20.500.12556/RUL-150353 Preučevanje mehanizmov odpornosti celic kronične limfocitne levkemije na sodobno terapijo Investigating the resistance mechanisms of chronic lymphocytic leukemia cells towards contemporary therapies Kronična limfocitna levkemija (KLL) predstavlja najpogostejšo obliko levkemije pri odraslih, za katero je značilno kopičenje limfocitov B v krvi, kostnem mozgu in limfatičnih organih. V sodobnem času temelji zdravljenje KLL na uporabi tarčnih zdravil. Mednje spadajo zaviralci Brutonove tirozin kinaze (BTK), kot sta ibrutinib in akalabrutinib, ter zaviralci fosfatidilinozitol 3-kinaze (PI3K), idelalizib in duvelizib. S tega vidika smo v okviru magistrske naloge preučili mehanizme odpornosti na zaviralce PI3K in BTK ter tako ponudili nove možnosti za njeno premagovanje pri KLL. Zaradi nezadostno razvitih modelov za preučevanje mehanizmov odpornosti smo uporabili novo razvite celične linije MEC-1, ki so odporne na ibrutinib, akalabrutinib, idelalizib in duvelizib. Na teh celicah smo preverili izražanje proteinov preživetvenih poti B-celičnega receptorja (BCR). Najprej smo z metodo prenosa western preverili izražanje antiapoptotičnih proteinov Mcl-1, Bcl-2 in Bcl-xL in ugotovili, da je protein Bcl-xL povišano izražen v odpornih celicah. Nato pa smo s testom PrestoBlue ugotovili, da s selektivnim zaviranjem Bcl-xL skupaj z zaviranjem PI3K sinergistično zmanjšamo metabolno aktivnost celic, odpornih na idelalizib in duvelizib, ter posledično premagamo odpornost. Nato smo vzroke za odpornost celic na zaviralce BTK in PI3K iskali v spremenjenem izražanju signalnih poti BCR. S prenosom western nismo zaznali sprememb v izražanju proteinov signalne poti BTK/PLCγ2. Z uporabo pretočne citometrije smo ugotovili konstitutivno povišano izražanje PI3K in c-Myc v odpornih celicah, s čimer smo izpostavili pomen povezave signalne poti PI3K in onkogenega proteina c-Myc pri odpornosti na idelalizib in duvelizib. Na koncu smo se osredotočili na vlogo signalne poti NF-κB pri odpornosti na zaviralce PI3K. V odpornih celicah smo zaznali povečano izražanje NF-κB. Povezavo med signalno potjo PI3K in NF-κB smo potrdili z uporabo aktivatorjev NF-κB (PMA/ionomicin, sCD40L) na celicah bolnikov s KLL, ki so zmanjšali citotoksičnost zaviralcev PI3K. Ker zaviralci proteasoma (cP) in imunoproteasoma (iP) zavirajo NF-κB, smo preverili ali lahko z njimi premostimo odpornost na zaviralce PI3K. Z uporabo selektivnega zaviralca cP karfilzomiba in zaviralca iP ONX-0914 v kombinaciji z zaviralci PI3K smo dosegli sinergistično citotoksičnost na celice MEC-1, odporne na idelalizib in duvelizib. Na podlagi tega rezultata smo sočasno uporabo zaviralcev PI3K in cP/iP predlagali kot novo možnost za premagovanje odpornosti pri KLL. Chronic lymphocytic leukemia (CLL) is the most common form of leukemia among adults and is characterized by the accumulation of B lymphocytes in the blood, bone marrow, and lymphatic organs. The contemporary treatment of CLL is based on targeted therapies. These include Bruton's tyrosine kinase (BTK) inhibitors, such as ibrutinib and acalabrutinib, and the phosphatidylinositol 3-kinase (PI3K) inhibitors idelalisib and duvelisib. In this Master's thesis, we studied the mechanisms of resistance to PI3K and BTK inhibitors and thus offered new treatment strategies for overcoming the resistance in CLL. Due to the lack of appropriate models for studying resistance mechanisms in CLL, we used de novo established ibrutinib-, acalabrutinib-, idelalisib-, and duvelisib-resistant MEC-1 clones. We first evaluated the expression of the anti-apoptotic proteins Mcl-1, Bcl-2 and Bcl-xL by western blotting and found that Bcl-xL protein was overexpressed in resistant cells. Using the PrestoBlue assay we showed that selective inhibition of Bcl-xL synergized with PI3K inhibitors in reducing the metabolic activity of idelalisib- and duvelisib-resistant cells, thus resulting in overcoming the resistance. Next, we investigated the causes of cell resistance to BTK and PI3K inhibitors in the altered expression of BCR signaling pathways. No changes in the expression of BTK/PLCγ2 signaling pathway proteins were detected by western blotting. Using flow cytometry, we found constitutively elevated expression of PI3K and c-Myc in resistant cells, thus highlighting the importance of the connection between the PI3K signaling pathway and the oncogenic protein c-Myc in resistance to idelalisib and duvelisib. In the end, we focused on the role of the NF-κB signalling pathway in resistance to PI3K inhibitors. We detected increased NF-κB expression in resistant cells. The link between the PI3K signalling pathway and NF-κB was confirmed by using NF-κB activators (PMA/ionomycin, sCD40L) on patient-derived CLL cells, which reduced the cytotoxicity of PI3K inhibitors. Since proteasome (cP) and immunoproteasome (iP) inhibitors inhibit NF-κB, we then tested whether they can overcome resistance to PI3K inhibitors. Selective cP inhibitor carfilzomib and the iP inhibitor ONX-0914 synergized with PI3K inhibitors against resistant MEC-1 clones. Based on these results, we proposed the combination of PI3K inhibitors and cP/iP inhibitors as a potential new therapeutic strategy for overcoming resistance in CLL. kronična limfocitna levkemija fosfatidilinozitol 3-kinaza PI3K NF-κB idelalizib duvelizib proteasom imunoproteasom karfilzomib ONX-0914 odpornost chronic lymphocytic leukaemia phosphatidylinositol 3-kinase PI3K NF-κB idelalisib duvelisib proteasome immunoproteasome carfilzomib ONX-0914 resistance true false false Slovenski jezik Angleški jezik Magistrsko delo/naloga 2023-09-16 07:45:30 2023-09-16 07:45:33 2024-09-17 07:33:15 0000-00-00 00:00:00 2023 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 2025-09-14 100862 18169.pdf 18169.pdf 1 F824836FA65AB4E247C471B4C4C083EA d1ad9f02f40e40c787cd50703779f8dd246da19808be039ebe97768a7b76fdc1 441b910a-5454-11ee-b1ae-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=174926 Fakulteta za farmacijo 0 0 0