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.
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