Vloga avtofagije v terapevtski občutljivosti celic kronične limfocitne levkemije

Jakoš Djordjevič, Alma Tana (author), Mlinarič-Raščan, Irena (mentor), Avsec, Damjan (comentor)

Kronična limfocitna levkemija (KLL) predstavlja 25 % vseh levkemij in je najpogostejše hematološko maligno obolenje pri odraslih. V zadnjih letih so nove majhne tarčne molekule ibrutinib (zaviralec BTK), idelalizib (zaviralec PI3K) in venetoklaks (antagonist Bcl-2) vodile v korenite spremembe na področju zdravljenja KLL. Kljub visoki učinkovitosti terapijo z novimi tarčnimi molekulami spremljajo številni neželeni učinki in pojav odpornosti. Iskanje novih terapij in terapevtskih tarč za zdravljenje KLL je zato v neprestanem teku. Ena takih tarč je avtofagija, katabolna pot razgradnje celici lastnih snovi, ki ima pomembno, a ne popolnoma definirano vlogo v KLL. V magistrski nalogi smo želeli preučiti protilevkemični potencial zaviranja avtofagije in njen vpliv na učinkovitost delovanja tarčnih zdravil v KLL. Na reporterski celični liniji THP1- Difluo hLC3 smo s pomočjo pretočnega citometra na zajetje slike dokazali, da ibrutinib in venetoklaks avtofagijo aktivirata, idelalizib pa na aktivnost avtofagije nima učinka. S testom PrestoBlue smo preverili citotoksične učinke različnih zaviralcev avtofagije na celicah 13 bolnikov s KLL. Uporabili smo zaviralce kinaze ULK1/2 (MRT68921), AMPK (dorzomorfin) in združitve avtofagosoma z lizosomom (klorokin). S programom GraphPad Prism smo izračunali vrednosti EC50 in prepoznali, da na celice KLL najbolj citotoksično deluje MRT68921. S primerjavo povprečnih vrednosti EC50 zaviralcev avtofagije po 24 in 48 urah smo potrdili, da zaviralci avtofagije delujejo časovno odvisno citotoksično. Tako kot učinkovitost je pri zdravljenju malignih obolenj pomembna tudi selektivnost terapije. Selektivnost zaviralcev avtofagije na maligne limfocite B smo ovrednotili s primerjavo učinkov zaviralcev avtofagije na metabolno aktivnost mononuklearnih celic periferne krvi (PBMC) in limfoblastoidnih celic (LCL) ter dokazali, da MRT68921, dorzomorfin in klorokin delujejo selektivno citotoksično na celice KLL. Vpliv zaviralcev avtofagije na učinkovitost tarčne terapije smo preverili na celicah KLL s testom PrestoBlue in potrdili sinergistično citotoksično delovanje MRT68921 v kombinaciji z ibrutinibom in venetoklaksom. Mehanizem sinergistične citotoksičnosti smo določili z analizo celičnega cikla in uporabo pankaspaznega zaviralca QVD-OPh s pomočjo pretočne citometrije. Potrdili smo antiproliferativne učinke MRT68921, saj le-ta inducira zastoj v G2/M fazi celičnega cikla. Prav tako smo pokazali, da je sinergistična citotoksičnost MRT68921 in tarčnih zdravil posredovana preko kaspaz. Avtofagijo smo prepoznali kot novo terapevtsko tarčo pri KLL, farmakološke zaviralce avtofagije pa kot potencialne nove učinkovine za zdravljenje bolnikov s KLL.

• Language: Slovenian
• Keywords: kronična limfocitna levkemija (KLL), ibrutinib, venetoklaks, avtofagija, MRT68921, sinergizem
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2021
• PID: 20.500.12556/RUL-126305
• Publication date in RUL: 16.04.2021

JAKOŠ DJORDJEVIČ, Alma Tana, 2021, Vloga avtofagije v terapevtski občutljivosti celic kronične limfocitne levkemije [online]. Master’s thesis. [Accessed 11 April 2025]. Retrieved from: https://repozitorij.uni-lj.si/IzpisGradiva.php?lang=eng&id=126305

Secondary language

• Language: English
• Title: The role of autophagy in the therapeutic susceptibility of chronic lymphocytic leukemia cells

Abstract

Chronic lymphocytic leukaemia (CLL) represents 25 % of all leukaemias and is the most common hematological malignancy in the world. In the recent years, BTK inhibitor ibrutinib, PI3K inhibitor idelalisib, and Bcl-2 antagonist venetoclax revolutionized the treatment of CLL. However, these therapies are presented with several drawbacks, such as severe adverse events and emergence of resistance. Therefore, the demand for novel targets is high. One of such targets is autophagy, a protective and degradative cell process that plays an important but still undefined role in CLL. The objective of this master's thesis was to elucidate the antileukemic potential of targeting autophagy and its effects on the efficacy of targeted therapy in CLL. Using the autophagy reporter cell line THP-1 Difluo hLC3 and imaging flow cytometry we demonstrated that ibrutinib and venetoclax but not idelalisib induce autophagy. Using PrestoBlue assay we evaluated the cytotoxic effects of different autophagy inhibitors on 13 samples of patient-derived CLL cells. ULK1/2 inhibitor MRT68921, AMPK inhibitor dorsomorphin and inhibitor of autophagosome-lysosome fusion chloroquine were used. We calculated and compared EC50 values of autophagy inhibitors and recognized that MRT68921 had the most potent cytotoxic effect on CLL cells. Mean EC50 values of autophagy inhibitors after 48 hours were lower than mean EC50 values after 24 hours, demonstrating that autophagy inhibitors act in a time-dependent manner. Selectivity of therapy is particularly important in cancer treatment. We evaluated the selective cytotoxicity of autophagy inhibitors by comparing their effects on metabolic activity of CLL cells, peripheral blood mononuclear cells (PBMC) and lymphoblastoid cells (LCL). Our results showed that MRT68921, dorsomorphin and chloroquine act selectively cytotoxic towards CLL cells. We evaluated the synergistic cytotoxicity of MRT68921 in combination with ibrutinib and venetoclax using PrestoBlue assay on CLL cells. Inhibiting autophagy using ULK1/2 inhibitor MRT68921 augmented the cytotoxicity of ibrutinib and venetoclax on primary CLL cells. To discern the mechanisms of synergistic effects of MRT68921 with ibrutinib and venetoclax we used pan-caspase inhibitor QVD-OPh and performed cell cycle assay using flow cytometry. MRT68921 had antiproliferative effect, causing cell cycle arrest at G2/M phase. We also confirmed that the combination of MRT68921 and targeted drugs acts synergistically cytotoxic through a caspase-dependent mechanism. We recognized autophagy as a new therapeutic target in CLL and inhibitors of autophagy as a potential new drug for the treatment of CLL.

• Keywords: chronic lymphocytic leukemia (CLL), ibrutinib, venetoclax, autophagy, MRT68921, synergism