In vitro modulation of O-GlcNAcylation and its impact on the function of selected immune and cancer cells : doctoral dissertation

Weiss, Matjaž (avtor), Gobec, Martina (mentor), Anderluh, Marko (komentor)

O-GlcNAcylation is a common posttranslational modification that demands many components, such as glucose, glutamine, uridine, acetyl-CoA and cellular energy. O-β-N-acetylglucosaminyl transferase (OGT) is a transferase that catalyses the transfer of N-acetylglucosamine from UDP-GlcNAc to serine and threonine residues of nuclear and cytoplasmic proteins. O-GlcNAcase (OGA) is a reciprocal enzyme to OGT that removes O-GlcNAc moieties from glycosylated proteins. Therefore, O-GlcNAcylation is a dynamic process in physiological environments, regulated by OGT and OGA expression levels and the availability of UDP-GlcNAc components. The number of discovered proteins modified by O-GlcNAcylation is increasing and includes several transcription factors, epigenetic regulators, kinases, cytoplasmic enzymes and mitochondrial proteins. The complexity of this posttranslational process arises within the cellular context, where the protein kinases compete with OGT for the same serine or threonine hydroxyl groups on targeted proteins (e.g. AKT). Another possibility of crosstalk exists at neighbouring amino acids (e.g. c-Myc), where binding of the first moiety, being either GlcNAc or a phosphate, affects binding of the second. Dysregulated O-GlcNAcylation is associated with several pathologies including cancers, cardiovascular and neurodegenerative diseases. It is basically a metabolic gauge and thus the highest expression of OGT is found in ß pancreatic cells where chronically high O-GlcNAcylation results in insulin resistance, so that diabetes correlates with the high level of O-GlcNAcylation. Elevated expression of OGT and increased levels of O-GlcNAcylated proteins are characteristic for some types of cancer. In the fight against malignant transformation or pathogens, the immune system plays a crucial role. The role of O-GlcNAcylation in macrophages, neutrophils, T and B cells has been studied in recent years, but there were no studies done on human dendritic (DC) and natural killer cells. Most cells obligatorily require adequate O-GlcNAcylation for their proper development and function. However, the exact role of O-GlcNAcylation in many biological systems is still not well understood. In recent years, some new potential inhibitors have been synthesized, to be used as molecular probes to study the role of OGT in biological systems. The most potent and widely used OGT inhibitors are the so-called OSMIs that, however, still have some shortcomings, such us scarce selectivity. In an effort to overcome these, we synthesized new quinolinone-based OGT inhibitors, targeting the uridine diphosphate (UDP) binding OGT pocket. To further 2 improve their inhibitory potency, we designed peptidic conjugates by attaching peptide substrates to low molecular OGT inhibitors. Conjugated moieties were connected via selected linkers and the effect of linker length and flexibility on inhibitory potency was evaluated as well. Unfortunately, none of the synthesized peptidic conjugates or low molecular OGT inhibitors was more potent than those previously reported in the literature. The most potent inhibitor from our series was the fragment-based 3b with IC50 = 116.0 μM. Aberrant O-GlcNAcylation of some protein substrates has been also involved in inappropriate immune response, but this remained underexplored. In our study, we investigated the effect of OGT inhibition on human dendritic and natural killer cells. We showed the immunosuppressive effects of OGT inhibitor in NK cells, by confirming their decreased cytotoxicity against K562 target cells by 33.4 ± 10.9%, when they were pretreated with OSMI-1 (20 μM). The reason for this phenomenon could be in decreased number of degranulating NK cells and the reduced release of inflammatory molecules which has been also observed in our experiments, yet the mechanisms how OGT inhibitors altered cytotoxicity remains unknown. In addition, we showed that OGT also plays a role in DCs, as OGT inhibitor, OSMI-1, has affected the functionality and phenotype of dendritic cells. The presence of OSMI-1 during the monocyte to DC differentiation process modulated the mTOR/AKT and MEK/ERK pathways both in immature as in mature monocyte derived DCs (moDCs). OGT inhibition also altered relevant surface marker profiles, where reduced expression of CD80 and DC-SIGN, as well as increased expression of CD14, CD86 and HLA-DR in immature monocyte derived DCs were observed. This suggests that inhibition of O-GlcNAcylation hampers the transition of monocytes into immature moDCs. Moreover, the presence of OSMI-1 during the moDC differentiation and activation process led to a decrease in IL-10 and an increase in IL-6 secretion by immature and mature moDCs. Additionally, diminished endocytosis was observed in immature moDCs. Inhibition of OGT also disrupted the maturation process of moDCs and impaired the proliferation of allogeneic T cells induced by the OSMI-1-treated mature moDCs, whereas no changes in the polarization of T cells could be observed compared to control. Taken together, we confirm that the OGT inhibitor, OSMI-1, alters the differentiation and function of moDCs under in vitro conditions. OGT inhibitors could potentially be used as a supportive therapy to already known chemotherapeutics in the treatment of cancer. Namely, we have observed a significantly 3 enhanced effect of OSMI-1 and regorafenib on metabolic activity of selected human cancer cell lines. The IC50 value of regorafenib on AMO-1 human plasmacytoma cell line was decreased 3.68-fold, when these were co-treated with non-cytotoxic concentration of OSMI-1 (10 μM). Further studies revealed a synergistic cytotoxic effect of both substances on the AMO-1 cell line, in which the percentage of dead cells increased from 11% to 38% when they were co-treated with 5 μM regorafenib and 10 μM OSMI-1, where apoptosis was not the predominant type of cell death. The proliferation of AMO-1 cells was evidently inhibited after the use of both inhibitors, as we observed a decreased number of cell divisions and an increased population of cells in the G0/G1 phase of the cell cycle. These effects seem to be linked to the inhibition of cyclin D-dependent activation of cyclin-dependent kinase 4 (CDK4), the inhibition of the retinoblastoma protein and the inhibition of S-phase gene transcription.

• Jezik: Angleški jezik
• Vrsta gradiva: Doktorska disertacija
• Tipologija: 2.08 - Doktorska disertacija
• Organizacija: FFA - Fakulteta za farmacijo
• Kraj izida: Ljubljana
• Založnik: [M. Weiss]
• Leto izida: 2022
• Št. strani: 171 str.
• PID: 20.500.12556/RUL-143832
• UDK: 615.4:54:616-006(043.3)
• COBISS.SI-ID: 97407491
• Datum objave v RUL: 13.01.2023

Sekundarni jezik

• Jezik: Slovenski jezik
• Naslov: Vpliv modulacije N-acetilglukozaminilacije na delovanje izbranih imunskih in rakavih celic in vitro

Izvleček

N-acetilglukozaminilacija je pogosta posttranslacijska sprememba, ki zahteva veliko komponent, kot so glukoza, glutamin, uridin, acetil-CoA in energija. O-β-N-acetilglukozaminil transferaza (OGT) je encim, ki katalizira prenos N-acetilglukozamina iz UDP-GlcNAc na serinske in treoninske ostanke jedrnih in citoplazemskih proteinov. Medtem pa encim O-GlcNAc hidrolaza (OGA) odstranjuje molekule O-GlcNAc iz teh proteinov in ima tako nasprotno vlogo kot OGT. N-acetilglukozaminilacija je torej dinamičen proces, ki ga uravnavajo tako sami encimi kot razpoložljivost sestavnih delov molekule UDP-GlcNAc. Število odkritih tarčnih proteinov, ki se lahko N-acetilglukozaminilirajo, narašča. Ti proteini so transkripcijski dejavniki, epigenetski regulatorji, različne kinaze, citoplazemski encimi in mitohondrijski proteini. Poleg tega je znano, da protein kinaze tekmujejo z OGT za iste serinske in treoninske hidroksilne skupine na tarčnih proteinih (npr. AKT). Drug način tovrstnih medsebojnih vplivov poteka s spreminjanjem bližnjih aminokislin (npr. c-Myc). Po vezavi prve posttranslacijske modifikacije (PTM), fosfata ali N-acetilglukozamina, ta nato vpliva na vezavo druge. Poleg tega pa je deregulacija N-acetilglukozaminilacije povezana tudi z določenimi boleznimi, vključno z rakavimi obolenji ter kardiovaskularnimi in nevrodegenerativnimi boleznimi. Največja izraženost encima OGT je bila zabeležena v β-celicah trebušne slinavke, zato ni presenetljivo, da kronično povišana N-acetilglukozaminilacija igra pomembno vlogo pri nastanku inzulinske rezistence in sladkorne bolezni. Povečana raven N-acetilglukozaminilacije in izraženost OGT sta prav tako značilni za nekatere vrste rakavih obolenj. Ker igra imunski sistem pomembno vlogo v boju proti malignim spremembam in patogenom, so vzadnjih letih proučevali vpliv N-acetilglukozaminilacije na delovanje makrofagov, nevtrofilcev ter limfocitov T in B, medtem ko do sedaj še ni na voljo izsledkov o vlogi tega procesa na človeške dendritične celice (DC) in naravne celice ubijalke (NK). Večina imunskih celic potrebuje ustrezno stopnjo N-acetilglukozaminilacije za pravilen razvoj in delovanje, vendar pa trenutno natančna vloga N-acetilglukozaminilacije v mnogih bioloških sistemih še vedno ni povsem pojasnjena. V zadnjih letih je bilo sintentiziranih kar nekaj potencialnih zaviralcev OGT, ki bi jih v obliki molekulskih prob lahko uporabili za proučevanje vloge OGT v bioloških sistemih. Spojine iz serije OSMI so najmočnejši in najpogosteje uporabljeni zaviralci, ki pa imajo še vedno nekaj pomankljivosti, kot je npr. slaba selektivnost. Da bi odpravili njihove pomanjkljivosti, 5 smo sintetizirali nove zaviralce OGT na osnovi kinolinona, z namenom ciljanja njihove vezave v vezavni žep OGT za uridin difosfat (UDP). Naši cilji so bili: izboljšati interakcije v omenjenem vezavnem žepu s sintezo peptidov, konjugiranih z nizkomolekularnimi zaviralci OGT; izboljšati interakcije s podaljšanjem nizkomolekularnih zaviralcev do vezavnega žepa O-GlcNAc in raziskati vpliv dolžine in fleksibilnosti uporabljenega distančnika na zaviralni učinek zaviralcev. Noben od sintetiziranih peptidnih konjugatov in nizko molekularnih zaviralcev ni bil močnejši zaviralec encima OGT od tistih, opisanih v literaturi. Med pripravljenimi zaviralci je sicer najmočnejši zaviralni učinek na izoliranem encimu izkazal fragment 3b, z IC50 = 116,0 μM. V dosedanjih maloštevilnih študijah, so spremenjen obseg N-acetilglukozaminilacije nekaterih proteinov, opazili v povezavi z nepravilnim imunskim odzivom. V naši študiji smo zato raziskali vpliv zmanjšane N-acetilglukozaminilacije na človeške DC, pripravljene iz monocitov in celice NK. Pri slednjih smo pričakovali imunosupresivne učinke zaviralcev OGT in jih tudi potrdili na osnovi opažene zmanjšane citotoksičnosti celic NK zoper tarčne rakave celice K562. Ta se je, v primeru, ko smo celice NK izpostavili OSMI-1 (20 μM), znižala za 33,4 ± 10,9%. Razlog za to bi lahko bilo ugotovljeno zmanjšano število celic NK, ki so sposobne degranulacije in zmanjšan obseg proizvodnje topnih vnetnih molekul. Nepojasnjeno pa ostaja, kako pride do teh učinkov. Poleg tega smo pokazali, da igra OGT pomembno vlogo tudi v dendritičnih celicah, pripravljenih iz monocitov (moDC) saj so zaviralci OGT vplivali tako na njihovo funkcionalnost kot tudi fenotip. Med postopkoma diferenciacije in dozorevanja DC je prisotnost zaviralca OSMI-1 tako v nezrelih kot tudi v zrelih moDC povzročila modulacijo aktivnosti signalnih poti mTOR/AKT in MEK/ERK. Prisotnost OSMI-1 je povzročila tudi spremembe v profilih izražanja značilnih molekulskih membranskih označevalcev DC, pri čemer smo opazili zmanjšani ekspresiji molekul CD80 in DC-SIGN ter povečano izražanje molekul CD14, CD86 in HLA-DR na nezrelih moDC. Ti izsledki kažejo, da zaviranje N-acetilglukozaminilacije ovira diferenciacijo monocitov v nezrele moDC. Poleg tega je prisotnost OSMI-1 v moDC povzročila zmanjšan obseg proizvodnje IL-10 in povečano izločanje IL-6. V nezrelih moDC je OSMI-1 zavrl tudi proces endocitoze. Zaviranje OGT je vplivalo tudi na dozorevanje oz. aktivacijo moDC, ki so posledično izkazovale večjo sposobnost sprožitve proliferacije alogenskih limfocitov T, pri čemer nismo opazili vidnega vpliva na njihovo polarizacijo. S tem smo potrdili, da OSMI-1 lahko vpliva na diferenciacijo in delovanje človeških moDC v pogojih in vitro. 6 Ugotovili smo tudi, da bi zaviralce OGT morda lahko uporabljali za podporno terapijo, skupaj z nekaterimi že znanim kemoterapevtiki, pri zdravljenju raka. Opazili smo namreč povečan zaviralni učinek kombinacije regorafeniba in OSMI-1 na metabolno aktivnost nekaterih človeških rakavih celičnih linij, in vitro. Vrednost IC50 za regorafenib se je, ob dodatku netoksične koncentracije OSMI-1 (10 μM) zmanjšala za 3,68-krat (celična linija AMO-1). Nadaljnji poskusi so razkrili tudi sinergistični citotoksični učinek na celično linijo AMO-1, ko je bila ta sočasno izpostavljena 5 μM regorafeniba in 10 μM OSMI-1, pri čemer se je odstotek mrtvih celic povečal z 11% na 38%, apoptoza pa v tem primeru ni predstavljala prevladujočega načina celične smrti. Poleg tega smo, v primeru uporabe obeh zaviralcev, opazili še zavrtje proliferacije celic AMO-1, pri čemer smo določili zmanjšano število celičnih delitev in povečano populacijo celic v fazi G0/G1 celičnega cikla. Ti rezultati so posledica zavrte aktivacije ciklina D in od ciklina odvisne kinaze 4 (CDK4), blokirane fosforilacije retinoblastoma ter zavrte transkripcije genov faze S celičnega cikla.

• Ključne besede: OGT, OGA, N-acetilglukozaminilacija, fosforilacija, regorafenib, zaviralci OGT, medsebojno sporazumevanje, posttranslacijske modifikacije, iz monocitov diferencirane dendritične celice, naravne celice ubijalke, sinergizem, celična linija AMO-1