Caenorhabditis elegans and humans, which is relevant to oncogenesis and aging.The coordinated activity of DNA replication factors is a highly dynamic process that involves ubiquitin-dependent regulation. In this context, the ubiquitin-directed ATPase CDC-48/p97 recently emerged as a key regulator of chromatin-associated degradation in several of the DNA metabolic pathways that assure genome integrity. However, the spatiotemporal control of distinct CDC-48/p97 substrates in the chromatin environment remained unclear. Here, we report that progression of the DNA replication fork is coordinated by UBXN-3/FAF1. UBXN-3/FAF1 binds to the licensing factor CDT-1 and additional ubiquitylated proteins, thus promoting CDC-48/p97-dependent turnover and disassembly of DNA replication factor complexes. Consequently, inactivation of UBXN-3/FAF1 stabilizes CDT-1 and CDC-45/GINS on chromatin, causing severe defects in replication fork dynamics accompanied by pronounced replication stress and eventually resulting in genome instability. Our work identifies a critical substrate selection module of CDC-48/p97 required for chromatin-associated protein degradation in both  Cdc48/p97 is a key component of the ubiquitin-proteasome system, acting as a ubiquitin-directed segregase to regulate multiple cellular functions. Here the authors identify UBXN-3/FAF1 as a crucial regulator of chromatin-associated protein degradation that recruits Cdc48/p97 to DNA replication forks. Duplication of the genomic information is a key task for dividing cells. The intricacy of DNA replication is reflected by the plethora of regulatory factors that promote different steps of DNA synthesis2345Caenorhabditis elegans15UFD-1/NPL-4 inactivation. Thus, CDC-48 coordinates chromatin association of different protein complexes with replication fork progression. Consequently, inhibition of CDC-48 results in severe S-phase progression defects, due to activation of the DNA replication checkpoint15Xenopus laevis and human cells identified a related requirement for CDT-1/Cdt1 mobilization and turnover, emphasizing a crucial function of CDC-48/p97 in eukaryotic DNA replication16The licensing factor CDT-1 (chromatin and DNA licensing factor 1) initiates the formation of a pre-replication complex (pre-RC) at replication origins once per cell cycle791113CDC-48/p97 is a key component of the ubiquitin/proteasome system, important for mobilization and targeting of ubiquitylated substrates for degradation by the 26S proteasome1820212216222324cdc-48 in C. elegans. Using an RNA interference (RNAi)-based approach, we identified UBXN-3 (ubiquitin regulatory X (UBX) domain-containing protein in nematodes 3) as a crucial factor that coordinates CDC-48-dependent DNA replication events. Embryos with reduced protein levels of both UBXN-3 and CDC-48 recapitulate phenotypes caused by complete depletion of CDC-48, including the accumulation of CDT-1 and GINS on mitotic chromatin. UBXN-3 binds to CDT-1, thus facilitating CDC-48 recruitment for substrate turnover and disassembly at the chromatin. Consequently, depletion of the human homologue FAS-associated factor 1 (FAF1) results in stabilization of CDT-1 protein, pronounced decrease in replication fork velocity, increased number of stalled forks, and newly fired origins, cumulating in the activation of the DNA damage response. In conclusion, our results demonstrate an evolutionarily conserved role of UBXN-3/FAF1 in orchestrating CAD of DNA replication factors to safeguard genome integrity, which is relevant to the emerging link between FAF1 deletion and cancer developmentTo address how CDC-48-dependent DNA replication is adjusted with substrate recruitment and cell-cycle progression, we screened for genetic interactors of cdc-48.1(tm544) deletion mutants, representing a sensitized background with a reduction of 80% in total CDC-48 protein levels in embryos  mutant (The ubiquitin-selective segregase CDC-48/p97 is a key regulator of numerous protein degradation pathways within eukaryotic cells18202122 embryos . The rem) mutant .UFD-1/NPL-4-depleted embryos exhibit a pronounced delay in S-phase progression caused by activation of the conserved DNA replication checkpoint kinases ATL-1/ATR and CHK-1/Chk1 (refs cdc-48.1(tm544); ubxn-3(RNAi) embryos, which is indicative of compromised DNA replication27ubxn-3-deletion mutants, excluding indirect effects on CDC-48UFD-1/NPL-4 complex integrity  mutant embryos lacking UBXN-3 towards the DNA replication inhibitor hydroxyurea (HU)ufd-1 and npl-4 RNAi defects (ubxn-3(RNAi) caused significant but less pronounced HU sensitivity in cdc-48.2(tm659) mutants, lacking only 20% of the overall CDC-48 protein level, as compared with the cdc-48.1(tm544) deletion (ubxn-3(tm6658) deletion mutant is hypersensitive towards npl-4(RNAi) conditions that do not markedly affect embryonic survival in the wt  deletion mutant further supports our observation that UBXN-3 is required for embryonic survival, particularly when DNA replication is compromised upon exposure to HU (ubxn-3 knockdown by RNAi (ubxn-3(tm6658) allele (ubxn-3(tm6658) deletion background complements sensitivity towards npl-4(RNAi) as well as HU  allele . Furtherll as HU . To prov as HU 31. Besides as HU 31. In concubxn-3(RNAi)  . In concUFD-1/NPL-4 complex causes increased levels of the DNA licensing factor CDT-1 on mitotic chromosomes as well as persistent chromatin association of CDC-45 and SLD-5, a subunit of the tetrameric GINS complexubxn-3 as a genetic interactor of cdc-48 and npl-4 important for DNA replication and genome stability, we next addressed whether UBXN-3 is involved in the regulation of these CDC-48 targets. Strikingly, depletion of UBXN-3 in the cdc-48.1(tm544) mutant resulted in elevated levels of CDT-1 on mitotic chromatin as revealed by immunostaining (ubxn-1 or ubxn-2 (RNAi) did not interfere with CDT-1 regulation in the absence of CDC-48.1 (1216cdc-48.1(tm544) background  experiments using h CDC-48 . Furtherh CDC-48 . Co-IP eh CDC-48 . Taken tUFD-1/NPL-4 complex (UFD-1/NPL-4 complex1516rbx-1(RNAi), which stabilizes CDT-1 specifically during S phase and consequently causes genomic instability, resulted in formation of UBXN-3-positive micronuclei rather than the characteristic UBXN-3 punctae inside the nucleus16UFD-1/NPL-4 complex.On the basis of these protein–protein interactions, we wondered whether the nuclear localization of UBXN-3 depends on integrity of the CDC-48 complex . Indeed, complex . In contin vitro binding assay using purified full-length or truncated variants of UBXN-3 (in vivo results shown in npl-4(RNAi), suggesting that the substrate-binding capacity of UBXN-3 is independent of the UFD-1/NPL-4 cofactor , urinary bladder cancer (T24) or kidney embryonic (HEK293) cells were successively pulse labelled with the nucleoside analogues 5-chloro-2′-deoxyuridine (CldU) and 5-iodo-2′-deoxyuridine (IdU), respectively, and progression of discrete replisomes was visualized by anti-CldU and anti-IdU immunofluorescence . OngoingTo show that CDT-1 is a substrate of FAF1 in human cells, we performed co-IP experiments using HA-FAF1 as bait. Nuclear lysates were prepared from HEK cells expressing the p97-E587Q substrate trap variantResearch during the past two decades led to the discovery of prominent cellular pathways regulated by the ubiquitin-selective ATPase CDC-48/p97 refs , 19. ObvUsing a genetic screening approach, we succeeded in finding a conserved UBX domain protein that provides a substrate selection module, triggering CDC-48/p97-dependent degradation at the chromatin. UBXN-3/FAF1 binds both the licensing factor CDT-1 and CDC-48/p97, and thus safeguards the timely regulation of target recognition at the replication fork. Replication stress emerging upon UBXN-3/FAF1 depletion provokes replication fork collapse and occurrence of genotoxic DNA breaks, activating the replication checkpoint and formation of DNA repair foci. Thus, our findings suggest that UBXN-3/FAF1 is a specific mediator of CAD, especially in the context of DNA replication since it is not essential for CDC-48/p97-mediated unfolded protein response pathways in the ER or mitochondria.The detailed analysis of DNA replication factors indeed revealed a crucial role of UBXN-3/FAF1 in the regulation of chromatin-loaded CDT-1, as well as chromatin association of CDC-45 and the GINS complex. Analysis of DNA fibre assays highlights the severe consequences of UBXN-3/FAF1 depletion on the DNA replication programme at single-origin resolution . DepletiC. elegans, the functional relevance of UBX proteins is scarcely defined. UBXN-2 is implicated in mitosis, by regulating centrosomal compositionubxn-3 does not affect the integrity of the CDC-48UFD-1/NPL-4 complex, excluding indirect effects on DNA synthesis. Conversely, the presence of CDC-48, UFD-1 and NPL-4 appears to be important for the correct nuclear localization of UBXN-3.UBXN-3/FAF1 is a member of the UBX family of CDC-48/p97 cofactors, which were proposed to provide substrate specificity43444542top-2 and cin-4 does not result in elevated CDT-1 levels on mitotic chromatin  were shown to be directly linked to accelerated aging and cancer susceptibility2255565758C. elegans nematodes were treated according to standard protocols at 20 °C, unless otherwise stated. The Bristol strain N2 was used as wt. Mutants and transgenic animals used in this study are listed in the following:cdc-48.1(tm544)II, ubxn-3(tm6658)II, ubxn-3(tm6658)II; hhIs149(unc-119(+); Ppie-1::mCherry::ubxn-3::pie-1-3′UTR), unc-119(ed3)III; hhIs149(unc-119(+); Ppie-1::mCherry::ubxn-3::pie-1-3′UTR), unc-119(ed3)III; hhIs177(unc-119(+), Ppie-1::LAP::mcm-7::pie-1-3′UTR), unc-119(ed3)III; gtIs65(unc-119(+), Ppie-1::LAP::cdc-45::pie-1-3′UTR), cdc-48.1(tm544)II; gtIs65, unc-119(ed3)III; gtIs67(unc-119(+), Ppie-1::LAP::sld-5::pie-1-3′UTR), cdc-48.1(tm544)II; gtIs67, unc-119(ed3)III; gtIs64(unc-119(+)), gtIs64, zcIs4(hsp-4::GFP)V, cdc-48.1(tm544)II; zcIs4(hsp-4::GFP)V, zcIs13(hsp-6::GFP)V, cdc-48.1(tm544)II; zcIs13(hsp-6::GFP)V.hhIs149 and hhIs177 were generated for this study. Briefly, ubxn-3 cDNA was cloned into the pAZCherry(N)mcm-7 cDNA was cloned into the pIC26 expression vectors, respectivelyC. elegans genome by particle bombardmentThe transgenic lines pcn-1 the respective cDNAs was subcloned into the pPD129.36 feeding vector. The empty feeding vector was used as control. Depletion of NPL-4 protein levels was achieved by induction of dsRNA corresponding to the npl-4.2a cDNA.RNAi-mediated depletion was achieved using the feeding methodubxn-3(tm6658) mutants was shifted by one day, respectively.HU sensitivity was monitored by growing worms in the presence of different HU concentrations. Worms were transferred to HU-containing medium after reaching the L4 larval stage to avoid developmental effects. Following RNAi and HU treatment until adulthood, five to six adult worms were kept for 5–6 h on nematode growth medium (NGM) plates for egg laying. The following day, hatched progeny and dead embryos were counted to calculate growth defects. Because of a developmental delay, the analysis of For lifespan assays, worms were synchronized by bleaching in sodium hypochlorite. Eggs were distributed on RNAi plates. For HU treatment, L4 larvae were transferred to HU-containing RNAi plates. In lifespan assays, first day of adulthood was defined as day 1. Worms were grown at 20 °C and monitored daily for survival by touch-provoked movement and pharyngeal pumping, until death. Animals that crawled off the plates or died owing to internal hatching of progeny or extruded organs were censored.For time-lapse microscopy, embryos were extruded from gravid hermaphrodites with the help of injection needles, transferred onto 3% agar pads in M9 buffer before microscopic analysis. An AxioImager.M1/Z1 microscope equipped with an AxioCam MRm camera (Carl Zeiss) was used for image acquisition. Time-lapse recordings in 90-s intervals were acquired using 2 × 2 mono binning to avoid photo bleaching and toxicity. To allow direct comparison of signal intensities, images were recorded under identical conditions. Analysis of time-lapse recordings was done in the AxioVision 4.7 software. Timing of cell division was estimated as follows: for the estimation of cell division timing distinct cell-cycle stages (nuclear envelope breakdown (NEBD), completion of mitotic furrow ingression (cytokinesis)) where determined morphologically from differential interference contrast (DIC) images. Time intervals in between theses cell-cycle stages were used to calculate the duration of mitosis (time elapsed between NEBD and cytokinesis of one-cell division) and duration of S phase (time elapsed between cytokinesis and NEBD of two subsequent divisions). The P1 delay refers to a combined estimation of the delay in cell-cycle progression of the P1 cell relative to the AB cell. The P1 delay represents the average delay calculated from the time difference between NEBD on one hand and the completion of cytokinesis in the AB and P1 cell on the other hand27cdt-1, npl-4.2 and ubxn-3b were cloned into the pET-21a expression vector (Addgene). Recombinant proteins were expressed in Escherichia coli strain BL21cp (Stratagene) and purified using the ÄKTApurifier system  or bench-top method using Ni-NTA agarose (Qiagen). His-tagged purified proteins were used for immunization of rabbits and anti-sera were affinity purified using respective Strep-II-tagged recombinant proteins (BioGenes).cDNAs encoding Immunostaining of early embryos was done essentially according to the ‘freeze-crack' protocol. Gravid worms were dissected onto polylysine-coated slides (Thermo Scientific) and frozen in liquid nitrogen, followed by incubation in methanol at −20 °C for 20 min and in acetone at −20 °C for 20 min. After rehydration in PBS and blocking in 5% BSA, embryos were incubated with primary antibody overnight at 4 °C , anti-FK2-ubiquitin 1:500 (Millipore) and anti-α-tubulin 1:200 ). Incubation with the fluorescently labelled secondary antibodies (Life Technologies) was done at room temperature for 1 h (1:400). Embryos were then mounted in DAPI Fluoromount-G medium (SouthernBiotech). For western blotting, purified proteins and worm lysates were separated by SDS–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to nitrocellulose membranes . Membranes were blocked in 3% milk solution and incubated with the primary antibodies overnight at 4 °C in Roti-Block , anti-tubulin 1:5,000 , anti-FK2-ubiquitin 1:2,000 (Millipore), anti-histone3 1:5,000 (Abcam) and anti-mCherry 1:2,500 (Abcam)). Incubation with fluorescently labelled secondary antibodies  was done at room temperature, before detection of signals using the Li-Cor Odyssey scanner. Quantification of signal intensities was done using the Odyssey V3.0 software (Li-Cor). The uncropped versions of western blots that have been used to assemble the main figures are collected in g for 5 min before SDS–PAGE analysis. Whole-worm lysates used for subsequent co-IP were prepared as follows: synchronized L1 larvae were grown on NGM-agar plates with OP50 bacteria until they reached adulthood. Worms were collected and washed once in lysis buffer (50 mM Tris (pH 7.2), 50 mM NaCl, 1 mM EDTA and protease inhibitors (Roche)) before shock-freezing in liquid nitrogen. Subsequently, thawed samples were subjected to sonication . Samples were centrifuged at 14,000g for 5 min and supernatant subsequently incubated with magnetic Dynabeads Protein A (Novex Life Technologies). Protein concentrations were calculated by extinction at 280 nm using the NanoDrop 8000 (Thermo Scientific).Synchronized L1 larvae were kept on control or RNAi plates at 15 °C until they reached adulthood. Embryos of gravid worms were collected by bleaching and re-suspended in 2 × Laemmli buffer. Embryos were sonicated twice for 15 s , incubated at 95 °C for 5 min, and centrifuged at 14,0002, 2 mM EDTA, 0.5 M sucrose, 1 mM dithiothreitol, 1 mM phenylmethyl sulphonyl fluoride and protease inhibitors (Roche)). Efficient lysis was monitored under the microscope. Cellular debris was collected by three subsequent centrifugation steps at 100g for 1 min at 4 °C. Nuclei were collected by centrifugation at 4,000g for 5 min at 4 °C. The supernatant contains represents the cytosolic fraction. The nuclei in the pellet were washed twice in detergent buffer  and collected as before at 4,000g centrifugation. After resuspension in one-fourth volume nuclear extraction buffer ) of the cytosolic fraction, the nuclear fraction was split in two halves. One half was kept on ice while the second half was subjected to sonication . Nucleoplasm and chromatin fraction were separated by centrifugation at 20,000g for 10 min at 4 °C. Supernatant contains soluble proteins, pellet contains chromatin-associated proteinsPurification of nuclei from embryos was achieved according to published protocols with some modifications67E. coli using Ni-NTA agarose (Qiagen) and PD10 gel-filtration columns . Isolated proteins were snap-frozen in liquid nitrogen and stored at −80 °C. Synchronized adult worms were lysed by sonication (Bandelin Sonopuls) in lysis buffer (50 mM Tris (pH 7), 150 mM NaCl, 0.5 mM EDTA, 5 mM MgCl2, 0.1% Igepal, 15 mM imidazole, protease inhibitors (Roche) and 5 mM iodoacetamide). Proteins coupled to Ni-NTA (50 μl slurry per 1 mg protein) or Ni-NTA agarose alone were incubated with worm lysate  for 30 min at room temperature. After three times washing with washing buffer (same as lysis buffer with 250 mM NaCl), bound proteins were eluted from agarose beads with elution buffer (50 mM Tris (pH 7), 8 M urea, 100 mM NaH2PO4 and 0.1% Igepal). Remaining proteins were eluted at 95 °C in Laemmli buffer.For co-IP analysis, magnetic Dynabeads Protein A (Novex Life Technologies) were used following the manufacturer's protocol. Dynabeads were mixed with the antibody of choice (10 μg), diluted in 200 μl of PBST (0.01% Tween 20), and crosslinked using BS3 (Thermo Scientific). After crosslinking, worm lysate  was added and incubated with the Dynabeads for 30 min at room temperature. After three times washing with washing buffer (50 mM Tris (pH 7.2), 1 mM EDTA, 150 mM NaCl, 10% glycerol and protease inhibitors (Roche)), bound proteins were eluted in Laemmli buffer without β-mercaptoethanol and heated for 10 min at 70 °C. β-Mercaptoethanol was added to the eluate just before SDS–PAGE analysis. For analysis of UBXN-3 functional domains, His-tagged proteins were expressed and purified from −1 final). In addition, cells were transiently transfected with a plasmid driving the expression of HA-tagged FAF1 or HA as control (pIRESpuro2b vector). HA beads were washed twice in IP buffer (50 mM Tris (pH 8.0), 150 mM NaCl and 0.05% NP-40) and incubated with nuclear lysates for 3 h at 4 °C. Nuclear lysates were preparedCo-IP analysis in human cells was achieved via anti-HA agarose beads following the manufacture's protocol. Twenty-four hours before collecting HEK293 cells, expression of the p97-E578Q mutant2 atmosphere at 37 °C. Cells were transfected with the indicated siRNAs  using Lipofectamine RNAiMAX (Invitrogen) transfection reagent according to manufacturer's instructions at 50–60% cell confluency.Human osteosarcoma (U2OS) cells, human embryonic kidney 293 (HEK293) cells and human bladder cancer (T24) cells  were cultured in six-well plates, 60- or 100-mm dishes in DMEM supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin in a 5% CON-ethylmaleimide (20 mM). The protein content was estimated using Bio-Rad protein assay reagents. Lysates were resolved in polyacrylamide gels using standard protocols. Membranes were blocked with 5% non-fat dry milk for 1 h with agitation at room temperature. The proteins were detected with respective primary and secondary antibodies and visualized on X-ray film.For preparation of lysates, cells were cultured in 60 or 100 mm dishes and collected 72 h after transfection. Cells were trypsinized, washed with PBS and the cell pellets were re-suspended in lysis buffer (50 mM Tris-HCl (pH 7.5), 120 mM NaCl, 20 mM sodium fluoride, 1 mM EDTA, 6 mM EGTA, 1% Nonidet P-40 and 10% glycerol), and supplemented with protease inhibitor , phosphatase inhibitor  and −1 final concentration). At the selected time points, the cells were collected and cell lysates were prepared as described above, followed by western blot analysis.To analyse protein CDT-1 turnover, HEK293 cells were grown in DMEM media before addition of CHX  for 30 min, washed three times with warm PBS and then 250 μM of IdU (Sigma-Aldrich) pulse were given for an additional 30 min. Labelling of replication forks was terminated by treating the cells with ice-cold PBS. Cells were lysed, and DNA fibres were spread onto glass slides, fixed with methanol and acetic acid, denatured with HCl, blocked with 2% BSA and stained with anti-rat and anti-mouse 5-bromo-2′-deoxyuridine (BrdU) that specifically recognize either CldU or IdU. Anti-rat Cy3  and anti-mouse Alexa 488  were used as the respective secondary antibodies. The analysis of DNA tract length is exclusively based on measurement of symmetric tracts, displaying no statistical differences in the lengths of respective CldU- and IdU-single tracts22siFAF1 #3 ; siFAF1 #5 ; siFAF1 #6 ; siFAF1 #7 ; siFAF1 3′UTR #2 ; siCdt1 ; and siNS .Rabbit anti-CDT-1 , rabbit anti-FAF1 , mouse anti-β-actin , mouse anti-p97 , mouse anti-ATM , rabbit anti-phospho ATM ser1941 , rabbit anti-ATR , rabbit anti-phospho ATR ser428 , mouse anti-CHK-1 , rabbit anti-phospho CHK-1 ser345 , rabbit anti-CHK2 , rabbit anti-phospho CHK2 Thr68 , rabbit anti-phospho H2AX Ser139 , rat anti-HA  and mouse anti-PCNA . Anti-rabbit and anti-mouse horse radish peroxidase-conjugated secondary antibodies were purchased from Sigma-Aldrich . Membranes were activated using Thermo Scientific SuperSignal West Pico and Dura substrates and developed accordingly using standard protocol.t-test. Statistical significance of C. elegans lifespan was estimated using the log-rank Mantel–Cox test. Asterisks indicate statistical significances according to common conventions.Statistical analysis was performed using Excel (Microsoft), Prism (GraphPad) and ImageJ software. Statistical significance was calculated with two-tailed paired Student's How to cite this article: Franz, A. et al. Chromatin-associated degradation is defined by UBXN-3/FAF1 to safeguard DNA replication fork progression. Nat. Commun. 7:10612 doi: 10.1038/ncomms10612 (2016).Supplementary Figures 1-8 and Supplementary Table 1


Caenorhabditis elegans, germline expression programs are actively repressed in somatic tissue by components of the synMuv (synthetic multi-vulva) B chromatin remodeling complex, which include homologs of tumor suppressors Retinoblastoma (Rb/35LIN-) and Malignant Brain Tumor (MBT/61LIN-). However, the full scope of pathways that suppress germline expression in the soma is unknown. To address this, we performed a mutagenesis and screened for somatic expression of GFP-tagged PGL-1, a core P-granule nucleating protein. Eight alleles were isolated from 4000 haploid genomes. Five of these alleles exhibit a synMuv phenotype, whereas the remaining three were identified as hypomorphic alleles of known synMuv B genes, lin-13 and dpl-1. These findings suggest that most suppressors of germline programs in the soma of C. elegans are either required for viability or function through synMuv B chromatin regulation.In  Cancer cells acquire a number of traits normally restricted to germline stem cells, including cellular immortality and the ability to self-renew. A subset of proteins exclusively found in the germ cells of the testis and/or ovary is overexpressed in most melanomas and is frequently found in breast, bladder, lung, and hepatocellular cancers . GermlinCaenorhabditis elegans is providing much needed insight into how germline programs are repressed in the soma. One remarkable discovery is that components of the synMuv B chromatin remodeling complex, which include homologs of the tumor suppressors Retinoblastoma (Rb or LIN-35) and Malignant Brain Tumor (MBT or LIN-61), actively repress the somatic expression of germline-specific ribonucleoprotein aggregates called germ granules 3xFLAG + ]I, MT10430lin-35(n745)I, and the CB4856 Hawaiian isolate were obtained from the Caenorhabditis Genetics Center (CGC). DUP10 [PGL-1::GFP]I; lin-13(sam4)III, DUP20 [PGL-1::GFP]I; lin-13(sam12)III, DUP21 [PGL-1::GFP]I; dpl-1(sam13)II, DUP6 [PGL-1::GFP]I; sam1, DUP14 [PGL-1::GFP]I; sam8, DUP15 [PGL-1::GFP]I; sam9, DUP16 [PGL-1::GFP]I; sam10, DUP25 [PGL-1::GFP]I; (sam17/+), DUP52 samEx4(WRM0614dE05 + pCFJ104); [PGL-1::GFP]I; dpl-1(sam13)II, and DUP53 samEx5(WRM064aA06 + pCFJ104); [PGL-1::GFP]I; lin-13(sam4)III were generated in this study.rotocols . TH206 [WRM064aA06 (20 ng/ul) to create DUP53, and DUP21 was injected with the fosmid WRM0614dE05 (20 ng/ul) to create DUP52. All injections used the myo-3p::mCherry coinjection marker pCFJ104 (10 ng/ul) (DUP10 was injected with the fosmid TH206 worms using the standard protocol (EMS mutagenesis was performed on protocol . Two thoCB4856 (Hawaiian) males were crossed into DUP10, DUP20, and DUP21 strains. F1 cross progeny were picked to new plates, and approximately 50 F2s with the somatic P-granule phenotype were selected from each cross. The progeny of these F2 animals were pooled and then whole genome–sequenced as previously described  were multiplexed with nine additional mutants (unpublished), and all 12 samples were sequenced in a single lane on an Illumina HiSeq2500. The CloudMap pipeline was used to analyze mutant genome sequences, obtain map data, and find mutations as previously described  were quantified for somatic PGL-1::GFP expression in each strain. A t-test was used to calculate the significance of enhancement or suppression from control RNAi.RNAi feeding constructs were obtained from the Ahringer library  multi-vulva (Muv) phenotype when combined with a mutation in a separate synMuv A class of genes. The class A synMuvs do not exhibit somatic P granules. Because screens for synMuv B mutants looking for the Muv phenotype have been completed to near saturation, we sought to distinguish mutations in the synMuv B pathway from those in a novel pathway. To do this, we used an RNAi feeding vector to knockdown expression of the synMuv A gene lin-15a in all eight of the new alleles. synMuv B mutants fed lin-15a RNAi exhibit a fully penetrant multi-vulva phenotype (lin-35/Rb mutant (positive synMuv B control) but not in wild-type worms , validating the specificity of our screen , making them likely to contain mutations in genes that act in parallel or downstream of the synMuv B pathway.Most components of the synMuv B heterochromatin complex antagonize P-granule accumulation in somatic cells  , suggesting that sam12 represents a stronger loss-of-function when compared to sam4 in an allelic series. On chromosome III, whole genome sequencing found four nonsynonymous mutations and one stop-gained mutation closely linked to sam12; however, RNAi depletion of these genes did not cause somatic P-granule expression (not shown). Sequence coverage was low and incomplete across lin-13 in sam12 worms, so we sequenced lin-13 and found a C to T mutation that introduced a stop codon (Q1585*) (lin-13(sam12) mutants prevented us from obtaining transgenic lines to test rescue. The predicted lin-13(n387) null allele is homozygous sterile in the absence of maternal LIN-13 (lin-13(sam12) worms exhibit very slow growth and low broods, sam12 is an unlikely null because it is possible to maintain homozygous mutants.On chromosome III, we found four nonsynonymous mutations closely linked to n LIN-13 . RNAi deansgene)  and obseame gene . sam4/sa(Q1585*) . Low brol LIN-13 . Althougsam13. The splice-donor mutation is a G to A base pair substitution in the first nucleotide of intron 2 in dpl-1  animals do not appear to have defects in ovulation, suggesting that this allele causes a splicing defect that only weakly compromises DPL-1 function.On chromosome II, we found one splice site donor, three frameshift, and three nonsynonymous mutations closely linked to in dpl-1 . Of the n  RNAi sensitive strain , lin-13(sam12), and dpl-1(sam13) mutants at 25°. At this temperature, Muv animals were still not observed with control or lin-15a RNAi on lin-13(sam4) and lin-13(sam12) worms (0/3 replicates). However, at 25° Muv worms were observed on 3/3 replicates of dpl-1(sam13) worms fed lin-15a RNAi , but not control RNAi (0/3 replicates). Collectively, these results suggest that sam4, sam12, and sam13 share features with previously described loss-of-function lin-13 and dpl-1 alleles, with the exception that they do not readily exhibit synMuv phenotypes.The two e strain . Like ot-44 RNAi , suggest alleles . Somatic mutants . Some aleratures , so lin-daf-2, a gene whose depletion was previously shown to cause ectopic expression of GFP::PGL-1 driven behind a pie-1 promoter , it is unclear how germ granules themselves may contribute to this process. Recently, it was discovered that stress granules, another ribonucleoprotein aggregate that shares some components with germ granules, are important for tumor progression; knockdown of the stress granule nucleator G3BP1 reduces local invasive capacity in tumor xenografts (C. elegans homolog, gtbp-1/K08F4.2, suppresses synMuv A/B phenotypes (Although it is assumed that germline components expressed in the soma induce germ cell characteristics that could lead to oncogenesis (


At ≤20°C, glp-1(ar202) is wild-type, whereas at 25°C it forms a germline stem cell⁄progenitor cell tumor reminiscent of human cancer. We identify a NOTCH tumor phenotype in which all tumor cells traffic rapidly to G2⁄M post-irradiation, attempt to repair DNA strand breaks exclusively via homology-driven repair, and when this fails die by mitotic death. Homology-driven repair inactivation is dramatically radiosensitizing. We show that these concepts translate directly to human cancer models.Mammalian NOTCH1-4 receptors are all associated with human malignancy, although exact roles remain enigmatic. Here we employ  C. elegans genetics to investigate potential pharmacologic approaches to NOTCH.Notch is a single-pass transmembrane glycoprotein receptor that plays key roles in lineage specification and differentiation processes during development, and in maintenance of stem cells in adult life . MammalsC. elegans contains two Notch family receptors, LIN-12 and GLP-1 . Thus the range of 120-480Gy appears appropriate for defining elements of C. elegans DNA damage response (DDR) and mechanisms of radiation lethality for this Notch-driven glp-1(ar202) tumor model.We first defined at 240Gy , left, sat 240Gy , right, glp-1(ar202) germline tumor cells exhibit rapid increase in nuclear size  gonads, was found in virtually all proliferative germ nuclei in both wild-type and glp-1(ar202) worms exposed to 480Gy. In wild-type worms, P-Tyr15-CDK-1 was present at 8h post-irradiation in 91% of proliferative zone nuclei, ending abruptly as germ cells entered meiotic prophase  as phenotypically similar to wild-type proliferative zone cells, P-Tyr15-CDK-1 was observed in 93% of post-irradiation tumor nuclei. Subsequent to G2⁄M cell cycle arrest, significant ar202 germ cell loss occurred, detected at 12h post 480Gy (young adult stage) .A hallmark of the eukaryotic DDR is cell cycle arrest, which facilitates coordinated deployment of multistage DNA repair systems or evolution of apoptotic death –38. Hereear size , 8h, wit p<0.01, , associa p<0.01, , consist elegans , suggestA damage , while nar202, we irradiated worms and examined germ cell apoptosis at 24h and 48h post radiation. However, there was little ar202 germ cell apoptosis after 240Gy (ced-3 is required for radiation-induced germline apoptosis [ced-3 was inactivated by 2 approaches in glp-1(ar202), either by generating a ced-3(n717);glp-1(ar202) double mutant or by RNAi, and germ cell number was scored after irradiation. Inactivation of caspase-mediated cell death by either approach did not alter ar202 radiation response , we employed RNAi knockdown of the conserved DDR repair machinery. Quantitative PCR confirmed RNAi knockdown efficiency , and the npp-15 ortholog of human NUP133, a mammalian nuclear pore component [rad-50 knockdown in mutant glp-1(ar202) does not enhance radiosensitivity in mitotic germline tumors, although rad-50 gene expression was reduced after RNAi by 81±8% in ar202  cell death, an outcome of failure of cycling cells to adequately repair DNA DSBs, usually by coordinate activation of NHEJ and HDR . To explficiency . Table 1omponent , conferrin ar202 , indicatrm cells .rad-51 and mre-11 revealed significantly-increased sensitivity of glp-1(ar202) germ cells between 60-300Gy, reducing 50% tumor control dose from 266 to 168Gy with rad-51 RNAi (p<0.01) and to 105Gy for mre-11 RNAi (p<0.01). Differences in tumor response were detectable at 24h after 210Gy (p<0.01), and at 120h rad-51-inactivated worms displayed 74% reduced germ cell number , while mre-11 inactivation nearly eradicated tumor. Furthermore, mre-11 RNAi treatment was associated with extension of ar202 lifespan post-irradiation, comparable to that of wild-type unirradiated worms (cku-80 and lig-4), cell cycle, DNA damage checkpoint, DNA replication and chromatin remodeling had no impact on ar202 germline tumor radiosensitivity , but not NHEJ (lig-4 and cku-80) genes.Detailed analysis of impact of inactivating er 210Gy ; p<0.01,ed worms . In contsitivity . RNAi coar202 germline tumors express NHEJ genes, we employed the temperature-sensitive germ cell-deficient mutant glp-4(bn2)[glp-4(bn2) animals are grown at the permissive temperature, and therefore contain a germ line, they express key NHEJ genes lig-4 and cku-80, as well as HDR genes mus-101, rad-51 and atl-1, at much higher levels than animals grown at the restrictive temperature, which lack a germ line. Gene expression levels in somatic tissue and germ line could also be affected by culturing animals at the different temperatures, although this is unlikely in our study. We conclude, therefore that NHEJ genes are, in fact, enriched in the germ line, while post-mitotic somatic cells in adult worms express minimal amounts. Consistent with these data, we recently reported mitotically-active cells of murine small intestinal crypts aggressively repair radiation DNA damage, while post-mitotic villus cells do not [To address whether lp-4(bn2). S1 Tabls do not .lig-4 RNAi plates were collected at 4h post egg laying, a time preceding vulval development, and irradiated with 120Gy. At 96h after 120Gy, minimal overall damage was detected in N2 worms even with rad-51 silencing, while lig-4 or cku-80 knockdown-worms displayed abnormal vulval development , with increased penetrance of somatic defects (lower panel) [To obtain functional evidence that RNAi feeding adequately inactivated respective NHEJ DSB repair genes, we examined consequence of inactivating NHEJ genes on somatic development in irradiated wild-type worms. For these studies, N2 embryos grown in r panel) ,48. Takecku-80 nor lig-4 RNAi impacted this post-radiation pattern . Irradiated CUTLL-1 cells display fewer cells in G1⁄S relative to G2 with G2 phase cells increasing from 9.2% at baseline to >55% at 24h after 4Gy, which persists for 48h , and minimal impact on Dq  mice, were irradiated at 100–150 mm3. Initial studies established the 50% tumor control dose (TCD50), a standard readout of radiotherapy effectiveness [RAD51-shRNA-expressing CUTLL-1 xenografts responded to 12Gy more robustly than non-silenced control CUTLL-1 tumors (p<0.001), with all RAD51 shRNA-expressing CUTLL-1 tumors showing complete responses by 10 days. Further, over 15 weeks, 83% of RAD51 shRNA-expressing CUTLL-1 tumors achieved autopsy-confirmed cure, while only 33% of CUTLL-1 tumors expressing non-silencing shRNA achieved cure  germline tumor cells display increased average volume in both distal and proximal tumorous germline . Asterisks indicate p<0.01.480Gy-treated (TIF)Click here for additional data file.S2 Figced-3 using RNAi does not alter ar202 response to radiation. Worms were irradiated at the L4 stage and germ nuclei counted in one gonad arm at 72h post radiation. Data (mean±s.e.m) represent number of germ nuclei per gonad from ≥10 worms per group. (B) As an RNAi assay control, radiation-induced germ cell apoptosis was measured in wild-type worms with ced-3 RNAi. Worms were irradiated at the L4 stage and apoptotic cells were scored at 30h post radiation. Data (mean±s.e.m) are from 8–11 worms per group.(A) Inactivation of (TIF)Click here for additional data file.S3 Figglp-1(ar202) was estimated for 8 genes in parallel with germline proliferation assays. Gene expression levels were analyzed by qPCR as in Methods. All samples were run in triplicate and standard deviations were <1.5%. Error bars indicate s.e.m from ≥3 independent experiments.Extent of RNAi-induced knockdown in (TIF)Click here for additional data file.S4 FigRAD51 GIPZ lentiviral-transduced CUTLL-1 cells display high-level GFP expression (200x magnification). (B) Level of RAD51 gene knockdown analyzed by qPCR. Control represents CUTLL-1 cells infected by non-silencing lentiviral shRNA. Error bar indicates s.e.m. collated from 3 independent experiments.(A) After puromycin selection, human (TIF)Click here for additional data file.S5 FigCUTLL-1 cells were treated with 0–100 μM Mirin for 1h before irradiation. Number of cell colonies (mean±s.e.m.) were scored on day 12.(TIF)Click here for additional data file.S6 FigXRCC4 lentiviral-transduced CUTLL-1 cells display high-level tdTomato expression (200x magnification). (B) Level of XRCC4 gene knockdown analyzed by qPCR. Control represents empty vector-treated CUTLL-1 cells. Error bars indicate s.e.m. collated from 3 independent experiments. (C) Clonogenic survival in CUTLL-1 cells expressing human XRCC4 shRNA. Surviving colonies (>50 cells) were scored at 11–14 days post irradiation.(A) After puromycin selection, human (TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


Oropharyngeal HNSCCa is frequently associated with HPV infection. DNA-based Immunotherapy with plasmids encoding HPV16 and HPV18 E6/E7 antigens has been shown to generate robust immune responses in women with HPV-driven high-grade cervical dysplasia. We hypothesize that HPV-specific immunotherapy with INO-3112 (VGX-3100 + INO-9012) in patients with HPV-associated HNSCCa will generate robust immunity which may contribute to disease stabilization or regression.Eligibility for this prospective Phase I/IIa trial included adults with HPV-positive (assessed by p16) HNSCCa, ECOG PS 0-1, and adequate organ function. Patients (pts) are enrolled into two cohorts. In Cohort 1, pts receive INO-3112 pre and post-surgery. In Cohort 2, pts receive INO-3112 after completion of cisplatin based chemoradiation. INO-3112 (6mg of VGX-3100 plus 1mg of INO-9012) is delivered IM followed by electroporation with the CELLECTRA® device, once every 3 weeks for a total of 4 doses. Pts are followed for 2 years. Primary and secondary endpoints are safety and immune responses. Exploratory endpoints include: anti-tumor effect and progression-free-survival. Assessment of post-immunotherapy surgical specimens is being done to evaluate vaccine-induced lymphocyte infiltration in tumor.As of June 2015, 19 pts have been enrolled. Complete safety data is available for 13 pts. Cohort 1: n=3, Cohort 2: n=10; 12 males; median age 57.7 years (range 39-76); cancers at base of tongue=6, tonsil=6, soft palate=1; never smoker=5, median follow-up is 104 days. INO-3112 was well tolerated with no Grade 3 or higher AEs. The most common AEs were injection site pain (n=11), local erythema (n=4) and hematoma/swelling . Two subjects had Grade 3 lymphopenia at baseline and no worsening during the trial. There was a Grade 2, unrelated SAE of post-surgical procedure hemorrhage. Enrollment and correlative analysis are ongoing; among samples tested to date, as compared to baseline, 4 of 5 evaluable pts showed elevated anti HPV16 and 18 E6/E7 antibody titers. Nine of 10 evaluable pts exhibited increased HPV-specific cellular responses by IFN-gamma ELISpot. Seven of 8 evaluable pts had HPV-specific CD8+ T cell activation concurrent with increased lytic proteins (granzymes and perforin) by flow cytometric analysis.These interim results demonstrate that this DNA-based immunotherapy (INO-3112) can safely generate HPV-specific CD8 T cell immunity in patients with HPV-related HNSSCa. All tested pts had positive cellular immune responses in at least one assay.This study (NCT02163057) is co-sponsored by Inovio and the Abramson Cancer Center at the University of Pennsylvania (5P30CA016520-39).ClinicalTrials.gov identifier NCT02163057.


Monocyte chemotactic protein 1 (MCP1) stimulates phosphorylation of cortactin on Y421 and Y446 residues in a time-dependent manner and phosphorylation at Y446 but not Y421 residue is required for MCP1-induced CDK-interacting protein 1 (p21Cip1) nuclear export and degradation in facilitating human aortic smooth muscle cell (HASMC) proliferation. In addition, MCP1-induced cortactin tyrosine phosphorylation, p21Cip1 degradation and HASMC proliferation are dependent on Fyn activation. Upstream to Fyn, MCP1 stimulated C-C chemokine receptor type 2 (CCR2) and Gi/o and inhibition of either one of these molecules using their specific antagonists or inhibitors attenuated MCP1-induced cortactin tyrosine phosphorylation, p21Cip1 degradation and HASMC proliferation. Cortactin phosphorylation at Y446 residue is also required for another G protein-coupled receptor (GPCR) agonist, thrombin-induced p21Cip1 nuclear export and its degradation in promoting HASMC proliferation. Quite interestingly, the receptor tyrosine kinase (RTK) agonist, platelet-derived growth factor-BB (PDGF-BB)-induced p21Cip1 degradation and HASMC proliferation do not require cortactin tyrosine phosphorylation. Together, these findings demonstrate that tyrosine phosphorylation of cortactin at Y446 residue is selective for only GPCR but not RTK agonist-induced nuclear export and proteolytic degradation of p21Cip1 in HASMC proliferation. Cell proliferation plays an essential role in the development of an organism and tissue repairing4710Post-translational modifications of cortactin especially acetylation and phosphorylation were shown to govern its interactions with other cytoskeletal proteins in the modulation of cell migration1213141517181414162124While the functional role of cortactin in cell migration and receptor endocytosis has been well studied, its role in cell proliferation is limited to a few studies. Overexpression of cortactin enhances serum- and epidermal growth factor-stimulated proliferation of head and neck squamous carcinoma cellsStudies from various laboratories including ours have demonstrated that cortactin plays an important role in cell migration915733Recently, we have reported that MCP1 induces serine phosphorylation of cortactin on S405 and S418 residues in enhancing F-actin stress fiber formation and HASMC migration17Since cortactin tyrosine phosphorylation is required for MCP1-induced p21Cip1 degradation and HASMC proliferation, we next wanted to identify the tyrosine residue(s) that are phosphorylated by MCP1. To gain insights into this, we mutated each of the five tyrosine residues to phenylalanine by site-directed mutagenesis and tested their effects on MCP1-induced cortactin tyrosine phosphorylation. While Y453F, Y470F, and Y486F cortactin mutants had no effect, the Y421F and Y446F cortactin mutants prevented MCP1-induced cortactin tyrosine phosphorylation, suggesting that MCP1 induces phosphorylation of cortactin at Y421 and Y446 residues . HoweverIt was reported that cortactin is a substrate for the Src family of the kinases (SFKs)12In our previous study we have demonstrated that Gαq/11 mediates MCP1-induced cortactin serine phosphorylation via PLCβ3-dependent PKCδ activationMCP1 mediates its effects via its receptors CCR2 and CCR4To find the specificity of cortactin tyrosine phosphorylation signaling in MCP1-induced HASMC proliferation, we first tested the effect of Y446F cortactin mutant on MCP1, thrombin and PDGF-BB-induced cortactin tyrosine phosphorylation. Surprisingly, overexpression of Y446F cortactin mutant prevented only MCP1 and thrombin but not PDGF-BB-induced cortactin tyrosine phosphorylation . These fMany studies have demonstrated a role for cortactin in the regulation of actin cytoskeleton remodeling and cell migration113840171814132425262713134547It is noteworthy that besides MCP1, thrombin but not PDGF-BB stimulates cortactin phosphorylation at Y446 residue and this event is required for thrombin-induced p21Cip1 degradation and HASMC proliferation. Although, PDGF-BB induced cortactin phosphorylation at other tyrosine residues, those sites appear to be not required for PDGF-BB-induced p21Cip1 degradation or HASMC proliferation. Thus, these findings indicate that the involvement of cortactin phosphorylation at Y446 residue in p21Cip1 degradation is selective to GPCR agonists. Previous studies have reported that PDGF-BB induces tyrosine phosphorylation of murine cortactin on Y421, Y466 and Y482 residues in the mediation of lamellipodium formation and cell migration1316In elucidating the upstream mechanisms of cortactin tyrosine phosphorylation by MCP1, we found that MCP1 activates Fyn more robustly than the other SFK members and depleting its levels inhibits MCP1-induced cortactin tyrosine phosphorylation, p21Cip1 degradation and HASMC proliferation. Fyn has also been shown to co-localize with cortactin in the mediation of metastatic murine melanoma cell migration16212291355In summary, as shown in 3H]-Thymidine (specific activity 20 Ci/mmol) was obtained from Perkin Elmer Life Sciences . Pertussis toxin from Bordetella pertussis was purchased from Sigma-Aldrich . PP1 (567809), PP2 (529573) and PP3 (529574) were bought from Calbiochem .CCR2 antagonist (SC-202525), CCR4 antagonist (SC-221406) HDAC6 inhibitor (SC-223877), HDAC8 inhibitor PCI-34051 (SC-364566), anti-cortactin (SC-11408), anti-Gαq (SC-393), anti-Gα11 , anti-GFP (SC-9996), anti-Fyn (SC-365913), anti-Lyn (SC-15), anti-p21Cip1 (SC-6246), anti-p27Kip1 (SC-528), anti-p57Kip2 (SC-1040), anti-pYes (SC-130182), anti-Yes (SC-8403) and anti-β-Tubulin (SC-9104) antibodies were obtained from Santa Cruz Biotechnology, Inc. . Anti-pSrc (2101) antibodies were purchased from Cell Signaling Technologies . Anti-phosphoserine/threonine antibody (ab17464), anti-proteasome 20Sα + β (ab22673), and pFyn (ab192172) were bought from Abcam . Anti-pLyn (04–375), anti-Src (05–184), anti-phosphotyrosine (05–777) antibodies were obtained from Millipore . Proteasome 20Sα1, 2, 3, 5, 6 & 7 subunits monoclonal antibody (BML-PW8195-0100) was obtained from Enzo Life Sciences . Goat anti-rabbit HRP (31460) and goat anti-mouse HRP (31437) antibodies, ambion silencer validated human Fyn siRNA (AM51331), BAPTA-AM (B6769), Lipofectamine 3000 transfection reagent (L3000-015), Hoechst 33342 (H3570), Prolong gold antifade mounting medium (P36930), Medium 231 (M231-500), smooth muscle growth supplements (S-007-25), and gentamicin/amphotericin solution (R-015-10) were obtained from Thermo Scientific . Protein A sepharose CL-4B (170780-01), Protein G sepharose Fast flow (17061801) and ECL Western blotting detection reagents (RPN2106) were received from GE Healthcare . Human cortactin siRNA (ON-TARGETplus SMARTpool L-010508-00-0005) and control non-targeting siRNA (D-001810-10) were purchased from Dharmacon RNAi Technologies . Anti-CCR2 antibody (NB110-55674) was purchased from Novus Biologicals . Rhodamine phalloidin (00027) was bought from Biotium . -thymidine incorporation as described previously and expressed as counts/min/dishDNA synthesis was measured by [After appropriate treatments, HASMC were fixed with 3.7% paraformaldehyde for 15 min, permeabilized in 0.3% Triton-X-100 for 15 min, blocked with 3% BSA for 1 hr and incubated with mouse anti-p21Cip1 (1:100) and rabbit anti-proteasome 20Sα + β (1:500) antibodies followed by incubation with Alexa Fluor 488-conjugated goat anti-mouse and Alexa Fluor 568-conjugated goat anti-rabbit secondary antibodies (1:500), respectively. After washing with PBS, cells were counterstained with Hoechst 33342 (1:2000) and mounted onto glass slides with Prolong Gold antifade mounting medium. Fluorescence images of cells were captured using an inverted Zeiss fluorescence microscope (AxioObserver Z1) with 40X/NA 0.6 objective and AxioCam MRm camera without any enhancements using the microscope operating and image analysis software AxioVision verstion 4.7.2 (Carl Zeiss Imaging Solutions GmbH).After appropriate treatments the cytoplasmic and nuclear fractions of HASMCs were prepared using NE-PER nuclear and cytolasmic extraction reagents as described in the manufacturer’s  instructions.An equal amount of protein from control and treatment samples was separated by SDS-PAGE and analyzed by Western blotting for the indicated molecules using their specific antibodies as described previously with minor modificationsHASMCs were transfected with non-targeted control or on-target siRNA at a final concentration of 100 nM using Lipofectamine 3000 transfection reagent according to the manufacturer’s instructions. Cells were transfected with plasmid DNAs at a final concentration of 2.5 μg/60 mm dish or 5 μg/100 mm dish using Lipofectamine 3000 transfection reagent according to the manufacturer’s instructions. After transfections, cells were growth-arrested for 36 hrs and used as required.All the experiments were repeated three times, and the data are presented as Mean ± S.D. One-way ANOVA was used to perform statistical analysis and the p values < 0.05 were considered statistically significant.How to cite this article: Janjanam, J. and Rao, G. N. Novel role of cortactin in G protein-coupled receptor agonist-induced nuclear export and degradation of p21Cip1. Sci. Rep. 6, 28687; doi: 10.1038/srep28687 (2016).


Upregulation of SESTRIN 2 (SESN2) has been reported in response to diverse cellular stresses. In this study we demonstrate SESTRIN 2 induction following endoplasmic reticulum (ER) stress. ER stress-induced increases in SESTRIN 2 expression were dependent on both PERK and IRE1/XBP1 arms of the unfolded protein response (UPR). SESTRIN 2 induction, post ER stress, was responsible for mTORC1 inactivation and contributed to autophagy induction. Conversely, knockdown of SESTRIN 2 prolonged mTORC1 signaling, repressed autophagy and increased ER stress-induced cell death. Unexpectedly, the increase in ER stress-induced cell death was not linked to autophagy inhibition. Analysis of UPR pathways identified prolonged eIF2α, ATF4 and CHOP signaling in SESTRIN 2 knockdown cells following ER stress. SESTRIN 2 regulation enables UPR derived signals to indirectly control mTORC1 activity shutting down protein translation thus preventing further exacerbation of ER stress. The mTORC1 complex is an important molecular sensor linking the cellular environment to key processes including proliferation and autophagy . CarefulStress-mediated induction of SESTRIN 2 has been predominantly associated with P53 signaling . HoweverSESTRIN 2 reduced stress driven autophagy and enhanced cell death. Intriguingly, our results suggest that enhanced stress-induced cell death observed in SESTRIN 2 knockdown cells is due to an exacerbation of ER stress rather than a decrease in autophagy.In this study we demonstrate that induction of ER stress following treatment with pharmacological agents or chemotherapeutics such as Methotrexate (Mtx) lead to a P53-independent increase in SESTRIN 2. Our data indicates both IRE1/XBP1 and PERK branches of the UPR contribute to ER stress-induced SESTRIN 2 expression. Knockdown of SESTRIN 2 mRNA levels  and Brefeldin A (BFA), two classical inducers of ER stress, was optimized to select doses which would permit examination of pro-survival and pro-death signaling over time (0.5 μg/ml BFA and 1 μM Tg) Figure . Tg and A levels  while, aA levels . SESTRINA levels . Likewis/+ cells . Increas/+ cells . CollectATF6, while successful, did not inhibit Tg-induced increase in SESTRIN 2 (−/−Xbp1 and −/−Perk mouse embryonic fibroblasts (MEF) cells. PERK-mediated translational inhibition is essential for cell survival following ER stress. Consequently, knockout of Perk renders cells exquisitely sensitive to ER stress-induced death [+/+Perk and −/−Perk MEF cells were treated with a lower dose (25nM) of Tg as described previously [−/−Perk MEF cells displayed lower SESTRIN 2 induction upon ER stress than their +/+Perk counterparts  was clearly evident in Tg-treated control siRNA cells. However, in SESTRIN 2 siRNA-transfected cells significantly less LC3-I to II conversion was detected in cells treated with both Tg and CQ  in BFA treated GFP-LC3 MCF7  cells with a range of chemotherapeutics detected elevated SESTRIN 2 expression following treatment with the anti-folate drug Mtx and the proteosomal inhibitor Btz  failed to sensitize cells to Mtx-induced death  phosphorylation following Mtx treatment  and EIF2α (EIF2S1) phosphorylation in Mtx-treated control versus SESTRIN 2 siRNA transfected cells. Prolonged activation of PERK signaling was observed in Mtx-treated cells transfected with SESTRIN 2 siRNA, suggesting SESTRIN 2 knockdown potentiates Mtx-induced ER stress in HCC1806 cells  were maintained in DMEM medium supplemented with 10% HI FBS, 2 mM L-glutamine, 1 mM sodium pyruvate (Sigma #S8636), non-essential amino acid solution (Sigma #M7145). +/+Perk and −/−Perk MEFs  were maintained in DMEM medium supplemented with 10% HI FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, non-essential amino acid solution, 55 μM β-mercaptoethanol (βME), 1% penicillin/streptomycin. All cells were cultured at 37°C, 5% CO2 in a humidified incubator. Cells were seeded at an appropriate number 24 h prior to treatment. 5-Fluorouracil (Sigma #F6627), Paclitaxel (Sigma #T7402), Doxorubicin (LC laboratories #D-4000), Melphalan (Sigma #M2011), Bortezomib , Methotrexate (Sigma #M9929), Brefeldin A (Sigma #B7651), Thapsigargin (Sigma #T9033), Etoposide (Sigma #E1383), Rapamycin (LC-laboratories #R-5000), IRE1 inhibitor, MKC-8866, (Mannkind Corporation), Spautin-1 (Sigma #SML0440) and PERK inhibitor, GSKG797800 .MCF7 (ATCC), HCC1806 (ATCC), K562 (ATCC) cells were cultured in either Dulbecco's modified Eagle's medium (DMEM) (Sigma #D6429) (MCF-7) or Roswel Park Memorial Institute medium (RPMI 1640) (Sigma #R0883)  supplemented with 10% heat inactivated fetal bovine serum , 100 U/ml penicillin/100 mg/ml streptomycin (Sigma #P0781) and 2 mM L-glutamine (Sigma #G7513). HCT116 colon cancer cells were cultured in McCOYs 5A medium modified (Sigma #M9309) supplemented with 10% HI FBS, 100 U/ml penicillin/100 mg/ml streptomycin. Cells were collected by trypsinization and subsequent centrifugation, resuspended in ice-cold PBS containing 0.7 μg/ml of propidium iodide (Sigma #P4170) and analysed using FACSCalibur flow cytometer (Becton Dickinson).Cells were washed once in ice-cold PBS and lysed in whole cell lysis buffer  and boiled at 95°C for 5 min. Equal amounts of protein samples were run on an SDS polyacrylamide gel. The proteins were transferred onto nitrocellulose membrane and blocked with 5% milk in PBS-0.1% Tween. For detection of protein expression the following antibodies were used: ACTIN (Sigma #A5060), LC3 (Sigma #L7543), SESTRIN 2 (Sigma #WH0083667M3), XBP1 (Abcam #619502), ATF4 (CST #11815), CHOP (CST #2895), MTOR (CST #2983) p-MTOR (CST #2974), PERK (CST #3192), p-P70 S6K (CST #9234). All the secondary antibodies were purchased from Jackson and the signal was visualized using Western Blotting Luminol Reagent (SantaCruz sc-2048).SESTRIN 2 (L-019134-02-0005); XBP1 (L-009552-00-0005); ATF6 (L-009917-00-0005); ATF4 (L-005125-00-0005); BECLIN1 (L-010552-00-0005); Non-coding siRNA (D-001810-01-20). Media was changed 4 h post-transfection and cells were left to recover for 24 h. After the recovery, cells were reseeded for the experiment that was performed 36 h post-transfection.MCF7 and HCC1806 cells were transfected using Lipofectamine 2000  according to the manufacturer's protocol. siRNAs (ON-TARGET plus smart pool) were obtained from Dharmacon versus diffuse cells (3 fields of 100 cells) were counted.GFP-LC3 plasmid was transiently transfected into MCF7 cells according to manufacturers protocol using Lipofectamine 2000 . 24 h post transfection cells were treated as indicated and GFP-LC3 positive punctate SESTRIN 2. GAPDH was used as an endogenous control using the forward primer 5′-ACCACAGTCCATGCCATC-3′ and reverse 5′-TCCACCACCTGTTGCTG-3′.Total RNA was isolated from cells using TRI Reagent (Invitrogen) according to the manufactures protocol. To synthesize cDNA, 2 μg of RNA was subjected to DNase treatment and incubated for 15 min at room temperature. The RNA was then reverse transcribed into cDNA using Superscript III first strand RT-PCR system and random hexamers (Invitrogen). The cDNA product was subjected to PCR using the forward primer 5′-CAGAGGGCACAGGAAAGAAG-3′ and the reverse primer 5′-GAACTAGGATTCGGGCAACA-3′ for the detection of human SESTRIN 2 and non-coding control siRNAs as described above. 36 h later they were treated with 20 μM of Mtx for 72 h. After the treatment, media was removed and cells were left to recover for 10 days. Colonies were stained with 2% crystal violet in 20% methanol.HCC1806 cells were transfected with *p < 0.05 is considered statistically significant, **p < 0.01, ***p < 0.001.Cell death and data is expressed as mean ± SD for three independent experiments. Real Time data is expressed as mean ± SEM for three independent experiments. Differences between the treatment groups were assessed using Graphpad's Two-tailed unpaired student's t-tests. The values with


Receptor tyrosine kinase-like orphan receptor-1 (ROR1) is expressed on sub-populations of B-cell malignancies and solid tumors, but not by healthy B cells or normal post-partum tissues. Thus, adoptive transfer of T cells specific for ROR1 has potential to eliminate tumor cells and spare healthy tissues. To test this hypothesis, we developed CARs targeting ROR1 in order to generate T cells specific for malignant cells. Two Sleeping Beauty transposons were constructed with 2nd generation ROR1-specific CARs signaling through CD3ζ and either CD28 (designated ROR1RCD28) or CD137 (designated ROR1RCD137) and were introduced into T cells. We selected for T cells expressing CAR through co-culture with γ-irradiated activating and propagating cells (AaPC), which co-expressed ROR1 and co-stimulatory molecules. Numeric expansion over one month of co-culture on AaPC in presence of soluble interleukin (IL)-2 and IL-21 occurred and resulted in a diverse memory phenotype of CAR+ T cells as measured by non-enzymatic digital array (NanoString) and multi-panel flow cytometry. Such T cells produced interferon-γ and had specific cytotoxic activity against ROR1+ tumors. Moreover, such cells could eliminate ROR1+ tumor xenografts, especially T cells expressing ROR1RCD137. Clinical trials will investigate the ability of ROR1-specific CAR+ T cells to specifically eliminate tumor cells while maintaining normal B-cell repertoire.T cells modified with chimeric antigen receptors (CARs) targeting CD19 demonstrated clinical activity against some B-cell malignancies. However, this is often accompanied by a loss of normal CD19 Both ROR1RCD28+ and ROR1RCD137+ T cells produced significant quantities of IFNγ when treated with PMA/Ionomycin  relative to mock-activated  cultures  to co-cultures with ROR1neg cells from EBV-transformed healthy donor B-cell lymphoblastoid cell lines (LCL), NALM-6, and parental EL4 cells  and NALM-6 cells  suggesting that the ROR1RCD28+ and ROR1RCD137+ T cells were more discriminant in their targeting of these B-ALL cell lines (neg T cells (No DNA) showed no lysis of these 4 cell lines indicating that the CAR responses were specific. ROR1RCD28+ and ROR1RCD137+ T cells generated from healthy donor PBMC also lysed allogeneic CLL patient B cells (ROR1+), but spared allogeneic LCL (ROR1neg) derived from healthy donor B-cells , while autologous T cells and parental EL4 (both ROR1neg) cells were spared  [in vivo. NOD.scid.γc-/- (NSG) mice were used because they lack functional adaptive immune systems and can therefore accept human tumor xenografts. Healthy, 6–12 week old female NSG mice were engrafted with Kasumi-2-ffLuc-mKate and distributed into 3 treatment groups (n = 5 per group): (i) no treatment, (ii) ROR1RCD28+ T-cell treated, and (iii) ROR1RCD137+ T-cell treated, where T cells were given at days 1, 8, and 15 post-tumor cell engraftment   for seriraftment . Untreatr tumors  top and BLI flux  middle ated mice  bottom, + T cells for investigational therapy of ROR1+ malignancies. We demonstrate that SB transposition achieved stable CAR expression in T cells and, in concert with co-culture on clone#1 AaPC, resulted in heterogeneous outgrowth of T-cell memory populations with ROR1-restricted anti-tumor activity. Currently, this process takes 28 days to achieve clinical numbers of T cells. Potentially shortening the production time for cell growth may (i) simplify the overall process, (ii) preserve more of the minimally differentiated subsets, and (iii) could take advantage of in vivo proliferation of CAR+ T cells thereby augmenting therapeutic efficacy. As SB transposition was used to express ROR1-specific CARs in naïve or minimally-differentiated T-cell populations present in quiescent PBMC, as has been done with other SB-based CAR studies [e.g., IL-15, can promote the survival of less differentiated T cells, and trans-presentation of IL-15 by IL15Rα has higher signaling potency than IL-15 alone [+ T cells expressing CD19RCD28 [e.g., ROR1) and molecules that promote T-cell survival , in concert with SB transposition can support the propagation of populations of T cells predicted to have sustained engraftment, i.e., TSCM and CD62L+ TCM populations [This work reveals the pre-clinical basis to support our current “first-in-human” Phase I clinical trial of CAR studies , 38, the15 alone , 46. ThuD19RCD28 , on the ulations , 48, whind generation CAR endodomain may impact the therapeutic activity of infused T cells. CD19-specific CARs activating T cells via chimeric CD28 or CD137 endodomains led to objective clinical responses [+ T cells displayed enhanced in vivo tumor clearance  and in vivo tumor clearance by CD8+ tumor-specific T cells has also been established for adoptive immunotherapy of melanoma, further supporting the notion that ex vivo culture conditions and memory phenotype of infused T cells are useful parameters to correlate with therapeutic efficacy [The choice of signaling motif within a 2learance  despite  T cells . The dec in vivo . CARs wi in vivo . Future efficacy . A side-+ malignancies. As a control for adverse events, conditional suicide genes, e.g., inducible Caspase9, could be co-expressed with CAR in order to eliminate T cells in vivo as needed [A major advantage of targeting ROR1 over the current T-cell therapies targeting CD19 is that recipients would not deplete B cells and develop hypogammaglobulinemia, thereby mitigating the risk for impaired humoral immunity , 51. RORs needed .+ T cells and a Phase I clinical trial is open to patients with CLL as of July 2014 (NCT02194374). Clinical-grade DNA plasmids coding for ROR1RCD137 and SB11 have been produced and a master-cell bank of AaPC clone#1 has been generated. Furthermore, regulatory documents and materials are in place to undertake the clinical trial. This will be the first time ROR1 on CLL has been targeted by T cells; therefore, the primary endpoint will be to determine toxicity and maximum tolerated T-cell dose. Future trials can then assess the clinical impact of targeting ROR1 on solid tumors.In summary, our data support the clinical application of ROR1-specific CARS1 Dataset+ T cells (middle), and multiparameter memory phenotype of T cells (bottom). (+ T cells following 6 hour co-culture with target cells. (+ T cells (top) and days of mouse euthanasia (bottom). tab Cellbottom).  tab MFI t cells.  tab 4-hot cells.  tab BLI (XLSX)Click here for additional data file.


The cyclic nucleotide-gated channels, CNGC19 and CNGC20, are the sole members of the highly isolated evolutionary group IV-A in Arabidopsis plants. Prior studies have shown that the expression of both CNGC19 and CNGC20 genes are induced by salinity and biotic stress. In this report, CNGC19 and CNGC20 were determined to localize to the vacuolar membrane. Co-expression of CNGC19 and CNGC20 increased the efficiency of vacuolar localization. CNGC19 and CNGC20 are, therefore, vacuolar membrane channels that are hypothesized to mediate plant response to salinity and biotic stress. 2+ signalling, and responses to biotic and abiotic stress. The 20 CNGC paralogues of Arabidopsis are divided into five evolutionary groups. Group IV-A is highly isolated and consists only of two closely spaced genes, CNGC19 and CNGC20. Prior studies have shown that both genes are induced by salinity and biotic stress. A unique feature of CNGC19 and CNGC20 is their long hydrophilic N-termini. To determine the subcellular locations of CNGC19 and CNGC20, partial and full-length fusions to GFP(S65T) were generated. Translational fusions of the N-termini of CNGC19 (residues 1–171) and CNGC20 (residues 1–200) to GFP(S65T) were targeted to punctate structures when transiently expressed in leaf protoplasts. In the case of CNGC20, but not CNGC19, the punctate structures were co-labelled with a marker for the Golgi. The full-length CNGC19-GFP fusion co-localized with markers for the vacuole membrane (αTIP- and γTIP-mCherry). Vacuole membrane labelling by the full-length CNGC20-GFP fusion was also observed, but the signal was weak and accompanied by numerous punctate signals that did not co-localize with αTIP- or γTIP-mCherry. These punctate structures diminished, and localization of full-length CNGC20-GFP to the vacuole increased, when it was co-expressed with the full-length CNGC19-mCherry. Vacuole membrane labelling was also detected in planta via immunoelectron microscopy using a CNGC20-antiserum on cryopreserved ultrathin sections of roots. We hypothesize that the role of group IV-A CNGCs is to mediate the movement of cations between the central vacuole and the cytosol in response to certain types of abiotic and biotic stress.Plant cyclic nucleotide-gated channels (CNGCs) are implicated in the uptake of both essential and toxic cations, Ca The subcellular localization of CNGCs has primarily been accomplished through the use of chimeric fluorescent reporter proteins. Fluorescent protein fusions indicate that at least four of the six CNGCs belonging to group I are targeted to the PM: CNGC3 . HoweverT_CHLORO .In this report, we utilized fluorescent reporter protein fusions and immunolelectron microscopy to determine the subcellular destinations of CNGC19 and CNGC20. We demonstrated that both channels are targeted to vacuolar membranes. Our findings suggest that group IV-A CNGCs mediate plant responses to salinity and pathogen infection by facilitating the movement of cations between the central vacuole and the cytosol.Arabidopsis CNGCs were performed using TargetP v1.1 , and the rice homologue OsCNGC-IVa1 (GenBank: BAD16877.1) were obtained using the NCBI BLAST server, while the moss homologue PpCNGC-IVa1 (COSMOSS v1.6: Pp1s204_120V6) was identified by a Phytozome BLAST search. Phylogenetic analysis with all 20 CNGCs from Arabidopsis confirmed that these CNGCs belong to group IV-A (data not shown).Subcellular localization predictions for :GFP(S65T)) was described previously (CNGC19FL-GFP) and pBL(35S:CNGC20FL-GFP) were created by replacing the EGFP coding sequence of plasmid pBL(35S:CNGC10-EGFP) (GFP(S65T), and the CNGC10 genomic DNA sequence with the full-length genomic DNA sequences of CNGC19 (At3g17690) or CNGC20 (At3g17700), respectively. The GFP(S65T) fragment was amplified from plasmid HBT95::sGFP(S65T)-NOS with primers GFP/NdeI (5′-CACCGACTAGTCATATGGTGAGCAAGGGCGAG-3′) and GFP/BstEII (5′-TACAGGGTCACCTTACTTGTACAGCTCGTCCATG-3′). CNGC19 was amplified from Arabidopsis thaliana (ecotype Col-0) genomic DNA using the PCR primer pair CNGC19/NcoI.F (5′-TAAACCATGGCTCACACTAGGACTTTCACT-3′) and CNGC19/NdeI.R (5′-ATCACATATGACGGTTGGAATTGGAGTGAG-3′), while CNGC20 was amplified using primers CNGC20/NcoI.F (5′-TGGTCAAGCCATGGCTTCCCACAACGA-3′) and CNGC20/NdeI.R (5′-AATACCATATGAAGGCTATAACTAGACTGAG-3′). The GFP(S65T) fragment was cloned between the NdeI and BstEII restriction sites of pBL(35S:CNGC10-EGFP), and the CNGC19 and CNGC20 fragments between NcoI and NdeI. It was necessary to ligate the CNGC20 amplicon sequentially as two separate fragments due to the presence of an internal NdeI site. Green fluorescent protein fusions to the N-terminal regions of CNGC19 and CNGC20 were developed as described above, except that the CNGC19N1 fragment was obtained using the primer pair CNGC19/NcoI.F and CNGC19(D86)/NdeI.R (5′-TTGAACATATGATCATCTTCAGGTGGAAC-3′), CNGC19N2 using primers CNGC19/NcoI.F and CNGC19(V166)/NdeI.R (5′-GTCCACATATGAACAAATTTGGAATGAGGA-3′), CNGC20N1 using primers CNGC20/NcoI.F and CNGC20(E117)NdeI.R (5′-TTCAACATATGCTCATCCTCTGAGGAGTT-3′) and CNGC20N2 using primers CNGC20/NcoI.F and CNGC20(V200)/NdeI.R (5′-GTCCACATATGAACCTCTTTGGCATGAGGA-3′). The fusion construct pBL(35S:CNGC19FL-mCherry) was generated by ligation of a PCR fragment containing both the coding sequence for mCherry and the nopaline synthase (nos) 3′ untranslated region (UTR) between the NdeI and SacI sites of pBL(35S:CNGC19FL-GFP). This fragment was amplified from plasmid pt-rk (GAGCTCCCGATCTAGTAACATAGA-3).All constructs used for protoplast transfection in this study were generated in cloning vector pBluescript KS(+). The generation of pBL(35SMan49-mCherry)], peroxisomes [pBL(35S:mCherry-SKL)] and tonoplast [pBL(35S:γTIP-mCherry)] are derivatives of the organelle markers developed by αTIP-mCherry), was generated by amplification of the full-length genomic DNA sequence of AtαTIP  with primers αTIP/XbaI.F (5′-AAGTTCTAGATCATAATGGCAACATCAGCTC-3′) and αTIP/BamHI.R (5′-GTTCGGATCCGTAATCTTCAGGGGCCAAG-3′), and subsequent ligation of the PCR product between the SpeI and BamHI sites of pBL(35S:γTIP-mCherry). The plastid envelope marker pBL(35S:NTT2-mCherry) was created in a similar manner, using primers NTT2/SpeI.F (5′-GTGCTACTAGTGAGATAGAGAGATGGAAGGT-3′) and NTT2/BamHI.R (5′-ATCATGGATCCAATGCCAGTAGGAGTAGATTTCT-3′) to amplify the genomic DNA sequence of AtNTT2 (At1g15500). pBL(35S:NTT2-GFP) was created by digesting pBL(35S:NTT2-mCherry) with KpnI and NcoI, and cloning the 3.1-kb fragment containing the cauliflower mosaic virus (CaMV) 35S promoter and AtNTT2 genomic DNA sequence between the KpnI and NcoI sites of pBL(35S:GFP(S65T)). The construct pBL(35S:BiP1-mCherry-HDEL) contains, in order, the CaMV 35S promoter, the AtBiP1 (At5g28540) genomic DNA sequence, the coding sequence of mCherry-HDEL and the nos terminator, inserted between the KpnI and SacI sites of pBluescript KS(+). The CaMV 35S promoter region was amplified with primers 35S/KpnI.F (5′-TTCAAGGTACCTTCATGGAGTCAAAGATTCA-3′) and 35S/XhoI.R (5′-ATCTACTCGAGTCAAGAGTCCCCCGTG-3′), the AtBiP1 gene sequence was amplified from A. thaliana (ecotype Col-0) genomic DNA with primers BiP1/XhoI.F (5′-GAGCTCGAGCGCAAAAGTTTCCGATATGGCTCGCTC-3′) and BiP1/XmaI.R (5′-AGACCCGGGGAGCTCATCGTGAGACTCATCTTC-3′), and the mCherry-HDEL coding sequence and nos 3′ UTR were amplified from plasmid ER-rk with primers GFP/XmaI.F (5′-AAACCCGGGCTTGTACAGCTCGTCCATGC-3′) and Nos/SacI.R.The constructs used for the transient expression of fluorescent markers for the Golgi  by the method described in An affinity-purified rabbit polyclonal antibody was raised against a unique region of CNGC20  by New England Peptide . For immunogold labelling, developing roots and leaves were preserved by high-pressure freezing/freeze-substitution techniques as described previously , and partially overlapping cyclic nucleotide-binding and calmodulin-binding domains situated near their C-termini , rice (Oryza sativa L. ssp. japonica) and moss (Physcomitrella patens ssp. patens). The rice and poplar orthologues have N-terminal hydrophilic regions that are comparable in length to CNGC19 and CNGC20 (183–209 aa), while the N-terminus of the P. patens orthologue is substantially shorter . Unlike their shorter counterparts, the longer CNGC19N2-GFP and CNGC20N2-GFP fusions displayed punctate GFP fluorescence patterns. However, these punctate signals did not overlap with chlorophyll autofluorescence . The corresponding chimeric proteins, designated as CNGC19FL-GFP) or CNGC20 (CNGC20FL-GFP) were examined. Several distinct localization patterns were observed in protoplasts transfected with the full-length CNGC19 and CNGC20 fusion constructs. The CNGC19FL-GFP fusion labelled elliptical structures  to the N-terminus of the monomeric red fluorescent protein (RFP) variant, mCherry. Protoplasts simultaneously expressing NTT2-mCherry in combination with either CNGC19FL-GFP or CNGC20FL-GFP did not display significant co-localizing GFP and RFP fluorescence -mCherry and γTIP-mCherry. Elliptical structures labelled by CNGC19rry Fig. A and γTIrry Fig. D. The CNnes Fig. E, in mannce Fig. B and C.FL-GFP with that of αTIP- or γTIP-mCherry revealed that CNGC20FL-GFP co-localized with the vacuole markers in both extended endomembranes  marker generated by fusing A. thaliana ER luminal binding protein 1 (BiP1) to a modified version of mCherry harbouring the C-terminal ER retention motif, HDEL. We also did not observe co-localization between CNGC20FL-GFP and markers for the mitochondria Supporting Information or peroxisomes [Supporting Information]. However, co-expression with the Golgi marker Man49-mCherry . Anti-CNGC20 labelling was observed at the membranes of vacuoles  when simultaneously expressed in protoplasts.In Arabidopsis transgenic plants stably expressing γTIP-GFP under the CaMV 35S promoter  do not display diffuse cytosolic labelling, but instead exhibit punctate labelling. This suggests that the N-terminal regions of CNGC19 and CNGC20 contain information that influences protein sorting, but are not sufficient to direct proteins to the vacuole. Interestingly, the N-terminal regions of group IV-A CNGCs contain a novel 19-aa conserved motif (LxxSGxLGxCxDPxCxxCP). Since GFP fusions harbouring shortened versions of the N-terminal region truncated just prior to this motif  exhibit diffuse labelling of the cytosol, we speculate that the 19-aa interval may play a role in protein sorting. Interestingly, in protoplast co-transfection experiments, the Golgi marker Man49-mCherry co-localized with CNGC20N2-GFP, but not with CNGC19N2-GFP. This suggests that differences exist between the protein sorting information contained within the N-terminal hydrophilic regions of CNGC19 and CNGC20. These differences may contribute, at least in part, to the dissimilar localization patterns of our full-length CNGC19FL-GFP and CNGC20FL-GFP fusions with regard to the absence or presence of punctate labelling (respectively).Protoplasts expressing GFP fused to the entire hydrophilic N-terminal region of CNGC19 or CNGC20  antiporters , suppressing cGMP accumulation with an inhibitor of guanylyl cyclases also diminishes the Ca2+ spike  and the Advances in Biosciences Education programme funded by the National Science Foundation (grant no. MCB-09-58107).This research was supported by a D.A.C. conceived the project, and D.A.C. and C.C.Y.Y. designed the protoplast transient expression experiments and developed all the new fusion constructs utilized in this study. D.A.C. performed all the immunoelectron microscopy experiments. C.C.Y.Y. conducted protoplast transfections and analysed the samples through confocal microscopy. D.A.C. and C.C.Y.Y. wrote the manuscript, and read and approved the final version for submission.None declared.The following Supporting Information is available in the online version of this article.File 1: Figure. Comparison of CNGC19FL- and CNGC20FL-GFP localization with a marker for the ER. Confocal laser scanning microscope images of leaf protoplasts co-transfected with the ER marker BiP1-mCherry-HDEL and (A) CNGC19FL-GFP or (B) CNGC20FL-GFP. Column 1, GFP signal (green); column 2, RFP signal (red); column 3, merged GFP and RFP signals; column 4, merged GFP and RFP signals with chlorophyll autofluorescence (blue). Scale bars represent 5 µm.File 2: Figure. Comparison of CNGC19FL- and CNGC20FL-GFP localization with a marker for mitochondria. Confocal microscopy images of leaf protoplasts transiently expressing (A) CNGC19FL-GFP or (B) CNGC20FL-GFP, and stained with MitoTracker Orange. Column 1, GFP signal (green); column 2, MitoTracker Orange signal (red); column 3, merged GFP and MitoTracker Orange signals; column 4, merged GFP and MitoTracker Orange signals with chlorophyll autofluorescence (blue). Scale bars represent 5 µm.File 3: Figure. Comparison of CNGC19N2- and CNGC20N2-GFP localization with a marker for peroxisomes. Confocal laser scanning microscope images of leaf protoplasts co-transfected with the peroxisome marker mCherry-SKL and (A) CNGC19N2-GFP or (B) CNGC20N2-GFP. Column 1, GFP signal (green); column 2, RFP signal (red); column 3, merged GFP and RFP signals; column 4, merged GFP and RFP signals with chlorophyll autofluorescence (blue). Scale bars represent 5 µm.File 4: Figure. Pre-immune serum staining of roots. Immunolabelling of Arabidopsis cryo-fixed thin root tissue sections with rabbit pre-immmune serum and anti-rabbit 15 nm gold-conjugated secondary antiserum as a negative control. Samples were viewed via TEM as described in the Methods. No labelling was observed. G, Golgi apparatus, Cy, cytoplasm; Vc, vacuole; ER, rough endoplasmic reticulum; Pm, plasma membrane; M, mitochondrion.File 5: Figure. Immunolabelling of leaves. Immunolabelling of Arabidopsis cryo-fixed thin leaf tissue sections with anti-CNGC20 antiserum and anti-rabbit 15 nm gold-conjugated secondary antiserum. Samples were viewed via TEM as described in the Methods. No labelling was observed in the chloroplasts (CT); some labelling was observed in the edges of vacuoles (V).File 6: Figure. Immunoblot analysis of CNGC20 in Arabidopsis total cellular proteins. Immunoblot analysis was performed using the anti-CNGC20-specific peptide antiserum on 40 μg of total seedling proteins (14-day-old) from wild type (WT) and the CNGC20 T-DNA mutant (MUT). The homozygous T-DNA insert (SALK_129133.22.05) is in the fourth exon of the CNGC20 locus (At3g17700). The antiserum detects a single band of ∼84 kDa in the wild-type protein sample, which is the predicted size of CNGC20, whereas no CNGC20 protein is detected in the mutant. This indicates that the antiserum is specific to CNGC20. Coomassie-stained proteins (COOM) are shown from a duplicate gel.


Excitotoxicity (the toxic overstimulation of neurons by the excitatory transmitter Glutamate) is a central process in widespread neurodegenerative conditions such as brain ischemia and chronic neurological diseases. Many mechanisms have been suggested to mediate excitotoxicity, but their significance across diverse excitotoxic scenarios remains unclear. Death Associated Protein Kinase (DAPK), a critical molecular switch that controls a range of key signaling and cell death pathways, has been suggested to have an important role in excitotoxicity. However, the molecular mechanism by which DAPK exerts its effect is controversial. A few distinct mechanisms have been suggested by single (sometimes contradicting) studies, and a larger array of potential mechanisms is implicated by the extensive interactome of DAPK.C. elegans to show that DAPK is an important mediator of excitotoxic neurodegeneration across a large evolutionary distance. We further show that some proposed mechanisms of DAPK’s action  do not have a major role in nematode excitotoxicity. In contrast, Pin1/PINN-1 (a DAPK interaction-partner and a peptidyl-prolyl isomerase involved in chronic neurodegenerative conditions) suppresses neurodegeneration in our excitotoxicity model.Here we analyze a well-characterized model of excitotoxicity in the nematode Our studies highlight the prominence of DAPK and Pin1/PINN-1 as conserved mediators of cell death processes in diverse scenarios of neurodegeneration. Despite our familiarity with the first few steps in excitotoxicity, our understanding of the steps following Ca2+ influx is very limited. Clinical trials using GluRs antagonists ended with disappointment, and recent data suggests that using antagonists to block GluR functions might be counterproductive, because Glu signaling includes both neurotoxic and pro-survival cascades  (IV); juEx1933 (the frIs7 insertion was later eliminated during our cross); Aldicarb assay control strains: sv-hyper-releasing/aldicarb-oversensitive RB1367: cpx-1(ok1552) (RRID:CGC_RB1367); sv-under-releasing/aldicarb-resistant NM791: rab-3(js49) (RRID:CGC_NM791): Autophagy modulatorunc-51: CB369: unc-51(e369) V (RRID:CGC_CB369); Autophagy label, DsRed::LGG-1:  II; glr-2(ak10) glr-1(ky176) III;DANGER-related protein MAB-21: EM128: mab-21(bx53) III  (RRID:CGC_EM128).CaMKK-like KO: ckk-1(ok1033) III (RRID:CGC_VC691);Pin1-like KO:pinn-1(tm2235) II (this 364 bp deletion runs the ORF into a stop codon after 8 codons). Most crosses were followed by PCR analysis to detect deletions, and by monitoring nuIs5’s GFP expression in glr-1-expressing interneurons using a high power fluorescence dissecting scope. dapk-1 over-expressing construct juEx1933 was followed by its coinjection marker, which produces RFP expression on AIY neurons. The unc-51 mutation was followed by uncoordinated phenotype and sequencing. DsRed::LGG-1 was followed by DsRed expression in cells throughout the body and by GFP expression in the pharynx (from the myo-2 promoter). We constructed the following strains: dapk-1 in excitotoxicity (by crossing VC432 and ZB1102) IMN26: dapk-1(gk219) I; glt-3(bz34) IV; nuIs5 V;DAPK-1 overexpression in excitotoxicity (by crossing ZB1102 and CZ9277) IMN25: glt-3(bz34) IV; nuIs5 V; juEx1933 ; Autophagy suppression in excitotoxicity (by crossing ZB1102 and CB369) IMN22: glt-3(bz34) IV; nuIs5 , unc-51(e369) V;Autophagy reporter in excitotoxicity IMN24: glt-3(bz34) IV; nuIs5 V; Ex;CaMKK deletion in excitotoxicity:ckk-1 (ok1033) III; glt-3(bz34) IV; nuIs5 V.DANGER-like deletion in excitotoxicity: (by crossing EM128 and ZB1102): mab-21(bx53) III; glt-3(bz34) IV; nuIs5 V; Pin1-like deletion in excitotoxicity: IMN30: pinn-1(tm2235) II; glt-3(bz34) IV ; nuIs5 V (± dapk-1(gk219) I).The following akis lab , strain nuIs5 animals under the glr-1 promoter. For heat-shock-induced overexpression of DAPK-1, animals carrying  were placed in a 35°C incubator for 2 hours to induce activation of heat shock promoter, left to rest for about 30 minutes and scored regularly for two days for the extent of swollen degenerating neurons .The level of necrotic neurodegeneration in head neurons was monitored using an inverted scope and Nomarski Differential Interference Contrast (DIC). Animals with no anesthetics were examined on a fresh chunk of agar of nematode culture flipped upside down over a slide. Swollen “vacuolated-looking” cells located in the area of the nerve ring were counted as head neurons undergoing necrosis, as described previously ,41. Brieup, UCSD ). ExperiEx ; glt-3(bz34) IV; nuIs5 V and Ex ; nuIs5 V were analyzed for the extent to which glr-1 expressing command interneuron (labeled with GFP) were undergoing autophagy . Animals were mounted on a 2% agar pad. To compare average DsRed intensity between worms we identified the cell with the highest DsRed intensity as 100% and then compared the other neurons to this cell  versus experimental  groups.Animals carrying 1 marker ). A few glt-3; nuIs5) or the DAPK-inhibited excitotoxicity strain  were incubated overnight with 10 mM 3-MA  dissolved in 1% DMSO  or 1% DMSO alone (control). Plates were supplemented with E. coli OP50. The extent of neurodegeneration was recorded 20–24 h after treatment.For 3-methyladenine (3MA) treatment, mixed stage animals from the excitotoxicity strain  . We did


This rescue in root growth occurs in the absence of a QC. Conversely, when the modified PHB, which is highly resistant to microRNA, was expressed throughout the stele of the wild-type root meristem, root growth became very similar to that observed in the shr; however, the identity of the QC was unaffected. Interestingly, a moderate increase in PHB resulted in a root meristem phenotype similar to that observed following the application of high levels of cytokinin. Our protoplast assay and transgenic approach using ARR10 suggest that the depletion of TA cells by high PHB in the stele occurs via the repression of B-ARR activities. This regulatory mechanism seems to help to maintain the cytokinin homeostasis in the meristem. Taken together, our study suggests that PHB can dynamically regulate TA cell activities in a QC-independent manner, and that the SHR-PHB pathway enables a robust root growth system by coordinating the stem cell niche and TA domain.Plant growth depends on stem cell niches in meristems. In the root apical meristem, the quiescent center (QC) cells form a niche together with the surrounding stem cells. Stem cells produce daughter cells that are displaced into a transit-amplifying (TA) domain of the root meristem. TA cells divide several times to provide cells for growth. SHORTROOT (SHR) and SCARECROW (SCR) are key regulators of the stem cell niche. Cytokinin controls TA cell activities in a dose-dependent manner. Although the regulatory programs in each compartment of the root meristem have been identified, it is still unclear how they coordinate one another. Here, we investigate how PHABULOSA (PHB), under the posttranscriptional control of SHR and SCR, regulates TA cell activities. The root meristem and growth defects in  shr mutant plants, PHB, which in the meristem is actively restricted to the central region of the stele by SHORTROOT (SHR) via miR165/6, suppresses root meristem activity leading to root growth arrest. A high concentration of PHB in the stele does this by modulating B-ARR activity through a QC-independent pathway. Accordingly, we observed a significant recovery of root meristem activity and growth in the shr phb double mutant, while the QC remained absent. However, the presence of QC may be required to sustain continuous root growth. On the basis of our results, we propose that SHR maintains root growth via two separate pathways: by modulating PHB levels in the root stele, and by maintaining the QC identity.Plant roots are programmed to grow continuously into the soil, searching for nutrients and water. The iterative process of cell division, elongation, and differentiation contributes to root growth. The quiescent center (QC) is known to maintain the root meristem, and thus ensure root growth. In this study, we report a novel aspect of root growth regulation controlled independently of the QC by PHABULOSA (PHB). In  Plants, unlike animals, grow continuously and dynamically adjust their architecture. Meristems at the apices of shoots and roots harbor stem cells and serve as the center of cell division and growth. In the root apical meristem (RAM), the quiescent center (QC) maintains a stem cell population to form a stem cell niche , 2]. Th. Th2]. TSHR or SCR is knocked out, QC cells are not maintained and root growth terminates prematurely. The essential role of SHR and SCR in QC specification was further evident in a regeneration study of QC cells. In contrast to wild-type plants, QC cells failed to recover in shr and scr mutants after laser ablation . U. UARR1, or genes . This prignaling . A recenignaling .HOMEODOMAIN-LEUCINE ZIPPER Class III (HD-ZIP III) genes are key regulators of shoot and root meristems , PHAVOLUTA (PHV), CORONA (CNA), REVOLUTA (REV), and ARABIDOPSIS THALIANA HOMEOBOX8 (ATHB8) ). ). HOMEOD (ATHB8) . These gA) 165/6 , 20]. S. SHOMEODA) 165/6 , 22]. S. SHOMEOD1 (DCL1) . In se mem fails . SHR andand 166B . HoweverIn summary, significant progress has been made to identify key transcription factors and signaling molecules in the function and maintenance of the root stem cell niche and TA cells. However, it remains unclear how these factors and molecules are integrated to control root growth. Here, we show that PHB, the concentration of which is controlled by SHR, governs TA cell activities by repressing B-ARRs. Such a repressive effect of PHB on B-ARR activities is enhanced by high cytokinin. Therefore, restricting the PHB level via the SHR-microRNA pathway is a critical step for maintaining root growth.miR165/6 in the ground tissue layer quantitatively and spatially restricts HD-ZIP III expression within the stele, which is necessary for normal xylem tissue patterning , . T. Tshr phArabidopsis root . T. TArabidsis root . shr phbhe stele , 61]. O. OArabidIPT3 and IPT7 expression in accordance with elevated levels of PHB, and vice versa. Direct regulation of IPT7 expression by PHB was recently demonstrated . F. FPHB-emg at 4°C), and the pellets were re-extracted in the same way. The combined supernatants were purified using a 1-g SCX column, evaporated to water phase in a vacuum followed by immunoaffinity chromatography using an immobilized wide-range anti-cytokinin monoclonal antibody as previously described [Cytokinin quantification was performed as previously described , with soescribed . The fraescribed . The datArabidopsis mesophyll protoplasts were isolated and transfected as previously described [4 cells) were transfected with 40 μg of plasmid DNA with different combinations of reporter (pTCS:fLUC), effector , and internal control (pUBQ:rLUC) plasmids , which were collected from protoplast transient assay. Total RNA extraction was performed with PicoPure RNA Isolation Kit (Arcturus). 20μL of reverse transcript reaction was performed for the first cDNA strand synthesis using 500ng of total RNAs and Superscript III reverse transcriptase (Invitrogen). For quantitative PCR reactions, a master mix was prepared using an iQ SYBR Green supermix (Bio-Rad) and PCR condition was programmed according to the manufacturer’s instructions for CFX96 Real-Time PCR machine (Bio-Rad). Three technical replicate reactions were performed. Primer information for RT-PCR is available in Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as internal control gene.escribed , and DNAescribed . Protoplplasmids . ProtoplArabidopsis Genome Initiative or GenBank/EMBL databases under the following accession numbers: SHR (At4G37650), PHB (At2G34710), PHV (At1G30490), CNA (At1G52150), ATHB8 (At4G32880), REV (At5G60690), SCR (At3G54220), PLT1 (At3G20840), PLT2 (At1G51190), S32 (At2g18380), AGL42 (At5g62165), IPT3 (At3G63110), IPT7 (At3G23630), ARR7 (At1G19050), ARR10 (At4G31920) and GAPDH (At3g26650).Microarray data have been submitted to the GEO database (accession number GSE33015). Sequence data from this article can be found in the S1 FigA) Restoration in root length in shr phb double mutants. New phb alleles; phb-15 & 16 also resulted in a significant recovery in the shr root growth. (B) Root meristem size in wild-type, shr-2, phb-6, shr-2 phb-6, scr-4, and scr-4 phb-6 plants (5 DAG). (C) Comparable levels of pCycB1.2:GUS expression in the proximal meristem cells in wild-type and shr phb roots (15 DAG). (D) Quantification of pCycB1.2:GUS expression is shown in the panel (C). White arrowheads marks the end of the meristem. Error bars represent the standard error (n = 15–30 plants). Scale bars: B, 50 μm; C, 20 μm.((TIF)Click here for additional data file.S2 FigA) Aniline blue staining of sieve plates in phloem sieve elements in wild-type, shr-2, and shr-2 phb-6 roots. Two sieve plates are organized in parallel in the wild-type root, whereas only one is found in the shr-2 and shr-2 phb-6 roots. (B) Expression patterns of known QC marker genes in wild-type, shr-2 and shr-2 phb-6 roots. Red arrowheads mark position of sieve plates and white arrowheads mark QC. Scale bars represent 25 μm.((TIF)Click here for additional data file.S3 FigA) Similar levels of pWOX5:erGFP expression in wild-type, shr-2, and shr-2 phb-6 embryos at the late heart stage. (B) A significant decrease in pWOX5:erGFP expression in shr-2 and shr-2 phb-6 embryos in contrast to the wild type at the bent-cotyledon stage. (C) Lugol’s staining shows precocious differentiation of columella stem cells in the shr-2 phb-6 roots (5 DAG). Black and red arrowheads indicate QC and columella stem cells respectively. Scale bars represent A and B, 25 μm; C, 20 μm.((TIF)Click here for additional data file.S4 FigA) Four synonymous mutations introduced into the miRNA 165/6 binding site of PHB via site-directed mutagenesis. Mutated bases are shown in purple. (B) A comparison of PHB-GFP expression domains in pPHB:PHB:GFP, pPHB:PHB-m:GFP and pPHB:PHB:GFP shr-2 roots (5 DAG). Scale bars represent 20 μm. (C) A comparison of root meristem size in wild-type, pWOL:PHB-m:GFPNLS and pWOL:PHB-em:GFPNLS plants . Scale bars represent 20 μm (upper panel) and 10 μm (lower panel). White arrowheads mark where meristem ends. Asterisks indicate cortex cells.  Expression of pWOX5:YFP (D) and pQC25:GUS (E) in 15 day-old pWOL:PHB-em:GFPNLS roots. (F) Aberrant cell divisions in the QC position of pWOL:PHB-em:GFPNLS roots (10 DAG). (G) WOX5 in situ suggests decline in its expression levels in the QC region of pWOL:PHB-em:GFPNLS roots. White arrows indicate QC cells undergoing abnormal cell divisions; Scale bars represent 25 μm for D-G.((TIF)Click here for additional data file.S5 FigpWOL:erGFP in the wild-type, shr-2, and shr-2 phb-6 roots. Scale bars represent 25 μm.Expression pattern of (TIF)Click here for additional data file.S6 FigA) Correlation coefficients (R2) between cell sorting/tiling array data. Biological replicates are highlighted in yellow.  Expression dynamics of differentially expressed genes between the shr and wild-type plants, summarized with PCA (B) and QT clustering (C). shr-2, SHR1 and 2; wild type, WT1, 2 and 3; shr-2 phb-6, DM1 and 2.((TIF)Click here for additional data file.S7 FigExpression patterns are classified by Hierarchical Clustering.(TIF)Click here for additional data file.S8 FigA) pDR5:YFPvenus shows a reduced signal in shr-2 mutants in comparison to wild-type and shr-2 phb-6 plants (7 DAG).  Reduced expression of pPIN1:PIN1:GFP in 5- (B) and 10- (C) day-old shr-2 roots in comparison to the wild type. Expression is partially restored in shr-2 phb-6. Scale bars represent 25 μm.((TIF)Click here for additional data file.S9 FigA) Cytokinin content in the wild-type, shr-2 and shr-2 phb-6 roots. tZ, Transzeatin; tZR, tZ riboside; cZ, cis-zeatin; cZR, cZ riboside; iP, N6-(Δ2-isopentenyl) adenine; iPR, iP riboside; DHZ, Dihydrozeatin; DHZR, Dihydrozeatin riboside; DHZ9G, DHZ-9-glucoside; iP9G, iP-9-glucoside; tZ9G, tZ-9-glucoside; cZ9G, cZ-9-glucoside. (B) Heat map showing expression levels of A-ARRs in the wild-type , shr (SHR1 and 2), and shr phb (DM1 and 2) root stele. (C) Relative mRNA levels of ARR5, ARR7 and ARR15 in the shr-2 and shr-2 phb-6 roots. Data are normalized to the wild-type plants. (D) Root growth in shr-2 phb-6 arr7 mutant. (E) Expression pattern of pTCS:erGFP in phb-7d roots. (F) pS32:erGFP expression in the wild-type, shr-2 and shr-2 phb-6 roots. Scale bars represent 25 μm. SD: standard deviation .((TIF)Click here for additional data file.S10 FigA) Quantitative comparison of TCS-GFP levels in the stele and root cap expressing the wild type (WT) and transgenic line expressing PHB-em fused to the glucocorticoid receptor (GR) under the PHB promoter. (B) Expression patterns of pTCS:erGFP in WT and transgenic line crossed with pPHB:PHBem:GR. All seedlings grew under the same conditions. After 5 DAG, seedlings were transferred to new media including no DEX or 10 uM DEX and were incubated for 3 h. GFP expression is reduced following treatment with 10 uM DEX in transgenic lines.((TIF)Click here for additional data file.S11 Fig(A) A high dosage of PHB (p35S:PHB-em) suppressed ARR10 activities under the high cytokinin. In contrast, PHB expressed by p35S:PHB was not effective on ARR10 activities. (B) PHB expression is analyzed by qRT-PCR using 100 nM BAP-treated protoplasts. The error bar represents standard deviation. BAP, 6-benzylaminopurine.(TIF)Click here for additional data file.S1 Table(XLS)Click here for additional data file.S2 Table(XLS)Click here for additional data file.S3 Table(XLS)Click here for additional data file.S4 Table(XLS)Click here for additional data file.S5 Table(XLS)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.


Treatment options for SCLC after failing platinum-based (PLT) chemotherapy (CT) are limited. Combined blockade of programmed death-1 (PD-1) and cytotoxic T-lymphocyte antigen-4 (CTLA-4) immune checkpoint pathways has anti-tumor activity with a manageable safety profile. Nivolumab is a fully human IgG4 PD-1 immune checkpoint inhibitor approved for melanoma and squamous NSCLC in the US and for melanoma in the EU and Japan. Interim efficacy and safety of nivolumab +/- ipilimumab, a CTLA-4 checkpoint inhibitor, in pretreated SCLC patients are reported.Patients with progressive disease (PD) after PLT first-line treatment were eligible, regardless of platinum sensitivity, tumor PD-L1 expression, or number of prior CT regimens. Patients were randomized to nivolumab 3 mg/kg IV Q2W or nivolumab+ipilimumab (1+1 mg/kg or 1+3 mg/kg) IV Q3W for 4 cycles, followed by nivolumab 3 mg/kg Q2W. Primary objective was objective response rate (ORR). Additional objectives included safety, progression-free survival (PFS), overall survival (OS), and biomarker analysis.Of 90 patients enrolled , 53% had ≥2 prior regimens. Efficacy results for evaluable patients are shown (Table In this PD-L1 unselected SCLC population with progression after PLT-CT, nivolumab monotherapy and nivolumab+ipilimumab were generally well tolerated with manageable toxicity. Rare severe toxicities will require close follow-up. Durable responses occurred with nivolumab monotherapy and in combination with ipilimumab.Clinical Trial Number: NCT01928394.