The official journal of INTERNATIONAL FEDERATION OF PIGMENT CELL SOCIETIES · SOCIETY FOR MELANOMA RESEARCH
PIGMENT CELL & MELANOMA Research Decatenation checkpoint-defective melanomas are dependent on PI3K for survival Kelly Brooks, Max Ranall, Loredana Spoerri, Alex Stevenson, Gency Gunasingh, Sandra Pavey, Fred Meunier, Thomas J. Gonda and Brian Gabrielli
DOI: 10.1111/pcmr.12268 Volume 27, Issue 5, Pages 813–821 If you wish to order reprints of this article, please see the guidelines here Supporting Information for this article is freely available here EMAIL ALERTS Receive free email alerts and stay up-to-date on what is published in Pigment Cell & Melanoma Research – click here
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Subscribe to PCMR and stay up-to-date with the only journal committed to publishing basic research in melanoma and pigment cell biology As a member of the IFPCS or the SMR you automatically get online access to PCMR. Sign up as a member today at www.ifpcs.org or at www.societymelanomaresarch.org
To take out a personal subscription, please click here More information about Pigment Cell & Melanoma Research at www.pigment.org
ORIGINAL ARTICLE
Pigment Cell Melanoma Res. 27; 813–821
Decatenation checkpoint-defective melanomas are dependent on PI3K for survival Kelly Brooks1,3, Max Ranall1, Loredana Spoerri1, Alex Stevenson1, Gency Gunasingh1, Sandra Pavey1, Fred Meunier2, Thomas J. Gonda1,4 and Brian Gabrielli1 1 Translational Research Institute, The University of Queensland Diamantina Institute, Brisbane, Qld, Australia 2 Clem Jones Centre for Ageing Dementia Research, Queensland Brain Institute, The University of Queensland, Brisbane, Qld, Australia 3 Present address: Cancer Research UK Manchester Institute, The University of Manchester, Wilmslow Road, Manchester, M20 4BX, UK 4 Present address: School of Pharmacy, The University of Queensland, Brisbane, Qld, Australia
KEYWORDS decatenation synthetic lethality/PI3K
checkpoint/melanoma/
PUBLICATION DATA Received 10 February 2014, revised and accepted for publication 25 May 2014, published online 29 May 2014
CORRESPONDENCE B. Gabrielli, e-mail: [email protected] doi: 10.1111/pcmr.12268
Summary Melanoma cell lines are commonly defective for the G2-phase cell cycle checkpoint that responds to incomplete catenation of the replicated chromosomes. Here, we demonstrate that melanomas defective for this checkpoint response are less sensitive to genotoxic stress, suggesting that the defective cell lines compensated for the checkpoint loss by increasing their ability to cope with DNA damage. We performed an siRNA kinome screen to identify kinases responsible and identified PI3K pathway components. Checkpoint-defective cell lines were three-fold more sensitive to small molecule inhibitors of PI3K. The PI3K inhibitor PF-05212384 promoted apoptosis in the checkpoint-defective lines, and the increased sensitivity to PI3K inhibition correlated with increased levels of activated Akt. This work demonstrates that increased PI3K pathway activation is a necessary adaption for the continued viability of melanomas with a defective decatenation checkpoint.
Introduction Melanoma is a common human malignancy, and while primary melanomas are generally easy to treat via excision, late-stage melanoma still proves a difficult target for therapeutic intervention. Late-stage melanoma is refractory to most treatments with the most common treatment dacarbazine, having a response rate of 15–20%, improving survival by only 3–6 months (Fang et al., 2004). One recent development in late-stage melanoma treatment has been the use of BRAF inhibitors for melanomas harbouring the BRAFV600E mutation (50–70% of melanomas) (Davies et al., 2002; Wan et al.,
2004). Pairing of these inhibitors with the BRAFV600E mutation has provided the most promising response rate to date of ~81% (Flaherty et al., 2010). Unfortunately, this response is only temporary in most cases, with the melanomas fast developing resistance via numerous methods (Johannessen et al., 2010; Villanueva et al., 2010). Such studies have demonstrated, however, that promising response rates are achievable in melanoma if the right target/subset and approach can be identified. Cell cycle defects, particularly in cell cycle checkpoints that normally respond to specific cellular stresses, are a common feature in many cancers (Malumbres and Barbacid, 2001). Such defects often confer a growth
Significance Melanomas are considered to be generally resistant to chemotherapy, although the basis for this is not clear. The correlation of the G2-phase decatenation checkpoint defect with reduced sensitivity to genotoxic stress suggests that adaptation to loss of the checkpoint may be responsible for this increased survival. Here we have demonstrated that increased PI3K pathway activation as a necessary adaptation for the continued viability of checkpoint-defective melanomas. This study provides evidence that targeting adaptations critical for continued viability in checkpoint-defective melanomas is a viable option for the selective destruction of tumours with this defect.
ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
813
Brooks et al.
Results Using ICRF-193 and etoposide to inhibit decatenation, the impact of failing to arrest at the decatenation checkpoint was assessed in terms of DNA damage and cell viability. Both checkpoint-functional (A2058) and -defective (MM576, HT144, SKMel13) cell lines showed little increase in the DNA damage marker cH2AX levels after 8-h ICRF-193 treatment; in all cases, there was a strong accumulation at 24-h and 48-h treatment (Figure 1A). All cell lines responded strongly to etoposide treatment. Long-term proliferation assays demonstrated that all cell lines experienced a similar decrease in viability after 814
A
Functional γH2AX H2AX
A2058
α Tubulin
Defective γH2AX MM576
H2AX α Tubulin γH2AX
HT144
H2AX α Tubulin γH2AX
SKMel13
H2AX 48 h
24 h
8h
Etop
Con
α Tubulin
ICRF-193 ICRF-193 60 40 20 0
MM415 A02 SKMel13 D25 MM383 MM576 MM96L A2058 MM603 MM329
% control
B
Defective
Functional
Etoposide 60 40 20 0
MM415 A02 SKMel13 D25 MM383 MM576 MM96L A2058 MM603 MM329
% control
advantages to the cancer (Hartwell and Kastan, 1994; Kaufmann and Paules, 1996; Paulovich et al., 1997); however, these checkpoints are protective mechanisms, functioning to ensure the health and viability of the cells. Their loss therefore can lead to an accumulation of DNA damage and replicative stress in these cells. To remain viable in the presence of such stress, it is reasonable to predict that these cells become more reliant on other signalling pathways, for example, anti-apoptotic, to compensate for these intrinsic stresses. These dependencies provide an opportunity to exploit the cell cycle checkpoint defects as selective targets, employing the concept of synthetic lethality. Many synthetic lethal approaches are attempts to target specific genetic lesions, for example, BRCA mutations in breast cancer. Recently, we reported that the decatenation checkpoint is defective in ~67% of melanoma cell lines (Brooks et al., 2013). The decatenation checkpoint is a G2-phase checkpoint important for monitoring the catenation of chromosomes, ensuring sister chromatids can be accurately segregated during mitosis (Damelin and Bestor, 2007; Deming et al., 2001; Kaufmann, 2006). Decatenation, the process of untangling catenated chromosomes is performed by topoisomerase II (TopoII) which can be inhibited using catalytic inhibitors such as ICRF-193 or poisons such as etoposide (Clifford et al., 2003; Kaufmann and Kies, 1998; Morris et al., 2000; Roca and Wang, 1994). We demonstrated that TopoII inhibition in decatenation checkpoint-defective cell lines resulted in increased genomic instability due to attempts to segregate catenated chromosomes during mitosis (Brooks et al., 2013), and attempts to segregate catenated chromosomes have previously been reported to increase DNA damage (Damelin and Bestor, 2007; Gorbsky, 1994; Hajji et al., 2003; Ishida et al., 1994), providing one example of intrinsic stress caused by a decatenation checkpoint dysfunction. The presence of at least one obvious source of intrinsic stress and the commonality of the decatenation checkpoint defect in melanoma make these cells a good candidate for investigating cell cycle checkpoint targeting in melanoma. This potential was investigated through siRNA screening of the kinome, and one such vital target was identified.
Defective
Functional
Figure 1. Decatenation checkpoint-defective cell lines do not have increased sensitivity to TopoII inhibition. (A) Whole cell lysates of cells treated with DMSO, 2 lM etoposide for 24 h, or 2 lM ICRF193 for 8, 24 or 48 h were immunoblotted for cH2AX as a marker for DNA damage. Total H2AX and a-tubulin were used as loading controls. B. Colony forming assay for cells treated for 24 h with 2uM ICRF-193 or 2lM etoposide. Colonies grown for 14 days and numbers normalized to DMSO-treated controls.
ICRF-193 treatment despite the severe chromosomal and mitotic aberrations occurring only in the checkpointdefective cell lines (Brooks et al., 2013), suggesting that these defective cells can compensate for this defect, showing no increased loss in viability compared with functional lines. Treatment with etoposide, which produced a similar level cH2AX at 24-h ICRF-193 treatment, had a much stronger effect on the checkpoint-functional ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
PI3K adapts to checkpoint defect
Z-Score
SKMel13 resazurin
6
Z-Score
4 2 0 –2 –4
B
0.5 0
Z-Score
–0.5 –1 –1.5 –2 –2.5
PL K1
B
8
K AP M T2 AK
K2
2A
C
C
K3
JA
PI
–3
K3
ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
A2058 resazurin
A 12 10 8 6 4 2 0 –2 –4
PI
cell lines (Figure 1B). This provides further evidence that cells which had lost decatenation checkpoint function were more resistant to the cytotoxic effects of DNA damage, likely reflecting an adaptation to loss of the checkpoint function. We reasoned that the adaptations in the checkpointdefective cell lines were likely to be required for the normal maintenance of viability of these cells but would not have a similarly critical role in checkpoint-functional cells. To identify these adaptations, an siRNA screen of the kinome (~700 genes) was performed. One decatenation checkpoint-functional (A2058) cell line and one decatenation checkpoint-defective (SKMel13) cell line were used. Assays for cell viability (Resazurin) and biomass (crystal violet) were performed for each siRNA in a 384-well format. Z-scores were calculated for each well compared with internal plate controls giving a spread of scores for both cell lines (Figure 2A and S1). Because the efficiency of knockdown cannot be determined for each well, an arbitrary cut-off was set for hit selection at a Z-score of 2.5, corresponding to a reduction in viability and biomass of at least 25%. Hits were then filtered as outlined (Figure S2). Potential hits that were only present in SKMel13 checkpoint-defective cell line were filtered to remove genes that were not expressed in the SKMel13 cell line, or were not expressed in a high proportion of the checkpoint-defective melanoma cell lines previously characterized (Brooks et al., 2013). This resulted in a list with the five top hits being PIK3CB, PIK3C2A, MAPK8, JAK2 and AKT2. Closer analysis of these revealed that PIK3CB was the strongest hit in both assays (Figure 2B and S1B) and provided a similar level of loss of viability in the checkpoint-defective cell line as Plk1, the positive control for loss of viability. Interestingly, the negative effect of knockdown was enhanced with ICRF193 treatment, particularly for PIK3C2A in the checkpoint-functional A2058 cells. ICRF193 had little effect on the other targets in either cell line. These five hits were validated using a different siRNA pool from the screening pool in an extended panel of cell lines. Two checkpoint-functional (A2058 and MM603) and two checkpoint-defective (MM415 and SKMel13) cell lines were used for validation. None of the checkpointfunctional cell lines demonstrated any reduction in viability upon knockdown of any of the five genes (Figure 3A). MAPK8 knockdown increased viability, with the greatest increase (1.6-fold) in the MM603 cell line. In the checkpoint-defective cell lines, no loss of viability was observed after knockdown of either ATK2 or JAK2. Knockdown of MAPK8 produced a selective loss of viability in the MM415 and SKMel13 decatenation checkpoint-defective cell lines (Figure 3A). However, this decrease was only small, 77–84% the viability of non-targeting controls. In contrast, knockdown of both PIK3CB and PIK3C2A produced a significant reduction in viability, reducing viability to
PIGMENT CELL & MELANOMA Research Decatenation checkpoint-defective melanomas are dependent on PI3K for survival Kelly Brooks, Max Ranall, Loredana Spoerri, Alex Stevenson, Gency Gunasingh, Sandra Pavey, Fred Meunier, Thomas J. Gonda and Brian Gabrielli
DOI: 10.1111/pcmr.12268 Volume 27, Issue 5, Pages 813–821 If you wish to order reprints of this article, please see the guidelines here Supporting Information for this article is freely available here EMAIL ALERTS Receive free email alerts and stay up-to-date on what is published in Pigment Cell & Melanoma Research – click here
Submit your next paper to PCMR online at http://mc.manuscriptcentral.com/pcmr
Subscribe to PCMR and stay up-to-date with the only journal committed to publishing basic research in melanoma and pigment cell biology As a member of the IFPCS or the SMR you automatically get online access to PCMR. Sign up as a member today at www.ifpcs.org or at www.societymelanomaresarch.org
To take out a personal subscription, please click here More information about Pigment Cell & Melanoma Research at www.pigment.org
ORIGINAL ARTICLE
Pigment Cell Melanoma Res. 27; 813–821
Decatenation checkpoint-defective melanomas are dependent on PI3K for survival Kelly Brooks1,3, Max Ranall1, Loredana Spoerri1, Alex Stevenson1, Gency Gunasingh1, Sandra Pavey1, Fred Meunier2, Thomas J. Gonda1,4 and Brian Gabrielli1 1 Translational Research Institute, The University of Queensland Diamantina Institute, Brisbane, Qld, Australia 2 Clem Jones Centre for Ageing Dementia Research, Queensland Brain Institute, The University of Queensland, Brisbane, Qld, Australia 3 Present address: Cancer Research UK Manchester Institute, The University of Manchester, Wilmslow Road, Manchester, M20 4BX, UK 4 Present address: School of Pharmacy, The University of Queensland, Brisbane, Qld, Australia
KEYWORDS decatenation synthetic lethality/PI3K
checkpoint/melanoma/
PUBLICATION DATA Received 10 February 2014, revised and accepted for publication 25 May 2014, published online 29 May 2014
CORRESPONDENCE B. Gabrielli, e-mail: [email protected] doi: 10.1111/pcmr.12268
Summary Melanoma cell lines are commonly defective for the G2-phase cell cycle checkpoint that responds to incomplete catenation of the replicated chromosomes. Here, we demonstrate that melanomas defective for this checkpoint response are less sensitive to genotoxic stress, suggesting that the defective cell lines compensated for the checkpoint loss by increasing their ability to cope with DNA damage. We performed an siRNA kinome screen to identify kinases responsible and identified PI3K pathway components. Checkpoint-defective cell lines were three-fold more sensitive to small molecule inhibitors of PI3K. The PI3K inhibitor PF-05212384 promoted apoptosis in the checkpoint-defective lines, and the increased sensitivity to PI3K inhibition correlated with increased levels of activated Akt. This work demonstrates that increased PI3K pathway activation is a necessary adaption for the continued viability of melanomas with a defective decatenation checkpoint.
Introduction Melanoma is a common human malignancy, and while primary melanomas are generally easy to treat via excision, late-stage melanoma still proves a difficult target for therapeutic intervention. Late-stage melanoma is refractory to most treatments with the most common treatment dacarbazine, having a response rate of 15–20%, improving survival by only 3–6 months (Fang et al., 2004). One recent development in late-stage melanoma treatment has been the use of BRAF inhibitors for melanomas harbouring the BRAFV600E mutation (50–70% of melanomas) (Davies et al., 2002; Wan et al.,
2004). Pairing of these inhibitors with the BRAFV600E mutation has provided the most promising response rate to date of ~81% (Flaherty et al., 2010). Unfortunately, this response is only temporary in most cases, with the melanomas fast developing resistance via numerous methods (Johannessen et al., 2010; Villanueva et al., 2010). Such studies have demonstrated, however, that promising response rates are achievable in melanoma if the right target/subset and approach can be identified. Cell cycle defects, particularly in cell cycle checkpoints that normally respond to specific cellular stresses, are a common feature in many cancers (Malumbres and Barbacid, 2001). Such defects often confer a growth
Significance Melanomas are considered to be generally resistant to chemotherapy, although the basis for this is not clear. The correlation of the G2-phase decatenation checkpoint defect with reduced sensitivity to genotoxic stress suggests that adaptation to loss of the checkpoint may be responsible for this increased survival. Here we have demonstrated that increased PI3K pathway activation as a necessary adaptation for the continued viability of checkpoint-defective melanomas. This study provides evidence that targeting adaptations critical for continued viability in checkpoint-defective melanomas is a viable option for the selective destruction of tumours with this defect.
ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
813
Brooks et al.
Results Using ICRF-193 and etoposide to inhibit decatenation, the impact of failing to arrest at the decatenation checkpoint was assessed in terms of DNA damage and cell viability. Both checkpoint-functional (A2058) and -defective (MM576, HT144, SKMel13) cell lines showed little increase in the DNA damage marker cH2AX levels after 8-h ICRF-193 treatment; in all cases, there was a strong accumulation at 24-h and 48-h treatment (Figure 1A). All cell lines responded strongly to etoposide treatment. Long-term proliferation assays demonstrated that all cell lines experienced a similar decrease in viability after 814
A
Functional γH2AX H2AX
A2058
α Tubulin
Defective γH2AX MM576
H2AX α Tubulin γH2AX
HT144
H2AX α Tubulin γH2AX
SKMel13
H2AX 48 h
24 h
8h
Etop
Con
α Tubulin
ICRF-193 ICRF-193 60 40 20 0
MM415 A02 SKMel13 D25 MM383 MM576 MM96L A2058 MM603 MM329
% control
B
Defective
Functional
Etoposide 60 40 20 0
MM415 A02 SKMel13 D25 MM383 MM576 MM96L A2058 MM603 MM329
% control
advantages to the cancer (Hartwell and Kastan, 1994; Kaufmann and Paules, 1996; Paulovich et al., 1997); however, these checkpoints are protective mechanisms, functioning to ensure the health and viability of the cells. Their loss therefore can lead to an accumulation of DNA damage and replicative stress in these cells. To remain viable in the presence of such stress, it is reasonable to predict that these cells become more reliant on other signalling pathways, for example, anti-apoptotic, to compensate for these intrinsic stresses. These dependencies provide an opportunity to exploit the cell cycle checkpoint defects as selective targets, employing the concept of synthetic lethality. Many synthetic lethal approaches are attempts to target specific genetic lesions, for example, BRCA mutations in breast cancer. Recently, we reported that the decatenation checkpoint is defective in ~67% of melanoma cell lines (Brooks et al., 2013). The decatenation checkpoint is a G2-phase checkpoint important for monitoring the catenation of chromosomes, ensuring sister chromatids can be accurately segregated during mitosis (Damelin and Bestor, 2007; Deming et al., 2001; Kaufmann, 2006). Decatenation, the process of untangling catenated chromosomes is performed by topoisomerase II (TopoII) which can be inhibited using catalytic inhibitors such as ICRF-193 or poisons such as etoposide (Clifford et al., 2003; Kaufmann and Kies, 1998; Morris et al., 2000; Roca and Wang, 1994). We demonstrated that TopoII inhibition in decatenation checkpoint-defective cell lines resulted in increased genomic instability due to attempts to segregate catenated chromosomes during mitosis (Brooks et al., 2013), and attempts to segregate catenated chromosomes have previously been reported to increase DNA damage (Damelin and Bestor, 2007; Gorbsky, 1994; Hajji et al., 2003; Ishida et al., 1994), providing one example of intrinsic stress caused by a decatenation checkpoint dysfunction. The presence of at least one obvious source of intrinsic stress and the commonality of the decatenation checkpoint defect in melanoma make these cells a good candidate for investigating cell cycle checkpoint targeting in melanoma. This potential was investigated through siRNA screening of the kinome, and one such vital target was identified.
Defective
Functional
Figure 1. Decatenation checkpoint-defective cell lines do not have increased sensitivity to TopoII inhibition. (A) Whole cell lysates of cells treated with DMSO, 2 lM etoposide for 24 h, or 2 lM ICRF193 for 8, 24 or 48 h were immunoblotted for cH2AX as a marker for DNA damage. Total H2AX and a-tubulin were used as loading controls. B. Colony forming assay for cells treated for 24 h with 2uM ICRF-193 or 2lM etoposide. Colonies grown for 14 days and numbers normalized to DMSO-treated controls.
ICRF-193 treatment despite the severe chromosomal and mitotic aberrations occurring only in the checkpointdefective cell lines (Brooks et al., 2013), suggesting that these defective cells can compensate for this defect, showing no increased loss in viability compared with functional lines. Treatment with etoposide, which produced a similar level cH2AX at 24-h ICRF-193 treatment, had a much stronger effect on the checkpoint-functional ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
PI3K adapts to checkpoint defect
Z-Score
SKMel13 resazurin
6
Z-Score
4 2 0 –2 –4
B
0.5 0
Z-Score
–0.5 –1 –1.5 –2 –2.5
PL K1
B
8
K AP M T2 AK
K2
2A
C
C
K3
JA
PI
–3
K3
ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
A2058 resazurin
A 12 10 8 6 4 2 0 –2 –4
PI
cell lines (Figure 1B). This provides further evidence that cells which had lost decatenation checkpoint function were more resistant to the cytotoxic effects of DNA damage, likely reflecting an adaptation to loss of the checkpoint function. We reasoned that the adaptations in the checkpointdefective cell lines were likely to be required for the normal maintenance of viability of these cells but would not have a similarly critical role in checkpoint-functional cells. To identify these adaptations, an siRNA screen of the kinome (~700 genes) was performed. One decatenation checkpoint-functional (A2058) cell line and one decatenation checkpoint-defective (SKMel13) cell line were used. Assays for cell viability (Resazurin) and biomass (crystal violet) were performed for each siRNA in a 384-well format. Z-scores were calculated for each well compared with internal plate controls giving a spread of scores for both cell lines (Figure 2A and S1). Because the efficiency of knockdown cannot be determined for each well, an arbitrary cut-off was set for hit selection at a Z-score of 2.5, corresponding to a reduction in viability and biomass of at least 25%. Hits were then filtered as outlined (Figure S2). Potential hits that were only present in SKMel13 checkpoint-defective cell line were filtered to remove genes that were not expressed in the SKMel13 cell line, or were not expressed in a high proportion of the checkpoint-defective melanoma cell lines previously characterized (Brooks et al., 2013). This resulted in a list with the five top hits being PIK3CB, PIK3C2A, MAPK8, JAK2 and AKT2. Closer analysis of these revealed that PIK3CB was the strongest hit in both assays (Figure 2B and S1B) and provided a similar level of loss of viability in the checkpoint-defective cell line as Plk1, the positive control for loss of viability. Interestingly, the negative effect of knockdown was enhanced with ICRF193 treatment, particularly for PIK3C2A in the checkpoint-functional A2058 cells. ICRF193 had little effect on the other targets in either cell line. These five hits were validated using a different siRNA pool from the screening pool in an extended panel of cell lines. Two checkpoint-functional (A2058 and MM603) and two checkpoint-defective (MM415 and SKMel13) cell lines were used for validation. None of the checkpointfunctional cell lines demonstrated any reduction in viability upon knockdown of any of the five genes (Figure 3A). MAPK8 knockdown increased viability, with the greatest increase (1.6-fold) in the MM603 cell line. In the checkpoint-defective cell lines, no loss of viability was observed after knockdown of either ATK2 or JAK2. Knockdown of MAPK8 produced a selective loss of viability in the MM415 and SKMel13 decatenation checkpoint-defective cell lines (Figure 3A). However, this decrease was only small, 77–84% the viability of non-targeting controls. In contrast, knockdown of both PIK3CB and PIK3C2A produced a significant reduction in viability, reducing viability to
