1. RESEARCH POSTER PRESENTATION DESIGN © 2011
www.PosterPresentations.com
Christina Murphy1, Berkley Gryder2, Javed Khan2, Jack Shern3
Figure 5. Potential combination strategies for targeting the
PAX3-FOXO1 fusion oncogene. In addition to the compounds
isolated in the drug screen, Bromodomain inhibitors had
previously been shown by our lab to specifically downregulate
the transcriptional output of PAX3-FOXO1. We hypothesize
that coupling Bromodomain inhibitors with our discovered
compounds will have synergistic toxicity toward fusion-
positive cell lines.
Potential combination strategies include:
Drug Screen
RNA Isolation IncuCyte Experiments
Discovery of candidate
compounds
A high-throughput drug screen identifies inhibitors of
PAX3-FOXO in pediatric Rhabdomyosarcoma
Rhabdomyosarcoma (RMS) is the most common soft tissue
sarcoma afflicting children, with an incidence of 4.5 cases per 1
million adolescents yearly.1 RMS arises from skeletal muscle
precursor cells, though its anatomic sites of origin are highly
varied and not restricted to striated muscle.
Histological differences between embryonal and alveolar
RMS subtypes were established as a basis for
clinicopathological correlation.2 Embryonal
rhabdomyosarcoma (ERMS) has earlier age of onset and better
prognosis,3 while alveolar rhabdomyosarcoma (ARMS) is
associated with different primary sites, is more aggressive,
usually presents with metastasis and shows severely lagging
5-year survival rates when compared to ERMS.1
The dawn of next-generation sequencing revealed 2 distinct
RMS genotypes, characterized by presence or absence of a gene
fusion resulting from PAX translocation with FOXO16.Fusion-
positive tumors carry significantly fewer genetic mutations
when compared to fusion-negative tumors7. Molecular
profiling of RMS has become invaluable in initial diagnosis, as
ARMS is typically identified by fusion-positive status8. The
fusion event yields a chimeric transcript consisting of the N-
terminus of PAX, which is a DNA-binding domain, and the C-
terminus of FOXO1, an activating domain9,10.The oncogenic
consequences of this fusion protein include heightened
activation of normal downstream PAX3 targets as well as
altered gene expression patterns11. Presence of PAX-FOXO
rather than histology seems to be the primary cause of poor
outcomes associated with the specific subtype.
BACKGROUND
PURPOSE
RESULTS
Figure 4. Development of a secondary screen to evaluate
candidate compounds. To evaluate the transcriptional effects
of the compounds discovered in the primary screen, we
developed an assay to rapidly elevated expression changes of
the genes downstream of PAX3-FOXO1. Cells were treated with
all candidate compounds at 1 uM for 6 hours and RNA was
isolated.
Gene selection. RNAsequencing data previously generated
from fusion-positive Rhabdomyosarcoma tumors was mined to
discover genes distinctly upregulated in P3F tumors.
RESULTS RESULTS
Figure 7. Potential for BET-inhibitor, topoisomerase-inhibitor
synergy.
CONCLUSION
ACKNOWLEDGEMENTS
Thanks to Dr. Jack Shern and the rest of the Shern lab,
including Dr. Fountaine and Dr. Sayers. Thanks also to the
National Institutes of Health for their support through the
Summer Internship Program.
1University of Notre Dame, Notre Dame, IN 60040, USA
2 Genetics Branch, NCI, NIH, Bethesda, MD, USA
3Pediatric Oncology Branch, Oncogenomics Section, Center for Cancer Research, National Institutes of Health, Gaithersburg, MD, 20877, USA
Mechanism
Analysis
Pre-Clinical
Validation
Clinical Trial
FUTURE DIRECTIONS
• RNA sequencing to determine genetic profile of drug-treated
RH4 cells.
• Mechanistic investigation of main classes of drugs pulled
down from drug screen.
• Optimization of a low dose BET-inhibitor, topoisomerase-
inhibitor combination treatment for clinical trial.
Figure 1. RH4 cell line and pGreenFire Reporter used for the
primary screen.
The chemotherapeutic backbone for treating RMS has
remained relatively unchanged since 1975, and intensified
regimen yields only marginal results for the most aggressive of
RMS cases. Significant toxicity and long-term morbidity are
especially concerning in these young patients. An enhanced
understanding of the tumor biology and discovery of new drugs
for precision targeting will improve outcomes for these children.
Being that fusion-positive RMS is associated with the cases
having some of the most grim outcomes, the PAX3-FOXO fusion
oncogene warrants further investigation as a therapeutic target.
EXPERIMENTAL DESIGN
Figure 2. P3F-dependent enhancer sequence used in the
reporter construct.
RNAseq espression of selected genes in primary
Rhabdomyosarcoma tumors and normal tissues.
Log2FPKM value of the genes selected for inclusion in Nanostring
assay design were obtained from RNAseq data on a panel of tumors
(left) and normal tissue samples (right).
Secondary screen of candidate
compounds: Nanostring
Expression Assay
Figure 3. Primary PAX3-FOXO1 inhibitor compound screen.
Figure 6. Real-time quantitative live-cell analysis by IncuCyte of
RH4 cell line 48h following drug treatment.
This high-throughput drug screen provided many
potential leads toward projects investigating PAX3-FOXO target
for development of future therapies. The Nanostring assay will
elucidate the different mechanisms of potentially specific, less
toxic compounds to be included in a new generation of fusion-
positive RMS chemotherapeutics. BET inhibitors in past studies
in our research group have shown to inhibit PAX3-FOXO1-
dependent transcription. Topoisomerase inhibitors were a
recurring class of drugs pulled down in the compound screen.
Our understanding of the PAX3-FOXO super enhancer leads us
to believe that topoisomerases are especially crucial to PAX3-
FOXO-driven transcription due to the torsional strain accrued
from extreme chromatin remodeling. Topoisomerases were
effective in treating RH4 cells, even at low nanomolar doses.
When combined with BET-inhibitors, a potentially synergistic
effect was observed that warrants further investigation. A
combination treatment of these two inhibitory drugs could
potentially provide a new low-dose option for chemotherapeutic
treatment. Both teniposide and PLX2 are commercially available
and could be rolled into clinical trial after further
experimentation and pre-clinical validation.
Topoisomerase Inhibitors
20
2
0.2
0.02
0.002
16
32
64
128
256
Concentration
NormalizedLuciferase
CMV
ALK
XTT
Teniposide
20
2
0.2
0.02
0.002
8
16
32
64
128
256
512
1024
Concentration
NormalizedLuciferase
CMV
ALK
XTT
AmonafideAmsacrine
20
2
0.2
0.02
0.002
8
16
32
64
128
Concentration
NormalizedLuciferase
CMV
ALK
XTT
OtherHDAC Inhibitors
1-alaninechlamydocin
20
2
0.2
0.02
0.002
2
4
8
16
32
64
128
256
512
Concentration
NormalizedLuciferase
CMV
ALK
XTT
20
2
0.2
0.02
0.002
16
32
64
128
256
Concentration
NormalizedLuciferase
CMV
ALK
XTT
Camptothecin
derivative
Menogaril
20
2
0.2
0.02
0.002
4
8
16
32
64
128
256
Concentration
NormalizedLuciferase
CMV
ALK
XTT
PsammaplinA
TetrocarcinA
20
2
0.2
0.02
0.002
0.125
0.25
0.5
1
2
4
8
16
32
64
128
Concentration
NormalizedLuciferase
CMV
ALK
XTT
20
2
0.2
0.02
0.002
0.03125
1
32
1024
Concentration
NormalizedLuciferase
CMV
ALK
XTT
Midostaurin
20
2
0.2
0.02
0.002
16
32
64
128
256
512
Concentration
NormalizedLuciferase
CMV
ALK
XTT
PD-407824
20
2
0.2
0.02
0.002
16
32
64
128
256
512
Concentration
NormalizedLuciferase
CMV
ALK
XTT
0 50 100
0
50
100
Hours
%Confluence
RH4 PLX2 dose response
10uM PLX2 +Teniposide
5uM PLX2 + 100nM Teniposide
2.5uM PLX2 +Teniposide
1.25uM PLX2 + Teniposide
0.625uM PLX2 +Teniposide
0.312uM PLX2 +Teniposide
0.156uM PLX2 + Teniposide
0.078uM PLX2 + Teniposide
0.039uM PLX2 + Teniposide
DMSO + 100nM Teniposide
10uM PLX2
5uM PLX2
2.5uM PLX2
1.25uM PLX2
0.625uM PLX2
0.312uM PLX2
0.156uM PLX2
0.078uM PLX2
0.039uM PLX2
DMSO alone
Dose Response curves of
normalized luciferase
values of the ALK enhancer
construct, a CMV only
promotor construct and
viability (DTT assay).
Bromodomain inhibitor + TOPO1 inhibitor Bromodomain inhibitor + HDAC inhibitor
Bromodomain inhibitor + TOPO2 inhibitor Bromodomain inhibitor + Kinase inhibitor
(Midostaurin)
Fusion Positive Tumors Normal Tissue Samples