pten opposes negative selection and enables … · hassan jumaa & markus müschen inventory:...

Supplementary information

PTEN opposes negative selection and enables oncogenic transformation of pre-B cells

Seyedmehdi Shojaee, Lai N. Chan, Maike Buchner, Valeria Cazzaniga, Kadriye Nehir Cosgun, Huimin Geng, Yi Hua Qiu, Marcus Dühren von Minden; Thomas Ernst, Andreas Hochhaus,

Giovanni Cazzaniga, Ari Melnick, Steven M. Kornblau, Thomas G. Graeber, Hong Wu, Hassan Jumaa & Markus Müschen

Inventory:• Supplementary Figures 1-10• Supplementary Tables 1-5

Nature Medicine: doi:10.1038/nm.4062

(a) Ptenfl/fl BCR-ABL1-transformed pre-B ALLcells were transduced with tamoxifen-induced Cre-ERT2 or an empty vector (EV) control. Cellviabilities were measured by flow cytometry usingpropidium iodide (PI). Numbers denote thepercentages of viable cells. Representative flowcytometry plots are shown (n = 3).

Figure S1: Pten-deletion in pre-B cells is synthetically lethal with transformation by BCR-ABL1

Day 0

Day 6

73 67

69 9

Ptenfl/fl +EV Ptenfl/fl +Cre

a

Day 1 Day 2 Day 3 Day 4 Day 5

Ptenfl/fl

+EV

Ptenfl/fl

+Cre

FSC

BC

R-A

BL1

-GF

P

b 19.2 31.6 44.0 49.9 53.6

16.5 22.8 15.7 9.1 9.1

FSC

PI

(b) IL7-dependent Ptenfl/fl pre-B cells were transduced with Cre-ERT2 or an empty vector (EV)control. Two days after tamoxifen-induced activation of Cre, cells were transduced with BCR-ABL1-GFP. Fractions of GFP positive cells were measured by flow cytometry. Representativeflow cytometry plots for GFP positive cells are shown (n = 3).


(c) One million Ptenfl/fl BCR-ABL1-transformed pre-B ALL cells carryingtamoxifen-inducible Cre or an empty vector(EV) were injected into NOD/SCID recipientmice (n = 7 per group). Pten deletion wasinduced by treatment of the cells for 24 hourswith tamoxifen (0.5 µmol/liter) beforeinjection. All mice in the Cre group were stillalive at the end of the experiment (day 70) andshowed no signs of disease.

MRD-PCR Controls: Ptenfl/fl

1 2 3 4 5 EV Cre H2O

PtenWT

Ptenflox

Ptendel

Mice at day 70 These mice were then sacrificed, bonemarrow mononucleated cells were isolatedand analyzed by genomic PCR. Ptenfl/fl pre-BALL cells with and without in vitro-deletionof Pten were used as positive controls as wellas water as negative control. PCRamplification of Pten genomic region frombone marrow of the NOD/SCID transplantrecipients allowed to detect wildtype, floxedand deleted Pten alleles as markers forminimal residual disease (MRD). In the bonemarrow of NOD/SCID transplant recipients,only wildtype Pten alleles and no signs ofMRD were detected.

Figure S1(continued)

c

PtenWT

Ptenflox

Ptendel

Colony H2O+/+ fl/fl

One colony of Ptenfl/fl pre-B ALL cellstransduced with Cre that had formed inmethylcellulose (Fig. 2b) was isolated forgenotyping by genomic PCR. Individualamplification products are detected for thefloxed and deleted alleles of Pten.

Figure S2: Genotyping of colonies Ptenfl/fl pre-B ALL cells growing in methylcellulose

Activation of Cre in vitro

1 2 3 4 5 Long-term surviving mice

EV

Cre


(Top) The frequencies of activatingmutations of AKT1, AKT3,PIK3CA, PIK3C2A, PIK3CD,PIK3R1, MTOR, RPS6KA2,RPS6KB2 genes that encodesignaling molecules in thePI3K/AKT pathway werecalculated for four types ofhematopoietic malignancies,namely pre-B ALL, B cell Non-Hodgkin’s Lymphoma (B-NHL),myeloid leukemia and T celllineage ALL. Depicted is thenumber of samples studied (Seq),the number of cases with activatingmutation (Mut). Mutationfrequencies (bold face) are given aspercentages of mutated versus totalnumber of cases sequenced.(Bottom) Red and green barsrepresent the frequencies ofactivating and inactivatingmutations, respectively. Mutationdata was obtained from Catalogueof Somatic Mutations in Cancerdatabase (COSMIC,http://cancer.sanger.ac.uk/cancergenome/projects/cosmic/).

-15

-10

-5

0

5

AK

T1,

AK

T3

PIK

3CA

, PIK

3C2A

, PIK

3CD

, PIK

3R1

MT

OR

, RP

S6K

A2,

RP

S6K

B2

PT

EN

Mut

atio

n fr

eque

ncie

s[%

]

AK

T1,

AK

T3

PIK

3CA

, PIK

3C2A

, PIK

3CD

, PIK

3R1

MT

OR

, RP

S6K

A2,

RP

S6K

B2

PT

EN A

KT

1, A

KT

3

PIK

3CA

, PIK

3C2A

, PIK

3CD

, PIK

3R1

MT

OR

, RP

S6K

A2,

RP

S6K

B2

PT

EN

AK

T1,

AK

T3

PIK

3CA

, PIK

3C2A

, PIK

3CD

, PIK

3R1

MT

OR

, RP

S6K

A2,

RP

S6K

B2

PT

EN

Pre-B ALL B-NHL Myeloid T cell lineageleukemia ALL

Figure S3: Frequency of activating PI3K-AKT pathway mutations in hematological malignancies


Figure S4: Correlation of PTEN expression with patient survival in T cell lineage vs pre-B ALL

0 2 4 6 8 0 1 2 3 0 5 10 15 0 2 4 6 8 10 12

T cell lineage ALL Pre-B ALL

P=0.0105; n=22 P=0.0759; n=18

PTENHIGH

PTENLOW

PTENHIGH

PTENLOW

Ove

rall

surv

ival

[%

]

Rel

apse

-fre

e su

rviv

al [

%]

Ove

rall

surv

ival

[%

]

P=0.101; n=155 P=0.0415; n=132

PTENHIGH PTENHIGH

PTENLOWPTENLOW

Rel

apse

-fre

e su

rviv

al [

%]

Follow up [years] Follow up [years] Follow up [years] Follow up [years]

100

50

0

100

50

0

100

50

0

100

50

0

Reverse phase protein array (RPPA) data for PTEN expression were analyzed for patients with T celllineage ALL (n = 22) and pre-B ALL (n = 155). Based on higher or lower than median expression levelsof PTEN, patients were divided into two groups (PTENHigh, PTENLow) and the overall survival as wellas relapse-free survival of these patients are plotted over a follow up period of 3 to 18 years. P valueswere calculated by Mantel-Cox log rank test.

Figure S5: Small molecule inhibitors of Syk, PI3K and AKT rescue Pten-deletion

FSC

GF

P

b

(b) Pten deletion was induced in BCR-ABL1-transformed pre-B ALL cells in the presence or absence ofAKT inhibitor AZD5363 (AZD, 3 µmol/liter), PI3K inhibitor BKM120 (BKM, 1 µmol/liter) and Sykinhibitor PRT060318 (PRT, 3 µmol/liter). Representative flow cytometry plots for GFP+ cells areshown. All plots are gated on viable cells.

Cre + AZD

Cre + Vehicle

Ptenfl/fl

BCR-ABL1-transformed Ptenfl/fl

NRASG12D-transformed

Cre + AZD

Cre + Vehicle

Day 0

Day 10

FSC

GF

P

a

85 87 42 40

3 32 2 17

EV +Vehicle

EV +BKM

EV +AZD

EV +PRT

Cre +Vehicle

Cre + BKM

Cre + AZD

Cre +PRT

67 70 70 69

66 71 68 65

37 41 40 38

0.9 2.8 3.2 25

D0

D12

(a) Ptenfl/fl pre-B cells transformedwith BCR-ABL1 or NRASG12D weretransduced with inducible Cre-GFP-ERT2 or a GFP empty vector (EV)control. Pten deletion was inducedwith tamoxifen in the presence orabsence of AKT inhibitor AZD5363(AZD, 3 µmol/liter). Representativeflow cytometry plots for GFP+ cells areshown. All plots are gated on viablecells


BCR-ABL1 Pten+/+, Pten+/fl and Ptenfl/fl ALL + Cre

Pten+/+

Pten+/fl

Ptenfl/fl Dis

ease

-fre

e su

rviv

al [

%] 100

0 50 100 150 Days after injection

Pten+/+

Pten+/+ vs Pten+/fl P = 0.0003

Pten+/+ vs Ptenfl/fl P = 0.0002

Pten+/fl

Ptenfl/fl

50

0

d

e

AKT-pT308

Ptenfl/fl Pten+/fl

EV Cre EV Cre

AKT

-actin

Day 8 Day 15 Day 20

291 ± 13 26 ± 4 8 ± 3

Ptenfl/fl + EV Pten+/fl + Cre Ptenfl/fl + Cre

Figure S6: Monoallelic deletion of Pten in pre-B ALL cells compromises leukemia initiation in vivo

a

P = 5.1× 10-6

P = 3.7 × 10-6

0 200 300 [Colonies/104]100Pten

fl/fl

+/-

-/-CFU/10,000 plated cells

2.5% ± 2% 20.5% ± 7% 37.7% ± 6%

Ptenfl/fl + EV Pten+/fl + Cre Ptenfl/fl + Cre

c

Pten

fl/fl

+/-

-/-

0 20 30 40 5010

P = 0.005

P = 0.0002

Senescence-associated-βgal+ cells [%]

S

G2/M

G0/G1

S S

7AAD

Brd

U

G0/G1 G0/G1

G2/M G2/M

Ptenfl/fl + EV Pten+/fl + Cre Ptenfl/fl + Creb

54%

45%

47%

Pten

fl/fl

+/-

-/-

0 20 40 60 80 100

P = 0.0007

P = 0.0008

G2/M phaseSG0/1

Pten+/fl vs Ptenfl/fl P > 0.9

To compare the effects of deletion of one vs both alleles ofPten, Ptenfl/fl and Pten+/fl BCR-ABL1-transformed pre-B cellswere transduced with tamoxifen-inducible Cre-ERT2 or anempty vector (EV) control. (a) Effects of Pten deletion oncolony forming ability of pre-B ALL were measured by plating10,000 cells after tamoxifen induction in methylcellulose. (b)Following 24 hours of tamoxifen-induced Pten deletion, cellcycle progression was assayed by measuring genomic BrdUincorporation which represents the cells in the S phase.

30,000 300,000p/sec/cm2 /sr


Legend (continued): (c) Cellular senescence was detected by measuring β-galactosidase activityunder acidic conditions (pH=5.5) after deletion of Pten. The percentages of cells stained withblue dots are depicted on the panels. Scale bars in (a) and (c) represent 1 mm and 50 µmrespectively. P values (a–c) were calculated by Student’s t test. (d) 105 luciferase-labeled Pten+/+,Pten+/fl and Ptenfl/fl BCR-ABL1-transformed pre-B ALL cells transduced with Cre-ERT2 wereinjected into NOD/SCID recipient mice (n = 7 per group). Pten deletion was induced bytreatment of the cells for 24 hours with tamoxifen (0.5 µmol/liter) before injection. P values werecalculated by Mantel-Cox log rank test. Scale bars = 3 cm (e) Western blot was performed fortotal and T308-phosphorylated AKT after 48 hours of Pten deletion in Ptenfl/fl and Ptenfl/+ BCR-ABL1 transformed pre-B ALL cells. β-actin was used as loading control. Representative blot, n =3.

Figure S6: Monoallelic deletion of Pten in pre-B ALL cells compromises leukemia initiation in vivo


µHCNA +Vehicle

µHCNA + PRT

µHCNA +AZD

µHCAuto +Vehicle

µHCAuto + PRT

µHCAuto +AZD

EV +Vehicle

EV +PRT

EV +AZDa

Day 1

Day 5

AKTCA + Vehicle

AKTCA + AZD

EV + Vehicle

EV + AZD

Day 0

Day 7

SykCA +Vehicle

SykCA + BKM

SykCA +AZD

SykCA +PRT

EV +Vehicle

EV +AZD

EV +PRT

EV +BKM

Day 0

Day 12

c

b

FSC

FSC

FSC

(b) BCR-ABL1-transformed pre-B ALL cells were transduced with retroviral vector encodingactive form of Syk (SykCA: SykY348E/Y352E) or a GFP empty vector (EV) control in the presence ofvehicle, Syk inhibitor PRT060318 (PRT, 3 µmol/liter), PI3K inhibitor BKM120 (BKM, 1µmol/liter) and AKT inhibitor AZD5363 (AZD, 3 µmol/liter). Representative flow cytometry plotsfor GFP positive cells are shown. All plots are gated on viable cells. (c) BCR-ABL1-transformedpre-B ALL cells were transduced with GFP tagged retroviral vector encoding myristoylated AKT(AKTCA) or a GFP empty vector (EV) control in the presence or absence of AKT inhibitorAZD5363 (AZD, 3 µmol/liter). Representative flow cytometry plots for GFP positive cells areshown. All plots are gated on viable cells.

Figure S7: Hyperactivation of PI3K-AKT pathway is toxic in pre-B ALL cells

69 70 70 69

69 68 71 67

10 12.3 14 13

1.6 12.4 11 11

74 72 56 58

80 81 1.1 13

31 38 35 30 35 17 23 2034

31 92 83 27 90 6 81 5280

GF

PG

FP

GF

P

(a) Pre-B ALL cells lacking the ability toexpress an endogenous pre-BCR weretransduced with a non- auto-reactive(µHCNA ) or an auto-reactive (µHCAuto )chimeric pre-BCR or an empty vector(EV) control. Cells were pre-treated withthe specific Syk inhibitor PRT060318(PRT, 3 µmol/liter) or AKT inhibitorAZD5363 (AZD, 3 µmol/liter) for twodays and tamoxifen was added to induceBCR signaling. Representative flowcytometry plots for GFP positive cells areshown. All plots are gated on viable cells.


0.0

0.5

1.0

1.5

2.0

2.5

0.5

1.0

1.5

2.0

0.0

0.5

1.0

1.5

2.0

EV Cre EV Cre EV Cre

- + - + Rapa - + - + Rapa - + - + Rapamycin

Glu

cose

con

sum

ptio

n [fo

ld]

Lact

ate

prod

uctio

n [fo

ld]

AT

P le

vels

[fo

ld c

hang

e]

P = 0.0165 P = 0.0151 P = 0.0004

Figure S8: Hyperactivation of PI3K-AKT as a consequence of Pten deletion induces exhaustion of glucose and energy reserves in pre-B ALL.

Following 24 hours pre-treatment with vehicle or Rapamycin (50 nmol/liter), Pten deletionwas induced by tamoxifen in Ptenfl/fl BCR-ABL1-transformed pre-B ALL cells transducedwith Cre-ERT2 (Cre) or ERT2 empty vector (EV) control. Glucose consumption, lactateproduction and ATP levels were measured on day 2 following tamoxifen-dependentinduction of Cre. Values obtained were normalized to number of viable cells and areshown as average relative levels. P values were calculated by Student’s t test. Mean valuesof 3 independent experiments are shown. Error bars represent S.D.


LAX7R KYO

KU812

Four human pre-B ALL (LAX7R, LAX2,ICN1 and ICN12) and three human CML celllines (KYO, KU812 and JURL-MK1) weretransduced with a scrambled control or twoshRNA against PTEN. Numbers denotepercentages of viable cells. Transduced cellswere selected based on puromycin resistance.Cell viability was measured by flowcytometry (DAPI staining and forwardscatter). Representative plots are shown ondays 0 and 8 after puromycin selection.

Pre-B ALL CMLScrambled PTEN sh-1 PTEN sh-2 Scrambled PTEN sh-1 PTEN sh-2

Ph+ Pre-B ALLScrambled PTEN sh-1 PTEN sh-2

Figure S9: PTEN-knockdown is selectively toxic in patient-derived pre-B ALL cells.

48 47 46 72 59 69

33 4.5 0.5 78 80 80

15 15 1122 19 23

16 0.5 4.572 66 61

38 34 35

43 2.0 12

41 43 35

63 27 16

FSC

Day 0

Day 8

Day 0

Day 8

Day 0

Day 8

Day 0

Day 8

Day 0

Day 8

Day 0

Day 8

JURL-MK1

68 63 69

78 85 70

Day 0

Day 8

FSC

DA

PI

DA

PI

FSC

DA

PI

ICN1

LAX2

ICN12


Vehicle SF1670 [2.5 µmol/liter]

1.7 1.8 7221

Vehicle SF1670 [2.5 µmol/liter]

Ptenfl/fl +EV Ptenfl/fl +Cre Vehicle SF1670-1 day SF1670-2 day

CD19

Vpr

eB

931.5

50.5

347

248

970.1

2.80.1

470.7

493.3

601

318

Figure S10: Small molecule inhibitor of PTEN recapitulates genetic deletion of Pten in pre-B ALL cells

a

b

(a) CML (KYO-1, left) and pre-B ALL (PDX2, right) cells were treated with vehicle orSF1670 (2.5 µmol/liter) and the viability/apoptosis were measured by Annexin V and 7-AAD staining. Representative flow cytometry plots are shown. Percentages of dead cellsare shown. (b) Changes in expression of pre-B cell antigens CD19 and VpreB werestudied by flow cytometry upon Pten deletion in mouse pre-B ALL by Cre (left) or uponPTEN inhibition in a human TCF3-PBX1+ pre-B ALL case with SF1670 (2.5 µmol/liter,right). Plots are representative of two independent experiments.

7-AAD

Ann

exin

V


Supplementary Table 1: Human samples and cell lines

Patient derived ALL samples _____________________________________ ___ __________________________ ____________ Case Cytogenetics Oncogene Clinical course Gender/Age _______________________ ____________________________ _______________________________ ____ _ LAX2 t(9;22)(q34;q11) BCR-ABL1; p210, T315I Relapse (Imatinib) m/38 BLQ1 FISH der(9), der(22) BCR-ABL1; p210, T315I Relapse (Imatinib) BLQ5 FISH der(9), der(22) BCR-ABL1; p190, T315I Relapse (Imatinib) f BLQ11 FISH der(9), der(22) BCR-ABL1; p210, T315I Relapse (Imatinib) m TXL2 t(9;22)(q34;q11) BCR-ABL1; p210, unmutated at diagnosis TXL3 t(9;22)(q34;q11) BCR-ABL1; p210, unmutated at diagnosis ICN1 t(9;22)(q34;q11) BCR-ABL1; p210, unmutated at diagnosis PDX2 der(9)(q10)t(9;22)(q34;q11) BCR-ABL1; n.d. at diagnosis f/52 ICN12 t(1;19)(q23;p13) TCF3-PBX1 f/8 LAX7R KRASG12V Relapse ________________________________________ ____________________________________ ___________ Notes: All primary samples are bone marrow biopsies, blast content >80%; LAX, Los Angeles; BLQ, Bologna; TXL, Berlin; SFO, San Francisco; ICN, Seoul; n.d., not done; f, female; m, male Primary CML chronic phase cases __________________________________________ __________________________________ Patient Age at Dx Age at last F/U Duration F/U (year) Therapy BCR-ABL1 % (IS)a __ ____________________________________________________________________________ CP1 73 75 2 Imatinib <0.0024 CP2 29 29 0.5 Imatinib <0.0149 CP3 66 67 1 Imatinib <0.0024 CP4 59 61 2 Imatinib+IFN <0.22 CP5 18 18 0 Imatinib < 0.11 __ ____________________________________________________________________________ Notes: CP, chronic phase; Dx, diagnosis; F/U, follow-up. aMinimal residual disease quantitative PCR results

Cell lines ___ ___________________________________ ________________________________________ Cell line Disease Genetic alteration/Oncogenic lesion Source _____________ ________________________________ _________________________________ JSC-1 Primary Effusion Lymphoma KSHV+ EBV+, HIV associated Dr. J. Jung, USC BCP-1 Primary Effusion Lymphoma KSHV+, HIV negative Dr. J. Jung, USC BCBL-1 Primary Effusion Lymphoma KSHV+, HIV associated, Dr. J. Jung, USC APK-1 Primary Effusion Lymphoma KSHV+ EBV+ Dr. J. Jung, USC KYO Chronic Myeloid leukemia Ph+ t(9;22)(q34;q11) DSMZ KU812 Chronic Myeloid leukemia Ph+ t(9;22)(q34;q11) DSMZ JURL-MK1 Chronic Myeloid leukemia Ph+ t(9;22)(q34;q11) DSMZ 697 Acute Lymphonlastic leukemia t(1;19)(q23;p13) → TCF3-PBX1 fusion DSMZ _______________________ ________________________________ ___________________________________ Supplementary Table 2: viral vectors Constitutive expression retroviral vector

Construct Expression of Purpose _________________ _____________________________________________________________ MSCV IRES-GFP GFP Empty vector control MSCV IRES-Neo Neomycin resistance Empty vector control MSCV BCR-ABL1 IRES-Neo BCR-ABL1 Leukemic transformation (Ph+ ALL, CML) MSCV SYK (Y348E/Y352E) IRES-GFP SYK (Y348E/Y352E) SykCA MSCV IRES- AKT-Myr-GFP Myristoylated AKT AKTCA pQCXI LUC-BLAST Firefly-Luciferase Luciferase bioimaging MSCV IRES-NRASG12D-Puro NRASG12D Leukemic transformation (ALL) _______________ _____________________________________________________________


Supplementary Table 2 (continued): viral vectors Constitutive expression lentiviral vectors pLKO-Scramble Control knockdown pLKO-PTEN-shRNA-1320 PTEN knockdown pLKO-PTEN-shRNA-3001 PTEN knockdown ___ ____________________________________________________________________ ___________________ Inducible retroviral vector _____________ ______________ ________________________________________________ Construct Overexpression of Purpose _______________________ ______________ _________________________________________ MSCV ERT2 IRES-Puro Puromycin resistance Empty vector control MSCV ERT2 IRES-GFP GFP Empty vector control pRetroX-Tet3G Tet-On Empty vector control MSCV Cre ERT2 IRES-Puro Cre; Puromycin resistance Inducible activation of Cre MSCV Cre ERT2 IRES-GFP Cre; GFP Inducible activation of Cre

pRetroX-TRE3G-C/EBPα Tet-On; C/EBPα Inducible expression of C/EBP _______________________________________________________ __________________________________ _

Supplementary Table 3: Mouse strains ____ ________________________________________________________________ ________ Mouse strain Source Stock # ____________________________________________________ _________________________ Ptenfl/fl Jackson Laboratories 004597

Tp53fl/fl Jackson Laboratories 008462

NOD/SCID Jackson Laboratories 001303 Mb1-Cre* Hassan Jumma, Germany ______________________________________________________ ________________________________ ____ * Hobeika E et al. Testing gene function early in the B cell lineage in mb1-cre mice., Proc Natl Acad Sci U S A. 2006 ep 12;103(37):13789-94.


Supplementary Table 4: Antibodies Flow cytometry antibodies _________________________ __________________________________ _________________ Surface antigen Clone ID Manufacturer __________________________ _________________________________ __________________ CD19 1D3 BD Biosciences B220 RA3-6B2 BD Biosciences µ-HC (Ighm) 11//41 BD Biosciences c-kit 2B8 BD Biosciences Sca-1 D7 BD Biosciences CD80 16-10A1 BD Biosciences IL7R (CD127) SB/199 BD Bioscience CD25 (IL2ra) 3C7 BD Biosciences Mac1 M1/70 BD Biosciences VpreB1 R3 Biolegend ________________________________ ______________________________________________________ ____

Western blot antibodies ____ ________________________________________________________________ ________ Antigen Clone ID Manufacturer Dilution factor ___________________________________________________ _________________________ β-actin ab8227 Abcam 1 in 2500 Arf ab80 Abcam 1 in 1000 CD19 ab31947 Abcam 1 in 1000 PTEN A2B1 Santa Cruz 1 in 1000 p21 C-19 Santa Cruz 1 in 1000 Pax5 D19F8 Cell Signaling 1 in 1000 p53 1C12 Cell Signaling 1 in 1000 p-p53 (S15) 16G8 Cell Signaling 1 in 500 Syk 2712 Cell Signaling 1 in 2000 p-Syk (Y352) 65E4 Cell Signaling 1 in 1000 AKT 9272 Cell Signaling 1 in 2000 p-AKT-(S473) 9271 Cell Signaling 1 in 1000 p-AKT-(Y308) 2965 Cell Signaling 1 in 1000 p-RPS6-(S235/6) 2F9 Cell Signaling 1 in 1000 RPS6 5G10 Cell Signaling 1 in 2500 C/EBPα D56F10 Cell Signaling 1 in 1000 ______________________________________________________ ________________________________ ____

Supplementary Table 5: Inhibitors ____ ________________________________________________________________ ________ Inhibitor Target Manufacturer ____________________________________________________ _________________________ SF1670 PTEN Biovision AZD5363 AKT Selleckchem BKM120 PI3K Selleckchem PRT060318 SYK Medkoo Biosciences Rapamycin mTOR Selleckchem ______________________________________________________ ________________________________ ____


pten opposes negative selection and enables … · hassan jumaa & markus müschen inventory:...

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