SUPPLEMENTARY INFORMATION
Supplementary Materials and Methods
Cell culture
HK and HS-5 cells were cultured in Iscove's modified Dulbecco's medium (IMDM) and
Dulbecco’s modified Eagle’s medium (DMEM, Life Technologies), respectively. HK cells
(0.75-1 x 105 cells/mL) and HS-5 cells (2 x 105 cells/mL) were plated overnight, and once
obtained a confluent stroma monolayer, medium was replaced by 2-5x105 CLL primary cells
and drugs were added for 72 hours. Afterward, CLL cells were collected by carefully rinsing the
wells without disturbing the stroma monolayer. When indicated, HK cells were replaced for
fresh ones and incubated for additional 72 hours. HUVEC cells, kindly provided by Dr Maria C.
Cid, were cultured in RPMI 1640 supplemented with 20% defined bovine calf serum (Thermo
Fisher Scientific, Waltham, MA, US), 0.2 mg/ml endothelial cell growth supplement (Becton
Dickinson), 5 units/ml sodium heparin (Merck Millipore), 2 mM glutamine, 100 units/ml
penicillin-streptomycin, 50 μg/ml gentamycin (Life Technologies) and 2.5 μg/ml fungizone
(Sigma-Aldrich).
Treatments and flow cytometry analysis of apoptosis
Cells were incubated with PF-03084014 (1 and 10 μM) and/or fludarabine (0.1, 0.5 and 1 μg/ml
i.e. 0.28, 1.37 and 2.74 μM) for 24 hours to 6 days. When indicated, cells were simultaneously
incubated with CD40L (1 μg/mL), IL4 (20 ng/mL; Sigma-Aldrich) and the drugs. Apoptosis
was quantified by double labeling with Annexin V–fluorescein isothiocyanate (FITC) and
propidium iodide (PI; eBioscience, San Diego, CA, US). To define the potential synergistic
effect of the drugs, the combination index (CI) was calculated with the CalcuSyn software
version 2.0 (Biosoft, Great Shelford, United Kingdom) according to the Chou and Talalay’s
algorithm. The interaction between 2 drugs was considered synergistic when CI < 1. For the
1
analysis of apoptosis in CD3+ and CD19+ populations, cells were labeled with anti-CD3PhycoerytrinCy5, anti-CD19-FITC (Becton Dickinson) and Annexin V-Pacific Blue (Life
Technologies). Twenty-five thousand cells per sample were acquired in an Attune acoustic
cytometer (Life Technologies).
Protein analysis
For protein immunodetection, the specific primary antibodies were used: cleaved Notch1
(Val1744; D3B8 clone) and phospho-IκBα (Ser32/36; 5A5 clone) (Cell Signaling Technology,
Danvers, MA, US), IκBα (Merck Millipore, Billerica, MA, US), β-actin and -tubulin (SigmaAldrich). Chemiluminescence was detected on a mini-LAS4000 Fujifilm device (GE
Healthcare, Little Chalfont, United Kingdom).
Gene expression profiling and gene set enrichment analysis (GSEA)
Total RNA was isolated from 107 CLL cells, previously exposed to drugs for 48 hours, using the
TRIzol reagent (Life Technologies) according to manufacturer’s instructions. cRNA obtained
from 150 ng of high quality RNA was fragmented and hybridized on the HT HG-U219
GeneChip (Affymetrix) following standardized protocols. Scanning was processed in the Gene
Titan instrument (Affymetrix) and analyzed with GeneChip Command Console Software
(AGCC) (Affymetrix). Raw data were normalized using the Robust Multichip Analysis (RMA)
algorithm implemented in the Expression Console Software v1.1 (Affymetrix). Top
differentially expressed genes associated with the combination compared to the other conditions
were obtained after setting up a 2-fold change cutoff in combination versus control. From this
list, the genes modulated less than 1.3-fold in combination versus fludarabine or PF-03084014
were removed. Finally, genes that were modulated in opposite senses between combination
versus PF-03084014 when compared to combination versus fludarabine were also discarded.
The final list of genes was displayed with Cluster (version 2.11) and TreeView (version 1.6)
softwares (Eisen Laboratory, Stanford University, CA, US). Significant gene signatures
modulated by each condition versus untreated cells were retrieved with gene set enrichment
2
analysis (GSEA) version 2.0 (Broad Institute at MIT) using the C2 (curated gene sets) collection
from the Molecular Signature Database v2.5 and experimentally-derived gene sets.1,2 A twoclass analysis with 1,000 permutations of gene sets and a weighted metric was used. Gene sets
with a false discovery rate (FDR) below 0.05 were considered as significant.
Statistical analyses
Nonparametric Wilcoxon signed rank test was used to compare the mean of a set of samples to a
theoretical value. Comparison between two groups of samples was evaluated by non-parametric
Wilcoxon paired t-test. Results were considered statistically significant when p-value < 0.05 (*
p< 0.05, ** p< 0.01, *** p< 0.001).
3
Supplementary Figures and Tables
Supplemental Figure S1
NOTCH1-mutated CLL
NOTCH1-unmutated CLL
ns
70
Cytotoxicity of fludarabine
at 72h (% of untreated)
Cytotoxicity of fludarabine
at 72h (% of untreated)
70
*
60
*
50
40
30
20
0
1
10
PF-03084014 (M)
ns
60
50
40
30
20
0
1
10
PF-03084014 (M)
Supplementary Figure S1. Combination of fludarabine plus GSI in cells from
chemoresistant CLL cases. Cells from NOTCH1-mutated (n=5) and NOTCH1–unmutated
(n=3) chemoresistant CLL patients were exposed to PF-3084014 and fludarabine (1 μg/mL).
Bars represent the mean ± SEM of all the samples tested. ns, not significant; * p< 0.05.
4
Supplemental Figure S2
0.5
**
0.75
0.50
0.25
-0
30
84
01
4
Fl
ud
ar
ab
in
C
e
om
bi
na
tio
n
PF
d
-0
30
84
01
4
Fl
ud
ar
ab
in
C
e
om
bi
na
tio
n
0.00
PF
-0
30
84
01
4
Fl
ud
ar
ab
in
C
e
om
bi
na
tio
n
0.0
PF
PF
mRNA relative level
0.25
1.0
***
1.00
nt
re
at
ed
0.50
mRNA relative level
0.75
nt
re
at
ed
nt
re
at
e
U
**
1.00
0.00
d
-0
30
84
01
4
Fl
ud
ar
ab
in
C
e
om
bi
na
tio
n
0.0
NR4A1
1.25
nt
re
at
e
0.5
THBS1
1.5
U
1.0
mRNA relative level
**
U
mRNA relative level
IL2RB
1.25
U
HCK
1.5
Supplementary Figure S2. Validation of gene expression profiling. NOTCH1-mutated CLL
patients (n=10) were exposed to PF-3084014 (10 μM) and fludarabine (1 μg/mL) for 48 hours
and mRNA levels of the selected genes were examined by quantitative real-time PCR. Relative
mRNA expression levels were referred to untreated controls. Bars represent the mean ± SEM of
all the samples tested. * p< 0.05, ** p< 0.01, *** p< 0.001.
5
Table S1- Genes modulated by the combination PF-03084014 plus fludarabine in CLL cells
Downregulated
genes
EGR1
SPP1
MYC
MMP9
NR4A1
ZBTB32
LPAR1
GPR114
GSTA4
PTPN6
GZMA
CPNE5
CD1D
TLR10
CST7
PRDM16
FBP1
IL32
TREML2
DUSP2
BCAT1
PVRIG /// STAG3
THBS1
ZMIZ1
IL2RB
COL15A1
ANXA6
TNNI2
TRIB2
A2M
DDIT4
TSPAN32
TUSC3
GLIS2
SERPINB2
FBN1
GZMK
PCGF2
SDC2
IL13RA2
GPR124
ASPH
USP9Y
PSAT1
ITM2C
MAGI1
GPR176
DPP4
ATF5
ARHGAP4
HLA-DQB1
TMC8
SH2D2A
ROBO1
TRD@
HK2
BNC2
--TIMP1
RASGEF1A
SLA
MAP1A
SGIP1
ZMYM6
MPDU1
CSGALNACT1
LY86
CTPS
GIMAP4
PTGES2
IL1R1
KIF21B
FBXL17
S100A9
PRICKLE1
PDE7B
CNTNAP1
HCK
RAC2
--SLC22A1
PDIA5
HFE
CARD9
CD9
SPON2
SIX4
KAT2B
PTPRK
MRAS
FGGY
TYROBP
AKAP5
FGL2
SH3BP2
PRSS16
SYNPO
SLC1A4
DOK3
Upregulated genes
SNIP1
MFSD9
SFTPC
PDLIM7
C1orf96
PLA2G16
ZEB1
NAPB
ZNF250
SNORD8
BHLHE41
MLL
REXO2
CPSF2
PCDHGA1
PMM2
RABGAP1L
HINT3
SYNE2
RGPD1
--SFRS18
WASL
SLFN5
TMPRSS5
PIM1
HGF
PAFAH1B2
QPRT
ZNF655
RRM2B
FSD1
EIF2AK1
SATB2
HIST2H2AC
GPLD1
OLFML3
C6orf35
--UBXN2A
SLC26A11
MXI1
TOR2A
PIGL
ZCCHC2
HSDL1
HRK
C17orf103
CRLF3
ZNF532
HTRA3
PTX3
POLDIP3
TRIO
SNORA28
UCN
NBR2
CSNK1G1
C11orf10
NR1D2
PTP4A1
CEACAM1
BDP1
PLEKHG1
KLHL24
KLHDC1
HIST1H2AC
TCTN1
EZH1
ATP6V1A
TWSG1
TMEM30A
JUN
LGMN
RPS27L
TNFRSF10D
FOXC1
GINS2
REST
ZNF667
TNFSF9
TUFT1
CCNG1
PLK2
IL13RA1
ABCB1
HIST1H2BN
TMEM123
HIST1H1E
GRAMD1C
CDKN1A
In bold: genes selected for qPCR validation
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SUPPLEMENTAL TABLE S2
Table S2.- Gene sets regulated by PF-03084014 plus fludarabine in CLL cells
GENE SIGNATURE (C2all)
MORI_MATURE_B_LYMPHOCYTE_UP
MARZEC_IL2_SIGNALING_UP
BIOCARTA_IL12_PATHWAY
Description
Up-regulated genes in the B lymphocyte developmental
signature, based on expression profiling of lymphomas
from the Emu-myc transgenic mice: the mature B.
Genes up-regulated by IL2 in cells derived from CD4+
cutaneous T-cell lymphoma.
IL12 and Stat4 Dependent Signaling Pathway in Th1
Development.
PID_IL6_7PATHWAY
IL6-mediated signaling events.
WU_CELL_MIGRATION
Genes associated with migration rate of 40 human bladder
cancer cells.
BIOCARTA_IL10_PATHWAY
IL-10 Anti-inflammatory Signaling Pathway.
RUTELLA_RESPONSE_TO_HGF_UP
GAL_LEUKEMIC_STEM_CELL_DN
RUTELLA_RESPONSE_TO_HGF_DN
Genes up-regulated in peripheral blood monocytes by
HGF.
Genes down-regulated in leukemic stem cells, defined as
CD34+CD38- cells from acute myeloid leukemia patients
compared to the CD34+CD38+ cells.
Genes down-regulated in peripheral blood monocytes by
HGF.
Gene set size
PF-03084014
FDR
Fludarabine
FDR
Combination
FDR
87
0.05
0.098
0.001
113
0.078
0.303
0.002
21
0.564
0.099
0.003
47
0.174
0.638
0.005
179
0.073
0.13
0.008
17
0.715
0.634
0.009
404
0.317
0.161
0.012
234
0.173
0.25
0.012
225
0.454
0.397
0.013
REACTOME_INTEGRIN_CELL_SURFACE_INTERACTIONS
Genes involved in Integrin cell surface interactions.
79
0.179
0.099
0.013
PLASARI_TGFB1_TARGETS_1HR_UP
Genes up-regulated in MEF cells upon stimulation with
TGFB1 for 1h.
34
0.122
0.507
0.028
REACTOME_IL_3_5_AND_GM_CSF_SIGNALING
Genes involved in Interleukin-3, 5 and GM-CSF signaling.
43
0.222
0.713
0.03
SCHOEN_NFKB_SIGNALING
Genes down-regulated in A375 cells treated with a small
molecule inhibitor of NFKB.
34
0.331
0.385
0.032
ROZANOV_MMP14_TARGETS_UP
Genes up-regulated in HT1080 cells over-expressing
MMP14 compared to those with knockdown of the gene
by RNAi.
253
0.089
0.491
0.04
PID_INTEGRIN1_PATHWAY
Beta1 integrin cell surface interactions.
66
0.22
0.29
0.042
LU_TUMOR_ANGIOGENESIS_UP
Up-regulated genes of putative pathways stimulated in
tumor endothelial cells by papillary serous ovarian
epithelial tumor cells.
25
0.083
0.559
0.043
FDR, False Discovery Rate
C2all motif gene signatures were obtained from the Molecular Signature Database v2.5
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References
1. Palomero T, Lim WK, Odom DT, Sulis ML, Real PJ, Margolin A, et al. NOTCH1 directly
regulates c-MYC and activates a feed-forward-loop transcriptional network promoting
leukemic cell growth. Proc Natl Acad Sci U S A 2006; 103: 18261-18266.
2. Rosenwald A, Chuang EY, Davis RE, Wiestner A, Alizadeh AA, Arthur DC, et al.
Fludarabine treatment of patients with chronic lymphocytic leukemia induces a p53dependent gene expression response. Blood 2004; 104: 1428-1434.
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