Supplementary Materials for · transduction inhibitors: 5nM AC220, 10µM AKT inhibitor VIII (AKTi,...

12
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

Transcript of Supplementary Materials for · transduction inhibitors: 5nM AC220, 10µM AKT inhibitor VIII (AKTi,...

Page 1: Supplementary Materials for · transduction inhibitors: 5nM AC220, 10µM AKT inhibitor VIII (AKTi, from Sigma-Aldrich), 10µM STAT5 inhibitor ... between FLT3 571-993 and Pim-2 recombinant

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

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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.

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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

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%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

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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

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5

10

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days after Dox

Cel

lnum

ber(

ratio

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)

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MOLM-14 sh Pim-2

0.0

0.5

1.0

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ativ

eC

ellV

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lity

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*

0.0

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ativ

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***

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.

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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.

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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

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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)

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50

100

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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.

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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

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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

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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-

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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

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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

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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).