Screening for regulators of methotrexate resistance in gestational trophoblastic disease
File(s)
Author(s)
Stokes, William Bernard
Type
Thesis
Abstract
Gestational trophoblastic disease (GTD) comprises a heterogeneous spectrum of pregnancy related conditions that arise from rapidly proliferating trophoblasts including the pre-malignant hydatidiform moles and the malignant disorders of invasive mole, choriocarcinoma and placental site trophoblastic tumour. GTD patients are categorised as low or high risk and in the case of the former are treated with methotrexate (MTX). However, one third of patients treated with MTX develop resistance.
To facilitate better understanding of development of MTX resistance, a siRNA screen targeting members of the kinome was undertaken in the choriocarcinoma cell lines, JEG-3 and JEG-3R, which are sensitive and resistant to MTX, respectively. The initial stages of this study focused firstly on characterising our MTX sensitive and resistant cell lines to understand the role of classical MTX resistance mechanisms in the choriocarcinoma cell lines as well as a normal placental cell line pair. Secondly, the conditions for the siRNA screen were established which included determining an appropriate cell number, MTX concentration and quality controls.
The screen revealed 19 targets that were identified following the performance of statistical analysis, literature review and creation of an association network. siRNAs targeting members of the DNA damage response pathway, namely ATR (Ataxia Telangiectasia and Rad3-Related), Wee1 (WEE1 G2 Checkpoint Kinase), and CHK1 (Checkpoint Kinase 1), were identified as sensitisers to MTX.
ATR (Ataxia telangiectasia and Rad3 related) accumulates at single strand DNA break sites where it phosphorylates targets which indirectly results in cell cycle arrest at the G2/M phase of the cell cycle. siRNA mediated knockdown of ATR sensitised both JEG-3 and JEG-3R cells to MTX. The reversible ATR inhibitors VE-821 and VX-970, also sensitised the choriocarcinoma cell lines to MTX. Using CRISPR-Cas9 technology to knock out ATR, we confirmed the findings using siRNA and inhibitors in JEG-3R cells, but we observed no different sensitivity in JEG-3 cells. Targeting the ATR pathway thus has a role in enhancing MTX sensitivity.
Secondly, targeting CHK1, a protein that is directly activated by ATR and has a role in activating the cell cycle checkpoint by inhibitory phosphorylation of CDC25 (Cell Division Cycle 25) and by activating Wee1 by phosphorylation, using siRNA sensitised JEG-3 and JEG-3R cells to MTX. The CHK1 inhibitor, LY-2603618, supported the findings using siRNA by sensitising JEG-3 and JEG-3R cells to MTX.
Thirdly, using siRNA to specifically target Wee1, a kinase that phosphorylates and thereby inactivates CDC2 (Cyclin Dependent Kinase 1), rendered JEG-3 and JEG-3R cells more sensitive to MTX. Thereafter, the specific Wee1 inhibitor, MK-1775, was shown to sensitise JEG-3 and JEG-3R cells to MTX.
Four siRNAs targeting CAMK1 (Calcium/Calmodulin Dependent Protein Kinase I), CAMKK1 (Calcium/Calmodulin Dependent Protein Kinase Kinase I), CDK2 (Cyclin Dependent Kinase 2) and TLK1 (Tousled like Kinase 1) were identified by the screen and validated as antagonistic to MTX sensitivity. Further work is necessary to elucidate the role of these proteins in MTX resistance, including determining whether any are elevated in MTX-resistant GTD and thus can act as a biomarker of MTX resistance.
Both phospho-CDC2 and phospho-Wee1 levels were decreased in JEG-3R and Plac-R cell lines compared with their sensitive counterparts, JEG-3 and Plac cells respectively, suggesting that a more robust DNA damage response is not responsible for increased MTX resistance in these cell lines. Future work will focus on further characterising the role of these validated hits identified in this study.
To facilitate better understanding of development of MTX resistance, a siRNA screen targeting members of the kinome was undertaken in the choriocarcinoma cell lines, JEG-3 and JEG-3R, which are sensitive and resistant to MTX, respectively. The initial stages of this study focused firstly on characterising our MTX sensitive and resistant cell lines to understand the role of classical MTX resistance mechanisms in the choriocarcinoma cell lines as well as a normal placental cell line pair. Secondly, the conditions for the siRNA screen were established which included determining an appropriate cell number, MTX concentration and quality controls.
The screen revealed 19 targets that were identified following the performance of statistical analysis, literature review and creation of an association network. siRNAs targeting members of the DNA damage response pathway, namely ATR (Ataxia Telangiectasia and Rad3-Related), Wee1 (WEE1 G2 Checkpoint Kinase), and CHK1 (Checkpoint Kinase 1), were identified as sensitisers to MTX.
ATR (Ataxia telangiectasia and Rad3 related) accumulates at single strand DNA break sites where it phosphorylates targets which indirectly results in cell cycle arrest at the G2/M phase of the cell cycle. siRNA mediated knockdown of ATR sensitised both JEG-3 and JEG-3R cells to MTX. The reversible ATR inhibitors VE-821 and VX-970, also sensitised the choriocarcinoma cell lines to MTX. Using CRISPR-Cas9 technology to knock out ATR, we confirmed the findings using siRNA and inhibitors in JEG-3R cells, but we observed no different sensitivity in JEG-3 cells. Targeting the ATR pathway thus has a role in enhancing MTX sensitivity.
Secondly, targeting CHK1, a protein that is directly activated by ATR and has a role in activating the cell cycle checkpoint by inhibitory phosphorylation of CDC25 (Cell Division Cycle 25) and by activating Wee1 by phosphorylation, using siRNA sensitised JEG-3 and JEG-3R cells to MTX. The CHK1 inhibitor, LY-2603618, supported the findings using siRNA by sensitising JEG-3 and JEG-3R cells to MTX.
Thirdly, using siRNA to specifically target Wee1, a kinase that phosphorylates and thereby inactivates CDC2 (Cyclin Dependent Kinase 1), rendered JEG-3 and JEG-3R cells more sensitive to MTX. Thereafter, the specific Wee1 inhibitor, MK-1775, was shown to sensitise JEG-3 and JEG-3R cells to MTX.
Four siRNAs targeting CAMK1 (Calcium/Calmodulin Dependent Protein Kinase I), CAMKK1 (Calcium/Calmodulin Dependent Protein Kinase Kinase I), CDK2 (Cyclin Dependent Kinase 2) and TLK1 (Tousled like Kinase 1) were identified by the screen and validated as antagonistic to MTX sensitivity. Further work is necessary to elucidate the role of these proteins in MTX resistance, including determining whether any are elevated in MTX-resistant GTD and thus can act as a biomarker of MTX resistance.
Both phospho-CDC2 and phospho-Wee1 levels were decreased in JEG-3R and Plac-R cell lines compared with their sensitive counterparts, JEG-3 and Plac cells respectively, suggesting that a more robust DNA damage response is not responsible for increased MTX resistance in these cell lines. Future work will focus on further characterising the role of these validated hits identified in this study.
Version
Open Access
Date Issued
2017-06
Date Awarded
2018-03
Advisor
Seckl, Michael
Pardo, Olivier
Publisher Department
Department of Surgery & Cancer
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)