High-grade serous ovarian carcinoma (HGSOC) is the most frequent epithelial histotype of ovarian cancer and causes more deaths than any other type of gynecological cancer. More than half of HGSOCs are characterized by a homologous recombination (HR) deficiency due to mutations in genes involved in this pathway, including Brca1 and Brca2. Olaparib is a poly(ADP-ribose) polymerase inhibitor (PARPi) that has shown striking in vitro activity in HR-depleted cells and has been approved as a first-line maintenance treatment after platinum-based treatment in patients with Ovarian cancer with BRCA mutation. Despite the clinical benefits, resistance to PARPi is starting to occur in clinical settings. In order to identify new therapeutic strategies to overcome this resistance, we used a syngeneic model of murine ovarian epithelial origin previously made resistant to Olaparib in our laboratory. On the newly introduced model we carried out a high-throughput screening of a library made up of more than 1400 compounds, approved by the FDA, in combination with a sub-toxic dose of Olaparib to identify compounds capable of overcoming resistance to Olaparib. We found that the combination of Olaparib and topoisomerase 1 inhibitors was very active in the Olaparib-resistant cell line. We subsequently carried out a study on the disruption of the cell cycle that the combination created, and we saw that the single treatment with Olaparib did not block the cell cycle in the G2 phase (which instead happens for the sensitive line), the treatment with the topoisomerase 1 inhibitor, however, it prevents the cells from entering the S phase, blocking the cell cycle in the G1 phase; the combination of the two drugs also presents a block in the G1 phase; furthermore, as the treatment time passed we were able to notice the increase in cells in the sub-G1 phase, a phase generally associated with apoptotic cells. We therefore investigated the mechanisms of cell death that allowed us to overcome this resistance and we found that the combination of the two drugs induces, in the cells, high values of caspase activity, inducing apoptosis, compared to the treatments of the two drugs alone, an increase in activity directly correlated with treatment time. Hypothesis which was also further confirmed by Western Blot analysis through which we looked for the expression levels of γH2A (a histone protein recruited when there is damage to the double strand of DNA).
Il carcinoma ovarico sieroso ad alto grado (HGSOC) è l'istotipo epiteliale più frequente di cancro ovarico e provoca più decessi di qualsiasi altro tipo di cancro ginecologico. Più della metà degli HGSOC è caratterizzata da un deficit di ricombinazione omologa (HR) dovuto alle mutazioni nei geni coinvolti in questa via, inclusi Brca1 e Brca2. Olaparib è un inibitore della poli (ADP-ribosio) polimerasi (PARPi) che ha mostrato in vitro un'attività sorprendente nelle cellule prive di HR ed è stato approvato in prima linea come trattamento di mantenimento dopo il trattamento a base di platino in pazienti con cancro ovarico con mutazione Brca. Nonostante i benefici clinici, in ambito clinico stanno iniziando a verificarsi fenomeni di resistenza ai PARPi . Al fine di identificare nuove strategie terapeutiche per superare questa resistenza, abbiamo utilizzato un modello singenico di origine epiteliale ovarica murina precedentemente resa resistente a Olaparib nel nostro laboratorio. Sul modello appena introdotto abbiamo effettuato uno screening highthroughput di una library formata da più di 1400 composti, approvati dalla FDA, in combinazione con una dose sub-tossica di Olaparib per identificare i composti in grado di superare la resistenza a Olaparib. Abbiamo riscontrato che la la combinazione di Olaparib e inibitori delle topoisomerasi 1 risulta essere molto attiva nella linea cellulare resistente a Olaparib. Successivamente abbiamo effetuato uno studio sulla perturbazione del ciclo cellulare che la combinazione andava a creare, e abbiamo visto che il trattamento in singolo con Olaparib non andava a bloccare il ciclo cellulare in fase G2 (cosa che invece avviene per la linea sensibile), il trattamento con l’inibitore delle topoisomerasi 1 invece impedisce l’ingresso in fase S delle cellule, bloccando il ciclo cellulare in fase G1, anche la combinazione dei due farmaci presenta un blocco in fase G1; inoltre, con il passare del tempo di trattamento abbiamo potuto notare l’incremento di cellule in fase sub-G1, fase generalmente associata a cellule apoptotiche. Abbiamo quindi indagato i meccanismi di morte cellulare che permettessero di superare tale resistenza e abbiamo riscontrato che la combinazione dei due farmaci induce, nelle cellule, alti valori di attività caspasica, inducendo apoptosi, rispetto ai trattamenti dei due farmaci in singolo, aumento di attività direttamente correlato con il tempo di trattamento. Ipotesi che è stata ulteriormente confermata anche dall’analisi Western Blot tramite la quale siamo andati a ricercare i livelli di espressione di γH2A (una proteina istonica reclutata quando si ha danno al doppio filamento di DNA).
Nuove strategie terapeutiche per superare la resistenza ad Olaparib nel Carcinoma Ovarico Sieroso ad Alto Grado
SIAS, FABIO
2022/2023
Abstract
High-grade serous ovarian carcinoma (HGSOC) is the most frequent epithelial histotype of ovarian cancer and causes more deaths than any other type of gynecological cancer. More than half of HGSOCs are characterized by a homologous recombination (HR) deficiency due to mutations in genes involved in this pathway, including Brca1 and Brca2. Olaparib is a poly(ADP-ribose) polymerase inhibitor (PARPi) that has shown striking in vitro activity in HR-depleted cells and has been approved as a first-line maintenance treatment after platinum-based treatment in patients with Ovarian cancer with BRCA mutation. Despite the clinical benefits, resistance to PARPi is starting to occur in clinical settings. In order to identify new therapeutic strategies to overcome this resistance, we used a syngeneic model of murine ovarian epithelial origin previously made resistant to Olaparib in our laboratory. On the newly introduced model we carried out a high-throughput screening of a library made up of more than 1400 compounds, approved by the FDA, in combination with a sub-toxic dose of Olaparib to identify compounds capable of overcoming resistance to Olaparib. We found that the combination of Olaparib and topoisomerase 1 inhibitors was very active in the Olaparib-resistant cell line. We subsequently carried out a study on the disruption of the cell cycle that the combination created, and we saw that the single treatment with Olaparib did not block the cell cycle in the G2 phase (which instead happens for the sensitive line), the treatment with the topoisomerase 1 inhibitor, however, it prevents the cells from entering the S phase, blocking the cell cycle in the G1 phase; the combination of the two drugs also presents a block in the G1 phase; furthermore, as the treatment time passed we were able to notice the increase in cells in the sub-G1 phase, a phase generally associated with apoptotic cells. We therefore investigated the mechanisms of cell death that allowed us to overcome this resistance and we found that the combination of the two drugs induces, in the cells, high values of caspase activity, inducing apoptosis, compared to the treatments of the two drugs alone, an increase in activity directly correlated with treatment time. Hypothesis which was also further confirmed by Western Blot analysis through which we looked for the expression levels of γH2A (a histone protein recruited when there is damage to the double strand of DNA).È consentito all'utente scaricare e condividere i documenti disponibili a testo pieno in UNITESI UNIPV nel rispetto della licenza Creative Commons del tipo CC BY NC ND.
Per maggiori informazioni e per verifiche sull'eventuale disponibilità del file scrivere a: unitesi@unipv.it.
https://hdl.handle.net/20.500.14239/17160