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

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1 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 1-1 Supplementary Tables 1-5

2 Figure S1: Pten-deletion in pre-b cells is synthetically lethal with transformation by BCR-ABL1 a Pten fl/fl +EV Pten fl/fl +Cre PI 69 9 Day 6 (a) Pten fl/fl BCR-ABL1-transformed pre-b ALL cells were transduced with tamoxifen-induced Cre- ER T2 or an empty vector (EV) control. Cell viabilities were measured by flow cytometry using propidium iodide (PI). Numbers denote the percentages of viable cells. Representative flow cytometry plots are shown (n =3). Day 1 Day 2 Day 3 Day 4 Day 5 b Pten fl/fl +EV BCR-ABL1-GFP Pten fl/fl +Cre (b) IL7-dependent Pten fl/fl pre-b cells were transduced with Cre-ER T2 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. Representative flow cytometry plots for GFP positive cells are shown (n =3).

3 Figure S1(continued) c EV Cre Long-term surviving mice (c) One million Pten fl/fl BCR-ABL1- transformed pre-b ALL cells carrying tamoxifen-inducible Cre or an empty vector (EV) were injected into NOD/SCID recipient mice (n = 7 per group). Pten deletion was induced by treatment of the cells for 24 hours with tamoxifen (.5 µmol/liter) before injection. All mice in the Cre group were still alive at the end of the experiment (day 7) and showed no signs of disease. Mice at day 7 These mice were then sacrificed, bone MRD-PCR Controls: Pten fl/fl marrow mononucleated cells were isolated EV Cre H 2 O and analyzed by genomic PCR. Pten fl/fl pre-b ALL cells with and without in vitro-deletion Pten flox of Pten were used as positive controls as well Pten WT as water as negative control. PCR amplification of Pten genomic region from Pten del bone marrow of the NOD/SCID transplant recipients allowed to detect wildtype, floxed and deleted Pten alleles as markers for minimal residual disease (MRD). In the bone marrow of NOD/SCID transplant recipients, only wildtype Pten alleles and no signs of MRD were detected. Figure S2: Genotyping of colonies Pten fl/fl pre-b ALL cells growing in methylcellulose Colony Activation of Cre in vitro +/+ fl/fl H 2 O Pten flox Pten WT One colony of Pten fl/fl pre-b ALL cells transduced with Cre that had formed in methylcellulose (Fig. 2b) was isolated for genotyping by genomic PCR. Individual amplification products are detected for the floxed and deleted alleles of Pten. Pten del

4 Figure S3: Frequency of activating PI3K-AKT pathway mutations in hematological malignancies Pre-B ALL B-NHL Myeloid T cell lineage leukemia ALL (Top) The frequencies of activating mutations of AKT1, AKT3, PIK3CA, PIK3C2A, PIK3CD, PIK3R1, MTOR, RPS6KA2, RPS6KB2 genes that encode signaling molecules in the PI3K/AKT pathway were calculated for four types of hematopoietic malignancies, namely pre-b ALL, B cell Non- Hodgkin s Lymphoma (B-NHL), myeloid leukemia and T cell lineage ALL. Depicted is the number of samples studied (Seq), the number of cases with activating mutation (Mut). Mutation frequencies (bold face) are given as percentages of mutated versus total number of cases sequenced. (Bottom) Red and green bars represent the frequencies of activating and inactivating mutations, respectively. Mutation data was obtained from Catalogue of Somatic Mutations in Cancer database (COSMIC, nome/projects/cosmic/). 5 Mutation frequencies[%] AKT1, AKT3 PIK3CA, PIK3C2A, PIK3CD, PIK3R1 MTOR, RPS6KA2, RPS6KB2 PTEN AKT1, AKT3 PIK3CA, PIK3C2A, PIK3CD, PIK3R1 MTOR, RPS6KA2, RPS6KB2 PTEN AKT1, AKT3 PIK3CA, PIK3C2A, PIK3CD, PIK3R1 MTOR, RPS6KA2, RPS6KB2 PTEN AKT1, AKT3 PIK3CA, PIK3C2A, PIK3CD, PIK3R1 MTOR, RPS6KA2, RPS6KB2 PTEN

5 Figure S4: Correlation of PTEN expression with patient survival in T cell lineage vs pre-b ALL T cell lineage ALL Pre-B ALL Overall survival [%] P=.15; n=22 P=.759; n= PTEN HIGH Relapse-free survival [%] 5 PTEN HIGH Overall survival [%] P=.11; n=155 P=.415; n= PTEN LOW Relapse-free survival [%] PTEN LOW PTEN LOW PTEN LOW PTEN HIGH PTEN HIGH Follow up [years] Follow up [years] Follow up [years] Follow up [years] Reverse phase protein array (RPPA) data for PTEN expression were analyzed for patients with T cell lineage ALL (n = 22) and pre-b ALL (n = 155). Based on higher or lower than median expression levels of PTEN, patients were divided into two groups (PTEN High,PTEN Low ) and the overall survival as well as relapse-free survival of these patients are plotted over a follow up period of 3 to 18 years. P values were calculated by Mantel-Cox log rank test. 5 5 Figure S5: Small molecule inhibitors of Syk, PI3K and AKT rescue Pten-deletion a GFP Pten fl/fl BCR-ABL1-transformed Pten fl/fl NRAS G12D -transformed Day 1 (a) Pten fl/fl pre-b cells transformed with BCR-ABL1 or NRAS G12D were transduced with inducible Cre-GFP- ER T2 or a GFP empty vector (EV) control. Pten deletion was induced with tamoxifen in the presence or absence of AKT inhibitor 5363 (, 3 µmol/liter). Representative flow cytometry plots for GFP + cells are shown. All plots are gated on viable cells b PRT BKM PRT BKM D GFP D12 (b) Pten deletion was induced in BCR-ABL1-transformed pre-b ALL cells in the presence or absence of AKT inhibitor 5363 (, 3 µmol/liter), PI3K inhibitor BKM12 (BKM, 1 µmol/liter) and Syk inhibitor PRT6318 (PRT, 3 µmol/liter). Representative flow cytometry plots for GFP + cells are shown. All plots are gated on viable cells.

6 Figure S6: Monoallelic deletion of Pten in pre-b ALL cells compromises leukemia initiation in vivo a Pten fl/fl + EV Pten +/fl + Cre Pten fl/fl + Cre 291 ± ± 4 8 ± 3 b BrdU Pten fl/fl + EV Pten +/fl + Cre Pten fl/fl + Cre S S S G 2 /M G /G 1 G 2 /M G /G 1 G 2 /M G /G 1 7AAD -/- Pten fl/fl 54% P =.7 +/- 45% -/- 47% P =.8 G/1 S G2/M phase c d Pten fl/fl + EV Pten +/fl + Cre Pten fl/fl + Cre 2.5% ± 2% 2.5% ± 7% 37.7% ± 6% BCR-ABL1 Pten +/+, Pten +/fl and Pten fl/fl ALL + Cre Pten fl/fl Pten [Colonies/1 4 ] fl/fl P = /- -/- CFU/1, plated cells P = / Senescence-associated-βgal + cells [%] P =.5 P =.2 Pten +/+ Pten +/fl Pten fl/fl Day 8 Day 15 Day 2 5 Disease-free survival [%]1 Pten +/fl Pten fl/fl Pten +/+ Pten +/+ vs Pten +/fl P =.3 Pten +/+ vs Pten fl/fl P =.2 Pten +/fl vs Pten fl/fl P > Days after injection e 3, p/sec/cm 2 /sr 3, Pten fl/fl Pten +/fl EV Cre EV Cre AKT-pT 38 AKT -actin To compare the effects of deletion of one vs both alleles of Pten, Pten fl/fl and Pten +/fl BCR-ABL1-transformed pre-b cells were transduced with tamoxifen-inducible Cre-ER T2 or an empty vector (EV) control. (a) Effects of Pten deletion on colony forming ability of pre-b ALL were measured by plating 1, cells after tamoxifen induction in methylcellulose. (b) Following 24 hours of tamoxifen-induced Pten deletion, cell cycle progression was assayed by measuring genomic BrdU incorporation which represents the cells in the S phase.

7 Figure S6: Monoallelic deletion of Pten in pre-b ALL cells compromises leukemia initiation in vivo Legend (continued): (c) Cellular senescence was detected by measuring β-galactosidase activity under acidic conditions (ph=5.5) after deletion of Pten. The percentages of cells stained with blue dots are depicted on the panels. Scale bars in (a) and(c) represent 1 mm and 5 µm respectively. P values (a c) were calculated by Student s t test. (d) 1 5 luciferase-labeled Pten +/+, Pten +/fl and Pten fl/fl BCR-ABL1-transformed pre-b ALL cells transduced with Cre-ER T2 were injected into NOD/SCID recipient mice (n = 7 per group). Pten deletion was induced by treatment of the cells for 24 hours with tamoxifen (.5 µmol/liter) before injection. P values were calculated by Mantel-Cox log rank test. Scale bars = 3 cm (e) Western blot was performed for total and T 38 -phosphorylated AKT after 48 hours of Pten deletion in Pten fl/fl and Pten fl/+ BCR- ABL1 transformed pre-b ALL cells. β-actin was used as loading control. Representative blot, n = 3.

8 Figure S7: Hyperactivation of PI3K-AKT pathway is toxic in pre-b ALL cells a PRT µhc NA + µhc NA + PRT µhc NA + µhc Auto + µhc Auto + PRT µhc Auto Day 1 GFP GFP b PRT BKM Syk CA + Syk CA + PRT Syk CA + BKM Syk CA Day 5 Day 12 c GFP AKT CA + AKT CA (b) BCR-ABL1-transformed pre-b ALL cells were transduced with retroviral vector encoding active form of Syk (Syk CA :Syk Y348E/Y352E ) or a GFP empty vector (EV) control in the presence of vehicle, Syk inhibitor PRT6318 (PRT, 3 µmol/liter), PI3K inhibitor BKM12 (BKM, 1 µmol/liter) and AKT inhibitor 5363 (, 3 µmol/liter). Representative flow cytometry plots for GFP positive cells are shown. All plots are gated on viable cells. (c) BCR-ABL1-transformed pre-b ALL cells were transduced with GFP tagged retroviral vector encoding myristoylated AKT (AKT CA ) or a GFP empty vector (EV) control in the presence or absence of AKT inhibitor 5363 (, 3 µmol/liter). Representative flow cytometry plots for GFP positive cells are shown. All plots are gated on viable cells. Day 7 (a) Pre-B ALL cells lacking the ability to express an endogenous pre-bcr were transduced with a non- auto-reactive (µhc NA )oranauto-reactive(µhc Auto ) chimeric pre-bcr or an empty vector (EV) control. Cells were pre-treated with the specific Syk inhibitor PRT6318 (PRT, 3 µmol/liter) or AKT inhibitor 5363 (, 3 µmol/liter) for two days and tamoxifen was added to induce BCR signaling. Representative flow cytometry plots for GFP positive cells are shown. All plots are gated on viable cells.

9 Figure S8: Hyperactivation of PI3K-AKT as a consequence of Pten deletion induces exhaustion of glucose and energy reserves in pre-b ALL. 2. P =.165 P =.151 P = Glucose consumption [fold] Lactate production [fold] ATP levels [fold change] EV Cre EV Cre EV Cre Rapa Rapa Rapamycin Following 24 hours pre-treatment with vehicle or Rapamycin (5 nmol/liter), Pten deletion was induced by tamoxifen in Pten fl/fl BCR-ABL1-transformed pre-b ALL cells transduced with Cre-ER T2 (Cre) or ER T2 empty vector (EV) control. Glucose consumption, lactate production and ATP levels were measured on day 2 following tamoxifen-dependent induction of Cre. Values obtained were normalized to number of viable cells and are shown as average relative levels. P values were calculated by Student s t test. Mean values of 3 independent experiments are shown. Error bars represent S.D.

10 Figure S9: PTEN-knockdown is selectively toxic in patient-derived pre-b ALL cells. Pre-B ALL CML Scrambled PTEN sh-1 PTEN sh-2 Scrambled PTEN sh-1 PTEN sh-2 LAX7R KYO Day 8 Day ICN12 KU812 DAPI DAPI LAX2 ICN Ph + Pre-B ALL Scrambled PTEN sh-1 PTEN sh Day 8 Day 8 Day 8 JURL- MK1 DAPI Four human pre-b ALL (LAX7R, LAX2, ICN1 and ICN12) and three human CML cell lines (KYO, KU812 and JURL-MK1) were transduced with a scrambled control or two shrna against PTEN. Numbers denote percentages of viable cells. Transduced cells were selected based on puromycin resistance. Cell viability was measured by flow cytometry (DAPI staining and forward scatter). Representative plots are shown on days and 8 after puromycin selection. Day 8 Day 8

11 Figure S1: Small molecule inhibitor of PTEN recapitulates genetic deletion of Pten in pre-b ALL cells a Annexin V SF167 [2.5 µmol/liter] SF167 [2.5 µmol/liter] b 7-AAD Pten fl/fl +EV Pten fl/fl +Cre SF167-1 day SF167-2 day VpreB CD19 (a) CML (KYO-1, left) and pre-b ALL (PDX2, right) cells were treated with vehicle or SF167 (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 cells are shown. (b) Changes in expression of pre-b cell antigens CD19 and VpreB were studied by flow cytometry upon Pten deletion in mouse pre-b ALL by Cre (left) or upon PTEN inhibition in a human TCF3-PBX1 + pre-b ALL case with SF167 (2.5 µmol/liter, right). Plots are representative of two independent experiments.

12 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; p21, T315I Relapse (Imatinib) m/38 BLQ1 FISH der(9), der(22) BCR-ABL1; p21, T315I Relapse (Imatinib) BLQ5 FISH der(9), der(22) BCR-ABL1; p19, T315I Relapse (Imatinib) f BLQ11 FISH der(9), der(22) BCR-ABL1; p21, T315I Relapse (Imatinib) m TXL2 t(9;22)(q34;q11) BCR-ABL1; p21, unmutated at diagnosis TXL3 t(9;22)(q34;q11) BCR-ABL1; p21, unmutated at diagnosis ICN1 t(9;22)(q34;q11) BCR-ABL1; p21, unmutated at diagnosis PDX2 der(9)(q1)t(9;22)(q34;q11) BCR-ABL1; n.d. at diagnosis f/52 ICN12 t(1;19)(q23;p13) TCF3-PBX1 f/8 LAX7R KRAS G12V Relapse Notes: All primary samples are bone marrow biopsies, blast content >8%; 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 CP Imatinib <.24 CP Imatinib <.149 CP Imatinib <.24 CP Imatinib+IFN <.22 CP Imatinib <.11 Notes: CP, chronic phase; Dx, diagnosis; F/U, follow-up. a Minimal 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) Syk CA MSCV IRES- AKT-Myr-GFP Myristoylated AKT AKT CA pqcxi LUC-BLAST Firefly-Luciferase Luciferase bioimaging MSCV IRES-NRAS G12D -Puro NRAS G12D Leukemic transformation (ALL)

13 Supplementary Table 2 (continued): viral vectors Constitutive expression lentiviral vectors plko-scramble Control knockdown plko-pten-shrna-132 PTEN knockdown plko-pten-shrna-31 PTEN knockdown Inducible retroviral vector Construct Overexpression of Purpose MSCV ER T2 IRES-Puro Puromycin resistance Empty vector control MSCV ER T2 IRES-GFP GFP Empty vector control pretrox-tet3g Tet-On Empty vector control MSCV Cre ER T2 IRES-Puro Cre; Puromycin resistance Inducible activation of Cre MSCV Cre ER T2 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 # Pten fl/fl Jackson Laboratories 4597 Tp53 fl/fl Jackson Laboratories 8462 NOD/SCID Jackson Laboratories 133 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. 26 ep 12;13(37):

14 Supplementary Table 4: Antibodies Flow cytometry antibodies Surface antigen Clone ID Manufacturer CD19 1D3 BD Biosciences B22 RA3-6B2 BD Biosciences µ-hc (Ighm) 11//41 BD Biosciences c-kit 2B8 BD Biosciences Sca-1 D7 BD Biosciences CD8 16-1A1 BD Biosciences IL7R (CD127) SB/199 BD Bioscience CD25 (IL2ra) 3C7 BD Biosciences Mac1 M1/7 BD Biosciences VpreB1 R3 Biolegend Western blot antibodies Antigen Clone ID Manufacturer Dilution factor β-actin ab8227 Abcam 1 in 25 Arf ab8 Abcam 1 in 1 CD19 ab31947 Abcam 1 in 1 PTEN A2B1 Santa Cruz 1 in 1 p21 C-19 Santa Cruz 1 in 1 Pax5 D19F8 Cell Signaling 1 in 1 p53 1C12 Cell Signaling 1 in 1 p-p53 (S15) 16G8 Cell Signaling 1 in 5 Syk 2712 Cell Signaling 1 in 2 p-syk (Y352) 65E4 Cell Signaling 1 in 1 AKT 9272 Cell Signaling 1 in 2 p-akt-(s473) 9271 Cell Signaling 1 in 1 p-akt-(y38) 2965 Cell Signaling 1 in 1 p-rps6-(s235/6) 2F9 Cell Signaling 1 in 1 RPS6 5G1 Cell Signaling 1 in 25 C/EBPα D56F1 Cell Signaling 1 in 1 Supplementary Table 5: Inhibitors Inhibitor Target Manufacturer SF167 PTEN Biovision 5363 AKT Selleckchem BKM12 PI3K Selleckchem PRT6318 SYK Medkoo Biosciences Rapamycin mtor Selleckchem