Sinergistično delovanje zaviralcev proteasoma in imunoproteasoma z zaviralci receptorske poti limfocitov B na celicah kronične limfocitne levkemije

Burnik, Tilen (avtor), Mlinarič-Raščan, Irena (mentor), Avsec, Damjan (komentor)

Kronična limfocitna levkemija (KLL) je najpogostejša oblika levkemije v zahodnem svetu. V zadnjem času je zdravljenje z majhnimi tarčnimi molekulami praktično v celoti nadomestilo kemoterapijo, vendar pa tudi pri tarčni terapiji prihaja do pojava neželenih učinkov in odpornosti. V magistrski nalogi smo se tako osredotočili na proteasom (cP) in imunoproteasom (iP) kot potencialni tarči pri zdravljenju KLL. Pokazali smo, da neselektivni zaviralci cP zavirajo tako cP kot tudi iP, selektivni zaviralci iP pa so iP zavrli od 10 (ONX-0914) do 300-krat (M3258) močneje kot cP. Zaviralci cP in iP na celičnih linijah ter na primarnih celicah KLL delujejo časovno in koncentracijsko citotoksično. Kot najbolj citotoksičen se je na celicah KLL izkazal zaviralec cP karfilzomib (CFZ), ki je vrednost EC50 po 48 urah dosegel pri 1,6 nM. Sledila sta mu ONX-0914 ter bortezomib z 8,5 nM, oziroma 9,8 nM. Zaviralci cP in iP na celice KLL delujejo neodvisno od genetskih sprememb in so primerljivo citotoksični tudi na celicah bolnikov z napovedno najbolj neugodnima spremembama delecijo 17p in nemutiranim genom IGHV. Njihovo delovanje je tudi dokazano selektivno, saj so na mononuklearnih celicah periferne krvi zdravih prostovoljcev vrednost EC50 izražali pri 2,5-krat (CFZ), oziroma 8-krat (ONX-0914) višji koncentraciji kot na celicah KLL. Pokazali smo, da zaviralci cP in iP prožijo od kaspaz odvisno celično smrt. Proženje programirane celične smrti se je izkazalo tudi kot njihov glavni mehanizem delovanja. Proteasom ima pomembno vlogo pri aktivaciji NFκB signalne poti, ki je pri KLL konstitutivno aktivna. Na poročevalskih celicah Ramos-Blue zaviralci cP in iP na koncentracijsko odvisen način zavirajo s TNF-α aktivirano signalno pot NFκB. To smo potrdili tudi na celičnih linijah ter primarnih celicah KLL, kjer sta CFZ in ONX-0914 uspešno zavrla prenos NFκB v jedro celic. Na koncu smo na celicah KLL pokazali tudi, da ob sočasnem tretiranju celic s tarčnimi zdravili ibrutinibom, akalabrutinibom, idelalizibom, duvelizibom in venetoklaksom ter zaviralci cP in iP CFZ, ONX-0914 in M3258 dosežemo tudi sinergistično citotoksičnost. Zaznali smo tudi, da zaviralci cP in iP ojačajo delovanje tarčnih zdravil na celicah bolnikov, ki se na terapijo s tarčnimi zdravili slabo odzivajo. Konstitutivni proteasom in imunoproteasom smo prepoznali kot pomembni novi tarči pri KLL ter zaviralce cP in iP kot potencialne nove učinkovine za zdravljenje bolnikov s KLL.

• Jezik: Slovenski jezik
• Ključne besede: kronična limfocitna levkemija, konstitutivni proteasom, imunoproteasom, karfilzomib, ONX-0914, NFκB, sinergizem
• Vrsta gradiva: Magistrsko delo/naloga
• Organizacija: FFA - Fakulteta za farmacijo
• Leto izida: 2022
• PID: 20.500.12556/RUL-141369
• Datum objave v RUL: 29.09.2022

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Synergistic activity of proteasome and immunoproteasome inhibitors with B-cell receptor pathway inhibitors in chronic lymphocytic leukemia cells

Izvleček

Chronic lymphocytic leukemia (CLL) is the most common type of leukemia in the Western countries. In recent years targeted treatment has almost completely replaced chemoimmunotherapy as a first line treatment. But even with targeted therapy the incidence of adverse effects and resistance remains high. In this master’s thesis we have thus focused on proteasome and immunoproteasome as potential targets in the treatment of CLL. We showed that the non-selective cP inhibitors inhibit both the cP and iP, while the selective iP inhibitors showed 10 (ONX-0914) to 300 (M3258) times stronger inhibition of iP compared to cP. We also showed that cP and iP inhibitors exhibit time and dose dependent cytotoxicity on both the CLL cell lines and primary CLL cells. Carfilzomib (CFZ) exhibited the strongest cytotoxic effect on primary CLL cells, reaching the EC50 of 1.6 nM after 48 hours. It was followed by ONX-0914 and bortezomib with 8.5 nM and 9.8 nM respectively. cP and iP inhibitors showed efficacy irrespective of genetic traits exhibited by the CLL cells and exhibited comparable cytotoxicity on CLL cells with the prognostically unfavorable del(17p) and unmutated IGHV. They also proved to be selectively cytotoxic, reaching EC50 values that were 2.5 times (CFZ) and 8 times (ONX-0914) higher on peripheral blood mononuclear cells of healthy volunteers compared to the CLL cells. We showed that cP and iP inhibitors trigger caspase-dependent cell death. Triggering cell death also appeared to be their main mechanism of action. Proteasome has an important role in activating the NFκB signaling pathway, which is constitutively active in CLL. On Ramos-Blue reporter cell line cP and iP inhibitors inhibit the TNF-α activated NFκB signaling pathway in a dose-dependent manner. We have further proved this theory on CLL cell lines and primary CLL cells, where CFZ and ONX-0914 successfully inhibited the translocation of NFκB into nucleus. Finally, we showed that treating primary CLL cells with both the targeted therapy drugs ibrutinib, acalabrutinib, idelalisib, duvelisib and venetoclax and cP/iP inhibitors CFZ, ONX-0914 and M3258 results in synergistic cytotoxicity. We also observed that cP and iP inhibitors express cytotoxic activity on cells that poorly responded to targeted therapy. We demonstrated the rationale of targeting cP and iP in the treatment of CLL and used in-vitro models to recognize the cP and iP inhibitors as sensible drugs in treatment of CLL.

• Ključne besede: chronic lymphocytic leukemia, proteasome, immunoproteasome, carfilzomib, ONX-0914, NFκB, synergism