Page 256 - 2014 Printable Abstract Book
P. 256
the response to platinum and radiation therapy we have performed a genome-wide RNAi high-throughput
screen via the transfection of human lung cancer cells (PC9) with a shRNA viral library containing approx.
60,000 individual shRNAs. After two weeks of cisplatin and/or radiation treatment of PC9 cells with shRNA
and controls, depleted versus enriched shRNAs were identified through library sequencing, hypothesizing
that depleted shRNAs in the treated versus control cells might represent genes whose function is crucial
for resistance to therapy-induced cell death. From the cisplatin screen, analysis of top 100 potential hits
(depleted shRNAs) interestingly showed several targets involved in epithelial-mesenchymal transition
(EMT) including Smurf1, Smurf2, YAP1, CEBPZ, and glycolytic pathway including PFKFB3. We further
validated the Smurf2 and PFKFB3 targets by survival assays using siRNA treated PC9 cells to successfully
down-regulate Smurf2 and PFKFB3 protein expression. MTT cell survival assay was performed by
transfecting 1500 PC9 cells with either Smurf2 and PFKFB3 siRNA in triplicate and then treated with
increasing dosage of cisplatin (0.1, 1 and 10 µM) or X-ray radiation (2, 4 and 6 Gy). Significantly decreased
cell survival was observed in Smurf2 and PFKFB3 siRNA transfected cells treated with cisplatin or radiation
compared to control transfected cells. By performing an unbiased genome wide for therapeutic
resistance, we have successfully identified and validated targets in glycolytic and EMT pathways. We are
further evaluating the radiation and concurrent chemoradiation screens to determine common
synergistic as well as distinct resistance pathways shared between these two DNA damage interventions.
(PS4-35) Utilizing Akt Inhibition with MK-2206 as a Radiosensitizer in Experimental Neuroblastoma.
1
1
1
1
2
Fauzia Shaikh, B.A. ; Youping Sun, PhD ; K. S. Chao, MD ; Tom K. Hei, PhD ; Eileen P. Connolly, MD Ph.D
1
Columbia University Medical Center- Department of Radiation Oncology, New York, NY and Columbia
University Medical Center- Center for Radiological Research, New York, NY 2
Purpose: Radiation therapy is integral to the treatment of high-risk neuroblastoma (NB). Its
efficacy is often limited by toxicity to normal tissue, particularly in the pediatric population. Thus, a
search for radiation enhancing strategies is underway. It is expected that targeting specific oncogenic
pathways may improve the therapeutic index of radiation therapy by sensitization of tumor cells. The
PI3K/Akt pathway is a key survival signaling pathway know to play a role in the process of acquiring
resistance to radiation therapy. It is aberrantly expressed in high-grade NB. Thus, we hypothesize that
targeted inhibition of Akt will enhance radiation sensitivity of NB both in vitro and in an invivo a
preclinical mouse model. Methods: Utilizing two established human neuroblasoma cell-lines, NGP, an
MYCN amplified line, and SY5Y, a MYCN non-amplified cell line. Cells were treated with MK-2206, a
potent allosteric pan-Akt inhibitor, alone or in combination with increasing doses of radiation (IR) from
0-8 Gy. In vitro studies performed included; cell survival assays and immunoblot analysis of key proteins.
Results: While single agent MK-2206 inhibited phospho-AKT signaling in a dose-dependent manner, it
did not impact in vitro cell survival of either cell line. Upon establishing the baseline radiosensitivity of
both cell lines, we found that the combination of IR and Akt inhibition by 2.5 uM MK-2206 led to
synergistic effects, particularly at low doses of IR between 2-4Gy. After 2 hours of pretreatment with
MK-2206, MK-2206 exhibited inhibition of radiation-induced Akt activation, while prolonged exposure
for 24 hours of MK-2206 exhibited inhibition of endogenous and radiation-induced Akt activation. Con-
clusions: These studies demonstrate that targeted inhibition of Akt effectively radiosensitizes both
MYCN amplified and nonamplified cells lines. Clonagenic assays are ongoing to confirm these results. In
addition we plan to further characterization of cell death, cell-cycle and DNA-repair mechanisms with
MK-2206 to better characterize the mechanism if radiosensitizaton with Akt inhibition. An in vivo trial of
254 | P a g e
screen via the transfection of human lung cancer cells (PC9) with a shRNA viral library containing approx.
60,000 individual shRNAs. After two weeks of cisplatin and/or radiation treatment of PC9 cells with shRNA
and controls, depleted versus enriched shRNAs were identified through library sequencing, hypothesizing
that depleted shRNAs in the treated versus control cells might represent genes whose function is crucial
for resistance to therapy-induced cell death. From the cisplatin screen, analysis of top 100 potential hits
(depleted shRNAs) interestingly showed several targets involved in epithelial-mesenchymal transition
(EMT) including Smurf1, Smurf2, YAP1, CEBPZ, and glycolytic pathway including PFKFB3. We further
validated the Smurf2 and PFKFB3 targets by survival assays using siRNA treated PC9 cells to successfully
down-regulate Smurf2 and PFKFB3 protein expression. MTT cell survival assay was performed by
transfecting 1500 PC9 cells with either Smurf2 and PFKFB3 siRNA in triplicate and then treated with
increasing dosage of cisplatin (0.1, 1 and 10 µM) or X-ray radiation (2, 4 and 6 Gy). Significantly decreased
cell survival was observed in Smurf2 and PFKFB3 siRNA transfected cells treated with cisplatin or radiation
compared to control transfected cells. By performing an unbiased genome wide for therapeutic
resistance, we have successfully identified and validated targets in glycolytic and EMT pathways. We are
further evaluating the radiation and concurrent chemoradiation screens to determine common
synergistic as well as distinct resistance pathways shared between these two DNA damage interventions.
(PS4-35) Utilizing Akt Inhibition with MK-2206 as a Radiosensitizer in Experimental Neuroblastoma.
1
1
1
1
2
Fauzia Shaikh, B.A. ; Youping Sun, PhD ; K. S. Chao, MD ; Tom K. Hei, PhD ; Eileen P. Connolly, MD Ph.D
1
Columbia University Medical Center- Department of Radiation Oncology, New York, NY and Columbia
University Medical Center- Center for Radiological Research, New York, NY 2
Purpose: Radiation therapy is integral to the treatment of high-risk neuroblastoma (NB). Its
efficacy is often limited by toxicity to normal tissue, particularly in the pediatric population. Thus, a
search for radiation enhancing strategies is underway. It is expected that targeting specific oncogenic
pathways may improve the therapeutic index of radiation therapy by sensitization of tumor cells. The
PI3K/Akt pathway is a key survival signaling pathway know to play a role in the process of acquiring
resistance to radiation therapy. It is aberrantly expressed in high-grade NB. Thus, we hypothesize that
targeted inhibition of Akt will enhance radiation sensitivity of NB both in vitro and in an invivo a
preclinical mouse model. Methods: Utilizing two established human neuroblasoma cell-lines, NGP, an
MYCN amplified line, and SY5Y, a MYCN non-amplified cell line. Cells were treated with MK-2206, a
potent allosteric pan-Akt inhibitor, alone or in combination with increasing doses of radiation (IR) from
0-8 Gy. In vitro studies performed included; cell survival assays and immunoblot analysis of key proteins.
Results: While single agent MK-2206 inhibited phospho-AKT signaling in a dose-dependent manner, it
did not impact in vitro cell survival of either cell line. Upon establishing the baseline radiosensitivity of
both cell lines, we found that the combination of IR and Akt inhibition by 2.5 uM MK-2206 led to
synergistic effects, particularly at low doses of IR between 2-4Gy. After 2 hours of pretreatment with
MK-2206, MK-2206 exhibited inhibition of radiation-induced Akt activation, while prolonged exposure
for 24 hours of MK-2206 exhibited inhibition of endogenous and radiation-induced Akt activation. Con-
clusions: These studies demonstrate that targeted inhibition of Akt effectively radiosensitizes both
MYCN amplified and nonamplified cells lines. Clonagenic assays are ongoing to confirm these results. In
addition we plan to further characterization of cell death, cell-cycle and DNA-repair mechanisms with
MK-2206 to better characterize the mechanism if radiosensitizaton with Akt inhibition. An in vivo trial of
254 | P a g e
