The role of autophagy in the resistance of chronic lymphocytic leukemia cells to the B-cell receptor pathway inhibitors

Ena izmed najpogostejših vrst levkemije v Sloveniji je kronična limfocitna levkemija (KLL). Zdravljenje je pred nekaj leti potekalo še z uporabo standardne kemoimunoterapije, sedaj pa kot prvo linijo zdravljenja uporabljajo tarčna zdravila. V magistrski nalogi smo se posvetili zaviralcem receptorske poti celic B, in sicer zaviralcem BTK (ibrutinib in akalabrutinib) ter zaviralcem PI3K (idelalizib in duvelizib). Zaradi pomanjkanja ustreznih modelov za preučevanje odpornosti na tarčna zdravila, smo na začetku raziskovanja vzpostavili klinično relevantne in vitro modele odporne KLL. S testom metabolne aktivnosti PrestoBlue in pretočno citometrijo smo potrdili, da so kloni selekcioniranih celic HG-3 odporni na tarčna zdravila. Ker smo želeli raziskati vzrok odpornosti celičnih linij, smo nato ovrednotili nivo bazalne avtofagije v dovzetnih in odpornih celicah HG-3. Z analizo proteinov avtofagije AMBRA 1, p62, LC3 in BECN-1 smo ugotovili, da je bazalna avtofagija v odpornih celicah HG-3 povečana. Na podlagi tega smo se nato odločili, da podrobneje proučimo receptorske poti, ki jih zavirajo tarčna zdravila, in avtofagijo. Ugotovili smo, da tarčna zdravila zavirajo signalni poti BTK/Akt/mTOR in PI3K/Akt/mTOR in tako aktivirajo avtofagijo. Dodatno smo s pretočnim citometrom na zajetje slike pokazali, da tarčna zdravila ne prožijo prenosa TFEB v jedro celic KLL, s tem pa ne vplivajo na prepis avtofagnih genov. Učinkovitost izbranih zaviralcev avtofagije, dorzomorfina, MRT68921, VPS34-IN 1 in klorokina, smo preverili na celičnih linijah MEC-1, MEC-2 in HG-3 ter na primarnih celicah KLL s testom metabolne aktivnosti. Na podlagi vrednosti EC50 smo ugotovili, da zaviralci avtofagije delujejo koncentracijsko in časovno-odvisno citotoksično, na celice KLL pa deluje najbolj citotoksično zaviranje ULK1 s spojino MRT68921. Zaviralci avtofagije delujejo na celice KLL neodvisno od genetskih sprememb tudi proti vzorcem z napovedno neugodnimi spremembami, npr. del(17p) in nemutiranim IGHV. S korelacijo vrednosti EC50 zaviralcev avtofagije in tarčnih zdravil smo v nadaljevanju ugotovili, da obstajajo povezave med delovanjem ibrutiniba, akalabrutiniba in duveliziba z VPS34-IN 1. Ker tarčna zdravila aktivirajo avtofagijo, smo na koncu ovrednotili še smiselnost uporabe zaviralcev avtofagije v kombinaciji s tarčnimi zdravili. Sinergizem zaviralcev avtofagije in tarčnih zdravil smo zasledili pri kombinacijah MRT68921, dorzomorfinu in klorokinu s tarčnimi zdravili, za VPS34-IN 1 pa zgolj pri kombinaciji z zaviralci BTK. S tem smo pokazali smiselnost ciljanja avtofagije za izboljšanje učinkovitosti tarčnih zdravil pri zdravljenju KLL.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\u27s 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