Supplementary Materials for · transduction inhibitors: 5nM AC220, 10µM AKT inhibitor VIII (AKTi,...
Transcript of Supplementary Materials for · transduction inhibitors: 5nM AC220, 10µM AKT inhibitor VIII (AKTi,...
advances.sciencemag.org/cgi/content/full/1/8/e1500221/DC1
Supplementary Materials for
Pim kinases modulate resistance to FLT3 tyrosine kinase inhibitors in
FLT3-ITD acute myeloid leukemia
Alexa S. Green, Thiago T. Maciel, Marie-Anne Hospital, Chae Yin, Fetta Mazed, Elizabeth C. Townsend,
Sylvain Pilorge, Mireille Lambert, Etienne Paubelle, Arnaud Jacquel, Florence Zylbersztejn,
Justine Decroocq, Laury Poulain, Pierre Sujobert, Nathalie Jacque, Kevin Adam, Jason C. C. So,
Olivier Kosmider, Patrick Auberger, Olivier Hermine, David M. Weinstock, Catherine Lacombe,
Patrick Mayeux, Gary J. Vanasse, Anskar Y. Leung, Ivan C. Moura, Didier Bouscary, Jerome Tamburini
Published 18 September 2015, Sci. Adv. 1, e1500221 (2015)
DOI: 10.1126/sciadv.1500221
This PDF file includes:
Fig. S1. Pim kinase expression in AML.
Fig. S2. Pim-2 regulation by downstream FLT3-ITD receptors.
Fig. S3. Pim-1 and Pim-2 regulation by FLT3-ITD receptors.
Fig. S4. Direct phosphorylation of FLT3 receptors by Pim-2.
Fig. S5. Dual inhibition of FLT3 and Pim kinases produces synergistic
cytotoxicity in AML.
Table S1. Genotyping of AML cell lines used in the current study.
Table S2. References of the antibodies used in the current study.
Materials and Methods
AML#
s1M
OLM
-14
MV4
-11
K562
Pim-2
β-actin
Pim-1
AML#
s2AM
L#s3
AML#
s4AM
L#s5
AML#
s6AM
L#s7
AML#
s8AM
L#s9
AML#
s10
Pim-3
Supplemental Figure 1
AML#
s11
Blue: FLT3 wiltypeRed: FLT3-ITD
Supplemental Figure 1. Pim kinases expression in AML. Protein extracts from AML cell lines (K562, MV4-11 and MOLM-14) and 11 primary AML samples (AML#s1 to AML#s11) were submitted to immunoblotting using anti-Pim-1, -Pim-2, -Pim-3 and –β-actin antibodies.
MOLM -14
GM-CSFG-CSF
SCF
OCI-AML3
IL-3
Pim-2
β-actin
p-Akt S473
Flt3-L
Akt
FCS 10%- - - -+-- - - --+
- + - ---
- - - +--- - + ---
- - - ---
--
-
--
+
- - - -+-- - - --+
- + - ---
- - - +--- - + ---
- - - ---
--
-
--
+ pAKT S473
pERK
pSTAT5
p4E-BP1 S65
--
--
-- + -+ -
-
-
+ --
+-
- - -
--
--
-- + -+ -
-
-
+ --
+-
- - -AKTiSTATiAZD8055
AC220
- - - +- - - - +- U0126----
-----
-
MOLM -14 OCI-AML3
p4E-BP1 T37/46
Pim-2
ERK
pAKT T308
pP70S6K
STAT5
P70S6K
AKT
β-actin4E-BP1
- + - + - + - +0
10
20
30
40
%an
nexi
nV
Dox
*** ***
Supplemental Figure 2A
B C
- + - +0
20
40
60
%an
nexi
nV
********
****
CTRmPim-2
MOLM-14 mPim-2
AC220 - + - +0
20
40
60
80
100
%an
nexi
nV
AC220
*****
*****
CTRAKT
MOLM-14 myrAKTm
yrA
KT
CTR
HA
β-actinPim-2
β-actin
Pim
2
CTR
D E
Supplemental Figure 2. Pim-2 regulation downstream FLT3-ITD receptors. (A) Annexin V staining in 4 AML cell lines (HL-60, OCI-AML3, MOLM-14 and MV4-11) transduced with Dox-inducible FLT3 shRNA using lentivirus. (B) MOLM-14 and OCI-AML3 cell lines were cultured without or with 10% fetal bovine serum (FBS) or starved from FBS and then stimulated for 0,5 h with different cytokines including 2.5µg/ml GM-CSF, 2.5µg/ml G-CSF, 20ng/ml interleukine-3 (IL-3), 10ng/ml stem cell factor (SCF) or 10ng/ml FLT3-ligand (FLT3-L). Western blots were done using anti-Pim-2, anti-phospho-AKT (S473) and anti-AKT. (C) MOLM-14 and OCI-AML3 cell lines were cultured without or with signal transduction inhibitors: 5nM AC220, 10µM AKT inhibitor VIII (AKTi, from Sigma-Aldrich), 10µM STAT5 inhibitor (STATi, CAS285986-31-4, from Calbiochem), 100nM of the mTOR inhibitor AZD8055 (from Selleckchem) and 10µM of the MEK inhibitor UO126 (from Selleckchem). Western blots were done using different antibodies for phosphorylated and non-phosphorylated proteins, as indicated. (D) MOLM-14 cells were transduced with Pim2 or control lentivirus and efficacy of transduction was assessed by western blotting using an anti-Pim-2 antibody. Cells were treated with vehicle (-) or 5nM AC220 (+). Apoptosis was evaluated by Annexin V staining using flow cytometry (n=4). (E) MOLM-14 cells were transduced with a control or a myrAKT expressing vector. myrAKT expression was detected by immunoblotting using an anti-HA antibody (Upper panel). Cells were treated with vehicle (-) or 5nM AC220 (+). Apoptosis was evaluated by Annexin V staining using flow cytometry (Lower panel, n=3). β-actin was used as the loading control on western blots. Results are expressed as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001.
HL-
60
OC
I-AM
L3
MO
LM-1
4
MV
4-11
Pim2
Pim1
Pim3
actin
sh P
im-2
#3
shSC
R
sh P
im-2
#5
sh P
im-2
#3
shSC
R
MOLM-14 CD34+
sh P
im-2
3
shSC
R
MV4-11
Pim-1
β-actinPim-2
+- Dox
MOLM-14 shPim-1
d2 d3 d4 d50
10
20
30
40
50
-DOX+DOX
%An
nexi
nV
MOLM-14 shPim-1
A B
D
Supplemental Figure 3
0 1 2 3 4 5 6 7 80
5
10
15
20
days after Dox
Cel
lnum
ber(
ratio
tod0
)
-DOX+DOX
MOLM-14 sh Pim-1
1 2 3 4 5 6 70
5
10
15
20
days after Dox
Cel
lnum
ber(
ratio
tod0
)
-DOX+DOX
MOLM-14 sh Pim-2
0.0
0.5
1.0
Rel
ativ
eC
ellV
iabi
lity
-DOX+DOX
*
0.0
0.5
1.0
Rel
ativ
eC
ellV
iabi
lity
-DOX+DOX
***
C
Supplemental Figure 3. Pim-1 and Pim-2 regulation by FLT3-ITD receptors. (A) MV4-11 and MOLM-14 AML cell lines and normal CD34+ hematopoietic progenitor cells were transduced with a scrambled (SCR) or a Pim-2 (two different clones shPim-2#3 and shPim-2#5) shRNA. Western blots were done using anti-Pim-1, -Pim-2 and -Pim-3 antibodies. (B) MOLM-14 cells were transduced with a Dox-inducible Pim-1 shRNA through lentivirus. After 48 h in the presence (+) or in the absence (-) of 200ng/ml Dox, protein extracts were submitted to immunoblotting using Pim-1 and Pim-2 antibodies. (C) Annexin V staining through time (day 2 to day 5) in MOLM-14 cells induced (+DOX) or not (DOX) for Pim-1 shRNA expression. (D) Cell proliferation assessment by daily counting in MOLM-14 cells expressing a Pim-1 (left panel) or a Pim-2 (right panel) shRNA upon treatment with 200ng/ml Dox (Upper panel) and cell viability assessed by an Uptiblue® assay in MOLM-14 cells after induction of a Pim-1 (left panel) or a Pim-2 (right panel) shRNA (Lower panel). β-actin was used as the loading control on western blots. Results are expressed as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001.
C
Supplemental Figure 4
123456
S977S938S937S953S895S864
H. sapiens SRKRPSFP M. musculus SRKRPSFP P. troglodytes SRKRPSFP R. norvegicus SRKRPSFP M. mulatta SRKRPSFP C. gallus DSRRRPSF C. lupus SRKRPSFP D. rerio NPVDRPCF B. taurus SRKRPSFP X. tropicalis SRKRPSFP
S935 S935
pFLT3 Y591
FLT3
Pim2
GST-PIM2GST-FLT3
-+ +
+
S-phosphate
pFLT3 Y591
pSTAT5 Y694
LGB321 - +
FLT3
Pim2GST
GST-PIM2GST-FLT3
++ +
+
In vitro Pim-2 / FLT3 kinase assaysA B
p-p70S6K
p-BAD
p-ERK1/2
p-STAT5
β-actin
MS
LGB
321
Baf/3-ITD
D
1
3
2
4
5
6
Supplemental Figure 4. Direct phosphorylation of FLT3 receptors by Pim-2. (A) In vitro kinase assays were performed between FLT3 571-993 and Pim-2 recombinant proteins (references F6432 and K3518 SIGMA from Sigma-Aldrich, respectively). Proteins were submitted to immunoblotting using an anti-thiophosphate ester antibody after alkylation according to the manufacturer instructions and an anti-phospho-FLT3 Y591 antibody. (B) Similar experiments were repeated after incubation with vehicle or 1µM LGB321 and proteins were submitted to immunoblotting using anti-phospho-STAT5 Y694 and anti-phospho-FLT3 Y591 antibodies. (C) Conservation of the S935 residue within FLT3 kinase domain through species according to the HomoloGene function of the PubMed.
E
ITD D835V F691L
p-STAT5(Y694)STAT5β-actin
- + - - + - - + - - - + - - + - - +
AC220 SGI1776
D
Supplemental Figure 5
p-STAT5(Y694)
STAT5
p-ERK1/2(T202/Y204)
ERK1/2
- +- + + +- - LGB321 (1µM)AC220 (nM)0 1 3 5 0 1 3 5
Ba/F3 ITD F
A
-12 -11 -10 -9 -8 -7 -60.0
0.5
1.0
log[AC220(M)]
Rel
ativ
e C
ell V
iabi
lity
Ba/F3
IC50parental
3.384e-007ITD
5.032e-010D835Y
3.158e-009F691L
2.017e-007
parentalITDD835YF691L
-12 -11 -10 -9 -8 -7 -60.0
0.5
1.0
log[LGB321(M)]
Rel
ativ
e C
ell V
iabi
lity
Ba/F3
IC50parental
3.028e-009ITD
1.213e-009D835Y
9.158e-010F691L
1.018e-009
parentalITDD835YF691L
0
10
20
30
40
50
60
70
Anne
xin
V (%
)
ITDD835YF691L
- - + ++ +--
LGB321AC220
***
*
**
*
* Vehicle vs AC220
* AC220 vs AC220+LGB321
B
Add
itivi
tyIn
hibi
tion=
45.0
LGB321 MV4-11 / 4.2 µM
AC
220
/ 2.9
nM 1.2
0.6
0
0 0.6 1.2
Combination therapySingle agent dose-responses
Inhi
bitio
n (%
)
LGB321 (µM)
0
50
100
0.1 1 10
Inhi
bitio
n (%
)
AC220 (nM)
0
50
100
0.1 1 10
MV4-11
0 0.54 1.1
0
0.54
1.1
Add
itivi
tyIn
hibi
tion=
40.0
LGB321 MOLM-13 / 1.2 µM
AC
220
/ 2.1
nM
Combination therapy
LGB321 (µM)0.1 1 10
Inhi
bitio
n (%
)
AC220 (nM)0
50
100
0.1 1 10
Single agent dose-responses
Inhi
bitio
n (%
)
0
50
100
MOLM-13
G
10-9
10-8
10-7
D83
5Y
F691
L
ITD
IC50
(M)
AC220AC220 + LGB321
C
Supplemental Figure 5. Dual FLT3 and Pim kinases inhibition produces synergistic cytotoxicity in AML. (A-B) Cell Title Glo ® assays in parental Ba/F3 cells and in Ba/F3 cells transduced with FLT3-ITD, FLT3-ITD-D835Y and FLT3-ITD-F691L alleles treated with log-dilutions of AC220 (A) or LGB321 (B). Results are presented using the log versus inhibitor four variables slope function of the GraphPad® software (Prism) and calculated IC50 are provided (Lower panel). (C) Ba/F3-ITD cells were treated with 1µM MLGB321 during 1 h. Western blot were done using anti-phospho-P70S6K (T389), anti-phospho-BAD (S112), anti-phospho-ERK (T202/Y204), anti-phospho-STAT5 (Y694) and �-actin antibodies. (D) Ba/F3 cells expressing FLT3-ITD, -D835Y and -F691L alleles were treated for 48 h with vehicle, 1µM LGB321, 5nM AC220 or combination, and apoptosis was evaluated by annexin V staining. (E) Ba/F3 ITD cells were treated without or with 1, 3 or 5nM AC220 and/or 1µM LGB321 during 1 h. Protein extracts were submitted to western blotting using anti-phospho-STAT5 (Y694), anti-phospho-ERK (T202/Y204), anti-STAT5 and anti-ERK antibodies. (F) Ba/F3 ITD, D835V and F691F cells were treated with vehicle, 5nM AC220 or with 1µM of the dual Pim and FLT3 kinase inhibitor SGI-1776 during 4 h. Protein extracts were submitted to western blotting using anti-phospho-STAT5 (Y694) and anti-STAT5 antibodies. (G) MV4-11 and MOLM-13 cell lines were treated with different concentrations of the drugs according to the layout for 72 h. PrestoBlue (Life Technologies) was added for fluorescence readout according to manufacturer’s instructions. Single-agent activity is depicted in each cell line separately as a percentage of viability inhibition relative to the vehicle condition (left panels). Synergy analysis resulting from AC220 and LGB321 combination-induced viability inhibition was performed using the Chalice software (Horizon CombinatoRx, Cambridge, MA, USA) and presented as isobologram (right panels). β-actin was used as the loading control on western blots. Results are expressed as mean ± SEM. *P<0.05, ***P<0.001.
SUPPLEMENTAL TABLES
Supplemental Table 1. Genotyping of AML cell lines used in the current study. FLT3-
ITD: FLT3 internal tandem duplication; FLT3-TKD: FLT3 tyrosine kinase domain mutation;
NPM1: nucleophosmine 1; IDH: isocitrate dehydrogenase; DNMT3A: DNA methyl
transferase 3A; F: technical failure; ND: not done.
FLT3-ITD FLT3-TKD NPM1 IDH1 IDH2 N-ras DNMT3A TP53
MOLM-14 pos0 neg neg Neg neg neg neg neg
MV4-11 pos1 neg neg Neg neg neg neg pos6
HL-60 neg neg neg Neg neg pos3 neg F
OCI-AML3 neg neg pos2 Neg neg pos4 pos5 neg
THP-1 neg neg neg ND ND ND ND ND
0 heterozygous 21bp insertion 1 homozygous 30bp insertion 2 exon 12 TCTG insertion 3 heterozygous Q61 mutation 4 homozygous Q61 mutation 5 heterozygous R882C mutation 6 heterozygous R248W mutation
Supplemental Table 2. References of the antibodies used in the current study.
Antibody Manufacturer Application Reference
Phospho-4E-BP1 (S65) Cell Signaling WB, IHC 9456
Phospho-p70S6K (T389) Cell Signaling WB 9205
Phospho-ERK (T202/Y204) Cell Signaling WB 9272
Phospho-STAT5a/b (Y694) Cell Signaling WB 9351
Phospho-BAD (S112) Cell Signaling WB 5284
Phospho-AKT (S473) Cell Signaling WB 9271
Phospho-AKT (T308) Cell Signaling WB 4056
Phospho-4E-BP1 (S65) Cell Signaling WB 9456
Phospho-4E-BP1 (T37/46) Cell Signaling WB 2855
4G10 (anti-p-Tyr) UBI WB
4E-BP1 Cell Signaling WB 9452
p70S6K Cell Signaling WB 9202
ERK Cell Signaling WB 9010
STAT5 Cell Signaling WB 9363
STAT5A Cell Signaling WB 4807
STAT5B Santa Cruz Bio. WB 1656
Bcl-xL Santa Cruz Bio. WB 8392
Pim-2 (human) Cell Signaling WB, IHC 4730
Pim-2 (mouse, human) Santa Cruz Bio. WB sc-13674
Pim-1 (12H8) Santa Cruz Bio. WB, IHC sc-13513
Pim-3 Cell Signaling WB 4165
FLT3/Flk-2 (S18) Santa Cruz Bio. WB, IP sc-480
FLT3/Flk-2 (C20, human) Santa Cruz Bio. IHC sc-479
β-actin Sigma WB A5441
Anti-thiophosphate Epitomics WB 2688
MATERIALS AND METHODS
Lentiviral and retroviral particle production
We used 293-T packaging cells to produce all of the constructs through co-transfection with
lentiviral protein-encoding plasmids and each of the plasmids listed below. Supernatants were
collected over three consecutive days beginning 48 h post-transfection, and stored at -80°C.
For retroviral production, Plat-E cells were transfected with pMX-FLT3-ITD or pMX vectors
(kindly donated by Dr. Patrice Dubreuil, CRCM, Marseille, France) using lipofectamine LTX
(Invitrogen, Carlsbad, CA, USA). Supernatants were collected 48 h after transfection and
used directly to transduce bone marrow cells with retronectin (Clontech, Mountain View, CA,
USA) following the manufacturer’s protocol.
Mammalian expression plasmids
Human Pim-2: PIM2. Human PIM2 cDNA was amplified in the presence of the template
pcDNA2.1deltaNotI (Thermo Scientific, Waltham, MA, USA) and oligonucleotide primers
2A and 2B (Supplemental Materials). The latter incorporated an HA-tag on the 3' end of
hPim2 cDNA. Human isoform 2 of the Pim-2 lentiviral expression vector was generated by
PCR cloning human PIM2 cDNA into the pLenti PGK Puro DEST vector (Addgene plasmid
19068, Cambridge, MA) (48) using the Gateway system recombination procedure (Life
Technologies, Carlsbad, CA) in the presence of LR clonase. The construct was then
sequenced for validation.
Mouse Pim-2: Pim2. An identical approach was used to construct the Pim2 lentiviral
expression vector from the commercially available murine pEN_Pim2 plasmid of mouse
origin (ATCC, LGC Standards, Teddington, Middlesex, UK).
Mouse Pim-2 kinase domain K61A mutant: Pim2KD. We used the Pim2 construct
described above as a template for targeted mutagenesis using the QuikChange II XL Site-
Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA) to generate a K61A
catalytic domain mutant (49) hereafter referred to as Pim2KD (for kinase-dead).
FLT3-ITD mutants: The FLT3-ITD gene was cloned into the pLKO.1-blast lentiviral
expression vector (Addgene Plasmid 26655) (50). Mutations including D835Y, F691L,
S935A and S935D were generated using the QuikChange II XL Site-Directed Mutagenesis
Kit (Agilent Technologies), in accordance with the manufacturer’s instructions using the
following (5’-3’) primers:
CTTTGGATTGGCTCGATATATCATGAGTGATTCCAAC (D835Y)
CAGGACCAATTTACTTGATTTTGGAATACTGTTGCTATGGTG (F691L)
TTTGACTCAAGGAAACGGCCAGCCTTCCCTAATTTG (S935A)
TGACTCAAGGAAACGGCCAGACTTCCCTAATTTGACTTCG (S935D)
AML xenografts in nude mice
MOLM-14 cells stably expressing the shPim-2 pLKO-Tet-On vector were subcutaneously
injected into nude mice. Briefly, 5x106 cells were mixed with Matrigel (1:1, vol/vol) and
subcutaneously injected into 8-week-old female athymic nude mice (Janvier SAS, France).
Mice were then treated (n=8) or not (n=8) with 200µg/ml of Dox administered ad libitum in
drinking water (with 1% sucrose) beginning one day after AML xenotransplantation. In
another set of experiments, MOLM-14 cells stably expressing a Pim2 allele following
lentiviral infection (n=8) or parental MOLM-14 cells (n=8) were subcutaneously injected into
nude mice which were treated with 1 mg/kg AC220 by oral gavage every 48 h, commencing
once the tumors reached 100 mm3 volume. Tumor growth was measured three times per week
in accordance with the equation: V = L X (S2)π/6, where L is the longer and S is the shorter
of the two dimensions. All experiments were conducted in accordance with the guidelines of
the Association for Assessment and Accreditation of Laboratory Animal Care International
and after approval of the local ethics committee. At the end of the experiment, mice were
sacrificed and their tumors excised. Samples were fixed in 4% formalin for
immunohistochemical analysis) or plated in liquid culture medium (10% FCS supplemented
MEM) to perform in vitro studies.
Immunohistochemistry
Paraffin-embedded spleen sections (4 m) were stained with hematoxylin-eosin or
hematoxylin erythrosine saffron (HES) for morphological analysis. For
immunohistochemistry, sections were processed for antigen retrieval as indicated by each
manufacturer and incubated overnight with primary antibodies. Following hour-long
incubation with biotinylated anti-species specific secondary antibodies at 1:500 dilution,
followed by thirty minutes in streptavidin-HRP (Vectastain, ABC kit; Vector Laboratories,
Burlingame, CA, USA), the immunoperoxidase reaction was visualized by addition of 3,3’-
diaminobenzidine (DAB) in Chromogen Solution (Dako, Carpinteria, CA, USA). Slides were
mounted with Eukitt mounting medium (Electron Microscopy Sciences, Hatfield, PA, USA)
and read with an upright microscope (Leica DM2000, Leica Microsystems, Solms, Germany)
at 200X magnification.
TUNEL assay
Apoptosis was assessed by the Terminal deoxynucleotidyl transferase mediated X-dUTP Nick
End Labelling (TUNEL) technique according to the manufacturer’s instructions (Roche
Applied Science, Penzberg, Germany). Slides were mounted with the Fluoroshield mounting
medium (AbCam, Cambridge, UK).I Images were acquired with a confocal microscope (Zeiss
LSM 510, Oberkochen, Germany) and analyzed using Imaris software.
Measurement of free intracellular calcium
Ba/F3 or Ba/F3 FLT3-ITD cells were transduced with control, Pim2 or Pim2KD alleles
through lentivirus. Free intracellular calcium content was measured using the Fluo-4 Direct™
Calcium Assay Kit (Invitrogen). Fifty thousand cells were plated in 96-well black plates with
clear bottoms (Corning Inc., Corning, NY, USA), pre-coated with 0,1% poly-L-lysine
(Sigma-Aldrich). Cells were cultured for 24 h in 10% FCS-supplemented RPMI then serum
starved for 4 hours (in presence of 10ng/ml IL-3). In some experiments cells were treated with
vehicle or 1 µM LGB321 as indicated. Intracellular calcium was measured with the Fluo-4
Direct™ Calcium Assay Kit (Invitrogen) by labeling the cells with Fluo-4 dye for 60 minutes
at 37°C as indicated by the manufacturer. Changes in cytosolic free calcium were quantified
as relative fluorescent units (RFU) using a microplate reader (Tecan Infinite M200 and
Magellan Software, Tecan, Männedorf, Switzerland). Wavelengths of excitation and emission
were 485 and 516 nm respectively. Measurements were carried out for 150 seconds. FLT3-L
(30ng/ml) was injected in each sample at t=50 seconds. Calcium mobilization was calculated
by the mean of the RFU at each time point and normalized by the basal levels (F0) of
fluorescence for each group (F/F0).