Preučevanje sinergističnih učinkov agonistov prostaglandinskega receptorja EP4 z venetoklaksom, akalabrutinibom in duvelizibom in vitro

Bejek, Nastja (avtor), Mlinarič-Raščan, Irena (mentor), Markovič, Tijana (komentor)

Kronična limfocitna levkemija (KLL) je kronično potekajoča maligna bolezen, za katero je značilno postopno in nenadzorovano pomnoževanje ter kopičenje morfološko zrelih, a nefunkcionalnih limfocitov B. Predstavlja najpogostejšo vrsto levkemije pri odraslih v Sloveniji. KLL je morfološko, imunološko in genetsko izjemno heterogena bolezen, kar se odraža tako pri njenem poteku kot pri odzivu na zdravljenje. Prostaglandinski receptor EP4 predstavlja potencialno tarčo za zdravljenje B celičnih levkemij in limfomov. V predhodnih študijah so dokazali, da aktivacija prostaglandinskega receptorja EP4 v limfocitih B inhibira signalno pot NF-ĸB, kar vodi v apoptozo malignih limfocitov B. V magistrski nalogi smo vrednotili sinergistične učinke tarčnih zdravil venetoklaksa, ibrutiniba, akalabrutiniba, idelaliziba in duveliziba z agonistom receptorja EP4, PgE1-OH, na celičnih linijah kronične limfocitne levkemije MEC-1, MEC-1-VER in HG-3. Ker se vse pogosteje pri zdravljenju KLL pojavi odpornost na tarčno zdravilo venetoklaks, smo uporabili klinično relevanten in vitro model na venetoklaks odpornih celic KLL MEC-1-VER. Na celičnih linijah smo najprej s testom metabolne aktivnosti PrestoBlue ovrednotili citotoksične učinke agonistov receptorja EP4 in tarčnih zdravil ter določili vrednosti EC50. Ugotovili smo, da imajo vse spojine od časa in koncentracije odvisno delovanje. Ob primerjavi vrednosti EC50 na linijah MEC-1 in MEC-1-VER smo zaključili, da ima PgE1-OH primerljivo delovanje na obe celični liniji. Iz rezultatov lahko sklepamo, da odpornost na venetoklaks ni vplivala na citotoksično delovanje agonista receptorja EP4, PgE1-OH, in ima potencial v premagovanju odpornosti na tarčna zdravila. Ugotovili smo, da so vse testirane kombinacije PgE1-OH s tarčnimi zdravili, vsaj pri nekaterih koncentracijah, delovale sinergistično. Na celični liniji MEC-1 sta najmočnejši sinergistični učinek imeli kombinaciji PgE1-OH z zaviralcema signalne poti PI3K, idelalizibom in duvelizibom. Rezultate smo potrdili na celični liniji, odporni na venetoklaks, MEC-1-VER. V nadaljevanju smo na reporterski celični liniji Ramos Blue pokazali, da je PgE1-OH zavrl s TNF-α posredovano aktivacijo signalne poti NF-κB. V okviru magistrske naloge smo ugotovili, da imajo agonisti receptorja EP4 citotoksično delovanje in izkazujejo sinergistične učinke s tarčnimi zdravili in vitro tudi na celicah odpornih na venetoklaks MEC-1-VER, kar bi lahko predstavljalo potencialne možnosti za zdravljenje KLL.

• Jezik: Slovenski jezik
• Ključne besede: kronična limfocitna levkemija (KLL), prostaglandinski receptor tipa 4 (EP4), L-902, 688, PgE1-OH, KAG-308, venetoklaks, ibrutinib, akalabrutinib, idelalizib, duvelizib, sinergizem
• Vrsta gradiva: Magistrsko delo/naloga
• Organizacija: FFA - Fakulteta za farmacijo
• Leto izida: 2023
• PID: 20.500.12556/RUL-150949
• Datum objave v RUL: 26.09.2023

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Evaluation of synergistic effect of prostaglandin receptor EP4 agonists with venetoclax, acalabrutinib and duvelisib in vitro

Izvleček

Chronic lymphocytic leukaemia (CLL) is a chronic malignancy characterised by the progressive and uncontrolled multiplication and accumulation of morphologically mature but non-functional lymphocytes B. It is the most common type of leukaemia in adults in Slovenia. CLL is a morphologically, immunologically and genetically extremely heterogeneous disease, which is reflected both in its course and in its response to treatment. The prostaglandin receptor EP4 represents a potential target for the treatment of B-cell malignancies. Previous studies have shown that activation of prostaglandin receptor EP4 on lymphocytes B inhibits the NF-ĸB signalling pathway, leading to apoptosis of malignant B cells. In this master thesis we evaluated the synergistic effects of the target drugs venetoclax, ibrutinib, acalabrutinib, idelalisib and duvelisib with the EP4 receptor agonist, PgE1-OH, on CLL cell lines MEC-1, MEC-1-VER and HG-3. As resistance to the target drug venetoclax is increasingly emerging in the treatment of CLL, we have used a clinically relevant in vitro model of venetoclax-resistant MEC-1-VER CLL cells. First, we evaluated the cytotoxic effects of the EP4 receptor agonists and target drugs on the cell lines using the PrestoBlue metabolic activity assay and determined the EC50 values. Time- and concentration-dependent cytotoxicity was detected for all of the tested compounds. Comparing the EC50 values on MEC-1 and MEC-1-VER lines, we concluded that PgE1-OH has comparable activity also when the resistance to venetoclax was established. The results suggest that the cytotoxic activity of the EP4 receptor agonist PgE1-OH was not affected by venetoclax resistance, suggesting that it has the potential to overcome resistance to the target drugs. We found that all tested combinations of PgE1-OH with the target drugs, at least at several concentrations tested, acted synergistically. On the MEC-1 cell line, the combinations of PgE1-OH with the PI3K signalling pathway inhibitors idelalisib and duvelisib had the strongest synergistic effect. These results were confirmed on the MEC-1-VER cell line. Using Ramos Blue reporter cell line, we have shown that PgE1-OH inhibited TNF-α-mediated activation of the NF-κB signalling pathway. In conclusion, EP4 receptor agonist PgE1-OH exhibited synergistic effects with target drugs in vitro also on venetoclax resistant CLL cells MEC-1-VER, which could represent potential options for the treatment of CLL.

• Ključne besede: chronic lymphocytic leukemia (CLL), prostaglandin receptor type 4 (EP4), L-902, 688, PgE1-OH, KAG-308, venetoclax, ibrutinib, acalabrutinib, idelalisib, duvelisib, synergy