Volume 105  Number 1S  Supplement 2019
Here, we examine the influence of TTFields on BBB integrity in pre-
clinical models.
Materials/Methods: Immortalized murine brain capillary endothelial cells
(cerebEND) were treated with TTFields at 100-300 kHz for up to 72 h and
the protein Claudin-5 was stained for the assessment of tight junction
integrity. In addition, transendothelial electrical resistance (TEER) was
applied to determine BBB intactness. To examine BBB permeability,
dextran was coupled to fluorescein isothiocyanate (FITC) for staining,
followed by flow cytometry. For in vivo analysis, quantification of vessel
permeability was achieved by utilizing i.v. injected Evans Blue (EB) in
healthy rats during TTFields application to the brain (100 kHz, 72 h). The
blood vessel structure was investigated by Claudin 5 staining of brain
cryosections. Furthermore, Dynamic Contrast Enhanced Magnetic Reso-
nance Images (DCE-MRI) were acquired before and after bolus adminis-
tration of gadolinium (Gd-DTPA, MW 545, Magnetol) using a 1T Icon
MRI system at the end of the TTFields treatment as well as 96 h after.
Results: In vitro, the BBB was disrupted by TTFields application as tight
junction proteins were delocalized from the cell boundaries to the cyto-
plasm with maximal effects at 100 kHz. In line with this, the BBB integrity
was significantly reduced by 65% and the BBB permeability for 4 kDa
large molecules was significantly increased. The cell morphology started
to recover 48 h post-treatment and recovery was completed after 96 h,
revealing a reversibility of the effect on the BBB by TTFields. In vivo,
application of TTFields to the rats head significantly increased average
accumulation of EB. Cryosections of the brain revealed a disturbed blood
vessel structure. Moreover, the time course of changes in brain tissue
contrast enhancement in DCE-MRI showed significant enhancement in-
crease in the TTFields treated rat compared to the control animals. This
effect disappeared at 96 h after treatment. These results demonstrate that
TTFields application lead to a transient increase in BBB permeability.
Conclusion: Utilizing TTFields to deliver drugs generally unable to cross
the BBB to the central nervous system might be an approach in the future,
as TTFields demonstrated to reversibly disrupt the BBB in preclinical
models. Based on the data presented on in vitro and in vivo application of
TTFields to permeabilize the BBB, we plan to investigate this concept in a
clinical pilot trial.
Author Disclosure: A.F. Kessler: Research Grant; Novocure. E. Salvador:
Research Grant; Novocure. D. Domröse: None. M. Burek: Research
Grant; Novocure. C. Schaeffer: None. C. Tempel Brami: None. T.
Voloshin: None. M. Giladi: None. R. Ernestus: None. M. Löhr: Research
Grant; Novocure. C. Förster: Research Grant; Novocure. C. Hagemann:
Research Grant; Novocure.
1103
Unbiased Drug Discovery Approaches to Identify Novel
Radiosensitizers from Cancer Therapy Evaluation Program
(CTEP) Portfolio of Drugs in Pancreatic Cancer
P.K. Singh,1 B.P. Venkatesulu,1,2 J. Symons,1 L.S.K. Mahadevan,3
K.L. Sanders,1 B.K. Kim,4 and S. Krishnan1; 1Department of Experimental
Radiation Oncology, The University of Texas MD Anderson Cancer Center,
Houston, TX, 2University of Texas, MD Anderson Cancer Center, Houston,
TX, 3James Comprehensive Cancer Center, Ohio State University,
Columbus, OH, 4Department of Experimental Radiation Oncology, The
University of Texas MD Anderson Cancer Center, Houston, TX
Purpose/Objective(s): Given that research and development of newer
molecules typically takes anywhere from 15 to 20 years and can be pro-
hibitively expensive, repurposing previously approved FDA drugs or drugs
that are already in clinical phase for new uses outside the arena of their
original indication is an attractive option. We evaluated active agents in the
CTEP portfolio as novel radiosensitizers in pancreatic cancer through an
unbiased screening approach.
Materials/Methods: We performed a high-throughput 96-well plate clo-
nogenic assay of 46 drugs from the CTEP portfolio in Panc1 (KRAS
mutant, P53 mutant) and HPAC (KRAS mutant, P53 wild) pancreatic
cancer cell lines. The cell dispensing was automated to ensure uniform
seeding of cell number and the drugs were added at six different
Mini Oral Sessions S163
concentrations ranging from 0.1nM to 10uM at three incubation time
points (1h, 2h, 4h) before or after 2 Gy exposure with or without capeci-
tabine (10 uM). Drugs demonstrating a radiation dose enhancement factor
(DEF) >1.5 at two or more time points, particularly at two or more
clinically achievable concentrations, were further validated in standard
format clonogenic assays, initially with a single dose of 2 Gy and subse-
quently, across a range of radiation doses to generate a full curve. Initially,
four concentrations ranging from 1-100 nM were evaluated 4h before or
after 2 Gy exposure with or without capecitabine (10 uM). The drugs that
showed a positive hit in the second clonogenic screen were further vali-
dated in a classical clonogenic assay 4h before or after 0, 2, 4, and 6 Gy
exposure with or without capecitabine.
Results: The high-throughput clonogenic assay yielded 14 candidate
molecules that showed potent radiosensitization (DEF >1.5). Selumetinib,
dabrafenib, and veliparib showed radiosensitization in HPAC cells; ali-
sertib, dasatinib, AZD8186, copanlisib, and VX-970 showed radio-
sensitization in Panc1 cells; and AMG 232, temsirolimus, and olaparib
showed radiosensitization in both Panc1 and HPAC cells. Amongst the 14
candidate molecules, four drugs temsirolimus, olaparib, copanlisib, and
VX-970 showed consistent radiosensitization in classical clonogenic assay
as well. Notably, many candidate molecules targeted the PI3K/mTOR or
DNA repair pathways. We are evaluating five of these shortlisted drugs
further in patient-derived xenograft and orthotopic models.
Conclusion: High-throughput clonogenic assays coupled with validation
by classical clonogenic assays may provide an unbiased in vitro approach
to compare putative radiosensitizers head-to-head and allow rational
advancement of select agents for additional evaluation for repurposing as
radiosensitizers.
Author Disclosure: P.K. Singh: None. B.P. Venkatesulu: None. J.
Symons: None. L.K. Mahadevan: None. K. Sanders: None. B. Kim:
None. S. Krishnan: Research Grant; NIH, DoD, Celgene, Cancer Pre-
vention and Research Institute of Texas. Royalty; Taylor and Francis
Group.
1104
Dino Is a DNA Damage-Induced Tumor Suppressing Long
Noncoding RNA
E.S. Anderson, C. Marney, M. Adnan, K.L. Peng, N. Weinhold,
and A. Schmitt; Memorial Sloan Kettering Cancer Center, New York, NY
Purpose/Objective(s): While the CKDN1A locus is frequently suppressed
in human cancers, the mechanism by which this finding contributes to
tumor suppression has not been fully elucidated. We hypothesized that
DINO, a novel DNA damage-induced long noncoding RNA encoded in the
CDKN1A promoter, is essential to p53-dependent tumor suppression.
Materials/Methods: We used a combination of in vivo mouse models, cell
culture experiments and human tumor profiling to examine the contribution
of DINO to p53-dependent tumor suppression. We crossed a Dino loss of
function mouse to the E-mu-Myc model of B-cell lymphoma to examine
p53-dependent, p21-independent tumor suppression. Rescue experiments to
restore Dino function in Dino-/- lymphoma cells were performed to confirm
its contribution to growth suppression. Finally, we analyzed methylation
patterns of the DINO/CDKN1A locus in TCGA datasets to investigate the
relationship of DINO inactivation and TP53 mutation in human cancers.
Results: We find that loss of Dino significantly accelerates tumorigenesis
in the E-mu-Myc mouse model of B-cell lymphoma in a haplo-insufficient
manner, while previous studies have shown that loss of Cdkn1a does not
impact tumor development. Complementation with Dino suppresses
growth of isolated Dino-/-, E-mu-Myc lymphoma cells and delays tumor
development when re-engrafted into mice. We show that each of these
effects requires an intact p53 signaling pathway. Human DINO and mouse
Dino can each functionally restore growth suppression in cell culture,
suggesting functional conservation through mammalian evolution. Finally,
we find that DINO is hypermethylated in multiple types of human cancer
and that DINO hypermethylation is correlated to DINO expression, but not
CDNK1A expression. Hypermethylation of DINO is mutually exclusive
with TP53 alteration in several specific human cancers.
S164
Conclusion: We provide direct evidence that the long noncoding RNA
DINO is a tumor suppressor transcribed from the CDKN1A promoter.
These findings provide insight into a mechanism by which human tumors
inactivate p53-dependent functions and describe a novel mechanism of
tumor suppression by a long noncoding RNA. We suggest that inactivation
of DINO presents a potential biomarker of tumors with altered p53 func-
tion, and provides a future basis for targeted therapy and personalized
treatment.
Author Disclosure: E.S. Anderson: None. C. Marney: None. M. Adnan:
None. K. Peng: None. N. Weinhold: None. A. Schmitt: None.
1105
Caloric Restriction Curtails Tumor Infiltrating Regulatory T
Cells and Enhances Effector T Cell Activity after Radiation
Therapy
G. Manukian, B. Simone, T. DeAngelis, C. Kivolowitz, K. Ko,
and N.L. Simone; Sidney Kimmel Medical College at Thomas Jefferson
University, Sidney Kimmel Cancer Center, Philadelphia, PA
Purpose/Objective(s): Enhancing the immunogenic properties of radia-
tion therapy (RT) is currently of keen interest. While RT can promote
immunogenic cell death resulting in increased anti-tumor T cell activity, it
can also stimulate suppressive regulatory T-cells (Tregs) to counter this
response. Caloric restriction (CR), defined as a 20 to 40% reduction in
calories, is a cost effective way to enhance the efficacy of RT and improve
treatment outcomes in both preclinical and clinical models. As changes to
the metabolic environment can affect immune homeostasis, we hypothe-
sized that CR augments the effects of RT by impairing Treg activity and
enhancing effector T cell function.
Materials/Methods: Twelve-week old female Balb/c mice were injected
with 5x104 4T1 triple-negative, murine breast cancer cells. Mice were
randomized to either an ad lib (AL) or CR diet upon development of
palpable tumor. Each cohort was also treated with either diet alone or diet
plus RT (12 Gy in 3 daily fractions). Mice were euthanized on day 21 and
primary tumors, spleen, and peripheral blood mononuclear cells (PBMC)
were harvested for analysis of immune activity via cDNA microarray, flow
cytometry (FC), and immunohistochemistry. To establish a clinical
correlate, a multi-plex cytokine array was run on pre- and post-CR serum
samples obtained from 19 breast cancer patients enrolled on an IRB
approved clinical trial.
Results: Microarray analysis of primary tumors revealed at least a 1.5 fold
increase in the expression of genes related to integrin signaling and
inflammation due to cytokine and chemokine signaling in CR mice both
with and without RT compared with AL controls (p<0.05). FC analysis of
tumor infiltrating lymphocytes (TIL) demonstrated a significant increase in
CD4+CD25+Foxp3+ Tregs after RT in AL (12.3%) but not in CR (5.1%)
fed mice (pZ0.01). Furthermore, the ratio of CD8:Tregs in TIL was
increased after RT in CR but not AL fed mice (0.85 and 0.22, respectively;
pZ0.002). Intracellular cytokine staining of PBMCs after re-stimulation
revealed that effector T cells from CR fed mice produced significantly
more interferon-y then AL controls (10.5% and 3.04% respectively,
pZ0.02). Serum samples from breast cancer patients on a CR diet revealed
that regulation of the adaptive immune response was among the top
pathways affected by CR via GO pathway analysis (pZ0.002) with a
specific decrease in interleukin-10 production (pZ0.01).
Conclusion: CR significantly reduces Tregs in the tumor microenviron-
ment and enhances effector T cell responses after RT compared with an-
imals on an AL diet. Clinically, patients exhibit significant changes in
adaptive immune response pathways and decreases in systemic immuno-
suppressive cytokine levels after CR. Together these findings suggest that
CR may be a cost effective way to create an environment conducive to
developing anti-tumor immune responses. Future clinical trials should
therefore be designed to evaluate CR in combination with RT and
immunotherapy to further improve treatment outcomes.
Author Disclosure: G. Manukian: None. B. Simone: None. T. DeAngelis:
None. C. Kivolowitz: None. K. Ko: None. N.L. Simone: None.
International Journal of Radiation Oncology  Biology  Physics
1106
PSA/PSMA Targeted Recombinant Adenovirus Vector with
Radiation-induced Promoter t-PA Mediated CircRNA_100385
Silencing and Its Mechanism for Regulating Radiosensitivity
in Prostate Cancer
N. Wu, Y. Tang, and G. Cheng; Department of Radiation Oncology, China-
Japan Union Hospital of Jilin University, Changchun, China
Purpose/Objective(s): Radiotherapy plays an important role in prostate
cancer therapy, and radioresistance is the main reason for the treatment
failure. Our previous studies have demonstrated that circRNA_100385 could
function as a miRNA sponge and regulate miR-452 negatively, which may
play a role in the radioresistance transformation in prostate cancer cells with
PI3K/Akt pathway. This research intends to confirm the modality for the
treatment of prostate cancer to overcome radioresistance by combination of
circRNA_100385 targeted interference with radiotherapy.
Materials/Methods: The construction of recombinant adenovirus vector
Ad.ptPA- si(circRNA_100385)- PSES was constructed using gene
recombination technique. The most significant reversal effect for radio-
resistance was induced by circRNA_100385 interference sequence
downstream of tissue-type plasminogen activator (t-PA) which is the
efficient radiation-sensitive promoter in prostate cancer cells. The infection
of prostate cancer cells was mediated with the recombined condition
replicating form adenovirus vector including mosaicism enhancer PSES
which mixed prostate specific antigen (PSA) and prostate specific mem-
brane antigen (PSMA) together. The expression of miR-452 and PI3K/Akt
pathway genes regulated by the circRNA_100385 interference sequence in
prostate cancer cell lines were observed. Immunodeficient mice subcuta-
neously implanted with prostate cancer cells were treated by conventional
radiotherapy and/or gene therapy. The relationship between the change of
circRNA_100385-miR-452-PI3K/Akt pathway and radioresistance of
prostate cancer was investigated.
Results: The recombinant adenovirus vector significantly increased
interference efficiency of circRNA_100385 in human prostate cancer cell
lines. We demonstrated that gene therapy combined with radiation greatly
improved treatment efficacy by repressing colony formation, inducing
more apoptosis, leading to the arrest of the G2/M phase, increased double-
strand break levels and less inactivation of cell cycle check point in
radioresistant prostate cancer cells. Cancer control was most significantly
improved in the group receiving local radiotherapy combined with gene
therapy as shown by prolongation of mean survival time by 84.5%,
reduction in average tumor weight by 90.2%, decrease in pulmonary
metastasis by 65.8% and decrease in intratumor angiogenesis by 74.3% as
compared to local radiotherapy alone (P < 0.05).
Conclusion: The experimental findings in the present study showed that
adenovirus vectors Ad.ptPA- si(circRNA_100385)- PSES in combination
with local radiotherapy could improve the tumor control. The
circRNA_100385 may be the potential target for increasing the radio-
therapy sensitivity of prostate cancer. These observations may set the stage
for developing clinical protocols with recombinant adenovirus-mediated
gene-radiotherapy in prostate cancer.
Author Disclosure: N. Wu: None. Y. Tang: None. G. Cheng: None.
1107
Improved Tumor Control Through T-cell Infiltration
Modulated by Ultra-High Dose Rate Proton FLASH Using a
Clinical Pencil Beam Scanning Proton System
N. Rama,1 T. Saha,1 S. Shukla,1 C. Goda,1 D. Milewski,1 A.E. Mascia,2
R.E. Vatner,3 D. Sengupta,4 A. Katsis,4 E. Abel,4 S. Girdhani,4
M. Miyazaki,4 A. Rodriguez,4 A. Ku,4 R. Dua,4 R. Parry,4 and T.V. Kalin1;
1
Cincinnati Children’s Hospital Medical Center, Cincinnati, OH,
2
University of Cincinnati and UC Health, Cincinnati, OH, 3Department of
Radiation Oncology, University of Cincinnati and Cincinnati Children’s
Hospital Medical Center, Cincinnati, OH, 4Varian Medical Systems,
Palo Alto, CA