Vrednotenje citotoksičnih učinkov zaviralcev signalne poti NOTCH v celičnih linijah kronične limfocitne levkemije

Krajšek, Zala (avtor), Mlinarič-Raščan, Irena (mentor), Markovič, Tijana (komentor)

Kronična limfocitna levkemija (KLL) je neozdravljiva maligna bolezen, za katero je značilna akumulacija zrelih, vendar nefunkcionalnih limfocitov B. Je najpogostejši tip levkemije pri odraslih zahodnega sveta. Signalna pot NOTCH predstavlja novo potencialno terapevtsko tarčo za zdravljenje KLL, saj je v celicah KLL konstitutivno aktivirana in omogoča njihovo preživetje, diferenciacijo, proliferacijo in obstoj tumorskih matičnih celic. Zanimalo nas je, ali zaviranje signalne poti NOTCH modulira preživetje malignih limfocitov B, saj bi to lahko omogočilo boljše obvladovanje bolezni ter prispevalo k iskanju terapevtskih pristopov za premagovanje odpornosti na terapijo. V magistrski nalogi smo vrednotili zaviralce signalne poti NOTCH, limantrafin, RIN1, ZLDI‑8 in FLI-06. Kot eksperimentalne modele smo uporabili celične linije MEC1 in HG3 ter celične linije odporne na venetoklaks in ibrutinib, MEC1VER in MEC1IBR. Najprej smo s testom metabolne aktivnosti ovrednotili citotoksično delovanje zaviralcev NOTCH. Na podlagi vrednosti EC50 izmerjenih izpostavitvi zaviralcem NOTCH smo ugotovili, da ima ZLDI‑8 najmočnejše citotoksično delovanje. Po 24 urah so bile vrednosti EC50 v celicah MEC1 3,2 μM. V celični liniji MEC1 so bile po 48 urah vrednosti EC50 limantrafina 34,7 μM, RIN1 11,5 μM, ZLDI‑8 3,3 μM in FLI-06 4,5 μM. Ugotovili smo, da vsi proučevani zaviralci NOTCH delujejo citotoksično tudi v celičnih linijah, odpornih na venetoklaks in ibrutinib. Proučevali smo tudi potencialno sinergistično delovanje zaviralcev NOTCH s tarčnima zdraviloma venetoklaks in ibrutinib in ugotovili, da je najizrazitejše sinergistično delovanje izkazoval RIN1. Ker je signalna pot NF-κB v celicah KLL konstitutivno aktivirana, smo preverili, ali zaviralci NOTCH vplivajo na NF-κB signalizacijo v celični liniji RamosBlue. S testom QUANTI-Blue smo ugotovili, da so vsi proučevani zaviralci NOTCH znižali aktivnost signalne poti NF-κB. Nadalje smo s pretočno citometrijo proučili vpliv ZLDI‑8, RIN1 in FLI-06 na apoptozo in celični cikel. Ugotovili smo, da je glavni mehanizem celične smrti apoptoza in da zaviralci NOTCH celični cikel zavirajo v različnih fazah. Zaviralci signalne poti NOTCH delujejo citotoksično na celice KLL ter izkazujejo sinergistično delovanje s tarčnimi zdravili, zato bi jih bilo smiselno nadalje ovrednotiti kot potencialne nove kandidatne učinkovine za zdravljenje kronične limfocitne levkemije.

• Jezik: Slovenski jezik
• Ključne besede: kronična limfocitna levkemija (KLL), signalna pot NOTCH, zaviralci NOTCH, limantrafin, RBPJ inhibitor-1 (RIN1), sinergistično delovanje
• Vrsta gradiva: Magistrsko delo/naloga
• Organizacija: FFA - Fakulteta za farmacijo
• Leto izida: 2026
• PID: 20.500.12556/RUL-180808
• Datum objave v RUL: 17.03.2026

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Evaluation of cytotoxic effects of NOTCH inhibitors in chronic lymphocytic leukemia cell lines

Izvleček

Chronic lymphocytic leukemia (CLL) is an incurable malignant disease characterized by the accumulation of mature but functionally impaired B lymphocytes. It is the most prevalent form of leukemia in adults in Western countries. The NOTCH signaling pathway represents a promising therapeutic target in CLL, as it is constitutively activated in leukemic cells and supports their survival, proliferation, differentiation, and maintenance of tumor stem cells. This study aimed to determine whether inhibition of the NOTCH signaling pathway affects the survival of malignant B lymphocytes, thereby potentially improving disease control and contributing to strategies for overcoming therapy resistance. In this master’s thesis, the NOTCH pathway inhibitors limantrafin, RIN1, ZLDI-8, and FLI-06 were evaluated. The MEC1 and HG3 cell lines were used as experimental models, along with venetoclax- and ibrutinib-resistant cell lines MEC1VER and MEC1IBR. Cytotoxic effects were first assessed using a metabolic activity assay. Based on EC50 values, ZLDI-8 was identified as the most potent inhibitor, with an EC50 of 3.2 μM in MEC1 cells after 24 hours. After 48 hours, EC50 values in MEC1 cells were 34.7 μM for limantrafin, 11.5 μM for RIN1, 3.3 μM for ZLDI-8, and 4.5 μM for FLI-06. All tested NOTCH inhibitors also showed cytotoxic activity in venetoclax- and ibrutinib-resistant cell lines. Potential synergistic effects of NOTCH inhibitors in combination with venetoclax and ibrutinib were investigated, with RIN1 demonstrating the strongest synergistic activity. Since the NF-κB signaling pathway is constitutively active in CLL cells, the effects of NOTCH inhibition on NF-κB signaling were examined in the RamosBlue cell line. Using the QUANTI-Blue assay, all NOTCH inhibitors reduced NF-κB activity. Furthermore, flow cytometry analysis revealed that ZLDI-8, RIN1, and FLI-06 primarily induced apoptosis and caused cell cycle arrest at different phases. In conclusion, NOTCH signaling pathway inhibitors exert cytotoxic effects on CLL cells and show synergistic activity with targeted therapies. These findings support further evaluation of NOTCH inhibitors as potential novel therapeutic candidates for the treatment of chronic lymphocytic leukemia.

• Ključne besede: Chronic lymphocytic leukemia (CLL), NOTCH signaling pathway, NOTCH inhibitors, limantrafin, RBPJ Inhibitor-1 (RIN1), synergistic action