Preučevanje mehanizmov odpornosti celic kronične limfocitne levkemije na venetoklaks

Škrlj Miklavčič, Marja (avtor), Mlinarič-Raščan, Irena (mentor), Avsec, Damjan (komentor)

Kronična limfocitna levkemija (KLL) je najpogostejše hematološko maligno obolenje odraslih v zahodnem svetu. V zadnjem desetletju smo priča velikemu napredku na področju razvoja novih, tarčnih zdravil za zdravljenju KLL. Velik potencial kažejo predvsem antagonisti antiapoptotičnih proteinov, med katerimi je trenutno na voljo le venetoklaks. Je zdravilo zadnje izbire pri bolnikih z agresivno in neodzivno obliko KLL. Za te bolnike je zato pojav odpornosti na venetoklaks lahko zelo neugoden. V magistrski nalogi smo za namen preučevanja mehanizmov odpornosti celic KLL na venetoklaks iz imortaliziranih celic KLL (MEC-1) vzpostavili in vitro model na venetoklaks odpornih celic (MEC-1 VER) in z določitvijo EC50 venetoklaksa dokazali njihovo odpornost. Neodzivnost na zdravljenje je lahko odraz kompleksnih mehanizmov, zato smo preučevali razlike v odzivu dovzetnih in odpornih celic z različnimi pristopi. Z imunofenotipizacijo in pretočno citometrijo z zajemom slike smo pokazali znižano izražanje CD20 in ZAP-70 ter povečano izražanje CD49d in Ki-67 pri celicah MEC-1 VER. Znižano izražanje CD20 smo povezali z manjšo učinkovitostjo protitelesa proti CD20 obinutuzumaba, ki se pogosto uporablja v kombinaciji z venetoklaksom. Spremembe v izražanju ZAP-70, CD49d in Ki-67 so nakazale na vpletenost tumorskega mikrookolja v odpornost celic KLL na venetoklaks. V nadaljevanju smo z metodo prenosa western preučili udeleženost preživetvenih signalnih poti in izražanje antiapoptotičnih proteinov pri pojavu odpornosti na venetoklaks. Signifikanten doprinos k odpornosti na venetoklaks smo potrdili samo za signalno pot p38 MAPK, ki ponovno nakazuje na udeleženost tumorskega mikrookolja. Poleg preučevanja mehanizmov, udeleženih pri odpornosti na venetoklaks, smo se osredotočili tudi na raziskovanje možnosti premostitve odpornosti. Na podlagi preteklih raziskav smo se usmerili na zaviranje imunoproteasoma, ki je povečano aktiven pri celicah KLL. Selektivni zaviralec ONX-0914 je deloval koncentracijsko odvisno citotoksično na celice MEC-1 preko proženja apoptoze po intrinzični poti in izkazal močno sinergistično delovanje z venetoklaksom. Odkrili smo, da pojav odpornosti na venetoklaks ne vpliva na delovanje ONX-0914, temveč ONX-0914 v celoti premosti odpornost celic na venetoklaks. To nakazuje na obetavno novo strategijo za premagovanje odpornosti pri KLL.

• Jezik: Slovenski jezik
• Ključne besede: kronična limfocitna levkemija (KLL), venetoklaks, odpornost na zdravljenje, imunoproteasom, ONX-0914
• Vrsta gradiva: Magistrsko delo/naloga
• Organizacija: FFA - Fakulteta za farmacijo
• Leto izida: 2021
• PID: 20.500.12556/RUL-127688
• Datum objave v RUL: 19.06.2021

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Investigation of venetoclax resistance mechanisms in chronic lymphocytic leukemia cells

Izvleček

Chronic lymphocytic leukemia (CLL) is the most common hematologic malignancy of adults in the Western world. Over the past decade, we have witnessed a great progress in the development of new, targeted drugs for the treatment of CLL. Antagonists of antiapoptotic proteins in particular show great potential, of which only venetoclax is currently available. It is often the last option for patients with an aggressive and unresponsive form of CLL. Therefore, the emergence of venetoclax resistance can be very unfavorable for these patients. In the Master's thesis, we investigated the mechanisms of venetoclax resistance of CLL cells. We established an in vitro model of venetoclax resistant cells (MEC-1 VER) from immortalized CLL cells (MEC-1) and confirmed the resistance by determining the EC50 of venetoclax. As venetoclax failure in CLL is a reflection of complex mechanisms, we studied the differences in responses of susceptible and resistant cells using different approaches. Decreased CD20 and ZAP-70 expression and increased CD49d and Ki-67 expression in MEC-1 VER cells were demonstrated by immunophenotyping and imaging flow cytometry. Decreased CD20 expression was associated with diminished efficacy of the anti-CD20 antibody obinutuzumab, which is often used in combination with venetoclax. The changes in the expression of ZAP-70, CD49d and Ki-67 indicate that tumor microenvironment plays a role in resistance of CLL cells to venetoclax. Subsequently, the involvement of prosurvival signaling pathways and the expression of antiapoptotic proteins in the occurrence of venetoclax resistance were studied by western blot. A significant contribution to venetoclax resistance was confirmed only for the p38 MAPK signaling pathway, which again suggests that the tumor microenvironment is implicated in venetoclax resistance in CLL. In addition to studying the mechanisms involved in venetoclax resistance, we also focused on exploring the possibility of overcoming the resistance itself. Based on previous studies, we focused on inhibiting the immunoproteasome, whose activity is elevated in CLL cells. The selective immunoproteasome inhibitor ONX-0914 exhibited concentration-dependent cytotoxicity on MEC-1 cells by inducing apoptosis via intrinsic pathway and showed strong synergistic activity with venetoclax. We found that the emergence of venetoclax resistance does not affect the activity of ONX-0914, instead ONX-0914 completely overcomes it. This points to a promising new strategy for overcoming resistance in CLL.

• Ključne besede: chronic lymphocytic leukemia (CLL), venetoclax, resistance to treatment, immunoproteasome, ONX-0914