Wee1-kinaasin toiminnan estäminen korkean pahanlaatuisuusasteen seroosin tyypin munasarjasyöpäsoluissa

Wee1-kinaasi on tärkeä solusyklin etenemisen säätelijä. Se estää solua etenemästä G2-tarkastuspisteestä mitoosiin, jos solussa on havaittu DNA-vaurioita. Wee1-kinaasi inhiboi mitoosissa tarvittavaa sykliinistä riippuvaista kinaasia, mikä johtaa solusyklin pysähtymiseen. Solusyklin pysähtyminen antaa solulle aikaa korjata DNA-vauriot. DNA-vaurioita voidaan korjata myös solusyklin G1-tarkastuspisteessä, jossa vaurioiden korjaus on riippuvaista TP53-geenin koodaamasta p53-proteiinista. Korkean pahanlaatuisuusasteen seroosin tyypin munasarjasyöpäsolut (HGSOC) ovat TP53-mutatoituneita, joten ne ovat riippuvaisia G2-tarkastuspisteestä DNA-vaurioiden korjaamiseksi. Jos Wee1-kinaasin toiminta estetään, HGSOC-solut etenevät mitoosiin vaurioituneella DNA:lla, mikä voi johtaa solun kuolemaan. Lisäksi noin 50 % HGSOC:ssä DNA-vaurioiden korjaaminen homologisen rekombinaation (HR) avulla on puutteellista mm. BRCA1/2-mutaatioiden takia. HR:n puutteellisuus voi herkistää solut G2-tarkastuspisteen inhibiittoreille. Wee1-kinaasin inhibiittorin (AZD1775) vaikutusta solujen elinkelpoisuuteen tutkittiin yksin sekä kahden HGSOC:n hoidossa käytettävän lääkeaineen, sisplatiinin ja olaparibin, kanssa kaupallisilla sekä potilasperäisillä HGSOC-solulinjoilla. Lääkeaineiden vaikutus solujen elinkelpoisuuteen määritettiin ATP:n määrän mittaamiseen perustuvalla CTG-analyysillä, joka ilmaisi solujen metabolisen aktiivisuuden määrän näytteessä. Tutkimukseen valittiin solulinjoja, joissa DNA-vaurioiden korjaus HR:n avulla toimii sekä HR-puutteellisia solulinjoja. HR:n tila testattiin immunofluoresenssiin perustuvalla funktionaalisella menetelmällä. Lisäksi tutkittiin AZD1775:n solujen DNA:han aiheuttamia katkoksia Comet assay- menetelmällä. Tutkimuksessa havaittiin sisplatiinin ja AZD1775:n yhdistelmän heikentävän HGSOC-solujen elinkelpoisuutta enemmän kuin sisplatiinin ja olaparibin tai olaparibin ja AZD1775:n yhdistelmän. Kaikkien HR-puutteellisten solulinjojen ei havaittu olevan herkempiä AZD1775:lle kuin solulinjojen, joissa HR on toimivaa. AZD1775:n havaittiin aiheuttavan DNA-katkoksia HR-puutteellisissa soluissa enemmän, kuin solulinjoissa, joissa HR oli toimivaa. Hoidon aikana lääkeaineille kehittyvä resistenssi on HGSOC:ssä ongelma, johon ratkaisua haetaan lääkeaineiden yhdistelmistä. Tulosten perusteella, AZD1775 on potentiaalinen lääkeaine HGSOC:n hoitoon yhdistelmänä sisplatiinin ja olaparibin kanssa

Effects of Wee1 inhibitor adavosertib on patient-derived high-grade serous ovarian cancer cells are multiple and independent of homologous recombination status

ObjectiveA major challenge in the treatment of platinum-resistant high-grade serous ovarian cancer (HGSOC) is lack of effective therapies. Much of ongoing research on drug candidates relies on HGSOC cell lines that are poorly documented. The goal of this study was to screen for effective, state-of-the-art drug candidates using primary HGSOC cells. In addition, our aim was to dissect the inhibitory activities of Wee1 inhibitor adavosertib on primary and conventional HGSOC cell lines. MethodsA comprehensive drug sensitivity and resistance testing (DSRT) on 306 drug compounds was performed on three patient-derived genetically unique HGSOC cell lines and two commonly used ovarian cancer cell lines. The effect of adavosertib on the cell lines was tested in several assays, including cell-cycle analysis, apoptosis induction, proliferation, wound healing, DNA damage, and effect on nuclear integrity. ResultsSeveral compounds exerted cytotoxic activity toward all cell lines, when tested in both adherent and spheroid conditions. In further cytotoxicity tests, adavosertib exerted the most consistent cytotoxic activity. Adavosertib affected cell-cycle control in patient-derived and conventional HGSOC cells, inducing G2/M accumulation and reducing cyclin B1 levels. It induced apoptosis and inhibited proliferation and migration in all cell lines. Furthermore, the DNA damage marker gamma H2AX and the number of abnormal cell nuclei were clearly increased following adavosertib treatment. Based on the homologous recombination (HR) signature and functional HR assays of the cell lines, the effects of adavosertib were independent of the cells' HR status. ConclusionOur study indicates that Wee1 inhibitor adavosertib affects several critical functions related to proliferation, cell cycle and division, apoptosis, and invasion. Importantly, the effects are consistent in all tested cell lines, including primary HGSOC cells, and independent of the HR status of the cells. Wee1 inhibition may thus provide treatment opportunities especially for patients, whose cancer has acquired resistance to platinum-based chemotherapy or PARP inhibitors.Peer reviewe

Effects of Wee1 inhibitor adavosertib on patient-derived high-grade serous ovarian cancer cells are multiple and independent of homologous recombination status

Objective: A major challenge in the treatment of platinum-resistant high-grade serous ovarian cancer (HGSOC) is lack of effective therapies. Much of ongoing research on drug candidates relies on HGSOC cell lines that are poorly documented. The goal of this study was to screen for effective, state-of-the-art drug candidates using primary HGSOC cells. In addition, our aim was to dissect the inhibitory activities of Wee1 inhibitor adavosertib on primary and conventional HGSOC cell lines.Methods: A comprehensive drug sensitivity and resistance testing (DSRT) on 306 drug compounds was performed on three patient-derived genetically unique HGSOC cell lines and two commonly used ovarian cancer cell lines. The effect of adavosertib on the cell lines was tested in several assays, including cell-cycle analysis, apoptosis induction, proliferation, wound healing, DNA damage, and effect on nuclear integrity.Results: Several compounds exerted cytotoxic activity toward all cell lines, when tested in both adherent and spheroid conditions. In further cytotoxicity tests, adavosertib exerted the most consistent cytotoxic activity. Adavosertib affected cell-cycle control in patient-derived and conventional HGSOC cells, inducing G2/M accumulation and reducing cyclin B1 levels. It induced apoptosis and inhibited proliferation and migration in all cell lines. Furthermore, the DNA damage marker γH2AX and the number of abnormal cell nuclei were clearly increased following adavosertib treatment. Based on the homologous recombination (HR) signature and functional HR assays of the cell lines, the effects of adavosertib were independent of the cells' HR status.Conclusion: Our study indicates that Wee1 inhibitor adavosertib affects several critical functions related to proliferation, cell cycle and division, apoptosis, and invasion. Importantly, the effects are consistent in all tested cell lines, including primary HGSOC cells, and independent of the HR status of the cells. Wee1 inhibition may thus provide treatment opportunities especially for patients, whose cancer has acquired resistance to platinum-based chemotherapy or PARP inhibitors.</p

Functional Homologous Recombination Assay on FFPE Specimens of Advanced High-Grade Serous Ovarian Cancer Predicts Clinical Outcomes

Peer reviewe