Prrx1-

Cre-P

ML+/+

A. Prrx

1-Cre

-PML

+/+

Prrx1-

Cre-P

MLF/F

A. Prrx

1-Cre

-PML

F/F0

5

10

15

20

Rel

ativ

e ex

pres

sion

Fa

bp4

mR

NA

Pml+/+

Pml-/-0.8

1.0

1.2

1.4 n.s.

Rel

ativ

e ad

ipoc

ytic

col

onie

s/to

tal c

olon

ies

Prrx1-

Cre-P

ML+/+

Prrx1-

Cre-P

MLF/F

0.0

0.5

1.0

1.5

2.0n.s.

Rel

ativ

e C

FU-F

num

bers

b.

Pmlf/f targeting strategy

a.

Muc1f/f targeting strategy

Muc1 allele

NdeI BgllI NsiI

5� probe

3� probe

Targeting Construct BglII-Loxp Nsi-Frt-Neo-Frt-Loxp

Flippase

Targeted

allele

Floxed Allele

1 2 3 4 5 6 7

1 2 3 4 5 6 7

1 2 3 4 5 6 7

1 2 3 4 5 6 7

BglII

NdeI BgllI NsiI BglII

NdeI BgllI NsiI BglII

a b Pml F/F Prrx1-cre

X

Prrx1-cre-Pml F/F

c

Pml+/+

Pml-/-

BM-MSCs

Osteoblasts ALP staining

Adipocytes Oil-Red-O staining

Pml+/+ Pml-/-

Pml+/+

Pml-/- 0 2 4 6 8 10 120.0

0.2

0.4

0.6

0.8

1.0 Pml+/+Pml-/-

days in culture

Adso

rban

ce

0 1 2 3 40.0

0.2

0.4

0.6

0.8

1.0Pml+/+Pml-/-

days in culture

Adso

rban

ce

early passages

late passages

d e f

Prrx1-C

re-PML+

/+

O. Prrx

1-Cre-

PML+/+

Prrx1-C

re-PMLF

/F

O. Prrx

1-Cre-

PMLF/F

0

5

10

15

20

25

Rel

ativ

e ex

pres

sion

Al

p m

RN

A

Supplementary Figure 1 | Pml regulates the biology of MSCs. (a) Schematic overview of the experimental design used to generate Pml-floxed mice. (b) Expression levels, assayed by RT-qPCR, of Pml mRNA in the different populations of mesenchymal cells of Prrx1-Cre-Pml+/+ or Prrx1-Cre-PmlF/F mice; HSCs are used as control. Cells collected from n=3 mice for each genotype were pooled together prior to RNA extraction. (c) CFU-F colonies forming the capacity of MSCs derived from Prrx1-Cre-PmlF/F or Prrx1-Cre-Pml+/+ mice (n=7). (d) Proliferation of MSCs derived from Pml+/+ or Pml-/- mice either at early passages in vitro, or at late passages. One representative experiment is shown. (e) Differentiation of MSCs derived from Pml+/+ or Pml-/- mice into osteoblasts or adipocytes. Osteoblasts (on the left) were stained with Alkalyne Phosphatase (ALP), while adipocytes (on the right) were stained Oil-Red-O. The chart on the right shows the quantification of Oil-Red-O positive colonies (n=4). (f) Capacity of Prrx1-Cre-Pml+/+ or Prrx1-Cre-PmlF/F MSCs to differentiate to adipocytes (upper chart, A= MSCs triggered to adipogenesis), or into osteoblasts (lower chart. O= MSCs triggered to osteogenesis) once triggered with specific factors. The chart on the upper right panel shows the relative expression of Fabp4, while the one on the bottom shows the expression of Alp. One representative experiment ± SEM is shown.

Prrx1-C

re Pml+

/+

Prrx1-C

re PmlF

/F

Prrx1-C

re Pml+

/+

Prrx1-C

re PmlF

/F

Prrx1-C

re Pml+

/+

Prrx1-C

re PmlF

/F0.0

0.5

1.0

1.5 MSCs CD51+Sca1+Stroma CD51-Sca1-HSCs

*

Rel

ativ

e ex

pres

sion

Pm

l mR

NA

CD150

CD

48

Prrx1-Cre-PmlF/F Prrx1-Cre-Pml+/+

Gated on CD34- KLS cells

CD34

C-k

it

Prrx1-Cre-PmlF/F Prrx1-Cre-Pml+/+

Gated on KLS cells

Prrx1-Cre-PmlF/F Prrx1-Cre-Pml+/+

Gated on Lin- cells

Sca1

C-k

it

b

BCR/ABL leukemic cells

0 20 40 60 800

50

100

150

ControlPrrx1-Cre-PMLF/F

days

% s

urvi

val

*

c

Sca-1

0.9% 0.4%

c-ki

t

Pml+/+ Pml-/- Endosteal BM (E-BM)

Pml+/+ Pml-/-

c-ki

t 21.7% 8.5%

Sca-1

Inner BM (I-BM)

BA+ GFP+ leukemic cells

1st Recipients

2nd Recipients

BCR/ABL leukemic cells

0 20 40 60 800

50

100

150

ControlPrrx1-Cre-PMLF/F

days

% s

urvi

val

1st Recipients

2nd Recipients

a

Prrx1-

Cre P

ml+/+

Prrx1-

Cre P

mlF/F0.0

0.5

1.0

1.5

2.0 n.s.

Rel

ativ

e nu

mbe

rs o

f C

D34

- KLS

cel

ls

Pml+/+

Pml-/-0.0

0.5

1.0

1.5

Rel

ativ

e to

tal

num

bers

of

BA

+GFP

+KLS

cel

ls

*

Pml+/+

Pml-/-0.0

0.5

1.0

1.5

Rel

ativ

e to

tal

num

bers

of

BA

+GFP

+KLS

cel

ls

*

Supplementary Figure 2 | Pml expressed in MSCs regulates only marginally non-cell-autonomously HSCs. (a) Representative plots showing the hematopoietic sub-populations in Prrx1-Cre-Pml+/+ or Prrx1-Cre-PmlF/F mice. The upper left plot shows CD150+CD48-CD34-KLS cells; the one on the right shows CD34-KLS cells, while the one on the bottom shows KLS cells. The chart on the right shows the quantification of CD34-KLS cells in Prrx1-Cre-Pml+/+ or Prrx1-Cre-PmlF/F mice (n=5 mice analyzed). (b) Relative percentages (representative plots on the left) and relative total numbers (charts on the right) of BA+GFP+KLS cells present at the endosteal bone marrow (E-BM), or within the inner cavity of the bone marrow (I-BM) in second recipients Pml+/+ or Pml-/- (n=2 mice analyzed). (c) Survival curves of serially transplanted Prrx1-Cre-PmlF/F or control mice with BA+GFP+ cells. The upper panel shows the survival of primary recipients, while the bottom panel shows the survival of secondary recipient mice.

Pml+/+

Pml-/-0

1

2

3

Rel

ativ

e nu

mbe

rs o

f G

FP+c

kit+

cel

ls

c-ki

t

MSCs Pml+/+ MSCs Pml-/-

GFP

a

Sorting MSCs

Day 0 Day 12

~ 10 passages

1% oxygen

10,000 10,000 leukemic cells

Total leukemic cells and LICs

Freshly isolated MSCs

wild type

Sca-1

c-ki

t

gated on Lin- cells

KLS cells Pre-leukemic cells

GFP+

Expansion in wild type recipients

MLL-­‐AF9  

HoxA9  Meis-­‐1  

GFP+ leukemic cells

SERIAL co-cultures with

MSCs (Pml+/+ or Pml-/-)

RV-Fusion gene cDNA

SCL-tTA/BCR-ABL

BA+KLS cells LV-GFP

b

MSCs Pml+/+ MSCs Pml-/-

Sca-1

c-ki

t 12% 4.9% 9.8% 6.1%

Sca-1

c-ki

t

MSCs Pml+/+ MSCs Pml-/-

c

Sca-1

c-ki

t

MSCs Pml+/+ MSCs Pml-/-

c-ki

t

MSCs Pml+/+ MSCs Pml-/-

GFP

2.5% 0.6%

Sca-1

c-ki

t

MSCs Pml+/+ MSCs Pml-/-

d e 3rd co-culture

1st co-culture

1st co-culture

Bcr/Abl

MLL/AF9

HoxA9+ Meis1

Pml+/+

Pml-/-0.0

0.5

1.0

1.5

Rel

ativ

e to

tal

num

bers

of

BA

+GFP

+ ce

lls

Pml+/+

Pml-/-0.0

0.5

1.0

1.5

Rel

ativ

e to

tal

num

bers

of

BA

+GFP

+KLS

cel

ls

Pml+/+

Pml-/-0.0

0.5

1.0

1.5

Rel

ativ

e nu

mbe

rs o

f M

LL-A

F9-G

FP+

cells

Pml+/+

Pml-/-0.0

0.5

1.0

1.5

2.0

2.5

Rel

ativ

e nu

mbe

rs o

f G

FP+c

kit+

cel

ls

Pml+/+

Pml-/-0.0

0.5

1.0

1.5

2.0

2.5

Rel

ativ

e nu

mbe

rs o

fH

oxA9

-Mei

s1+G

FP+

cells

Supplementary Figure 3 | Pml expressed in MSCs regulates non-cell-autonomously LICs. (a) Schematic overview of MSCs culturing conditions, and of the co-cultures with MSCs and leukemic cells. (b) Schematic overview of the experimental design to generate leukemic cells that express HoxA9-Meis1, MLL/AF9, or GFP+BCR/ABL. (c) Relative numbers of BA+GFP+ cells, HoxA9+Meis1 leukemic cells and MLL/AF9 leukemic cells derived from the first co-cultures with MSCs Pml+/+ or Pml-/- (n≥4). (d) Relative numbers and percentages of BA+GFP+KLS cells, HoxA9-Meis1 GFP+c-kit+ cells, and MLL/AF9 GFP+c-kit+ cells in the first co-cultures with Pml+/+ or Pml-/-

MSCs. Quantifications are represented in the charts on the right, while representative plots are shown on the left (n≥3). (e) Representative plots showing percentages of BA+GFP+KLS cells, HoxA9-Meis1 GFP+c-kit+ cells, and MLL/AF9 GFP+c-kit+ cells in serial co-cultures with Pml+/+ or Pml-/- MSCs.

From co-cultures with MSCs Pml+/+

From co-cultures with MSCs Pml-/-

10x 10x

40x

From co-cultures with MSCs Pml+/+

From co-cultures with MSCs Pml-/-

10x 10x

60x

HoxA9-Meis1

MLL/AF9

b

c

Pml+/+Pml-/-

0.0

0.5

1.0

1.5

*

Rel

ativ

e nu

mbe

r of

colo

nies

Pml+/+Pml-/-

0.0

0.5

1.0

1.5

*

Rel

ativ

e nu

mbe

r of

colo

nies

0 5 10 15 200

50

100

150 PML+/+PML-/-

days

% s

urvi

val

0 10 20 30 400

50

100

150

1000cells PML+/+1000cells PML-/-20000cells PML+/+20000cells PML-/-

days

% s

urvi

val

a

Methyl cellulose

Untrea

ted

AS 2O3

shSCR

shPml

0.0

0.5

1.0

1.5

* **

Rel

ativ

e nu

mbe

r of

colo

nies

Unt

reat

ed

AS

2O3

Pml

Β-actin

* Pml

Β-actin

shS

CR

shP

ml

de

Supplementary Figure 4 | Pml expressed in MSCs regulates leukemic cells in a non-cell-autonomous manner. (a) Relative number of colonies in methyl-cellulose, generated by leukemic cells derived from co-cultures with MSCs Pml+/+ or Pml-/-. HoxA9-Meis1 leukemic cells are shown on the left, while MLL/AF9 leukemic cells are shown on the right. One experiment ± SEM is shown. (b) H&E of leukemic blasts in the blood of mice transplanted with HoxA9-Meis1 leukemic cells after co-culture with Pml+/+ or Pml-/- MSCs. The survival curve of these mice is shown on the right. (c) H&E of leukemic blasts in the blood of mice transplanted with MLL/AF9 leukemic cells after co-culture with Pml+/+ or Pml-/- MSCs. The survival curve of these mice is shown on the right. (d) Western blot analysis of Pml expression in MSCs upon knock-down with shRNA and upon treatment with AS2O3. (e) Methyl-cellulose assay performed with leukemic cells (MLL/AF9) which have been co-cultured with MSCs, while treating with AS2O3, or performed with leukemic cells co-cultured with MSCs transduced with an shRNA targeting Pml. One experiment ± SEM is shown.

b

H+M

BCR/ABL

MLL/AF9

Treatment: Anti-CXCR2

Anti-IL6R

1st co-culture 2nd co-culture

H+M

BCR/ABL

MLL/AF9

a

Start 1st co-culture

Treatment End 1st co-culture: LICs

Start 2nd co-culture

Analysis of LICs

Day 0 Day 1 Day 4 Day 8

HoxA9+Meis1 MLL/AF9 BCR/ABL

c BCR/ABL

d HoxA9+Meis1 MLL/AF9

N.T.

Sca-1

c-ki

t

aCXCR2 aIL6R

15% 9.89% 10.2% 6.9%

aCXCR2+aIL6R

GFP

aCXCR2+aIL6R N.T.

c-ki

t 16% 9%

e

aCXCR2+aIL6R N.T.

c-ki

t

GFP

12% 6%

f Pml+/+

Anti-CXCR2 Anti-IL6R

1st co-culture 2nd co-culture

Pml-/-

Pml+/+

Pml-/-

Not Treated (N.T.)

CXCL1+IL6 recombinant

Pml+/+

Pml-/-

Pml+/+

Pml-/-

MSCs Pml+/+ N.T

MSCs Pml+/+ aCXCR2+aIL6R

GFP

c-ki

t c-

kit

MSCs Pml-/- N.T

MSCs Pml-/- CXCL1+IL6

15.4% 7.9%

17.3% 6.2%

NT

αCXCR2αIL

6R

αCXCR2+

αIL6R

0

100

200

300

400 **

*x103

Tota

l num

bers

of

leuk

emic

cel

ls

NT

αCXCR2αIL

6R

αCXCR2+

αIL6R

0

100

200

300 *x103

Tota

l num

bers

of

leuk

emic

cel

ls

NT

αCXCR2αIL

6R

αCXCR2+

αIL6R

0

200

400

600

800*

x103

Tota

l num

bers

of

leuk

emic

cel

ls

NT

αCXCR2αIL

6R

αCXCR2+

αIL6R

0.0

0.5

1.0

1.5

**

*

Rel

ativ

e nu

mbe

rs o

f G

FP+S

ca1+

ckit+

cel

ls

NT

αCXCR2αIL

6R

αCXCR2+

αIL6R

0.0

0.5

1.0

1.5

*

Rel

ativ

e nu

mbe

rs o

f G

FP+c

kit+

cel

ls

NT

αCXCR2αIL

6R

αCXCR2+

αIL6R

0.0

0.5

1.0

1.5

*

Rel

ativ

e nu

mbe

rs o

f G

FP+c

kit+

cel

ls

Pml+/+

Pml-/-

Pml+/+ aC

XCR2+aIL

6R

Pml-/- CXCL1

+IL6

0.0

0.5

1.0

1.5

**

*

Rel

ativ

e to

tal

% o

f GFP

+cki

t+ c

ells

Supplementary Figure 5 | Pml regulates leukemic cells non-cell-autonomously through IL6/IL6R and CXCL1/CXCR2 pathways. (a) Schematic overview of the experimental design for treatment of the co-cultures with anti-IL6R and anti-CXCR2 antibodies. (b) Total number of leukemic cells not treated (NT) or treated with anti-Il6R, anti-CXCR2 singularly or in combination. One representative experiment ± SEM is shown. (c) Total numbers of BA+GFP+KLS cells after the treatment with anti-IL6R or/and anti-CXCR2, singularly or in combination. Not treated cells (NT) were used as control. The chart on the left shows the quantification, while representative plots are shown on the right. One representative experiment ± SEM is shown. (d) Chart on the left shows the numbers of HoxA9-Meis1+GFP+ckit+ cells after the first co-culture and treatment with anti-IL6R or/and anti-CXCR2 antibodies (singularly or in combination) One representative experiment ± SEM is shown; representative plots are shown on the right. (e) Chart on the left shows the numbers of MLL-AF9+GFP+ckit+ cells after the first co-culture and treatment with anti-IL6R or/and anti-CXCR2 antibodies (singularly or in combination) One representative experiment ± SEM is shown; representative plots are shown on the right. (f) Schematic overview of the experimental design is shown on the left. Co-cultures of MSCs Pml+/+ and HoxA9-Meis1+GFP+ leukemic cells were untreated (NT) as control, or treated with anti-CXCR2 in combination of anti-IL6R. Co-cultures with MSCs Pml-/- and HoxA9-Meis1+GFP+ leukemic cells were untreated as control, or treated with recombinant Il6 and Cxcl1 proteins. Leukemic cells were then re-plated onto the new MSCs (Pml+/+ or Pml-/-); secondary co-cultures were analyzed. Percentages of GFP+ckit+ cells in the different conditions are shown in the plots and the chart on the right. One experiment ± SEM is shown.

Supplementary Figure 6 | Original blots of Figure 5c