Chronic lymphocytic leukemia (CLL) is the most common type of leukemia found among adults in Slovenia. For many years, the treatment of CLL was based on standard chemoimmunotherapy, however, nowadays target therapies constitute the first line of treatment. In this master's thesis, we focused on inhibitors of the B-cell receptor pathway, namely BTK inhibitors (ibrutinib and acalabrutinib) and PI3K inhibitors (idelalisib and duvelisib).
Due to the lack of appropriate models for studying resistance to targeted therapies, we first established clinically relevant in vitro models of resistant CLL. The PrestoBlue assay and flow cytometry confirmed that clones of selected HG-3 cells were resistant to targeted therapies. To investigate what drives resistance of CLL cells to therapies, we evaluated the level of basal autophagy in susceptible and resistant HG-3 cells. By analyzing the expression of selected autophagic proteins AMBRA 1, p62, LC3, and BECN-1, we found that basal autophagy is increased in HG-3 resistant cells. Based on this, we then took a closer look at the connection between receptor pathways inhibited by targeted therapies and autophagy. We found that targeted therapies inhibit the BTK/Akt/mTOR and PI3K/Akt/mTOR signaling pathways and thus activate autophagy. Additionally, using imaging flow cytometry we showed that the targeted therapies do not trigger translocation of TFEB into the nucleus of CLL cells and thus do not affect the transcription of autophagic genes.
The efficacy of selected autophagy inhibitors, dorsomorphin, MRT68921, VPS34-IN 1, and chloroquine, was tested on MEC-1, MEC-2, and HG-3 cell lines and primary CLL cells by using the metabolic activity assay. Based on the EC50 values, we found that autophagy inhibitors had concentration- and time-dependent action. The most cytotoxic inhibitor was found to be ULK1 inhibitor MRT68921. Autophagy inhibitors acted on CLL cells independently of genetic traits and were also active against samples carrying prognostically unfavorable genetic aberrations, such as del(17p) and unmutated IGHV.
By correlating the EC50 values of autophagy inhibitors with the EC50 values of targeted therapies, we found that cytotoxicities of ibrutinib, acalabrutinib, and duvelisib correlate with cytotoxicity of VPS34-IN 1. Since targeted therapies activate autophagy, we finally evaluated the feasibility of using autophagy inhibitors in combination with targeted therapies. Synergistic effects of combining autophagy inhibitors and targeted therapies were observed for combinations of MRT68921, dorsomorphin, and chloroquine with targeted therapies, and for VPS34-IN 1 only in combination with BTK inhibitors. This demonstrated the rationale for targeting autophagy to improve the efficacy of targeted therapies in the treatment of CLL.
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