical and statistical analysis was performed using GraphPad Prism 5.01 software. Statistical significance (More details on the ‘Methods' section are described in n = 33), ABC–DLBCL (n = 190), GCB–DLBCL (n = 212) and unclassified DLBCL patients (n = 67). The gene expression levels of Cdc20 were significantly increased in all DLBCL subtypes compared with the B-cell samples. In contrast, the Cdh1 gene expression levels were not significantly different in the GCB–DLBCL and ABC–DLBCL patients compared with the B-cell samples  datasets. The expression of Cdc20 and Cdh1 mRNA was assessed in DLBCL patients, using GEP data from B-cell samples   and molecular DLBCL subtypes. Univariate COX analysis demonstrated that all these factors have a prognostic value in DLBCL Table . In the 42) Fig. . Consist42) Fig.  nor doub42) Fig. . TogetheNext, the APC/C was investigated as a potential target in DLBCL and MCL using human cell lines. Therefore, the Cdc20 and Cdh1 expression was first determined using gene expression profiling data of a large panel of DLBCL and MCL cell lines. Similar to the primary patient samples, we found a significantly higher Cdc20 mRNA expression in both the DLBCL and MCL cell lines compared with B-cell samples and a lower Cdh1 mRNA expression .ine Fig. . In contine Fig. . To furtine Fig. . These f35 The effect of proTAME treatment on the expression and phosphorylation of Mcl-1, Bcl-2 and Bcl-xL was evaluated during mitotic arrest by western blot. No clear difference in the total Bcl-2, Bcl-xL and Mcl-1 protein was observed. Nevertheless, consistent with the prolonged activation of Cdk1, an increase in phosphorylation of Bcl-2 and Bcl-xL was observed during the mitotic arrest  of both agents were used to test the combinatory effect 44 and apcin.45 The small molecule proTAME is the only true/selective APC/C inhibitor known so far.26 ProTAME has already been described to cause a prolonged metaphase in cancer cells.26 Consistent with these observations, synchronised DLBCL and MCL cells released into proTAME treatment were found to accumulate in the G2/M phase. Western blot analysis of these cells demonstrated an increase in cyclin B1 protein levels during this mitotic arrest, while Skp2 levels are not or only slightly affected. The induced G2/M-phase arrest was further investigated, and a significant increase in the percentage of cells in the metaphase was observed. It is known that cells in a prolonged metaphase can either undergo cell death or re-enter the cell cycle to exit mitosis. Cdc20 is believed to be a critical factor in this decision-making. When Cdc20 levels increase during the metaphase, cyclin B1 will be degraded, resulting in Cdk1 inactivation and mitotic exit.46 On the other hand, a prolonged activation of the cyclin B1–Cdk1 complex occurs when cells are arrested in the metaphase, resulting in the phosphorylation and inactivation of Bcl-2 and Bcl-xL, and subsequent activation of the intrinsic apoptotic pathway.48 Consistent with the above, the proTAME-induced prolonged metaphase in DLBCL and MCL cells is accompanied by a dose-dependent decrease in viability and increase in caspase-mediated apoptosis. Western blot analysis confirmed the phosphorylation of both Bcl-2 and Bcl-xL. In contrast, Mcl-1 and Cdc4 levels remained mostly unchanged during the mitotic arrest, indicating that the Cdc4–Mcl-1 axis is most likely not involved in proTAME-induced cell death. These findings provide evidence that APC/C targeting using proTAME induces a metaphase arrest in DLBCL and MCL cells, resulting in the accumulation of cyclin B1 and a prolonged activation of Cdk1, followed by phosphorylation of Bcl-2 and Bcl-xL and eventually cell death.The therapeutic potential of targeting the APC/C, either by blocking the activation of the complex or the co-activators, has been examined in cancer using proTAME,36 ProTAME blocks the interaction between the APC/C and its co-activators, while apcin inhibits the interaction between the co-activators and their substrates.36 This combination resulted in a synergistic anti-lymphoma effect in all cell lines. Other studies in myeloma also showed a combinatory effect with the anti-myeloma agents melphalan, vincristine, etoposide and the topoisomerase II inhibitor doxorubicin.27 Topoisomerase IIα is identified as a Cdh1 substrate and both proTAME and knockdown of Cdh1 significantly enhance the sensitivity of cancers cells to topoisomerase II inhibitors.49 Consistent with these findings, proTAME was also found to sensitise all lymphoma cell lines tested to doxorubicin. Combining proTAME with rituximab did not demonstrate any additional effect. This could be explained by the minor effects that rituximab alone had on the different cell lines in vitro. Consistently with our data, others also observed no or only minor effects when using rituximab as monotherapy in DLBCL cell lines.50 The anti-lymphoma effect of proTAME combined with a novel agent in clinical development in DLBCL and MCL, namely the selective Bcl-2 inhibitor venetoclax, was also tested. ProTAME strongly and significantly potentiated the anti-lymphoma activity of venetoclax in all cell lines tested, even in the highly sensitive ABC–DLBCL cell line U2932.To further ensure that cells will not re-enter the cell cycle after the metaphase arrest induced by proTAME, combination studies were performed. Previously, we and others demonstrated that inactivation of the APC/C is more effective when multiple protein interactions are simultaneously disrupted using proTAME and apcin.26 These enzymes can be present in the bloodstream, thus limiting the in vivo use of proTAME. A possible solution could be to pack (pro)TAME in a liposomal vesicle, such as the vesicle used for liposomal doxorubicin in the treatment of aggressive B-cell lymphoma.51 Another general concern is that inhibition of the APC/C will most likely affect all dividing cell types. To avoid these off-target effects, liposomal (pro)TAME could be altered, so that it can only be recognised by CD20+B cells. This would give the advantage of specifically targeting the lymphoma cells like rituximab.To further validate the therapeutic potential of targeting of APC/C in DLBCL and MCL, we acknowledge that in vivo studies are required. Unfortunately, when it comes to the in vivo use of proTAME, there are some concerns. ProTAME has not yet been optimised for in vivo studies, since this prodrug is activated intracellularly by esterases and the active form (TAME) is not cell permeable.In conclusion, this study demonstrates that high Cdc20 expression correlates with a poor prognosis in the aggressive B-cell malignancy DLBCL. We also provide evidence that APC/C targeting in DLBCL and MCL cells results in a prolonged metaphase followed by apoptosis and sensitises the lymphoma cells to the clinically relevant agents doxorubicin and venetoclax. Together, this study suggests that the APC/C is a potential new target in these cancer types, making it a promising target for further preclinical research.Supplementary information


For decades, retinoids and their synthetic derivatives have been well established anticancer treatments due to their ability to regulate cell growth and induce cell differentiation and apoptosis. Many studies have reported the promising role of retinoids in attaining better outcomes for adult or pediatric patients suffering from several types of cancer, especially acute myeloid leukemia and neuroblastoma. However, even this promising differentiation therapy has some limitations: retinoid toxicity and intrinsic or acquired resistance have been observed in many patients. Therefore, the identification of molecular markers that predict the therapeutic response to retinoid treatment is undoubtedly important for retinoid use in clinical practice. The purpose of this review is to summarize the current knowledge on candidate markers, including both genetic alterations and protein markers, for retinoid resistance and sensitivity in human malignancies. Defective or aberrant cell differentiation is a hallmark of many human malignancies. The initial step in an aberrant tumor cell phenotype involves various mutations that alter signaling pathways, epigenetic modifiers, and transcription factors, leading to the deregulated expression of proteins required for cell differentiation.During the 1970s and 1980s, as an elegant alternative to killing cancer cells by cytotoxic therapies, several scientific achievements popularized the strategy of inducing malignant cells to overcome differentiation inhibition and to enter apoptotic pathways .In general, differentiation therapy aims to reactivate the endogenous differentiation program in transformed cells to resume the mutation process and eliminate the tumor phenotype. Thus, this strategy offers the prospect of a less aggressive treatment that limits damage to the normal cells in the organism.trans retinoic acid (ATRA), 9-cis retinoic acid (9-cis-RA), and 13-cis retinoic acid (13-cis-RA), have been tested for potential use in cancer therapy and chemoprevention  functions as a transcriptional activator targeting PLZF-binding sites, leading to cellular retinoic acid-binding protein 1 (CRABP1) upregulation. The CRABP1 protein is structurally similar to the cellular retinol-binding proteins, sequesters retinoic acid to limit its access to the nucleus [PLZF-RARA fusion transcript only were sensitive to ATRA treatment under in vitro conditions, and these results correlated with clinical remission after ATRA administration in this patient [Molecular analysis of the possible mechanisms of retinoid resistance suggested that the reciprocal  nucleus , and is  nucleus –51. Simi nucleus , 52, 53. patient . Moreove patient . Further patient . NeverthPML-RARA fusion gene and the therapeutic response to ATRA [PML sequence, which contains heterogeneous breakpoint cluster regions (bcrs) at three different sites E6(−) isoform with exons 5 and 6 deleted, is associated with the ATRA-resistant phenotype [PML-RARA fusion gene might be helpful for predicting a patient’s response to ATRA treatment.Sequencing analysis of the henotype . A subsePML and RARA mRNA fusion partners. Subsequent in vitro studies confirmed these results, revealing that spacer deletion restored ATRA sensitivity [Similarly, APL cells with the V-type splicing isoform, characterized by exon 6 truncation, were also reported to be less sensitive to ATRA treatment. In this group of APL patients, a subset with lower ATRA sensitivity presented with a relatively long “spacer” with a cryptic coding sequence inserted into the joining sites between the truncated sitivity .RARβ expression is downregulated in PDAC [The vitamin A metabolism disturbances that result in a decreased intracellular ATRA concentration were originally described in pancreatic ductal adenocarcinoma (PDAC)  and late in PDAC –68, whichighCRABP2null PDAC lines were resistant to ATRA-mediated growth inhibition and apoptosis and also exhibited an increased migration and invasion phenotype. In contrast, FABP5nullCRABP2high cell lines retained ATRA sensitivity. These results were also confirmed in vivo using xenograft models [ATRA typically induces cell differentiation and growth arrest in most epithelial cell types. However, experiments in Capan-1 cell line have shown that in addition to an antiproliferative effect, retinoids increase cell migration, resulting in an invasive phenotype . This eft models . Immunoht models . Since tBreast carcinoma is a heterogenous disease classified into subtypes according to the expression of biological markers, such as estrogen receptor (ER), progesterone receptor (PR) and epidermal growth factor receptor 2 (HER2) –74. AccoIndeed, several studies have demonstrated that the average RARα receptor level is significantly higher in ATRA-sensitive than ATRA-resistant breast carcinoma cell lines –77. FurtERBB2 transfection in ATRA-sensitive breast carcinoma cells induced ATRA resistance. When Her2/neu was blocked by trastuzumab, the cells exhibiting induced ATRA resistance became ATRA sensitive again. This study also hypothesized that Her2/neu may induce ATRA resistance in breast carcinoma cells by suppressing RARA expression and/or by deregulating the G1 checkpoint of the cell cycle.Another potential marker of ATRA resistance was suggested by a study describing Her2/neu-induced ATRA resistance in breast cancer cell lines . ERBB2 tFABP5 is preferentially upregulated in estrogen receptor-negative (ER-) and triple-negative breast carcinoma cells (TNBC), and an increased FABP5 mRNA level is associated with poor patient prognosis and high tumor grade [FABP5 normalized signal intensity scores were categorized into high versus low using cut-off point of 0.768. In this cohort, 61% of patients showed high FABP5 expression and these patients had a significantly decreased survival rate if compared with those with low FABP5 expression. Moreover, FABP5 silencing in Hs578T breast carcinoma cell line resulted in approximately 40% reduction in proliferation activity. However, although breast cancer cells with an increased FABP5/CRABP2 ratio present with increased ATRA resistance, this ratio does not always accurately predict the breast cancer cell response to ATRA, indicating that other factors are also involved in the mechanism of retinoid resistance development. Another recent study identified CRABP1 as the third key player that potentially influences the breast cancer cell response to ATRA. This protein has been identified as a retinoid inhibitor and probably sequesters retinoic acid in the cytoplasm, thereby preventing RAR activation in the nucleus. Similarly to FABP5, CRABP1 is also preferentially expressed in ER- and TNBC tumor tissues that are prone to ATRA resistance [As described in the PDAC section in this review, the abundance of the intracellular retinoic acid transporters CRABP2 and FABP5 within the cell can indicate breast carcinoma cell response to ATRA, since these molecules have been shown to play opposing roles in mediating the cellular response to retinoids . Accordior grade . In thissistance . AccordiThese findings provide molecular tools to predict and eventually overcome ATRA resistance in breast carcinoma therapy. CRABP1 and FABP5 co-expression may serve as a predictive biomarker of ATRA resistance in this tumor type, and the downregulation may be a key step in (re)sensitizing breast carcinoma cells to retinoid therapy. A novel mechanism for resensitizing ATRA-resistant cells to ATRA-mediated apoptosis was recently introduced: the phytochemical curcumin is able to upregulate CRABPII, RARβ and RARγ expression in TNBC cell lines and thereby sensitizes cells to ATRA-induced apoptosis. This reversed resistance to ATRA-induced apoptosis in TNBC cells was dependent on the curcumin dose and treatment length . OverallAnother recent study compared the phosphoproteome and transcriptome of established ATRA-sensitive and ATRA-resistant cell lines derived from breast carcinoma . One of the most interesting results was that ATRA did not regulate the phosphorylation of the same proteins in both cell lines, i.e., the ATRA-resistant cell line exhibited a deregulated kinome. High-throughput sequencing experiments revealed that 80% of the genes regulated by ATRA in MCF7 cells were not regulated in BT474 cells and vice versa. Additionally, 40% more genes were regulated by ATRA in the MCF7 cells than in the BT474 cells. Moreover, this study indicates that ATRA induced RARα phosphorylation in resistant cell lines only, which may cause kinome deregulation and consequences in other intracellular metabolic pathways .Neuroblastoma (NBL) is a neuroectodermal tumor arising from elements of the neural crest and represents the most common extracranial solid tumor in children. In a subset of high-risk NBL patients with minimal residual disease, retinoid administration was proven effective as a part of postconsolidation therapy after intensive multimodal treatment. Unfortunately, approximately 50% of this patient population is resistant to this treatment or develops resistance during therapy . Moreovecis-RA, PBX1 mRNA and protein expression levels are both induced in 13-cis-RA-sensitive cell lines only. After treatment with 13-cis RA, all 6 RA-sensitive cell lines showed a significant increase in PBX1 expression, whereas RA-resistant cell lines exhibited no such effect. These studies also revealed that reduced PBX1 protein levels result in an aggressive growth phenotype and 13-cis-RA resistance. Finally, the authors demonstrated that in primary NBL tumor tissue, PBX1 expression correlated with the histological NBL subtype, with the highest PBX1 expression in benign ganglioneuromas and the lowest expression in high-risk NBL [PBX1 belongs to the three-amino-acid loop extension (TALE) family of atypical homeodomain proteins and interacts with other homeodomain-containing nuclear proteins, such as HOX and MEIS, to form heterodimeric transcription complexes. PBX1 is involved in a variety of biological processes including cell differentiation and tumorigenesis , 87. Recrisk NBL .HOXC9 promoter is epigenetically primed in an active state in ATRA-sensitive NBL cell lines and in a silenced state in ATRA-resistant NBL cell lines. Moreover, HOXC9 protein levels were significantly higher in differentiated NBL cells than in NBL cells undergoing ATRA-induced differentiation [Homeobox (HOX) proteins function as regulators of morphogenesis and cell fate specification and are key mediators of retinoid action in nervous system development. Among members of the HOX family of transcription factors, HOXC9 seems to play an important role in neuronal differentiation. A recent study revealed that the ntiation .NF1 knockdown continued to proliferate when exposed to RA in contrast to the control cells. Subsequent experiments showed downregulation of RA target genes in NF1 knockdown cells. These results may indicate the role of NF1 in maintaining RA resistant phenotype.The protein neurofibromin 1 (NF1) is known to antagonize the activation of RAS proteins but is also involved in other signaling pathways, such as the cAMP/PKA pathway . NF1 conNF1 gene were found in 6% of primary NBL representing a subset of cases where the loss of NF1 gene could be caused by gene mutation.In further research, genomic aberrations of the cis-RA (isotretinoin) treatment. Moreover, decreased NF1 and ZNF423 gene expression, reflecting hyperactivated RAS/MAPK signaling, is correlated with a very poor clinical outcome in NBL patients and was detected in 78% of patients with relapsed NBL [ZNF423 mRNA levels and the sensitivity of MPNST cells to ATRA [A connection between NF1-RAS-MEK signaling and retinoic acid action was demonstrated by the finding that the NF1-RAS-MEK cascade suppresses ZNF423 protein expression, which functions as a RAR/RXR coactivator. Additionally, tumors with activated RAS signaling and low ZNF423 expression present with a poor response to 13-psed NBL , whereaspsed NBL . Another to ATRA . These rHMGA2 is completely absent in adult organisms; its expression is restricted to rapidly dividing embryonic cells and tumors with epithelial and mesenchymal origins [HMGA2 was also detected in some retinoid-resistant NBL cell lines. In NBL cell lines, a causal link between HMGA2 expression and retinoid-induced growth arrest inhibition was proven using exogenous HMGA2 expression, which was sufficient to convert HMGA2-negative, retinoid-sensitive cells into retinoid-resistant cells [HMGA1 was found to be expressed at different levels in all NBL cell lines [High-mobility group A (HMGA) proteins function as ancillary transcription factors and regulate gene expression through direct DNA binding or protein-protein interactions and play important functions in controlling cell growth and differentiation.  origins . HMGA2 wnt cells . In contll lines , indicatUNC45A gene, a member of UNC45-like genes, which are evolutionarily highly conserved, and the resulting protein products are involved in muscle development and myosin assembly [UNC45A, another potential marker of retinoid resistance, is a protein encoded by the assembly . The UNCassembly . In NBL assembly . When UNThis review was aimed to summarize the current knowledge, both clinical and experimental, on predictive markers in human cancers that are treated with retinoids as a part of the therapeutic regimen. This review demonstrated that each described cancer type seems to have a unique pattern of altered signaling pathways, resulting in a set of predictive biomarkers that indicate retinoid resistance or sensitivity, which is typical for this malignancy. Many of the research studies mentioned in this review are only initial, and the acquired results require further detailed investigation and clinical validation of the proposed predictive biomarkers. However, these studies demonstrate the promising future for differentiation therapies that use retinoids, especially in identifying reliable markers that predict the response of each individual patient to this type of treatment. Hopefully, the personalized approach will be a new milestone in anticancer differentiation therapy.


The t6A pathway uses two sequentially acting enzymes, YRDC and OSGEP, the latter being a subunit of the multiprotein KEOPS complex. We recently identified mutations in genes encoding four out of the five KEOPS subunits in children with Galloway-Mowat syndrome (GAMOS), a clinically heterogeneous autosomal recessive disease characterized by early-onset steroid-resistant nephrotic syndrome and microcephaly. Here we show that mutations in YRDC cause an extremely severe form of GAMOS whereas mutations in GON7, encoding the fifth KEOPS subunit, lead to a milder form of the disease. The crystal structure of the GON7/LAGE3/OSGEP subcomplex shows that the intrinsically disordered GON7 protein becomes partially structured upon binding to LAGE3. The structure and cellular characterization of GON7 suggest its involvement in the cellular stability and quaternary arrangement of the KEOPS complex.N 6-threonylcarbamoylated adenosine 37 in tRNA (t6A) involves the YRDC enzyme and the KEOPS complex. Here, the authors report mutations in YRDC and the KEOPS component GON7 in Galloway-Mowat syndrome and determine the crystal structure of a GON7-containg subcomplex that suggests a role in KEOPS complex stability.The biosynthesis of N The majority of the complex modifications are concentrated in the anticodon region of the tRNAs and are crucial for accuracy of protein synthesis. The threonylcarbamoylation of the N6 nitrogen of the adenosine at position 37 (t6A) of most ANN-accepting tRNAs represents one of the very few nucleotide modifications that exists in every domain of life3. The t6A biosynthesis pathway consists of two steps: firstly, the YRDC enzyme (Sua5 in yeast) synthesizes an unstable threonylcarbamoyl-AMP intermediate (TC-AMP) and in a second step, the KEOPS protein complex transfers the TC-moiety from TC-AMP onto the tRNA substrate4. Enzymes that synthesize TC-AMP exist in two versions depending on the organism: a short form which only has the YrdC domain (such as human YRDC) and a long form which has an extra Sua5 domain (yeast SUA5 for instance). The eukaryotic KEOPS complex contains five subunits GON7/LAGE3/OSGEP/TP53RK/TPRKB that are arranged linearly in that order8. OSGEP is the catalytic subunit that carries out the TC-transfer reaction and its orthologs are present in virtually all sequenced genomes9. The other subunits are essential for the t6A modification of tRNA, but their precise roles are as yet unknown, especially that of GON7, an intrinsically disordered protein (IDP), which was only recently identified in humans7. Fungal Gon7 was shown to be an IDP that adopts a well-defined structure covering 50% of its sequence upon complex formation with Pcc1 (LAGE3 homolog)8. In humans, GON7 was recently shown to be also structurally disordered in absence of the other KEOPS complex subunits. GON7 was proposed to be a very remote homolog of the yeast Gon7 protein although its structure upon complex formation remains unknown7.Transfer RNAs (tRNA) are subject to multiple post-transcriptional modifications that are important for the stabilization of their ternary structure and the precision of the decoding process10. We recently identified autosomal recessive mutations in genes encoding four of the five subunits of human KEOPS complex, namely LAGE3, OSGEP, TP53RK, and TPRKB in patients with Galloway-Mowat syndrome . GAMOS is a rare neuro-renal disorder characterized by the co-occurrence of steroid-resistant nephrotic syndrome (SRNS) with microcephaly and neurological impairment11. GAMOS is clinically heterogeneous, reflecting a genetic heterogeneity. Indeed, disease-causing mutations have been identified in eight genes to date: four in KEOPS genes and four in other unrelated genes, WDR73, WDR4, NUP133, and NUP107 (refs. 17). SRNS is typically detected in the first months of life and most often rapidly progresses to end-stage renal disease (ESRD) within a few months; however, there are rarer cases with preserved renal function in adulthood18. Kidney lesions range from minimal change disease, to focal segmental glomerulosclerosis (FSGS) that might be of the collapsing type, or diffuse mesangial sclerosis (DMS). Cerebral imaging findings include cerebral and cerebellar atrophy, and gyration and/or myelination defects. These anomalies are associated with neurological deficits such as psychomotor impairment, hypotonia, seizures, and more rarely sensorineural blindness and deafness. Affected children may also present with facial and/or skeletal dysmorphic features. The prognosis of GAMOS is poor, and most affected children die before 6 years of age.tRNA modifications have been demonstrated to play a role in the development of the brain and nervous system, and an increasing number of defects in these modifications are now being linked to various human neurodevelopmental disordersGON7  and YRDC, both genes encoding proteins involved in the biosynthesis of the t6A modification. These data, together with our previous work, show that mutations in genes encoding all the proteins involved in the two chemical steps of t6A lead to GAMOS. Furthermore, we determine the crystal structure of the GON7/LAGE3/OSGEP KEOPS subcomplex showing that GON7 becomes structured upon binding to LAGE3. The structure also explains our observations that GON7 stabilizes the remainder of the KEOPS complex and directs its quaternary organization.Here we present 14 GAMOS-affected individuals from seven families, with mutations in GON7 gene in 11 affected individuals from 5 unrelated families and in the YRDC gene in 3 affected individuals from 2 unrelated families  carried the same homozygous nonsense mutation  which causes a stop codon at position 7 of the protein. These families, all originating from the same region of Algeria, shared a common haplotype at the GON7 locus indicating a founder effect  and a 4-base pair deletion leading to a frameshift . In Family G, we identified a homozygous in-frame deletion of Leucine 265 . For both families, western blot and qPCR analysis on cell extracts from affected children showed the presence of YRDC transcripts and proteins  structural model of human YRDC using the structure of the YRDC domain of the archaeal Sua5  and mapped these mutations onto this in silico model  , and myoclonia. This clinical picture is highly reminiscent of that observed in GAMOS-affected individuals with mutations in LAGE3, OSGEP, TP53RK, and TPRKB13. However, individuals with GON7 mutations presented with milder neurological and renal manifestations, always with post-natal microcephaly and no gyration defects, later onset of proteinuria (median age 18 months vs. 1) and slower progression to ESRD (median age 49 months in 8/11 children vs. 5 months in 3/3 children), and a longer survival compared to the YRDC cases.Early-onset proteinuria was observed in all affected children, with first detection ranging from between birth and 5 years. All but three children reached ESRD between 1.5 months and 6 years of age. All individuals carrying -2) Fig. . Extra-rYRDC mutations, we first used a yeast heterologous expression and complementation assay as previously performed for OSGEP mutations identified in GAMOS individuals13. Indeed, the deletion of SUA5, the YRDC ortholog in S. cerevisiae leads to a very severe growth defect, similarly to the deletion of each of the genes encoding the five KEOPS subunits21. We therefore heterologously expressed the human YRDC cDNAs encoding wild-type (WT) and mutant proteins in the Δsua5 strain to evaluate their ability to rescue the slow growth phenotype. Since the catalytic activity of YRDC does not require protein partners, the WT YRDC protein efficiently complemented the Δsua5 growth defect  and amorphic  alleles, as has been previously shown for OSGEP mutations13. A similar approach could not be applied for GON7 mutations since GON7 failed to complement the growth defect of the Δgon7 yeast strain (see ref. 7 and our data). We therefore measured the t6A content in fibroblasts from two individuals with the p.Tyr7* GON7 mutation, three individuals with YRDC mutations and one individual with the p.Arg325Gln OSGEP mutation. The t6A levels were significantly decreased in both YRDC- and OSGEP-mutated fibroblasts, and to a lesser extent in GON7-mutated fibroblasts  of human KEOPS components tes Fig. . DespiteRNA Fig. . The imping Fig. . By meassis Fig. . Since llls Fig. , even inted Fig. . Altoget6). Based on very weak sequence similarity, it was proposed, that GON7 is a remote homolog of yeast Gon7 (ref. 7). We first investigated the structure of GON7 in solution by collecting a 2D 1H-15N Band-Selective Optimized Flip Angle Short Transient Heteronuclear Multiple-Quantum Correlation (SOFAST-HMQC) NMR spectrum of a 15N-labeled GON7 sample. The poor spectral dispersion in the 1H dimension of the 2D correlation spectrum showed that GON7 lacks well-defined structure, confirming the conclusions of Wan et al.7. Adding non-labeled LAGE3 to the sample, caused the shift and/or disappearance for many crosspeaks, suggesting GON7 interacts with LAGE3 , one can assess qualitative information on the degree of compactness of the scattering object27. The plateau observed for GON7 is characteristic of a fully disordered protein, possibly with very short elements of secondary structure . LAGE3 contains 60 residues at the N-terminus that are absent in the Pcc1 orthologs from yeast and archaea and that are predicted to be disordered. We did not observe any electron density for this N-terminal peptide, confirming this region indeed lacks a stable structure. We cannot exclude however that partial proteolysis removed this peptide during the long crystallization process. LAGE3 is at the center of the complex, binding on one side to OSGEP and on the other to GON7, which does not directly interact with OSGEP  with the scattering curve calculated on the all-atom model built using the program BUNCH from the crystal structure (see Methods)  or good quality 3D models  for all of the KEOPS subunits at our disposal, except for GON7  Fig. . We therGON7 mutations. We first confirmed by mass spectrometry analysis that in a human podocyte cell line stably overexpressing either 2HA-tagged GON7 or V5-tagged LAGE3, the four additional KEOPS subunits significantly co-purified with GON7 or LAGE3, respectively, thus confirming that a five-subunit KEOPS complex is present in these renal glomerular cells  and/or affect the stability of the KEOPS complex (GON7). These results complement our previous findings and establish that mutations in all the genes involved in this pathway lead to GAMOS.In this study, we identified mutations in two genes encoding proteins involved in tGON7 or YRDC mutations present with the clinical features of GAMOS, similarly to the individuals previously reported to have mutations in the genes encoding the four other KEOPS subunits. In addition, we have expanded the GAMOS phenotype spectrum by describing congenital hypothyroidism to be associated with YRDC mutations. Although the two GON7 mutations encode truncated non-functional proteins, we noticed that they unexpectedly result in a less severe clinical outcome compared to that of individuals affected by mutations in other KEOPS subunit genes or in YRDC, for which biallelic null mutations were not found. This suggests that the absence of GON7 has less severe consequences for cell life compared to the other components of the t6A biosynthesis pathway, where four out of six are encoded by genes considered to be essential28. This less severe clinical outcome correlates with our data showing that GON7 loss of function and depletion in fibroblasts and podocytes, respectively, have globally a weaker effect on t6A levels, proliferation, apoptosis, and protein synthesis compared to that of other KEOPS subunits and YRDC mutations or depletion. However, although our data have confirmed that GON7 is a functional homolog of Gon7, the effect of their absence in human and yeast, respectively, is markedly different since in the absence of Gon7, the yeast KEOPS complex has only very low t6A activity and cell growth is dramatically affected3. Altogether, our data in humans suggest that GON7 is not as essential in humans as in yeast for t6A biosynthesis.All individuals bearing 7. The latter stoichiometry has also been observed for the archaeal KEOPS complex, for which no fifth subunit similar to Gon7 has yet been discovered. The Pcc1 subunit constitutes the dimerization unit of archaeal KEOPS29 and this is also very likely the case for the LAGE3 subunit of human KEOPS in absence of GON7 (ref. 7). In line with these results, our structure of the GON7/LAGE3/OSGEP complex shows that GON7 competes with LAGE3 for dimerization, explaining the different stoichiometries of the KEOPS complex observed in the absence or presence of GON7. Indeed, GON7 covers a large hydrophobic surface of LAGE3  quaternary structure as evidenced by the decrease in KEOPS subunits protein levels observed in GON7 patient cells. Taken together, our data demonstrated that GON7 impacts the stability of the KEOPS complex therefore having an effect on its enzymatic activity. This is in line with the in vitro data of Sicheri’s group showing that in presence of GON7, KEOPS t6A activity is potentiated29 and with our in vivo data showing that t6A levels in GON7-mutated patient fibroblasts are slightly decreased compared to YRDC- and OSGEP-mutated fibroblasts.Our biochemical and structural data provide a molecular framework to understand the pathophysiological effects of the GAMOS-associated mutations. The structure of the GON7/LAGE3/OSGEP complex shows that GON7 is bound exclusively to the non-catalytic LAGE3 subunit, distant from the catalytic center of OSGEP. It has been shown in vitro that the intact human KEOPS complex has a 1:1:1:1:1 stoichiometry, in contrast with the complex lacking GON7 which has a 2:2:2:2 stoichiometrygon7 deletion mutant7. Comparison of the GON7/LAGE3 and Gon7/Pcc1 complexes shows that the hydrophobic character of the residues at the interface is very well conserved  methyltransferase that is responsible for the highly conserved m7G modification on a specific subset of tRNAs33. Interestingly, it has been shown that the absence of m7G tRNA modification leads to impaired cell proliferation, neural differentiation, and a decrease in global translation with a less efficient translation of mRNAs involved in cell division and brain development, consistent with the microcephaly and brain anomalies found in individuals with WDR4 mutations34. Similarly, as a consequence of the decrease in t6A levels observed in affected individual’s cells, perturbed protein translation could impact the translation of specific mRNA involved in kidney and brain development and/or podocyte/neuron maintenance. It is likely that the requirement for t6A-modified tRNAs levels is dependent of the cell-type and/or cell cycle as has been previously shown in D. melanogaster where highly proliferative cells of the wing imaginal discs are more affected by the absence of t6A modification than fully differentiated photoreceptors35. Neuronal progenitors that have high mitotic activity probably have higher demands for protein translation, making them more vulnerable to any perturbation in the tight regulation of tRNA modifications. Furthermore, another potential regulatory step to spatiotemporally modulate these tRNA modifications and thus protein translation is the tissue- and developmental stage-specific expression of the tRNA-modifying enzymes37. YRDC and KEOPS subunits could be differentially expressed between specific cell-types in the brain  and the kidney (podocytes) and/or during development/differentiation explaining the tissue involvement and the course of the disease as well as its clinical outcome. Further studies on neuronal/renal progenitors and neurons/podocytes differentiated from induced pluripotent stem cells obtained from individuals with mutations in YRDC and KEOPS subunits will probably provide further insights into the pathogenesis of GAMOS.An increasing number of mutations are being identified in genes encoding tRNA-modifying enzymes that are linked to human neurodevelopmental disorders. Very recently, mutations in 6A biosynthesis pathway in the pathogenesis of GAMOS. Further investigations are needed to fully characterize all the KEOPS mutations at the biochemical, structural, and enzyme activity levels to better understand their impact on KEOPS complex-dependent t6A biosynthesis activity and how they influence the clinical phenotypes. Genes encoding components of the t6A biosynthesis pathway have to be added to the growing list of translation-associated proteins whose loss of function are responsible for rare genetic disorders.Together, our data strongly emphasize the importance and relevance of the tWritten informed consent was obtained from participants or their legal guardians, and the study was approved by the Comité de Protection des Personnes “Ile-De-France II.” Genomic DNA samples were isolated from peripheral blood leukocytes using standard procedures.We performed whole-exome sequencing using Agilent SureSelect All Exon 51 Mb V5 capture-kit on a HiSeq2500 (Illumina) sequencer (paired-end reads: 2 × 100 bases). Sequences were aligned to the human Genome Reference Consortium Human Build 37 (GRCh37) genome assembly with the Lifescope suite from Life Technologies. Variant calling was made using the Genome Analysis Toolkit pipeline. Then, variants were annotated using a pipeline designed by the Paris Descartes University Bioinformatics platform. We assumed the causal variant: (i) segregates with the disease status, (ii) is novel or has a minor allele frequency <1/1000 in gnomAD, (iii) was not found in >10/2352 projects of our in-house database. Missense variant pathogenicity was evaluated using in silico prediction tools . Sanger sequencing was used to validate the variant identified by exome sequencing and to perform segregation analysis in the families. Sequence were analyzed with the Sequencher software  and positions of mutations were numbered from the A of the ATG-translation initiation codon. For Family G, WES, and SNP-array were performed according to standard diagnostic procedures and WES quality criteria at the UMC Utrecht, the Netherlands. The patient–parent quartet WES with sibling-sharing analysis focused on the regions of homozygosity determined by SNP-array (parents are consanguineous in the eighth degree).NheI restriction site, and either two copies of the Human influenza hemagglutinin (HA) tag or one copy of the V5 epitope tag allowing epitope-tagging at the N-terminal of the encoded protein. Human full-length GON7, LAGE3, and YRDC cDNA  were amplified by PCR from IMAGE cDNA clones , and subcloned into the modified pLEX-MCS plasmid using either SpeI and XhoI (for GON7 and LAGE3) or NheI and XhoI (for YRDC). Human YRDC cDNA was also subcloned into the BamHI and SalI sites of pESC-TRP. Site-directed mutagenesis  was used to generate the mutations used in this study. An adapted cloning protocol was used to obtain the C-terminal extension found for the YRDC p.Val241Ilefs*72 mutant. For gene silencing, the shRNA sequences described in Supplementary Table AgeI and EcoRI restriction sites. This vector contains a cassette conferring puromycin resistance. All constructs were verified by Sanger sequencing.The following expression vectors were used in this publication: LentiORF pLEX-MCS (Open Biosystems), pESC-TRP with a c-myc tag (Agilent), and pLKO.1-TRC Cloning vector . The LentiORF pLEX-MCS plasmid was modified by site-directed mutagenesis  to insert one 2 in RPMI 1640 medium supplemented with 10% fetal bovine serum, insulin-transferrin-selenium, glutamine, and penicillin and streptomycin , and human primary fibroblasts, obtained from patient skin biopsies, were cultured in OPTIMEM medium supplemented with 10% fetal bovine serum, sodium pyruvate, glutamine, fungizone, and penicillin and streptomycin  at 37 °C with 7% CO2. Obtention and culture of lymphoblastoid cell lines are detailed in Supplementary Methods. Human podocytes stably overexpressing 2HA-GON7 or V5-LAGE3, or transiently depleted for GON7, LAGE3, OSGEP, or YRDC were obtained by transduction with lentiviral particles and subsequent puromycin selection (2 μg/ml). HEK293T cells (ATCC CRL-3216) were transiently transfected using Lipofectamine® 2000 (ThermoFisher Scientific).The human immortalized podocyte cell line (AB8/13) provided by M. Saleem  was grown at 33 °C with 7% COThe following antibodies were used in the study: mouse anti-α-tubulin , mouse anti-actin , mouse anti-HA , rabbit anti-GON7 , rabbit anti-LAGE3 , rabbit anti-TPRKB , rabbit anti-OSGEP , and mouse anti-GAPDH  from Sigma-Aldrich; mouse anti-V5  from Bio-Rad; rabbit anti-YRDC  from ThermoFisher Scientific; rabbit anti-LAGE3  and mouse anti-OSGEP  from Novus Biologicals; rabbit anti-TP53RK  from Abgent. Secondary antibodies for immunoblotting were sheep: anti-mouse and donkey anti-rabbit HRP-conjugated antibodies , and IRDye 800CW Donkey anti-rabbit (926-32213) and IRDye 680RD Donkey anti-mouse (926-68072) antibodies (LI-COR). Cycloheximide (C7698), Nuclease P1 (N8630), phosphodiesterase I from snake venom (P3243), and alkaline phosphatase (P4252) were purchased from Sigma-Aldrich.38. Media were supplemented with 2% agar for solid media. The S. cerevisiae W303 derived strain, Δsua5::KanMX(YCplac33-SUA5)39, was used as the host for the complementation assay. For each pESC-TRP plasmid derivative to be tested, three independent clones were selected after transformation and grown on GLU-TRP media. Clones were then streaked onto GAL-TRP containing 0.1% 5-fluoroorotic acid (5-FOA) to counter-select the YCplac33-SUA5 plasmid (containing URA3). After two rounds of selection, clones were checked for their acquired Ura- phenotype, their plasmid content was confirmed by sequencing after plasmid rescue before being finally evaluated for fitness by a 10-fold serial dilution spotted onto GAL-TRP minimal supplemented media. Empty pESC-TRP, pESC-TRP-SUA5, and pESC-TRP-SUA5-myc were used as negative and positive controls, respectively.Yeast cells were grown at 28 °C in standard rich medium YEPD  or minimal supplemented media . Cells were transformed using the lithium acetate method® Kit and treated with DNase I. One microgram total RNA was reverse-transcribed using Superscript II, according to the manufacturer’s protocol (Life Technologies). The relative expression levels of the mRNA of interest were determined by real-time PCR using Power SYBR Green ROX Mix (ThermoFisher Scientific) with specific primers listed in Supplementary Table Hgprt). Data were analyzed using the 2−ΔΔCt method.Total mRNA from knocked down podocytes, primary skin fibroblasts, and LCLs was isolated using Qiagen Extraction RNeasy600nm of approximately 3 × 107 cells/ml) with phenol induced cell permeabilization, LiCl-selective precipitation, and subsequent ion exchange-chromatography purification on an AXR-80 column , according to the manufacturer’s instructions. For human primary fibroblasts, the two-step protocol that was applied is detailed in Supplementary Methods. Ten micrograms of yeast tRNAs and approximately 1 µg of human fibroblast tRNAs were then enzymatically hydrolyzed into ribonucleosides with nuclease P1, phosphodiesterase, and alkaline phosphatase, deproteonized by filtration, and finally dried under vacuum according to the protocol of Thuring et al.40. t6A ribonucleoside was analyzed using the quantitative LC/MS-MS protocol of Thüring et al.40. Quantification of t6A was performed by integration of the peaks of interest and expressed relative to the total area of the peaks corresponding to the four canonical unmodified ribonucleosides assessed in the same sample for normalization. tRNA extracted from three independent samples were each measured twice . Detailed information are provided in Supplementary Methods.Yeast tRNAs were extracted and purified from actively growing cells (at ODTM protease inhibitors (Roche), as in Serrano-Perez et al.41. Fifty micrograms of proteins were loaded onto acrylamide gels and blotted onto nitrocellulose membranes (Amersham). The membranes were blocked in 1× Tris-buffered saline, 0.1% Tween 20 (TBST) with 5% bovine serum albumin or in Odyssey (LI-COR Bioscience) blocking buffer. Membranes were then incubated with the indicated primary antibodies, washed, and then incubated with either HRP-conjugated or LI-COR IRDye secondary antibodies. Signals were detected using ECL reagents (Amersham Biosciences) and acquired in a Fusion Fx7 darkroom (Vilber Lourmat) or acquired with Odyssey CLx near-infrared fluorescent imaging system (LI-COR Bioscience). Densitometry quantification was performed either using Bio-1D software or using Image studio lite software (version 5.2). Uncropped and unprocessed blots are provided in the Source Data file.Proteins from KD podocytes, primary fibroblasts, and LCLs were extracted in lysis buffer containing 150 mM NaCl, 50 mM Tris-HCl pH 7, 0.5% Triton-X100 with CompleteTM Epitope Tag Protein Isolation Kit (Miltenyi Biotec). Briefly, fresh lysates (1–1.5 mg of protein) were incubated either with mouse anti-V5 antibodies, followed by a 30-min incubation with magnetic beads coupled to protein A, or directly with magnetic beads coupled to an HA antibody. Immunoprecipitated proteins were isolated using µMACS® Separation Columns in a magnetic µMACS separator and subsequently eluted with 1× Laemmli buffer. Lysates and immunoprecipitated samples were subjected to immunoblot41. To assess rates of protein degradation, HEK293T cells transiently expressing either 2HA-GON7 or V5-LAGE3 alone, or co-expressing both proteins were incubated with cycloheximide at a final concentration of 100 µg/ml for the indicated time periods . Total protein extracts and immunoblotting were performed as described above. Anti-HA and anti-V5 antibodies were used to reveal GON7 and LAGE3, respectively. Relative GON7 and LAGE3 protein amounts were normalized to those of α-tubulin at each time point.For immunoprecipitation, HEK293T cells were transiently transfected with the adequate plasmids  and/or mutants) using calcium phosphate. Forty-eight hours post transfection, cells were lysed in 150 mM NaCl, 25 mM Tris-HCl pH 8, 0.5% Triton with protease inhibitors and HA-tagged GON7 was immunoprecipitated using the µMACSCell proliferation, apoptosis level, and rates of protein synthesis were assessed in KD podocytes using the CellTiter 96 Aqueous Non-Radioactive Cell Proliferation Assay (MTT) (Promega), the Caspase-3/7 Green detection Reagent , and the Click-iT HPG Alexa Fluor 488 Protein Synthesis Assays , respectively, according to the manufacturer’s instructions. Detailed information are provided in Supplementary Methods.13. Two groups (control IP versus IP HA or IP V5), each containing three biological replicates, were used for statistical analysis. Only proteins that were identified at least three times out of six were retained. A t-test was performed, and the data were represented in a volcano plot .Human podocyte cell lines stably expressing either 2HA-GON7 or V5-LAGE3 were used to perform proteomic studies. 2HA-GON7 and V5-LAGE3 were immunoprecipitated as described in the section above. Eluates were processed according to Braun et al.42.Measurement of the length of the terminal restriction fragments was performed by Southern blotting according to Touzot et al.All structural work was done using the full-length proteins of GON7, LAGE3, and OSGEP.15N-labeled his-tagged GON7 (vector “pET24d-C14_hisTEV_op”) whose sequences are shown in Supplementary Table 15N-GON7 and subcomplex LAGE3/15N-GON7 preparation are described in detail in Supplementary Methods.For NMR experiments, two vectors were ordered from Genscript  for the expression of either unlabeled his-tagged LAGE3 (vector “pET21a-LAGE3_hisTEV_op”) or For SAXS or crystallogenesis experiments, preparation of unlabeled GON7 and GON7/LAGE3/OSGEP subcomplex, and co-expression and purification of the KEOPS subunits are detailed in Supplementary Methods and Supplementary Figs. 43. X-ray diffraction data collection was carried out on beamline Proxima1 at the SOLEIL Synchrotron  at 100 K. Data were processed, integrated, and scaled with the XDS program package44. The crystal belonged to space group P43. The OSGEP and LAGE3 subunits were positioned by molecular replacement with the programs PHASER45 and MOLREP, implemented in the CCP4 suite46 using the structures of MjKae1 (PDB ID: 2VWB) and ScPcc1 (PDB ID: 4WX8) as search models. Residual electron density showed clearly the presence of the GON7 subunit, which was constructed using the program BUCCANEER46. The initial structure was refined using the BUSTER program47 and completed by interactive and manual model building using COOT48. The correctness of the assigned GON7 sequence was verified by omit mFo-DFc, 2mFo-DFc, Prime-and-switch electron density maps49 . To isolate the various species in solution, SAXS data were collected on samples eluting from an online size exclusion high-performance liquid chromatography (SEHPLCBio-SEC3Agilent) column and directly connected to the SAXS measuring cell. sixty-five microliters of GON7/LAGE3/OSGEP and GON7 samples concentrated at 1.5 and 6.7 mg/l, respectively, were injected into the column pre-equilibrated with a buffer composed of 20 mM MES pH 6.5, 200 mM NaCl, and 5 mM 2-mercaptoethanol. Flow rate was 300 µl/min, frame duration was 1.0 s, and the dead time between frames was 0.5 s. The protein concentration was estimated by UV absorption measurement at 280 and 295 nm using a spectrometer located immediately upstream of the SAXS measuring cell. A large number of frames were collected before the void volume and averaged to account for buffer scattering. SAXS data were normalized to the intensity of the incident beam and background (i.e. the elution buffer) subtracted using the program FoxTrot50, the Swing in-house software. The scattered intensities were displayed on an absolute scale using the scattering by water. Identical frames under the main elution peak were selected and averaged for further analysis. Radii of gyration, maximum particle dimensions, and molecular masses were determined using PrimusQT51 . An ultimate adjustment was performed using the program CRYSOL54. The modeling was repeated 10 times and the best model was deposited in SASBDB55 with codes SASDFK8, SASDFM8, and SASDFL8 for GON7, GON7/LAGE3, and GON7/LAGE3/OSGEP, respectively.SAXS experiments were carried out at the SOLEIL synchrotron SWING beamline . The sample to detector (Aviex CCD) distance was set to 1500 mm, allowing reliable data collection over the momentum transfer range 0.008 Ån = 3 independent experiments. For statistical analysis, data sets comparing more than three conditions (to a control group) were analyzed with ANOVA followed by Dunnett’s multiple comparisons test or by using Kruskal–Wallis test followed by a Dunn’s multiple comparisons test. Data sets with only two conditions to compare were analyzed using an unpaired t-test or a Mann–Whitney test. P < 0.05 was considered statistically significant. A standard confidence interval of 95% was applied in all analyses. Displayed in the figures are the mean values of all technical replicates for each of the independent experiments (displayed as single data points). Black lines indicate the mean values of all independent experiments.GraphPad Prism 8.0 software was used for the graphical representation and statistical analysis of cell-based data. Results are presented as mean ± s.e.m. of at least Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


Myrica rubra (MEO), which has significant antiproliferative effect in various cancer cell lines. In the present study, we compared the antiproliferative effect of these sesquiterpenes alone and in combination with the cytostatic drug doxorubicin (DOX) in cancer cell lines with different sensitivity to DOX. Two ovarian cancer cell lines (sensitive A2780 and partly resistant SKOV3) and two lymphoblast cancer cell lines (sensitive CCRF/CEM and completely resistant CEM/ADR) were used. The observed effects varied among sesquiterpenes and also differed in individual cell lines, with only VAL being effective in all the cell lines. A strong synergism of DOX with NER was found in the A2780 cells, while DOX acted synergistically with HUM and CAO in the SKOV3 cells. In the CCRF/CEM cells, a synergism of DOX with CAO and NER was observed. In resistant CEM/ADR cells, sesquiterpenes did not increase DOX efficacy, although they significantly increased accumulation of DOX (up to 10-times) and rhodamine-123 (substrate of efflux transporter ABCB1) within cancer cells. In conclusion, the tested sesquiterpenes were able to improve DOX efficacy in the sensitive and partly resistant cancer cells, but not in cells completely resistant to DOX.β-caryophyllene oxide (CAO), α-humulene (HUM), trans-nerolidol (NER) and valencene (VAL) are constituents of the essential oil of  Sesquiterpenes, defined as 15-carbon compounds formed from 3 isoprenoid units, represent an extremely diverse, heterogeneous and large group of natural compounds. Sesquiterpenes are secondary metabolites, formed mainly in plants, fungi, bacteria and marine invertebrates ,3. TogetMyrica rubra essential oil, α-humulene (HUM), caryophyllene oxide (CAO), trans-nerolidol (NER) and valencene (VAL), with doxorubicin (DOX) in the CaCo-2 cancer cell line [In addition, several sesquiterpenes were able to enhance the efficacy of classical anticancer drugs in vitro. For example, a combination of the α-methylene sesquiterpene lacton eremophila-110)-1113)-dien-12, 8β-olide with cisplatin or paclitaxel increased the effect of these cytostatics in the ovarian cell lines SKOV3, OVCAR-3, JC and JCpl . Oxalipl-11(13)-d-dien-12,ell line . Resistaell line ,16.The present study was designed to test and compare the effects of sesquiterpenes HUM, CAO, NER and VAL on cell proliferation and DOX efficacy in ovarian and lymphoblast cancer cells with different sensitivity to DOX. Moreover, the ability of these sesquiterpenes to enhance DOX concentration within cells and to inhibit efflux transporters was also tested. Two ovarian cancer cell lines—A2780 (sensitive to DOX) and SKOV3 (partly resistant to DOX)—and two lymphoblast cancer cell lines—CCRF/CEM (DOX-sensitive) and CEM/ADR (DOX-resistant)—were used for this purpose. The amount of the ABCB1 efflux transporter in these cell lines was quantified and compared.Myrica rubra essential oil and its sesquiterpene components HUM, CAO, NER and VAL to inhibit proliferation of colon cancer cell lines as well as to increase the efficacy of DOX treatment; this is probably via increased production of reactive oxygen species (ROS) and DOX accumulation in these cells. On the other hand, no cytotoxic effect or amelioration of DOX toxicity was observed in rat hepatocytes as a model of non-cancerous cells [The frequent occurrence of resistance of cancer cells to DOX underscores the necessity of determining compounds which are able to suppress this resistance. These compounds might be detected among sesquiterpenes, as these substances are able to enhance drug uptake via increased membrane permeability. In addition, some sesquiterpenes themselves have been shown to have anti-proliferative effects in cancer cells as well as to act synergistically with classic cytostatics . In a prus cells ,17,18. I50 values for individual sesquiterpenes and DOX alone were calculated as presented in Laurus nobilis L. in this cancer cell line [Juniperus, Pinus and Cedrus were able to inhibit cell proliferation in both CCRF/CEM and CEM/ADR lines [The effect of selected sesquiterpenes  on the number of viable cells after 72-h treatment was tested using a neutral red uptake (NRU) or Alamar Blue (AB) assay in ovarian cell lines and lymphoblast cell lines, respectively. The results are demonstrated in ell line . On the DR lines .The use of drugs in combinations with natural compounds can cause significant shifts in efficacy in both positive and negative ways. The present study was aimed at testing and comparing the effect of selected sesquiterpenes on DOX efficacy in cancer cell lines with different sensitivity to DOX. The software CalcuSyn based on the Chou-Talalay method was used to evaluate the effect of DOX + sesquiterpene combinations . ExperimThe cancer cell lines used in our study significantly differed in sensitivity to DOX see . IncreasThe accumulation of DOX within the cells after their treatment with DOX alone and with combinations of DOX + sesquiterpenes is compared in Cell line CEM/ADR (as a DOX-resistant line with high level of ABCB1) was chosen to test the inhibitory effect of sesquiterpenes on Rhodamine 123 efflux. This substrate of the ABCB1 transporter is often used in activity assays in various tissues and cancer cells ,26. In oN-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES) buffer, sodium pyruvate, d-glutamine, resazurin, rhodamine 123 (RHO 123) and neutral red were supplied by Sigma-Aldrich . Fetal bovine serum and gentamicin sulfate were purchased from Invitrogen  and bovine serum albumin (BSA) from Fluka . Doxorubicin and pure sesquiterpenes  were supplied by Sigma-Aldrich. Stock solutions of sesquiterpenes were prepared in dimethyl sulfoxide (DMSO) and stored in the dark at 4 °C. All other chemicals used were of HPLC or analytical grade and provided by Sigma-Aldrich.McCoy’s modified medium, RPMI-1640, d-glutamine, and CCRF/CEM and CEM/ADR were cultured in RPMI-1640 with 10% FBS, 1% HEPES, 1% sodium pyruvate. The resistance of CEM/ADR was maintained by incubation in 5 mM DOX for 24 h every week.Cell lines SKOV3 and A2780 were purchased from ATCC . Suspension cell lines CCRF/CEM and CEM/ADR5000 (CEM/ADR) were a gift from Prof. Wink, University of Heidelberg, DE. The cells were multiplied in three passages, frozen in aliquots and stored in liquid nitrogen. The absence of mycoplasma in all the cell lines used in the laboratory was periodically checked by Generi Biotech . For every set of experiments (each lasting 3–9 weeks) new batch of stored cells was used. SKOV3 were cultured in McCoy’s modified medium with 10% FBS, A2780 in RPMI-1640 with 10% FBS and 2 mM The sesquiterpenes and DOX were pre-dissolved in DMSO or in pure ethanol. The cells were exposed to various concentrations of the tested compounds in the culture medium. The final concentration of organic solvent in medium was 0.1%. Cells cultured in medium with pure DMSO or ethanol without the tested compounds were used as control samples. 10% DMSO served as a positive control. The number of viable cells was assayed after 72 h using the neutral red uptake test for SKOV3 and A2780 or Alamar blue for the suspension cell lines.The cells were cultured in 96-well plates. After 72 h exposure, the medium was removed and 200 μL of neutral red-containing medium was added into each well, and the plates were incubated for an additional 3 h at 37 °C. The cells were washed with 100 μL of PBS, then fixed in a solution of 0.5% formaldehyde/1% calcium chloride for 15 min. The neutral red dye was released from the viable cells with a lysing solvent (50% ethanol/1% acetic acid) by shaking for 30 min at room temperature. The absorbance of solubilized dye was measured using a spectrophotometer  at 540 nm. Each sample was assayed in 6 parallels and 3 independent experiments were performed. The viabilities of the treated cells were expressed as a percentage of untreated controls (100%). The effect of sesquiterpenes on DOX efficacy was evaluated using CalcuSyn software.The cells were cultured in 96-well plates, followed by 72 h exposure with 10 µL of Alamar blue solution (0.1 mg resazurin/1 mL PBS). After 1 h incubation at 37 °C, fluorescence was measured  using a fluoresce reader .g, 10 min, 4 °C). The supernatant was filtered through a 0.22 μm filter and the filtrate was used for analysis.A suspension of SKOV3 and A2780 cells  was set to a 12-well plate. After 48 h, 600 μL of solution in medium of the tested substances was added. The cells were treated with DOX (6 μM) alone, or in combination with individual sesquiterpenes (10 or 50 μg/mL) for 3 h. At the end of the experiment, the medium was removed and cells were washed with 500 μL of PBS. 100 μL of lysing buffer  added, at which time the cells were lysed for 15 min, then mixed with 300 μL of ice-cold methanol and centrifuged  equipped with a Zorbax C18 Eclipse Plus  column with a 1290 Infinity inline filter (Agilent). The HPLC method used by Skarka  was adapThe suspension of CEM/ADR cells (106/mL) was incubated with RHO123 . After incubation the cells were washed by PBS and incubated with the tested compounds in serum free medium, with verapamil (20 µM) used as a control. After a 2 h incubation, the cells were washed by PBS and fluoresce measured by a flow cytometer ; excitation was undertaken with a by 488 nm argon laser, 530 nm emission filter. Flow cytometry data were analyzed by Flowing Software 2.The cells were cultivated in 60 mm dishes, washed with PBS and lysed. Whole cell lysates were centrifuged . Supernatant was normalized for protein by BCA assay. For separation, 7.5% acrylamide gel was used. Proteins were transferred to a PVDF membrane by wet blot  and probed with anti-ABCB1 antibody  and anti-ACTB antibody . Secondary antibody with horseradish peroxidase was used and the blots were imaged by chemiluminescence captured on film.p < 0.05. The concentration inducing a 50% decrease of cell viability as compared to control (IC50) was calculated using GraphPad Prism 6.0 nonlinear regression.The data are presented as means ± S.D. of a given number of experiments. Statistical significance  was acceptable at a level of 50 than is normally achievable due to the lower solubility of the compounds. Only VAL had a measurable IC50 in all the tested cell lines, but in combination with DOX did not show a strong synergism in any cell line. Only NER showed a degree of synergism in all cell lines where it was possible to test for it. The effect from the combination of DOX with sesquiterpenes was not different from the effect of sesquiterpenes alone in the CEM/ADR cell line, despite the appearance of the largest shift of intracellular DOX concentrations. Further tests suggested the interaction of sesquiterpenes with efflux transporters as the possible mechanism of synergism with DOX.In summary, drug interactions of sesquiterpenes with DOX were cell line specific. Some of the sesquiterpenes had a higher estimated IC