International Journal of Radiation Oncology � Biology � PhysicsS656

by more than three thousand-fold after LOFU treatment. HSPA6 and

HSPA7 had slight expression in the untreated groups but had robust up-

regulation after LOFU irrespective of cell types.

Conclusions: Our study provides evidence that ultrasound induced an

acute cellular stress response with up-regulation of protein folding genes in

PCa cells. We also identified two ubiquitously up-regulated and tightly

regulated heat shock proteins after LOFU. Our study demonstrated LOFU

robustly induces expression of HSPA6 and HSPA7. The promoters of

HSPA6 and HSPA7 have important implications for cancer gene therapy as

targeted switches. Future analysis will explore the potential use of LOFU

as an externally delivered controlled activation.

Author Disclosure: J. Tang: None. Y. Liu: None. Z. He: None. H. Zhang:

None. H. Gao: None. D.J. Leong: None. R.J. Majeska: None. C. Guha:

None. H.B. Sun: None.

3186Protein Arginine Methyltransferase-5 as a Novel Epigenetic Targetin Lung CancerM.X. Welliver,1 K. Shilo,1 S. Sharma,1 X. Wu,1 S. Sif,1 A. Chakravarti,1

C. Li,2 R. Baiocchi,1 and G.A. Otterson1; 1The Ohio State University

James Cancer Center, Columbus, OH, 2The Ohio State University College

of Pharmacy, Columbus, OH

Purpose/Objective(s): PRMT5 is an arginine methyltransferase that

regulates a host of cellular events and has an impact on cell growth and

transformation pathways by modulation of E-cadherin, CDKN2A and

cyclin E proteins. In this study, we investigated the role of PRMT5 in lung

cancer by studying its expression pattern and the effect of its inhibition.

Materials/Methods: We studied the expression of PRMT5 using immu-

nohistochemistry from resection specimens obtained with an IRB-

approved protocol. Immortalized lung cancer cells and normal bronchial

airway cells were employed for in vitro experiments. Western blot, cell

cycle analysis, MTT and clonogenic survival assays were performed using

standard techniques. A novel and specific inhibitor of PRMT5 (CPD5)

developed in house was applied to in vitro culture systems.

Results: All 9 lung cancer resection specimens (3 adenocarcinomas, 3

squamous cell carcinomas, 2 small cell lung carcinomas, and 1 large cell

neuroendocrine carcinoma) showed diffuse cytoplasmic and variable

nuclear PRMT5 expression. PRMT5 expression was also seen in reactive

type 2 pneumocytes and respiratory epithelium adjacent to the tumors but

not in normal alveolar parenchyma, fibroblasts or endothelial cells. Using

Western immunoblot, PRMT5 was highly expressed in lung cancer cell

lines (A549, H1299 and H520) compared with normal lung cells

(HPAEpiC and HBEpiC). The p53 null H1299 cell line showed the highest

PRMT5 expression, whereas A549 with WT p53 had 1/5 PRMT5

expression comparing to that in H1299. CPD5 can induce cell death in

lung cancer cell lines with IC50 approximately 50 mM at 24 hr. At this

concentration, the PRMT5 specific mark of histone H4R3 methylation was

inhibited, and increases in early apoptotic population (3.5% to 12.8%)

were seen in H719 cells. There is a modest decrease in clonogenic survival

when A549 cells were treated with 10 mM or 25 mM CPD5 prior to and

during radiation treatment suggesting that PRMT5 inhibition may enhance

radiation effect in this cell line. Cell cycle changes were observed in A549

and H1299 cells treated with CPD5 for 24 hr and 48 hr. At 24 hours, the

percentage of cells in G0/G1 was 57.1% and 66.4% in DMSO versus

CPD5 treated A549 cells, and 52.5% and 70.9% in DMSO versus CPD5

treated H1299 cells. Using Western blot, the expression of p21 was found

to be increased while cyclin E1 was decreased in A549 cells treated with

CPD5 by western blot.

Conclusions: PRMT5 is expressed in lung cancer cells compared to

normal lung parenchyma and its level may be inversely related to

endogenous WT p53. The PRMT5 inhibitor, CPD5 inhibits proliferation

and induces cell death by cell cycle inhibition and apoptosis.

Author Disclosure: M.X. Welliver: None. K. Shilo: None. S. Sharma:

None. X. Wu: None. S. Sif: None. A. Chakravarti: None. C. Li: None.

R. Baiocchi: None. G.A. Otterson: None.

3187Evaluation of Novel DNA Polymerase Beta Inhibitors as PotentialRadiosensitizers of Prostate Cancer CellsM. Hedayati,1 D. Arian,2 H. Zhou,1 Z. Bilis,1 K. Chen,1 M. Greenberg,2

and T. DeWeese1; 1Department of Radiation Oncology and Molecular

Radiation Sciences, School of Medicine, Johns Hopkins University,

Baltimore, MD, 2Department of Chemistry, Johns Hopkins University,

Baltimore, MD

Purpose/Objective(s): DNA polymerase beta (Pol b) is an essential

enzyme that plays a critical role in the repair of the DNA base damage and

single stranded breaks (SSBs) commonly produced by ionizing radiation

(IR). If left unrepaired clusters of such lesions can result in double strand

breaks (DSBs), the major cause of cell death due to IR. Inhibitors of poly

ADP ribose polymerases (PARP) another key family of proteins involved

in the repair of SSBs have become important drugs in treatment of many

cancers when combined with DNA damaging agents such as radiation or

alkylating agents. Recently, it was reported that an oxidized abasic lesion

produced by several antitumor antibiotics that damage DNA can irre-

versibly inhibit Pol b in vitro. This finding led us to design several novel

irreversible inhibitors of Pol b. Here, we report the effectiveness of one

such inhibitor in potentiating the cytotoxicity of a prostate cancer (PCa)

cell line treated either with IR or methylmethane sulfonate (MMS).

Materials/Methods: A leading candidate identified by screening a library

of potential compounds for inhibiting the enzymatic activity of Pol b in

vitro was further developed into a prodrug (DA2114). DU145 PCa cells

were treated with a range of DA2114 concentrations to obtain an inhibitory

concentration at which 50% of the cells were able to survive (IC50).

Survival was determined by clonogenic assay. DA2114 was added to the

cells prior to treatment with either IR or MMS and remained present

throughout the assay.

Results: Treatment of DU145 cells with Pol b inhibitor DA2114 resulted

in IC50 of 15.2 � 2.0 mM. We then used DA2114 (at near IC50) and treated

cells in combination with either IR or MMS. The resulting survival fraction

(sf) after treatment with 250 mM MMS alone was 0.35 � 0.01 (compared

to untreated, p < 0.0001). In the presence of 15 mM DA2114 the

normalized sf was reduced to 0.21 � 0.03 after treatment with 250 mMMMS (compared to the expected sf of 0.35, p < 0.05). IR treatment alone

at 2 Gy and 4 Gy resulted in sfs of 0.75 � 0.11 and 0.52 � 0.15 respec-

tively. In the presence of 20 mM DA2114, the resulting normalized sfs

remained statistically unchanged after 2 Gy and 4 Gy treatments (0.84 �0.15 and 0.49 � 0.14 respectively, p > 0.2).

Conclusions: We show that a novel irreversible inhibitor of Pol b designed

on in vitro inhibition of Pol b enzymatic activity is effective in the treat-

ment of DU145 PCa cells. Furthermore, we report that this inhibitor

significantly potentiates the effect of the alkylating agent, MMS, but not IR

treatments. These results are important in understanding the repair of IR

lesions and suggest that evaluation of these novel inhibitors in conjunction

with alkylating agents and PARP inhibitors should be pursued.

Author Disclosure: M. Hedayati: None. D. Arian: None. H. Zhou: None. Z.

Bilis: None. K. Chen: None. M. Greenberg: None. T. DeWeese: None.

3188Investigation of Low-Dose Fractionated Radiation Therapy asa Chemopotentiator in Advanced/Metastatic GastrointestinalCarcinomaF. Carrier, D. Nguyen, E. Chang, and N. Sharma; University of Maryland,

Baltimore, Baltimore, MD

Purpose/Objective(s): Whole abdominal radiation therapy (WART) has

been used in cases of GI cancer with disseminated intra-abdominal disease.

However, the main shortcoming of WART is the inability to combine it

with full-dose chemotherapy, due to the increased toxicity to normal tissue.

Recent findings have suggested the possibility of a novel treatment para-

digm allowing the use of full dose systemic chemotherapy safely in

combination with Low Dose Fractionated radiation therapy (LDFRT).

Volume 87 � Number 2S � Supplement 2013 Poster Viewing Abstracts S657

Traditionally, the cell survival curve suggested that fractionated radiation

doses of greater than 120 cGy were required to overcome the initial

shoulder where low doses of radiation produced negligible effects on cell

survival. However, preliminary data have shown a low-dose hyper-radio-

sensitivity (HRS) phenomenon in which cells die from hypersensitivity to

15 cGy of radiation. The primary goal of this study is to identify various

biomarkers and cellular pathways that are responsive to low-dose radiation

and determine their contribution to chemopotentiation.

Materials/Methods: Treatment includes a combination of LDFRT and

modified Docetaxel, Cisplatin, 5-fluorouracil (mDCF). Metastatic gastric

carcinoma 5822 and gastric adenocarcinoma 1739 cells were treated with

three consecutive daily doses of radiation and 5-FU and docetaxel on day

1, 5-FU on day 2 and 5-FU and cisplatin on day 3. RNAwas extracted 24 h

later or cells were analyzed by clonogenic survival assay 10 days later.

Results: Our data indicate that three consecutive daily fractions of 15 cGy

are sufficient to induce HRS in gastric cancer cells and potentiate mDCF.

RT-PCR analysis of cellular pathways suggest that the combination of

mDCF with LDFRT induces HRS in gastric cancer cells by down regu-

lating the DNA repair machinery (ATM, p53, BRCA1, MDM2) and up

regulating the production of hydrogen peroxide (DUOX2).

Conclusions: LDFRT can potentiate mDCF through mechanisms different

than the conventional DNA damage response and could be considered for

WART in combination treatments for patients with advanced and meta-

static GI cancers.

Author Disclosure: F. Carrier: None. D. Nguyen: None. E. Chang: None.

N. Sharma: None.

3189Pim Kinase Inhibitor AZD1208 for Treatment of c-MYC-DrivenProstate CancerA.N. Kirschner,1 E.K. Keeton,2 D. Huszar,2 and S.A. Abdulkadir1;1Vanderbilt University, Nashville, TN, 2AstraZeneca R&D, Waltham, MA

Purpose/Objective(s): High grade prostate cancers often overexpress

PIM1 kinase and c-MYC. PIM1 and c-MYC are thought to synergize in

a manner dependent on PIM1 kinase activity to induce prostate cancer in

humans, based on studies in mice. Radiation and hypoxia are known to

increase PIM1, thereby reducing the efficacy of treatment. Targeted

therapy specific for PIM1 kinase is a novel method for inhibiting prostate

cancer growth and enhancing its treatment. In mice, deficiency of Pim

kinases is well tolerated, suggesting that Pim kinase inhibition is an

appealing therapeutic approach for prostate cancer.

Materials/Methods: In a c-MYC/Pim1-transduced prostate cancer tissue

recombination mouse model and in an aggressive mouse prostate cancer

Myc-CaP xenograft model, we examine the effect of a daily oral PIM

kinase inhibitor AZD1208 on prostate tumorigenesis and tumor growth.

Results: PIM inhibition significantly decreases c-MYC/Pim1 graft growth

by more than 50%, decreases cellular proliferation by nearly 50%, and

increases apoptosis by approximately 300%, likely in a phospho-

Bad(S112) mediated process. In addition, AZD1208 significantly

decreases the growth of aggressive mouse prostate cancer Myc-CaP

xenografts by decreasing proliferation and increasing apoptosis.

Conclusions: These results indicate that PIM kinase inhibition by an orally

available drug is a valid treatment for c-MYC/Pim1-driven and aggressive

c-MYC-overexpressing prostate cancers. This research supports efforts

aimed at enhancing systemic and radiation therapy for prostate cancer in

humans. AZD1208 is currently in phase 1 clinical trials.

Author Disclosure: A.N. Kirschner: None. E.K. Keeton: A. Employee;

AstraZeneca R&D. D. Huszar: A. Employee; AstraZeneca R&D. S.A.

Abdulkadir: None.

3190Preclinical Investigation of a Novel Redox Modulator, Parthenolide,as a Cytotoxic and Potential Radiosensitizing Agent inGlioblastoma MultiformeA.J. Smith, F.F. Xu, C. Wang, M.E. Stevens, Y. Xu, D.K. St. Clair,

and W.H. St. Clair; University of Kentucky, Lexington, KY

Purpose/Objective(s): Previous work has shown that the natural product

sesquiterpene parthenolide (PN) is cytotoxic to cancer cells in vitro and

in vivo while sparing normal cell counterparts. The cytotoxic effect of

PN in cancer cells is mediated, in part, through inhibition of the NF-kB

pathway, which also radiosensitizes by down-regulating the primary

antioxidant enzyme manganese superoxide dismutase (MnSOD). GBM

tumors resistant to O6-alkylators, such as temozolomide (TMZ), have

a high level of NF-kB activity, and inhibition of this activity by gene

therapy sensitizes GBM to O6-alkylators. The purpose of the current

study is to investigate PN as a potential cytotoxic, radiosensitizing, and

chemosensitizing agent in GBM cell lines as a precursor to clinical

exploration.

Materials/Methods: A bioinformatics analysis was carried out using an

Oncomine data set obtained from 514 patients to review the expressions

of proteins in the NF-kB pathway in GBM. In vitro work was done

using the GBM cell lines LN229 and U87, with MGMT promoter

methylated, and LN18, with MGMT promoter unmethylated. Cell

survivals were measured by MTT assay experiments with DMSO

control, PN, and TMZ, with and without radiation (RT) of up to 6 Gy.

Colony-forming assays (CFA) were carried out on the LN229 cell line

with DMSO control, PN, and TMZ with RT doses of 0-6 Gy. Surviving

fraction (SF) was plotted against RT dose and fitted to a linear quadratic

equation allowing for calculation of a dose-enhancement factor, DEF0.1.

This is the ratio of RT required to achieve 90% cell kill in the absence

versus presence of PN.

Results: The bioinformatics analysis indicates that the levels of MnSOD

mRNA were significantly higher in GBM versus normal tissues (p <

0.001). Importantly, the overall survival rate is inversely related to the

level of MnSOD (p < 0.05). MTT assays revealed that PN was cyto-

toxic to all GBM cell lines tested in a concentration-dependent manner.

U87 and LN229 were sensitive to TMZ, while LN18 was resistant to

TMZ up to 100 uM. While the combination of RT and PN is only an

additive in the two TMZ sensitive cell lines, low concentrations of PN

led to a synergistic effect of PN and RT in the TMZ resistant LN18

cells. CFA experiments in LN229 also showed dose-dependent cyto-

toxicity of PN. The data also suggest a radiosensitization effect of

parthenolide, with a DEF0.1 of 1.35 for 0.25 uM PN versus 0.1%

DMSO.

Conclusions: These preclinical data suggest that parthenolide should be an

attractive therapeutic agent in GBM. The MTT and CFA experiments show

that PN enhances the cytotoxic effect of radiation even when TMZ, the

current standard chemotherapy, does not. These promising data will be

followed up with further in vivo experiments towards a potential future

clinical trial.

Author Disclosure: A.J. Smith: None. F.F. Xu: None. C. Wang: None. M.E.

Stevens: None. Y. Xu: None. D.K. St. Clair: None. W.H. St. Clair: None.

3191Differential Cellular Response to Nanoparticle Docetaxel andDocetaxel at Subtherapeutic Dose RangeE.C. Wang, R. Sinnott, M.E. Werner, M. Sethi, A.W. Whitehurst,

and A.Z. Wang; University of North Carolina at Chapel Hill, Chapel Hill,

NC

Purpose/Objective(s): Nanoparticle (NP) formulations of taxanes

overcome the need for excipients and improve the delivery of taxane

therapeutics to tumors. These advantages have resulted in rapid clinical

translation of NP taxanes and high interest in preclinical and clinical

development of novel NP taxanes. However, the effects of NP taxanes

on tumor cells compared to their small molecule counterparts have not

been extensively evaluated. Existing NP taxanes are polymeric NPs and

release their cargo in a slow and controlled fashion. Due to this

controlled release, the taxane concentration level and exposure time to

a cell at any given time from NP taxanes are different from that of

small molecule taxanes. Thus, we hypothesized that the cellular

response to polymeric NP taxanes is different from that of small

molecule taxanes.