Page 307 - 2014 Printable Abstract Book
P. 307
(PS5-34) A robotic cancer cell line screening platform identifies TP53 as a biomarker for
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radiosensitzation with the mTOR inhibitor everolimus. Qi Liu, PhD ; Ashley Kern ; Meng Wang ; Cyril
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Benes ; Kathryn Held ; Jason Efstathiou ; and Henning Willers , MGH, Charlestown, MA and MGH,
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Boston, MA
High-throughput screening (HTS) of large panels of annotated cancer cell lines with molecular
targeted drugs is a powerful tool for the identification of sensitive tumor subsets and associated genomic
biomarkers. We have previously shown that non-clonogenic cell survival as used in these screening
platforms can be used for the study of radiosensitizing drugs. Here, we report further optimization of this
platform to include robotic handling suitable for HTS. 2 Gy single dose irradiation with 1h pretreatment
of drugs was used as the treatment regimen for cells in a 96-well format, with effects measured by the
ATP-based metabolic assay Cell-titer Glo after incubation for 5 days. We screened eight lung cancer cell
lines and 16 targeted drugs (123 combinations) where 43 combinations were validated by a clonogenic
survival assay (CSA). Short-term radiosensitization factors for 2 Gy (SRF2Gy) were calculated as well as
standard dose enhancement factors (DEFSF10) from CSA. A high accuracy in terms of predicting CSA-based
radiosensitization by this platform was demonstrated, with a sensitivity and specificity of 84% and 83.3%,
respectively (receiver-operating characteristic test: p<0.0001). To test whether the screening can be used
to identify genomic biomarkers of radiosensitization (Wang et al., Cancer Research 2014, in press; Liu et
al., JNCI 2014, submitted), we selected the mTOR inhibitor everolimus. In a panel of 14 lung cancer cell
lines, the subset of TP53 wild-type (wt) lines showed an increased SRF2Gy compared to the TP53 mutant
(mut) cell lines: SRF2Gy of 1.10 vs 0.95, respectively (p=0.03). It was further confirmed in an isogenic cell
pair expressing either wt or mut p53, with the SRF2Gy values as 1.18 vs 0.96, respectively (p=0.03).
Interestingly, cell inactivation following radiation/ everolimus treatments was correlated with premature
senescence and autophagy induction, but only in p53 wt cells. These data further highlight the ability of
screening platforms to identify radiosensitizing drugs and genomic biomarkers. We suggest that a
paradigm change regarding the utility of non-clonogenic survival assays in precision radiation medicine is
needed.
(PS5-35) Evaluation of pediatric patients with severe combined immunodeficiency (SCID) on the basis
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of cellular radiosensitivity. Pavel Lobachevsky ; Lisa Woodbine ; Sharon Choo ; Kuang-Chih Hsiao ; Jai
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Smith ; Nickala Best ; Chris Fraser ; Paul Gray ; Roger Martin ; Penny Jeggo ; Olga Martin , Peter
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MacCallum Cancer Centre, East Melbourne, Australia ; University of Sussex, Falmer Brighton, United
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Kingdom ; Royal Children’s Hospital, Melbourne, Australia ; Queensland Health, Herston, Australia ; and
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Sydney Children’s Hospital, Sydney, Australia
The SCID is a primary immune deficiency that results in the inability to produce T-lymphocytes
and immunoglobulin receptors. Approximately 30% of SCID patients have V (D) J recombination defects
leading to T and B cell lymphocytopenia. Mainly such defects arise from mutations in RAG1 and RAG2,
however, a subgroup of patients are deficient in NHEJ (non-homologous end joining) genes, such as LIGIV,
Cernunnos-XLF, XRCC4, Artemis-DCLRE1C, and DNA-Pkcs. Cells deficient in NHEJ are unable to effectively
repair DNA damage and, therefore, are radiosensitive (RS). SCID patients are treated by haematopoietic
stem cell transplantation (HSCT) that involves “conditioning” treatment with radiomimetic drugs.
According to the guidelines for HSCT, SCID patients with RS disorders receive a very specific conditioning
protocol and a higher transplantation priority. There is a dearth of reports of the application of functional
assays to detect RS SCID patients, but here we demonstrate that meticulous analysis of the cellular
response to radiation provides clear evidence of the important potential of functional RS screening. We
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radiosensitzation with the mTOR inhibitor everolimus. Qi Liu, PhD ; Ashley Kern ; Meng Wang ; Cyril
1
1
2
1
2
Benes ; Kathryn Held ; Jason Efstathiou ; and Henning Willers , MGH, Charlestown, MA and MGH,
2
Boston, MA
High-throughput screening (HTS) of large panels of annotated cancer cell lines with molecular
targeted drugs is a powerful tool for the identification of sensitive tumor subsets and associated genomic
biomarkers. We have previously shown that non-clonogenic cell survival as used in these screening
platforms can be used for the study of radiosensitizing drugs. Here, we report further optimization of this
platform to include robotic handling suitable for HTS. 2 Gy single dose irradiation with 1h pretreatment
of drugs was used as the treatment regimen for cells in a 96-well format, with effects measured by the
ATP-based metabolic assay Cell-titer Glo after incubation for 5 days. We screened eight lung cancer cell
lines and 16 targeted drugs (123 combinations) where 43 combinations were validated by a clonogenic
survival assay (CSA). Short-term radiosensitization factors for 2 Gy (SRF2Gy) were calculated as well as
standard dose enhancement factors (DEFSF10) from CSA. A high accuracy in terms of predicting CSA-based
radiosensitization by this platform was demonstrated, with a sensitivity and specificity of 84% and 83.3%,
respectively (receiver-operating characteristic test: p<0.0001). To test whether the screening can be used
to identify genomic biomarkers of radiosensitization (Wang et al., Cancer Research 2014, in press; Liu et
al., JNCI 2014, submitted), we selected the mTOR inhibitor everolimus. In a panel of 14 lung cancer cell
lines, the subset of TP53 wild-type (wt) lines showed an increased SRF2Gy compared to the TP53 mutant
(mut) cell lines: SRF2Gy of 1.10 vs 0.95, respectively (p=0.03). It was further confirmed in an isogenic cell
pair expressing either wt or mut p53, with the SRF2Gy values as 1.18 vs 0.96, respectively (p=0.03).
Interestingly, cell inactivation following radiation/ everolimus treatments was correlated with premature
senescence and autophagy induction, but only in p53 wt cells. These data further highlight the ability of
screening platforms to identify radiosensitizing drugs and genomic biomarkers. We suggest that a
paradigm change regarding the utility of non-clonogenic survival assays in precision radiation medicine is
needed.
(PS5-35) Evaluation of pediatric patients with severe combined immunodeficiency (SCID) on the basis
1
3
3
2
of cellular radiosensitivity. Pavel Lobachevsky ; Lisa Woodbine ; Sharon Choo ; Kuang-Chih Hsiao ; Jai
1
1
1
4
1
2
5
Smith ; Nickala Best ; Chris Fraser ; Paul Gray ; Roger Martin ; Penny Jeggo ; Olga Martin , Peter
1
MacCallum Cancer Centre, East Melbourne, Australia ; University of Sussex, Falmer Brighton, United
2
3
4
Kingdom ; Royal Children’s Hospital, Melbourne, Australia ; Queensland Health, Herston, Australia ; and
5
Sydney Children’s Hospital, Sydney, Australia
The SCID is a primary immune deficiency that results in the inability to produce T-lymphocytes
and immunoglobulin receptors. Approximately 30% of SCID patients have V (D) J recombination defects
leading to T and B cell lymphocytopenia. Mainly such defects arise from mutations in RAG1 and RAG2,
however, a subgroup of patients are deficient in NHEJ (non-homologous end joining) genes, such as LIGIV,
Cernunnos-XLF, XRCC4, Artemis-DCLRE1C, and DNA-Pkcs. Cells deficient in NHEJ are unable to effectively
repair DNA damage and, therefore, are radiosensitive (RS). SCID patients are treated by haematopoietic
stem cell transplantation (HSCT) that involves “conditioning” treatment with radiomimetic drugs.
According to the guidelines for HSCT, SCID patients with RS disorders receive a very specific conditioning
protocol and a higher transplantation priority. There is a dearth of reports of the application of functional
assays to detect RS SCID patients, but here we demonstrate that meticulous analysis of the cellular
response to radiation provides clear evidence of the important potential of functional RS screening. We
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