A 2.8-Angstrom-Resolution Cryo-Electron Microscopy Structure of Human Parechovirus 3 in Complex with Fab from a Neutralizing Antibody

Human parechovirus 3 (HPeV3) infection is associated with sepsis characterized by significant immune activation and subsequent tissue damage in neonates. Strategies to limit infection have been unsuccessful due to inadequate molecular diagnostic tools for early detection and the lack of a vaccine or specific antiviral therapy. Toward the latter, we present a 2.8-angstrom-resolution structure of HPeV3 in complex with fragments from a neutralizing human monoclonal antibody, AT12-015, using cryo-electron microscopy (cryo-EM) and image reconstruction. Modeling revealed that the epitope extends across neighboring asymmetric units with contributions from capsid proteins VP0, VP1, and VP3. Antibody decoration was found to block binding of HPeV3 to cultured cells. Additionally, at high resolution, it was possible to model a stretch of RNA inside the virion and, from this, identify the key features that drive and stabilize protein-RNA association during assembly. IMPORTANCE Human parechovirus 3 (HPeV3) is receiving increasing attention as a prevalent cause of sepsis-like symptoms in neonates, for which, despite the severity of disease, there are no effective treatments available. Structural and molecular insights into virus neutralization are urgently needed, especially as clinical cases are on the rise. Toward this goal, we present the first structure of HPeV3 in complex with fragments from a neutralizing monoclonal antibody. At high resolution, it was possible to precisely define the epitope that, when targeted, prevents virions from binding to cells. Such an atomic-level description is useful for understanding host-pathogen interactions and viral pathogenesis mechanisms and for finding potential cures for infection and disease.Peer reviewe

BRAF immunohistochemistry predicts sentinel lymph node involvement in intermediate thickness melanomas

Background Sentinel node biopsy (SNB) is an important step in melanoma staging and prognostication. It is commonly performed for patients with intermediate thickness melanomas, based on clinicopathological features. However, only 20-25% of patients eventually demonstrate nodal involvement. The aim of this study was to evaluate whether tissue biomarkers with links to melanoma biology, together with clinicopathological parameters, could aid in the prediction of sentinel node involvement and improve selection of patients for SNB. In addition, we examined the role of these clinical or biological markers in disease outcome. Methods We collected a case-control cohort of 140 intermediate thickness (Breslow 0,9-4,0mm) melanoma patients with or without SNB involvement matched for age, gender, Breslow thickness and location. From this cohort, we tested the predictive value of common clinicopathological parameters (ulceration, mitotic count and tumor regression) and FMNL-2, ezrin and BRAF V600E immunoreactivity, for sentinel node involvement and survival. We further analyzed the correlations in the superficial spreading melanoma subtype. Results Based on our case control analysis, of the markers, BRAF V600E status (p = 0.010) and mitotic count (p = 0.036) correlated with SNB involvement. SNB status was a strong independent prognosticator for recurrence free survival (RFS p Conclusions These results demonstrate that BRAF immunohistochemistry could serve as a useful addition to a marker panel for selecting intermediate thickness melanoma patients for SNB.Peer reviewe

BRAF immunohistochemistry predicts sentinel lymph node involvement in intermediate thickness melanomas

Background Sentinel node biopsy (SNB) is an important step in melanoma staging and prognostication. It is commonly performed for patients with intermediate thickness melanomas, based on clinicopathological features. However, only 20-25% of patients eventually demonstrate nodal involvement. The aim of this study was to evaluate whether tissue biomarkers with links to melanoma biology, together with clinicopathological parameters, could aid in the prediction of sentinel node involvement and improve selection of patients for SNB. In addition, we examined the role of these clinical or biological markers in disease outcome.Methods We collected a case-control cohort of 140 intermediate thickness (Breslow 0,9-4,0mm) melanoma patients with or without SNB involvement matched for age, gender, Breslow thickness and location. From this cohort, we tested the predictive value of common clinicopathological parameters (ulceration, mitotic count and tumor regression) and FMNL-2, ezrin and BRAF V600E immunoreactivity, for sentinel node involvement and survival. We further analyzed the correlations in the superficial spreading melanoma subtype.Results Based on our case control analysis, of the markers, BRAF V600E status (p = 0.010) and mitotic count (p = 0.036) correlated with SNB involvement. SNB status was a strong independent prognosticator for recurrence free survival (RFS pConclusions These results demonstrate that BRAF immunohistochemistry could serve as a useful addition to a marker panel for selecting intermediate thickness melanoma patients for SNB.</p

Aggressive and recurrent ovarian cancers upregulate ephrinA5, a non-canonical effector of EphA2 signaling duality

Erythropoietin producing hepatocellular (Eph) receptors and their membrane-bound ligands ephrins are variably expressed in epithelial cancers, with context-dependent implications to both tumor-promoting and-suppressive processes in ways that remain incompletely understood. Using ovarian cancer tissue microarrays and longitudinally collected patient cells, we show here that ephrinA5/EFNA5 is specifically overexpressed in the most aggressive high-grade serous carcinoma (HGSC) subtype, and increased in the HGSC cells upon disease progression. Among all the eight ephrin genes, high EFNA5 expression was most strongly associated with poor overall survival in HGSC patients from multiple independent datasets. In contrast, high EFNA3 predicted improved overall and progression-free survival in The Cancer Genome Atlas HGSC dataset, as expected for a canonical inducer of tumor-suppressive Eph receptor tyrosine kinase signaling. While depletion of either EFNA5 or the more extensively studied, canonically acting EFNA1 in HGSC cells increased the oncogenic EphA2-S897 phosphorylation, EFNA5 depletion left unaltered, or even increased the ligand-dependent EphA2-Y588 phosphorylation. Moreover, treatment with recombinant ephrinA5 led to limited EphA2 tyrosine phosphorylation, internalization and degradation compared to ephrinA1. Altogether, our results suggest a unique function for ephrinA5 in Eph-ephrin signaling and highlight the clinical potential of ephrinA5 as a cell surface biomarker in the most aggressive HGSCs.Peer reviewe

Adaptive RSK-EphA2-GPRC5A signaling switch triggers chemotherapy resistance in ovarian cancer

Metastatic cancers commonly activate adaptive chemotherapy resistance, attributed to both microenvironment-dependent phenotypic plasticity and genetic characteristics of cancer cells. However, the contribution of chemotherapy itself to the non-genetic resistance mechanisms was long neglected. Using high-grade serous ovarian cancer (HGSC) patient material and cell lines, we describe here an unexpectedly robust cisplatin and carboplatin chemotherapy-induced ERK1/2-RSK1/2-EphA2-GPRC5A signaling switch associated with cancer cell intrinsic and acquired chemoresistance. Mechanistically, pharmacological inhibition or knockdown of RSK1/2 prevented oncogenic EphA2-S897 phosphorylation and EphA2-GPRC5A co-regulation, thereby facilitating a signaling shift to the canonical tumor-suppressive tyrosine phosphorylation and consequent downregulation of EphA2. In combination with platinum, RSK inhibitors effectively sensitized even the most platinum-resistant EphA2(high), GPRC5A(high) cells to the therapy-induced apoptosis. In HGSC patient tumors, this orphan receptor GPRC5A was expressed exclusively in cancer cells and associated with chemotherapy resistance and poor survival. Our results reveal a kinase signaling pathway uniquely activated by platinum to elicit adaptive resistance. They further identify GPRC5A as a marker for abysmal HGSC outcome and putative vulnerability of the chemo-resistant cells to RSK1/2-EphA2-pS897 pathway inhibition.Peer reviewe

Aggressive and recurrent ovarian cancers upregulate ephrinA5, a non-canonical effector of EphA2 signaling duality

Erythropoietin producing hepatocellular (Eph) receptors and their membrane-bound ligands ephrins are variably expressed in epithelial cancers, with context-dependent implications to both tumor-promoting and-suppressive processes in ways that remain incompletely understood. Using ovarian cancer tissue microarrays and longitudinally collected patient cells, we show here that ephrinA5/EFNA5 is specifically overexpressed in the most aggressive high-grade serous carcinoma (HGSC) subtype, and increased in the HGSC cells upon disease progression. Among all the eight ephrin genes, high EFNA5 expression was most strongly associated with poor overall survival in HGSC patients from multiple independent datasets. In contrast, high EFNA3 predicted improved overall and progression-free survival in The Cancer Genome Atlas HGSC dataset, as expected for a canonical inducer of tumor-suppressive Eph receptor tyrosine kinase signaling. While depletion of either EFNA5 or the more extensively studied, canonically acting EFNA1 in HGSC cells increased the oncogenic EphA2-S897 phosphorylation, EFNA5 depletion left unaltered, or even increased the ligand-dependent EphA2-Y588 phosphorylation. Moreover, treatment with recombinant ephrinA5 led to limited EphA2 tyrosine phosphorylation, internalization and degradation compared to ephrinA1. Altogether, our results suggest a unique function for ephrinA5 in Eph-ephrin signaling and highlight the clinical potential of ephrinA5 as a cell surface biomarker in the most aggressive HGSCs

EfnA5 on epätyypillinen Eph-reseptoriligandi ja epäsuotuisa tekijä korkean pahanasteisuusluokan seroosikarsinoomassa

Eph-reseptorit (engl. Erythropoietin producing hepatocellular) ja niiden solukalvolle ankkuroidut efriiniligandit muodostavat nisäkkäiden suurimman reseptorityrosiinikinaasien proteiiniperheen. Yksilönkehityksen aikana Eph-efriiniviestintä säätelee yksilönkehityksen aikana muun muassa solujen migraatiota ja aksonien ohjautumisen kaltaisia toimintoja, erilaistuneissa kudoksissa esimerkiksi haavojen parantumista ja kudosrajojen ylläpitoa. Koska efriiniligandit sijoittuvat solukalvolle, Eph-efriiniviestintä pystyy kulkemaan kahteen suuntaan: eteenpäin Eph-reseptoria ilmentävään soluun, sekä taaksepäin efriiniligandia ilmentävään soluun. Tärkeiden yksilönkehityksen ja homeostaasin ylläpidon fysiologisten toimintojen lisäksi Eph-efriiniviestinnän on todettu olevan yhteyksissä useisiin sairauksiin, kuten moniin syöpiin, joissa Eph-efriiniviestinnän tasapaino on häiriintynyt. Esimerkkinä onkogeeniksi tunnistettu EphA2-reseptori, jolla on todettu olevan kaksi viestintätapaa, joista toinen on kasvua rajoittava ja toinen onkogeeninen. Näitä viestintätapoja säädellään reseptorin fosforylaatiotiloilla. Korkean pahanasteisuusluokan seroosikarsinooma on yleisin munasarjasyövän tyyppi, sekä tappavin gynekologinen syöpä, jonka viiden vuoden selviytymisennuste on hyvin huono. Korkean pahanasteisuusluokan seroosikarsinoomat eivät alkuvaiheissa oireile, mutta alkavat muodostaa nopeasti etäpesäkkeitä vatsaontelon alueelle suoraan alkuperäisestä kasvaimesta irtoavien syöpäsolujen takia. Pienellä potilasjoukolla tehty tutkimus on antanut viitteitä Eph-efriiniviestinnän merkityksestä korkean pahanasteisuusluokan seroosikarsinooman selviytymisennusteessa. Tämän tutkimuksen tarkoituksena oli tutkia Eph-efriiniviestintää korkean pahanasteisuusluokan seroosikarsinoomassa. Suuren potilasjoukon mRNA-sekvensointiaineistosta analysoitiin Eph-efriiniperheen geenien ekspression vaikutus potilaiden selviytymiseen. Tämän analyysin pohjalta löytyneiden mielenkiintoisten proteiinien toimintaa tutkittiin solulinjakokeilla. Lisäksi kliinisten näytteitä tutkittiin mielenkiintoisia proteiineja värjäämällä sekä tuoreita potilasnäytteitä että The Cancer Genome Atlaksen 428-potilaisen korkean pahanasteisuusluokan seroosikarsinoomanäyteryhmän mRNA-mikrosiruaineistosta analysoitiin kaikkien Eph-efriiniperheen geenien ilmennyksen korrelaatio selviytymisennusteeseen Kaplan-Meier-estimaattorilla. Kokeet solulinjoilla tehtiin rekombinanttiefriinikäsittelyillä ja siRNA-hiljennyksillä. Data analysoitiin Western blot-tekniikalla ja immunofluoresenssivärjäyksillä. Turun yliopistollisesta keskussairaalasta saatuja potilasnäytteitä analysoitiin immunohistokemiallisilla värjäyksillä, immunofluoresenssivärjäyksillä ja mRNA-sekvensoinnilla. Korkealla EfnA5:llä oli merkittävä korrelaatio potilaiden huonon selviytymisennusteen kanssa, toisin kuin usein efriineillä syövissä. Matala EfnA3 korreloi merkittävästi huonon selviytymisennusteen kanssa. Tunnettujen onkogeenien EphA2 ja EphA4 korkea ilmentyminen korreloi merkittävästi huonon selviytymisennusteen kanssa. Solulinjojen käsittely EfnA5:llä johti suurempaan määrään onkogeenistä EphA2-viestintää kuin tyypillinen Eph-efriiniviestintä EfnA1-käsittelyllä. EfnA5:n hiljentäminen lisäsi kasvua rajoittavan EphA2-viestintää, kun taas EfnA1-hiljennys lisäsi onkogeenistä EphA2-viestintää. Useita munasarjatyyppejä sisältävällä kliinisellä näytekokoelmalla tehty immunohistokemiavärjäys osoitti EfnA5:n assosioituvan merkittävästi korkean pahanlaatuisuusasteen seroosikarsinoomaan. EfnA5:n ilmentyminen myös kasvoi merkittävästi korkean pahanlaatuisuusluokan seroosikarsinooman edetessä. Eph-efriiniviestintä on yhdistetty useiden syöpien selviytymisennusteeseen, mutta viestinnän mekanismit syövissä ovat toistaiseksi pysyneet melko tuntemattomina, poislukien esimerkiksi EphB4-EfnB2-välitteinen angiogeneesi. Tämä tutkimus tarjoaa uutta tietoa onkogeenisestä Eph-efriiniviestinnästä, osoittaen että EphA2:n onkogeenistä viestintää voivat säädellä myös efriinit jo entuudestaan tunnetun kinaasivälitteisen aktivaatioreitin lisäksi. EfnA5:n epätyypillistä toimintaa korostaa sen välittämän viestinnän onkogeenisuus tyypilliseen Eph-efriiniviestintään verrattuna, sekä EfnA5:n assosiaatio korkean pahanasteisuusluokan seroosikarsinoomaan, sekä EfnA5:n kasvu tämän hyvin aggressiivisen syövän edetessä. Vaikka tässä tutkimuksessa takaperin etenevää Eph-efriiniviestintää ei tutkittu, korostaa tämä tutkimus efriinien tärkeyttä syövässä tapahtuvasssa Eph-efriiniviestinnässä. Syövissä tapahtuvaa Eph-efriiniviestintää tutkittaessa ei pitäisi keskittyä pelkästään Eph-reseptoreihin, vaan huomioida myös efriinien mahdollinen vaikutus.Eph (Erythropoietin producing hepatocellular) receptors and their membrane-bound ligands, ephrins, form the largest family of receptor tyrosine kinases in mammals. They regulate functions such as cell migration and axon guidance during development, and wound healing and tissue boundary maintenance in mature tissues for example. Due to the membrane-bound nature of the ephrin ligands, Eph-ephrin signalling can proceed in two directions: forward (Eph-expressing cell) and reverse (ephrin expressing cell). In addition to its critical physiological functions in development and tissue homeostasis, the Eph-ephrin system has also been implicated in multiple diseases, including several cancers, in which the aberrant expression of Eph receptors and ephrins are often present. The EphA2 receptor for example has been identified as an oncogene with a dual mode of action as either tumor suppressor or an oncogene through distinct phosphorylation statuses of the receptor. High-grade serous ovarian cancer is the most common subtype of ovarian cancer, and the deadliest gynaecological malignancy with a dismal five-year survival rate. High-grade serous ovarian cancer does not present symptoms at an early stage, yet it quickly progresses into forming peritoneal metastases by cell shedding from the primary tumour. Small patient cohort studies have given indications of the correlation between the Eph-ephrin system and survival in ovarian cancer. This aim of this study was to investigate the impact of the Eph-ephrin system in high-grade serous ovarian cancer using a large patient cohort mRNA expression dataset to obtain survival association data of proteins of interest used with cell-based studies and the analysis of clinical samples in the form of tumor microarrays and fresh primary samples to investigate the functions of the found proteins of interest. A 428-patient The Cancer Genome Atlas high-grade serous ovarian cancer microarray mRNA dataset was analysed using the Kaplan-Meier estimator for each Eph-ephrin family member. Cell based studies were performed with recombinant ephrin treatments and ephrin knockdowns. These data were analysed using Western blotting and immunofluorescence stainings. Clinical high-grade serous ovarian cancer samples obtained from Turku University Hospital were analysed using immunohistochemistry, immunofluorescence stainings, and mRNA sequencing. EfnA5 had a significant correlation of high expression and poor survival, which is atypical to ephrins. Low EfnA3 correlated with poor survival. High levels of known oncogenes EphA2 and EphA4 also correlated with poor survival. EfnA5 treatment resulted in increased oncogenic EphA2 signaling in comparison with canonical Eph-ephrin signalling mediated by EfnA1. Knockdown of EfnA5 increased canonical, tumour suppressive EphA2 signaling, while EfnA1 knockdown increased oncogenic EphA2 signaling. Immunohistochemical analysis of tumour microarrays with multiple ovarian cancer subtypes displayed an association between the highly malignant high-grade serous subtype and EfnA5 expression. In addition to this, EfnA5 expression was increased during high-grade serous ovarian cancer progression. The Eph-ephrin system is implicated in the survival associations of multiple cancers, but the exact functions facilitated by Eph-ephrin signalling in cancer have remained relatively unknown, with the exception of EphB4-EfnB2 driven angiogenesis. This study offers insights into oncogenic Eph-ephrin signalling in ovarian cancer, displaying that oncogenic EphA2 functions can be altered by ephrins in addition to the known kinase crosstalk pathway. The noncanonical nature of EfnA5 is highlighted by its oncogenic functions in comparison to typical Eph-ephrin signalling, and the significant increase of EfnA5 expression during high-grade serous ovarian cancer progression and association with this highly malignant subtype of ovarian cancer. Although the reverse signalling effects of EfnA5 were not studied, this study highlights the importance of ephrins in Eph-ephrin signalling in cancer, presenting that the focus should not be only on the Eph receptors when studying the oncogenic signalling facilitated by the Eph-ephrin system

Efficacy and Safety of Glycosphingolipid SSEA-4 Targeting CAR-T Cells in an Ovarian Carcinoma Model

Chimeric antigen receptor (CAR) T-cell immunotherapies for solid tumors face critical challenges such as heterogeneous antigen expression. We characterized stage-specific embryonic antigen-4 (SSEA-4) cell-surface glycolipid as a target for CAR T-cell therapy. SSEA-4 is mainly expressed during embryogenesis but is also found in several cancer types making it an attractive tumor-associated antigen. Anti-SSEA-4 CAR-T cells were generated and assessed preclinically in vitro and in vivo for antitumor response and safety. SSEA-4 CAR-T cells effectively eliminated SSEA-4-positive cells in all the tested cancer cell lines, whereas SSEA-4-negative cells lines were not targeted. In vivo efficacy and safety studies using NSG mice and the high-grade serous ovarian cancer cell line OVCAR4 demonstrated a remarkable and specific antitumor response at all the CAR T-cell doses used. At high T-cell doses, CAR T cell-treated mice showed signs of health deterioration after a follow-up period. However, the severity of toxicity was reduced with a delayed onset when lower CAR T-cell doses were used. Our data demonstrate the efficacy of anti-SSEA-4 CAR T-cell therapy; however, safety strategies, such as dose-limiting and/or equipping CAR-T cells with combinatorial antigen recognition should be implemented for its potential clinical translation.Peer reviewe