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SUPPLEMENTAL FIGURE LEGENDS
Figure S1. shRNA screen in orthotopic xenograft tumors. A. Classes of epigenetic regulators
and their associated genes contained in the shRNA library.
B-C. Enrichment score for
individual shRNAs specific for BRD2 (blue), BRD3 (orange), BRD4 (green) and BRDT (purple)
from tumors derived from cell lines 1312 (B) and 1275 (C). The relative enrichment/depletion in
the tumor is plotted in log scale on the y-axis.
Figure S2.
Expression of BET family members in normal pancreas and throughout the
histological progression to cancer. A tissue microarray stained with antibodies specific to BRD2
(A), BRD3 (B), and BRD4 (C) was scored for the nuclear expression of these proteins. Staining
was scored as very weak or negative (0), weak (1), and positive (2).
Figure S3. Effects of BET bromodomain inhibition on PDAC cell growth. A. PDAC cell lines
were exposed to increasing amounts (0-6.4 M) of BETi and analyzed for changes in growth
after six days. The average (±SEM) GI50 of each cell line is shown. B. Dose response curves of
cell lines exposed to increasing amounts (0-6.4 M) of the BET bromodomain inhibitor JQ1 for
six days. C. Expression of BET family members in high, intermediate, and low sensitivity PDAC
cell lines. Western blots were performed using 25 g of whole cell lysate from each cell line.
Results are representative of three independent experiments.
D. Expression of p27 Kip1,
phophorylated Rb (Ser780), total Rb were determined by Western blots using whole cell lysates
from each cell line after 72 hrs of treatment with 1.6 M BETi or control (CPI440). The
expression of -actin served as a loading control. Results are representative of at least three
independent experiments.
E. Relative viability of PDAC cells after exposure to various
concentrations of BETi for six days.
Figure S4. Regulation of MYC and GLI by BET bromodomains. A. Genome wide transcription
analysis of PDAC cell lines after BET inhibition was performed. The average fold changes at
the 10 hour time point for 1108, 1312, BxPc3, and CFPAC-1 cells were used for GSEA. The top
10 programs identified in the c6 collection in MSIGDB by GSEA analysis (C6) are shown. B.
Heat map demonstrating relative gene expression of downstream effectors of GLI that are
altered in at least two cell lines after treatment with BETi. High (red) and low (blue) gene
expression are indicated. C. qPCR analysis (average ± SD) of representative GLI target genes
altered after treatment with BETi for 10 hours. D. Relative GLI-luciferase reporter activity in
BxPc3 cells after exposure of cells to BETi or control CPI440 for 24 hours. E. Relative GLIluciferase reporter activity in PDAC cells after exposure of cells to JQ1 or vehicle control for 24
hours.
Figure S5. Pharmacological inhibition of BET bromodomains increases GLI protein half-life in
PDAC cells. A. Expression of GLI1 and GLI2 mRNA in PDAC cells after treatment with 1.6 M
BETi or control (CPI440) for 24 hours.
The averages (± SD) from three independent
experiments are shown. B. Time course of GLI1 and GLI2 mRNA expression in PDAC cells
after treatment with 1.6 M BETi for the indicated times. The averages (± SD) from three
independent experiments are shown. C. Western blot analysis using cell lysates from BETi or
control treated PANC-1 cells after inhibition of protein synthesis with cyclohexamide (CHX) for
the indicated times.
A global change in protein stability was not observed as only a 10%
difference in the expression of tubulin is observed between control and BETi treated cells after
24 hours. D. Western blot of GLI3 and actin in PDAC cells after treatment with 1.6 M BETi or
control (CPI440) for 24 hours.
Figure S6. BET family members interact with of GLI in PDAC cells. A. Immunoprecipitations
with cell lysates from PANC-1 cells ectopically expressing GLI1 were performed with control IgG
and antibodies specific for GLI1. Western blot detecting co-precipitated BRD4 is shown. B.
Immunoprecipitation experiments performed with control IgG or antibodies specific to BRD2,
BRD3, and BRD4 and cell lysates from 1108 cells ectopically expressing GLI2. Representative
Western blot detecting the co-immunoprecipitation of GLI2 is shown. C. Immunoprecipitation
experiments performed with control IgG or antibodies specific to BRD2, BRD3, and BRD4 and
cell lysates from 722 cells ectopically expressing GLI2. Representative Western blot detecting
the co-immunoprecipitation of GLI2 is shown. D. Immunoprecipitations with cell lysates from
PANC-1 cells were performed with control IgG and antibodies specific for BRD4. Western blot
detecting the co-precipitation of endogenous GLI2 is shown. E. Immunoprecipitations with cell
lysates from PANC-1 cells were performed with control IgG and antibodies specific for BRD4 in
the presence of control (CPI-440) or BETi.
Western blot detecting the co-precipitation of
endogenous GLI2 is shown.
Figure S7. BET bromodomain inhibition selectively alters the tumor stroma. A. Representative
H&E images of control and BETi treated xenograft tumors derived from cell line 1300 and
tumors from KRASG12D;p53-/- mice are shown. Scale bars = 100 M. B. Heterotopic xenograft
tumors derived from the PDAC cell line 1300 and KRASG12D;p53-/- mice treated with BETi or
vehicle control were analyzed for their collagen content by Mason trichrome staining and
vascular content by immunohistochemistry for CD31. Representative images from tumors are
shown. Scale bars = 100 M. Quantitative analysis of SMA and Ki67stained sections are
shown below the corresponding images.
C. qPCR analysis of BRD2, BRD3, and BRD4
expression in the mouse stroma of heterotopic xenograft tumors formed from PDAC cell line
1300. The expression of each gene is normalized to that of the housekeeping gene Elf1. D.
Representative immunohistochemistry for BRD2, BRD3, and BRD4 in normal human pancreas
(top) and PDAC (bottom). BRD2, BRD3, and BRD4 are widely expressed in the stroma of
PDAC and their relative intensity mimics that observed in the cancer cells.
BRD4 is
occasionally detected in interstitial fibroblasts of normal pancreatic parenchyma while
expression of BRD2 and BRD3 are largely absent in these cells. Each of these BET proteins
are more frequently detected in peri-ductal fibroblasts (shown). Scale bars = 50 M in main
images and 20 M in insets for top panels and 50 M in main images and 10 M in insets for
bottom panels. Locations of the inset images within the main images are indicated by asterisks.
Figure S8. Regulation of MYC, SHH, and GLI by BET bromodomains in murine PDAC cells.
A. qPCR analysis of MYC expression in NB494 cells treated with BETi or control CPI440 for 10
hours. The average (± std. dev.) of three experiments is shown. Asterisk indicates changes in
gene expression with p<0.001. B. qPCR analysis of SHH expression in NB494 cells treated
with BETi or CPI440 for 10 hours. The average (± std. dev.) of three experiments is shown.
Asterisk indicates changes in gene expression with p<0.001. C. Relative GLI-luciferase reporter
activity in NB494 cells after exposure of cells to BETi or CPI440 for 24 hours. The average (±
std. dev.) of three experiments is shown. Asterisk indicates p<0.002
Figure S9. shRNA-mediated reduction of BET protein expression. A and B. PDAC cell lines
722 (panel A) and 1108 (panel B) were infected with lentiviruses expressing shRNAs targeting
BRD2, BRD3, and BRD4. Representative Western blots demonstrating reduced expression of
each BET protein relative to cells expressing a non-targeting control shRNA (NTC) are shown.
-actin served as a loading control. Specific shRNAs are indicated above each panel.
Figure S10. Selective regulation of MYC, SHH, and GLI by individual BET proteins. A and B.
Western blot analysis of MYC expression in cell lines 722 (panel A) and 1108 (panel B)
expressing BET-specific shRNAs.
-actin served as a loading control.
Results are
representative of at least 2 independent infections. C and D. qPCR analysis of SHH expression
in cell lines 722 (panel C) and 1108 (panel D) expressing BET-specific shRNAs. The average
(± SEM.) of three experiments is shown. Single and double asterisks indicate changes in gene
expression with p<0.003 and p<0.03, respectively. E and F. Relative GLI-luciferase reporter
activity in cell lines 722 (panel E) and 1108 (panel F) expressing BET-specific shRNAs. The
average (± SEM) of three experiments is shown. Single and double asterisks indicate changes
with p<0.006 and p<0.04, respectively. G and H. qPCR analysis of PLEKHA2 expression in cell
lines 722 (panel G) and 1108 (panel H) expressing BET-specific shRNAs. The average (±
SEM.) of at least three experiments is shown. Asterisks indicate changes in gene expression
with p<0.03.
Figure S11. BET proteins are important for PDAC tumor growth. A and B. PDAC cell lines 722
(panel A) and 1108 (panel B) expressing BET-specific shRNAs were cultured in 96 well plates
for 6 days and their growth measured using a resazurin-based assay. The growth of each cell
is presented relative to the growth of cells expressing a non-targeting control shRNA (NTC).
The average (± SEM) of at least four independent experiments is shown. Single and double
asterisks indicate changes in growth with p<0.001 and p<0.05, respectively.
C and D.
Immunodeficient mice were implanted with 1108 cells expressing BET-specific shRNAs (n=3 for
each shRNA) on one side of the dorsal flank, and with cells expressing NTC on the opposite
side. Tumor volume (panel C) and weight (panel D) was measured at necropsy and compared
to that of tumors formed from cells expressing NTC in the same mice. E. Representative H&E
images from tumors formed by 1108 cells expressing NTC and expressing BET-specific
shRNAs. Scale bar = 100 M.
Figure S12. Paracrine regulation of the tumor stroma by individual BET proteins. A. Tumors
derived from 1108 cells expressing NTC and expressing BET-specific shRNAs were analyzed
by immunofluorescence for their proliferative index and the amount of cancer associated
fibroblasts. Representative images using a human specific Ki67 antibody (left panels) and an
antibody specific to smooth muscle actin (SMA, right panels) from tumors are shown. Scale
bars = 50 M. B. Quantitative analysis of Ki67 stained sections. Asterisk indicates p<0.05. C.
Quantitative analysis of SMA stained sections. Single and double asterisks indicate changes in
gene expression with p<0.005 and p<0.02, respectively.