The role of proteasome, immunoproteasome and autophagy in the therapy of chronic lymphocytic leukemia

Avsec, Damjan (author), Mlinarič Raščan, Irena (mentor), Podgornik, Helena (co-mentor)

Chronic lymphocytic leukemia (CLL) is a disease of accumulating malignant mature-appearing monoclonal B-lymphocytes in blood, bone marrow, and lymphatic tissues. The incidence of CLL in Slovenia is among the Europe’s highest, and with current demographic changes the burden of the disease is expected to increase in the future. In the last decade, a series of small, targeted molecules made it from bench-to-bedside in CLL. Despite the remarkable clinical success of BCR pathway inhibitors (ibrutinib, acalabrutinib, idelalisib, and duvelisib) and Bcl-2 antagonist (venetoclax), we are now faced with concerning drawbacks of these therapies, including high rates of discontinuation, severe adverse events, and the inevitable emergence of resistance. The primary purpose of our work was to address the unmet clinical need to improve the treatment of patients with CLL by delineating the role of proteasome (cP), immunoproteasome (iP), and autophagy in the therapy of CLL. We discovered that different cP/iP inhibitors suppress the prosurvival NF?B signaling and trigger apoptotic cell death independently of genetic background also in hard-to-treat patient-derived CLL cells carrying prognostically unfavorable del(11q), del(17p) and unmutated IGHV. These inhibitors (n=11) are selectively cytotoxic toward malignant CLL cells in low nanomolar concentrations, with cP inhibitor carfilzomib (EC50, 48h 2 nM) and iP inhibitor ONX-0914 (EC50, 48h 10 nM) being investigated in detail. They synergized with targeted therapies by perturbing the prosurvival BCR and NF?B pathways, thus culminating in a synergistic promotion of apoptosis. Moreover, they demonstrated a remarkable resistance-overcoming activity in both de-novo established resistant CLL clones and ex-vivo identified targeted therapy insensitive patient-derived CLL cells. We identified the molecular background to venetoclax resistance in CLL, including IFN?-mediated cytoprotection and iP formation, along with altered apoptotic pathways (downregulation of Bak and Bax, increased p-Bcl-2). The use of ONX-0914 successfully tackled IFN?-mediated cytoprotection and eradicated resistance in venetoclax resistant CLL clone. Delineating the role of autophagy in CLL, we found that targeted therapies activate autophagy via BTK/Akt, AMPK/ULK1/Beclin-1 and PI3K/Akt pathways. Block on autophagy at AMPK (dorsomorphin, EC50, 48h 4.6 µM), ULK1/2 (MRT68921, EC50, 48h 2.1 µM), VPS34 (VPS34-IN1, EC50, 48h 7.9 µM), and autolysosome formation (chloroquine, EC50, 48h 13.6 µM) induced apoptosis independently of the genetic background in hard-to-treat CLL cells carrying prognostically unfavorable del(11q), del(17p), and unmutated IGHV. Activation of autophagy was not cytotoxic to CLL cells, thus establishing that autophagy has a prosurvival role in CLL. De-novo established targeted therapy resistant CLL clones overexpressed several autophagic proteins. Autophagy inhibitors overcame this resistance and synergized with targeted therapies against hard-to-treat CLL cells. Several autophagy proteins were overexpressed in CLL cells compared to healthy controls, which defined the selective cytotoxicity of autophagy inhibitors against CLL cells. The levels of p62/SQSTM1 were higher in patients with unmutated IGHV, thus linking the p62/SQSTM1 to an adverse prognosis. In conclusion, we elucidated the role of cP, iP, and autophagy in the therapy of CLL and put forth a solid rationale to consider the use of cP, iP, and autophagy inhibitors for the treatment of relapsed/refractory patients with CLL.

• Language: English
• Keywords: CLL, proteasome, immunoproteasome, autophagy, targeted therapy, resistance, ibrutinib, idelalisib, venetoclax, NFκB, B-cell receptor
• Work type: Doctoral dissertation
• Organization: FFA - Faculty of Pharmacy
• Year: 2023
• PID: 20.500.12556/RUL-151332
• Publication date in RUL: 05.10.2023

Secondary language

• Language: Slovenian
• Title: Vloga proteasoma, imunoproteasoma in avtofagije v terapiji kronične limfocitne levkemije

Abstract

Kronična limfocitna levkemija (KLL) je bolezen kopičenja malignih, na videz zrelih monoklonskih B-limfocitov v krvi, kostnem mozgu in limfnem tkivu. Pojavnost KLL je v Sloveniji med najvišjimi v Evropi, ob trenutnih demografskih spremembah pa pričakujemo, da se bo breme bolezni v prihodnje še povečevalo. V zadnjem desetletju so v klinično uporabo pri KLL vpeljali vrsto majhnih tarčnih molekul. Kljub izjemnemu kliničnemu uspehu zaviralcev signalne poti B-celičnega receptorja (BCR) (ibrutinib, akalabrutinib, idelalizib in duvelizib) in antagonista Bcl-2 (venetoklaks) sedaj opažamo pereče pomanjkljivosti teh zdravil, vključno z visokimi stopnjami prekinitev zdravljenja, resnimi neželenimi učinki in neizogibnim pojavov odpornosti. Glavni namen našega dela je bil nasloviti neizpolnjeno klinično potrebo po izboljšanju zdravljenja bolnikov s KLL, pri čemer smo se osredotočili na vlogo proteasoma (cP), imunoproteasoma (iP) in avtofagije pri zdravljenju KLL. Odkrili smo, da različni zaviralci cP/iP zavirajo preživetveno signalno pot NFκB in sprožijo apoptotično celično smrt neodvisno od genetskega ozadja tudi v neobčutljivih celicah KLL, ki nosijo napovedno neugodne del(11q), del(17p) in nemutirani IGHV. Ti zaviralci (n=11) so selektivno citotoksični za maligne celice KLL v nizkih nanomolarnih koncentracijah, pri čemer smo podrobno raziskali zaviralec cP karfilzomib (EC50, 48h 2 nM) in zaviralec iP ONX-0914 (EC50, 48h 10 nM). S tarčnimi zdravili sta delovala sinergistično citotoksično z zaviranjem preživetvenih poti BCR in NFκB, kar je vodilo v sinergistično proženje apoptoze. Poleg tega so zaviralci cP/iP pokazali izjemno aktivnost pri premagovanju odpornosti tako pri novo vzpostavljenih odpornih klonih KLL kot pri ex-vivo identificiranih na tarčna zdravila neobčutljivih celicah bolnikov s KLL. Odkrili smo molekularno ozadje odpornosti na venetoklaks, vključno s citoprotektivnimi učinki IFNγ, ki inducira nastanek iP, in spremenjenimi apoptotičnimi potmi (znižano izražanje Bak in Bax, povečano izražanje p-Bcl-2). ONX-0914 je uspešno zavrl citoprotektivne učinke IFNγ in izkoreninil odpornost klona KLL na venetoklaks. Pri raziskovanju vloge avtofagije pri KLL smo ugotovili, da tarčna zdravila aktivirajo avtofagijo preko signalnih poti BTK/Akt, AMPK/ULK1/Beclin-1 in PI3K/Akt. Zaviranje avtofagije z delovanjem na AMPK (dorzomorfin, EC50, 48 h 4,6 µM), ULK1/2 (MRT68921, EC50, 48 h 2,1 µM), VPS34 (VPS34-IN1, EC50, 48 h 7,9 µM) in na tvorbo avtolizosomov (klorokin, EC50, 48 h 13,6 µM) sproži apoptozo neodvisno od genetskega ozadja v celicah KLL, ki nosijo napovedno neugodne del(11q), del(17p) in nemutirani IGHV. Aktivacija avtofagije ni bila citotoksična za celice KLL, kar pomeni, da ima avtofagija preživetveni pomen pri KLL. Novo vzpostavljeni na tarčna zdravila odporni kloni KLL so prekomerno izražali več proteinov avtofagije. Zaviralci avtofagije so premostili to odpornost in delovali sinergistično citotoksično s tarčnimi zdravili proti neobčutljivim celicam KLL. Več proteinov avtofagije je bilo prekomerno izraženih v celicah KLL v primerjavi z zdravimi kontrolami, kar je tudi razlog selektivne citotoksičnosti zaviralcev avtofagije na celice KLL. Ravni p62/SQSTM1 so bile višje pri bolnikih z nemutiranim IGHV, kar povezuje p62/SQSTM1 z neugodno napovedjo poteka bolezni. Če povzamemo, v doktorski disertaciji smo razjasnili vlogo cP, iP in avtofagije pri zdravljenju KLL in predstavili trdno utemeljitev za uporabo zaviralcev cP, iP in avtofagije pri zdravljenju bolnikov s ponovljeno/neodzivno KLL.

• Keywords: KLL, proteasom, immunoproteasom, avtofagija, tarčna zdravila, odpornost, ibrutinib, idelalizib, venetoklaks, NFκB, B-celični receptor