Supporting InformationPfeifer et al. 10.1073/pnas.1305656110SI Materials and MethodsCell Culture, Retroviral Constructs, and Transductions. Human dif-fuse large B-cell lymphoma (DLBCL) cell lines were cultured inRPMI (Invitrogen) with 10% (vol/vol) FCS (Sigma), except forOCI-Ly1, OCI-Ly2, OCI-Ly4, OCI-Ly7, OCI-Ly10, OCI-Ly19,and TMD8, which were cultured in Iscove’s modified Dulbeccomedium supplemented with either 20% human plasma or 10%FCS. All cell lines were maintained at 37 °C with 5% CO2.For efficient retroviral transductions, cell lines were engineered

to express the murine ecotropic receptor as previously described(1). Additionally, these cell lines were engineered to express thebacterial tetracycline repressor, allowing doxycycline-inducibleshRNA or cDNA expression. shRNA-mediated RNA inter-ference was performed as described (1). The targeting sequencesofMYC shRNAs 1 and 2 were CGATTCCTTCTAACAGAAATGand CCTATGAACTTGTTTCAAATG, respectively. As a negativecontrol, we used a previously described shRNA directed againstMSMO1 (2). PTEN (NM_000314.4),MYC (NM_002467.2), and aconstitutive active AKT cDNA (NM_005163.2) (3) were insertedinto either a modified version of the inducible pRetroSuper dualpromoter vector or a noninducible pMSCV vector as previouslydescribed (4). PTEN- and mutant PTEN-induced toxicity wasassessed as previously described (4). Briefly, PTEN or mutantPTEN cDNA was transduced using retroviruses. The expressionvector coexpressed GFP, allowing us to monitor the proportion ofGFP+ vs. GFP− cells over time as a measure of toxicity of the co-expressed PTEN cDNA. Rescue experiments were performed aspreviously described (2). Each experiment was completely repro-duced at least two to eight times for each cell line.

Gene Expression Profiling. Gene expression profiling after reex-pression of PTEN cDNA was performed in the PTEN-deficientgerminal center B-cell-like (GCB) DLBCL cell line HT. HT cellswere transduced with PTEN cDNA and selected with puromycin,and PTEN cDNA expression was induced with doxycycline; 6, 12,18, and 24 h after PTEN induction, total RNA was isolated usingthe NucleoSpin RNA II Kit (Macherey & Nagel) according tothe manufacturer’s protocol. RNA was amplified and labeledwith the TotalPrep RNA Amplification Kit (Illumina), and la-beled samples were hybridized on HumanHT-12 v4 ExpressionBeadChips (Illumina) using the manufacturer’s protocol. Thegene expression data have been deposited in the Gene Expres-sion Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo/;accession no. GSE45495).PTEN-induced changes in gene expression were measured in

two completely independent biologic replicates, and gene ex-pression changes in PTEN-transduced cells were compared withcells that were transduced with the empty vector alone. The in-dependent measurements were preprocessed and normalizedin the following manner. Data were imported on raw bead level,and subsequently, a bead-level spot filter was applied for eachmicroarray based on the fitted density mode for the backgroundintensities. Afterward, bead intensities of all measured micro-arrays were quantile-normalized, and beads were grouped bymeasured sequence to form bead sets. For genes having morethan one measured sequence, additional merged bead sets werecreated. Bead sets with more than 50% of their beads excludedby the spot filter were also excluded. Additional analyses wasperformed on gene level using median aggregation.Differentially expressed genes were identified in the following

manner. A one-tailed paired t test was used to calculate P valuesfor every gene based on the eight microarray pairs. Additionally,

we used the Benjamini and Hochberg method to calculate a falsediscovery rate (FDR) for every α-significance threshold. Gen-erally, more genes were up-regulated (546 genes; P < 0.01 andFDR < 0.11) than down-regulated (279 genes; P < 0.01 andFDR < 0.11) by PTEN. Using tighter cutoffs, we identified 72genes that were significantly down-regulated (P < 0.0025; FDR <0.06) and 82 genes that were up-regulated (P < 0.00025; FDR <0.02) across all time points after PTEN induction (paired t testbased on two independent replicates) (Fig. 4B and Table S6).To obtain a better understanding of the gene expression changes,

we performed an unbiased gene set enrichment analysis (GSEA) aspreviously described using a previously curated gene expressionsignature database (5, 6). The most enriched gene signatures fromthis query (enrichment score >0.5) are reported in Table S7.Additionally, we performed gene expression profiling of 34

primary DLBCL patient samples using Affymetrix GeneChipHuman Exon 1.0 ST v2 microarrays. Samples from this cohortwere later used as the basis for PTEN staining. These microar-rays were preprocessed with robust multiarray average (RMA)background subtraction and quantile normalization. Probes wereaggregated by median polish on gene level based on the v14.1 ofthe Entrez Gene CDF for Affymetrix GeneChip Human Exon1.0 ST v2 (7).The DLBCL samples were classified into activated B-cell-like

(ABC) and GCB DLBCL as previously described (8). We used tworeference samples from a previous cohort (9) to transfer the molec-ular subtypeprediction toAffymetrixGeneChipHumanExonarrays.

PCR Amplification and Sequencing. PCR amplification and sequenc-ing were performed as previously described (10). The sequencesfor primers applied to amplify PTEN exons are summarized inTable S4.

Array CGH. Array comparative genomic hybridization (aCGH) inDLBCL cell lines was performed as previously described (11).aCGH data from 10 DLBCL cell lines were obtained from GEOthrough GEO accession no. GSE43272. The aCGH data fromthe remaining cell lines have been deposited in the GEO data-base (accession no. GSE45495).

Quantitative Genomic PCR.To determine the DNA copy number ofthe PTEN locus, we used a predesigned assay according to themanufacturer’s protocol (Invitrogen).

FISH. Primary DLBCL patient samples from cohorts 1 and 2 wereinvestigated for the presence of MYC translocations using FISHas previously described (12, 13).

Cell Viability Assay, Annexin-V Staining, and SNARF-1 ProliferationAssay. GCB DLBCL cells were incubated with DMSO or differ-ent concentrations of the pan-PI3K inhibitor Ly294002 (CaymanChemicals). Cell viability was measured after incubation for 4 dusing the Cell Proliferation Kit II (Roche) according to themanufacturer’s recommendations.Annexin-V PE (BDPharmingen) staining was performed in the

GCB DLBCL cell lines BJAB, HT, and K422 2 d after PTENcDNA transduction according to the manufacturer’s protocol.Annexin-V staining of GFP-positive cells was measured by flowcytometry.SNARF-1 (carboxylic acid, acetate, and succinimidyl ester; Life

Technologies) staining was performed in the DLBCL cell linesBJAB,HT, andK422 beforePTEN cDNA transduction. SNARF-1dilutions were measured 2 and 6 d after PTEN transduction.

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SNARF staining was assessed by flow cytometry according to themanufacturer’s protocol.

Western Blotting. Western blotting was performed as previouslydescribed (2). In brief, whole-cell protein lysates were harvestedfrom DLBCL cell lines or primary patient samples in Phospho-safe lysis buffer (EMD Millipore), and protein was quantifiedusing the BCA assay (Thermo Scientific). Subsequently, lysateswere separated by SDS/PAGE on 12% polyacryalmide gels and

transferred to polyvinylidene difluoride membranes (Millipore).All antibodies used in this study were obtained from Cell Signal-ing, except for PTEN (Santa Cruz), MYC (Epitomics), p-MYC(Abcam), actin, and α-tubulin (Sigma).Western blotting for p-AKT was performed in DLBCL cell

lines and 16 primary GCB DLBCL samples from cohort 1. Inaddition, four GCB DLBCL samples with known PTEN sta-tus, for which frozen material was available, were analyzed forp-AKT levels.

1. Ngo VN, et al. (2006) A loss-of-function RNA interference screen for molecular targetsin cancer. Nature 441(7089):106–110.

2. Wenzel SS, et al. (2013) MCL1 is deregulated in subgroups of diffuse large B-celllymphoma. Leukemia 27(6):1381–1390.

3. Kharas MG, et al. (2010) Constitutively active AKT depletes hematopoietic stem cellsand induces leukemia in mice. Blood 115(7):1406–1415.

4. Hailfinger S, et al. (2011) Malt1-dependent RelB cleavage promotes canonical NF-kappaB activation in lymphocytes and lymphoma cell lines. Proc Natl Acad Sci USA 108(35):14596–14601.

5. Shaffer AL, et al. (2006) A library of gene expression signatures to illuminate normaland pathological lymphoid biology. Immunol Rev 210:67–85.

6. Subramanian A, et al. (2005) Gene set enrichment analysis: A knowledge-basedapproach for interpreting genome-wide expression profiles. Proc Natl Acad Sci USA102(43):15545–15550.

7. Dai M, et al. (2005) Evolving gene/transcript definitions significantly alter theinterpretation of GeneChip data. Nucleic Acids Res 33(20):e175.

8. Wright G, et al. (2003) A gene expression-based method to diagnose clinically distinctsubgroups of diffuse large B cell lymphoma. Proc Natl Acad Sci USA 100(17):9991–9996.

9. Hummel M, et al. (2006) A biologic definition of Burkitt’s lymphoma fromtranscriptional and genomic profiling. N Engl J Med 354(23):2419–2430.

10. Lenz G, et al. (2008) Oncogenic CARD11 mutations in human diffuse large B celllymphoma. Science 319(5870):1676–1679.

11. Lenz G, et al. (2008) Molecular subtypes of diffuse large B-cell lymphoma arise bydistinct genetic pathways. Proc Natl Acad Sci USA 105(36):13520–13525.

12. Obermann EC, Csato M, Dirnhofer S, Tzankov A (2009) Aberrations of the MYC genein unselected cases of diffuse large B-cell lymphoma are rare and unpredictable bymorphological or immunohistochemical assessment. J Clin Pathol 62(8):754–756.

13. Kwanhian W, et al. (2012) MicroRNA-142 is mutated in about 20% of diffuse large B-cell lymphoma. Cancer Med 1(2):141–155.

85 86 87 88 89 90 91 92 93 94

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number

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ABC DLBCL

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Fig. S1. PTEN is deregulated in GCB DLBCL. (A) OCI-Ly1 and K422 cells are characterized by heterozygous deletions of the PTEN locus. (B) Analysis of PTEN DNAcopy number using quantitative PCR. Each bar represents a DLBCL cell line. (C) OCI-Ly1 cells are characterized by an internal PTEN deletion affecting exons 6–9.NTC, nontemplate control.

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AY68H SUDHL-10Wildtype OCI-Ly7 Wildtype OCI-Ly7 D162H SUDHL-10

B Wildtype OCI-Ly7 N276S, T277A RL

CWildtype OCI-Ly7 L320* OCI-Ly4 T321 Frame shift OCI-Ly4

DWildtype OCI-Ly7 Splice acceptor mutation Ex 3 K422

EG209FS patient_019 tumor DNA Germ-line DNA patient_019

G

ΔY177 patient_028 tumor DNA Germ-line DNA patient_028F

Fig. S2. PTEN is deregulated by somatic PTEN mutations in GCB DLBCL. (A) SUDHL-10 cells are characterized by two different PTEN missense mutations. Arrowsindicate mutations compared with WT sequence detected in OCI-Ly7 cells. (B) In RL cells, two different PTEN missense mutations are detectable. Arrows indicatemutations compared withWT sequence detected in OCI-Ly7 cells. (C) OCI-Ly4 cells are characterized by a nonsense and a frame shift PTENmutation. Arrows indicatemutations compared with WT sequence detected in OCI-Ly7 cells (depicted are cloned and sequenced PCR products). (D) K422 cells harbor a splice acceptor mu-tation. Arrows indicate the mutation compared with WT sequence detected in OCI-Ly7 cells. (E) Three base pair deletion detected in the GCB DLBCL patient sample028. Arrows indicate the position of the first nucleotide of the deletion compared with WT sequence detected in the patients’ germ-line DNA. (F) Frame shiftmutation detected in the ABC DLBCL patient sample 019. Arrows indicate aberration compared with WT sequence detected in the patients’ germ-line DNA. (G) GCBDLBCL patient sample 028, which is characterized by a three base pair PTEN deletion, does not express detectable PTEN protein levels by immunohistochemistry.

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PTEN

FLAG

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Fig. S3. PTEN-deficient GCB DLBCL cell lines are addicted to PI3K/AKT signaling. (A) PTEN-deficient (BJAB and HT) and PTEN-positive (OCI-Ly19) DLBCL cell linesexpress similar levels of exogenous PTEN after retroviral transduction. (B) Reexpression of PTEN induces apoptosis in BJAB but not in HT measured by anincrease in cleaved caspases 3 and 9 by Western blotting. (C) PTEN mutants induce reduced or no toxicity in the PTEN-deficient GCB DLBCL cell line RL comparedwith PTEN WT and no toxicity in the PTEN-positive cell line OCI-Ly19. (D) Phosphatase-inactive PTEN mutant C124S does not induce toxicity in DLBCL cell lines.

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MYC

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Fig. S4. PTEN loss up-regulates MYC in GCB DLBCL. (A) PI3K/AKT inhibition using the pan-PI3K inhibitor Ly294002 down-regulates MYC protein expression inPTEN-deficient GCB DLBCL cell lines BJAB, HT, and K422. (B) Exogeneous MYC protein expression in BJAB and HT cells to perform the MYC rescue experiment(Fig. 4F). (C) MYC shRNA 1 and 2 significantly down-regulate MYC protein measured by Western blotting. (D) shRNA-mediated MYC knockdown (MYC shRNA 2)is toxic to PTEN-positive and -deficient GCB DLBCL cell lines. A negative control shRNA does not induce toxicity. (E) Expression of an MYC cDNA rescues BJAB,HT, and K422 cells transduced withMYC shRNAs 1 and 2 (targeting the MYC 3′UTR) from toxicity. Representative results from two independent replicates areshown. (F) MYC protein expression in ABC and GCB DLBCL cell lines determined by Western blotting.

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Table S1. PTEN expression in subtypes of DLBCL determined byimmunohistochemistry using a cutoff level of <10% of positivelymphoma cells

Molecular subtype andpatient cohort PTEN positive PTEN negative n

GCB DLBCL cohort 1 9 9 18Non-GCB DLBCL cohort 1 12 4 16GCB DLBCL cohort 2 29 42 71Non-GCB DLBCL cohort 2 88 55 143GCB DLBCL combined 38 51 89Non-GCB DLBCL combined* 100 59 159

*P = 0.003.

Table S2. Summary of PTEN genomic analyses in DLBCL cell lines

Cell line DLBCL subtypePTEN mutation

status PTEN DNA copy number PTEN inactivation patternPTEN proteinexpression

p-AKTstatus

HBL-1 ABC WT 2 No aberration detectable + +OCI-Ly10 ABC WT 2 No aberration detectable + +TMD8 ABC WT 2 No aberration detectable + +DB GCB WT 1 Monoallelic inactivation + −OCI-Ly2 GCB WT 2 No aberration detectable + −OCI-Ly7 GCB WT 2 No aberration detectable + −OCI-Ly19 GCB WT 2 No aberration detectable + −OCI-Ly1 GCB ΔExons 6-9 1 exons 1-5, 0 exons 6-9 Biallelic inactivation − +OCI-Ly4 GCB L320*, T321FS 4 Monoallelic inactivation − +BJAB GCB WT 3 No aberration detectable − +HT GCB WT 2 No aberration detectable − +RL GCB N276S, T277A 2 Monoallelic inactivation − −SUDHL-10 GCB Y68H, D162H 1 Biallelic inactivation − +K422 GCB SA Exon3 1 Biallelic inactivation − +

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Table S3. Summary of PTEN genomic analyses in primary DLBCL patient samples of cohort 1

Patient IDDLBCLsubtype

PTEN mutationstatus

PTEN DNA copynumber

PTEN inactivationpattern

PTEN proteinexpression

p-AKTstatus

MYC translocationstatus

DLBCL_017 ABC WT 2 No aberration detectable + n.a. NegativeDLBCL_019 ABC G209FS 2 Monoallelic inactivation + n.a. NegativeDLBCL_041 ABC WT 1 Monoallelic inactivation − n.a. NegativeDLBCL_050 ABC WT 2 No aberration detectable + n.a. NegativeDLBCL_069 ABC WT 3 No aberration detectable + n.a. NegativeDLBCL_073 ABC WT 2 No aberration detectable + n.a. NegativeDLBCL_092 ABC WT 2 No aberration detectable + n.a. NegativeDLBCL_111 ABC WT 2 No aberration detectable + n.a. NegativeDLBCL_016 GCB WT 3 No aberration detectable + − NegativeDLBCL_021 GCB WT 3 No aberration detectable − + NegativeDLBCL_025 GCB WT 2 No aberration detectable + n.a. NegativeDLBCL_028 GCB ΔY177 1 Biallelic inactivation − + NegativeDLBCL_034 GCB WT 2 No aberration detectable + − NegativeDLBCL_037 GCB WT 2 No aberration detectable + n.a. NegativeDLBCL_054 GCB WT 1 Monoallelic inactivation + − NegativeDLBCL_058 GCB WT 2 No aberration detectable + − PositiveDLBCL_059 GCB WT 2 No aberration detectable + + NegativeDLBCL_061 GCB WT 2 No aberration detectable − + NegativeDLBCL_071 GCB WT 1 Monoallelic inactivation − + NegativeDLBCL_076 GCB WT 2 No aberration detectable − + NegativeDLBCL_079 GCB WT 2 No aberration detectable − + NegativeDLBCL_085 GCB WT 2 No aberration detectable − + PositiveDLBCL_090 GCB WT 3 No aberration detectable + + n.a.DLBCL_104 GCB WT 2 No aberration detectable − + NegativeDLBCL_114 GCB WT 2 No aberration detectable − + n.a.DLBCL_099 GCB WT 3 No aberration detectable + − NegativeDLBCL_006 Unclassified WT 2 No aberration detectable + n.a. NegativeDLBCL_013 Unclassified WT 1 Monoallelic inactivation + n.a. NegativeDLBCL_040 Unclassified WT 2 No aberration detectable + n.a. PositiveDLBCL_042 Unclassified WT 1 Monoallelic inactivation + n.a. NegativeDLBCL_057 Unclassified WT 2 No aberration detectable − n.a. n.a.DLBCL_063 Unclassified WT 1 Monoallelic inactivation − n.a. n.a.DLBCL_081 Unclassified WT 2 No aberration detectable + n.a. NegativeDLBCL_107 Unclassified WT 2 No aberration detectable + n.a. Negative

n.a., not available.

Table S4. Sequences of primers used for amplification of PTENexons

Exon Primer name Sequence

1 PTEN_1_f 5′-TTTCCATCCTGCAGAAGAAG-3′1 PTEN_1_r 5′-CATTTTCGCATCCGTCTACT-32 PTEN_2_f 5′-CTCCAGCTATAGTGGGGAAAACTTTC-3′2 PTEN_2_r 5′-CCCCTGAAGTCCATTAGGTACGG-3′3 PTEN_3_f 5′-ATATTCTCTGAAAAGCTCTGG-3′3 PTEN_3_r 5′-TTAATCGGTTTAGGAATACAA-3′4 PTEN_4_f 5′-TTGAAAAAGGTGATCGTTGG-3′4 PTEN_4_r 5′-ATTGTTATGACAGTAAGATACAGTCTATCG-3′5 PTEN_5_f 5′-GACCTATGCTACCAGTCCGTA-3′5 PTEN_5_r 5′-CACCTCAATAAAACTGAAGGAAAAA-3′6 PTEN_6_f 5′-AATGTATATATGTTCTTAAATGGCTACGA-3′6 PTEN_6_r 5′-GCTTCAGAAATATAGTCTCCTGCAT-3′7 PTEN_7_f 5′-CAGATACAGAATCCATATTTCGTG-3′7 PTEN_7_r 5′-AAGCAAAACACCTGCAGATCTAATA-3′8 PTEN_8_f 5′-AAATGCAACAGATAACTCAGATTG-3′8 PTEN_8_r 5′-AGCAAGTTCTTCATCAGCTGTACT-3′9 PTEN_9_f 5′-GTTTAAGATGAGTCATATTTG-3′9 PTEN_9_r 5′-TGGTGTTTTATCCCTCTTGAT-3′

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Table S5. Patient samples analyzed by Western blotting for p-AKT

Sample no. inWestern blot Patient ID

PTEN protein status byimmunohistochemistry DLBCL subtype

1 DLBCL_8862 0 GCB2 DLBCL_10483 0 GCB3 DLBCL_5332 0 GCB4 DLBCL_085 0 GCB5 DLBCL_104 0 GCB6 DLBCL_028 0 GCB7 DLBCL_061 0 GCB8 DLBCL_071 0 GCB9 DLBCL_076 0 GCB10 DLBCL_114 0 GCB11 DLBCL_021 0 GCB12 DLBCL_079 0 GCB13 DLBCL_18143 1 GCB14 DLBCL_090 1 GCB15 DLBCL_054 1 GCB16 DLBCL_058 1 GCB17 DLBCL_059 1 GCB18 DLBCL_016 1 GCB19 DLBCL_034 1 GCB20 DLBCL_099 1 GCB

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Table S6. Up- and down-regulated genes after PTEN induction

Signature nameGenesymbol Probe set name Gene ID Gene description

Down-regulated genes AATF ILMN_1703743 26574 Apoptosis antagonizingtranscription factor

Down-regulated genes ACTL8 ILMN_1704078 81569 Actin-like 8Down-regulated genes ACTN4 ILMN_1725534 81 Actinin, α4Down-regulated genes APOL3 ILMN_1756862 80833 Apolipoprotein L, 3Down-regulated genes BRMS1 ILMN_2398432 25855 Breast cancer metastasis

suppressor 1Down-regulated genes CACTIN 1) ILMN_2350421, 2) ILMN_2262462 58509 Cactin, spliceosome C complex

subunitDown-regulated genes CASP1 1) ILMN_2326509, 2) ILMN_2326512 834 Caspase 1, apoptosis-related

cysteine peptidaseDown-regulated genes CD48 ILMN_2061043 962 CD48 moleculeDown-regulated genes CDC42P2 ILMN_3282321 643336 Cell division cycle 42

pseudogene 2Down-regulated genes CDK18 ILMN_2284222 5129 Cyclin-dependent kinase 18Down-regulated genes CEACAM1 1) ILMN_2371724, 2) ILMN_1716815, 3) ILMN_1664330 634 Carcinoembryonic antigen-

related cell adhesionmolecule 1 (biliaryglycoprotein)

Down-regulated genes CENPN ILMN_1720526 55839 Centromere protein NDown-regulated genes CHN1 1) ILMN_1679638, 2) ILMN_1678493 1123 Chimerin (chimaerin) 1Down-regulated genes COL5A1 ILMN_1706505 1289 Collagen, type V, α1Down-regulated genes CORO1C ILMN_1745954 23603 Coronin, actin binding

protein, 1CDown-regulated genes DHX37 1) ILMN_2192683, 2) ILMN_1805742 57647 DEAH (Asp-Glu-Ala-His) box

polypeptide 37Down-regulated genes EPSTI1 ILMN_2388547 94240 Epithelial stromal interaction

1 (breast)Down-regulated genes FAM136BP ILMN_3241756 387071 Family with sequence similarity

136, member B, pseudogeneDown-regulated genes FAM153C ILMN_1765002 653316 Family with sequence similarity

153, member C, pseudogeneDown-regulated genes FAM203B ILMN_3243302 728071 Family with sequence similarity

203, member BDown-regulated genes FAM207BP ILMN_3306019 729535 Family with sequence similarity

207, member B, pseudogeneDown-regulated genes FAM53B 1) ILMN_2053490, 2) ILMN_1704571 9679 Family with sequence similarity

53, member BDown-regulated genes FOXD4 ILMN_2173524 2298 Forkhead box D4Down-regulated genes FYN ILMN_1781207 2534 FYN oncogene related to

SRC, FGR, YESDown-regulated genes GCFC1 1) ILMN_1682896, 2) ILMN_2331197, 3) ILMN_1712936 94104 GC-rich sequence DNA-binding

factor 1Down-regulated genes GEMIN4 ILMN_1770206 50628 Gem (nuclear organelle)

associated protein 4Down-regulated genes GPR97 ILMN_1765941 222487 G protein-coupled receptor 97Down-regulated genes GUSBP3 ILMN_1763628 653188 Glucuronidase, β-pseudogene 3Down-regulated genes HLA-DRA 1) ILMN_2157441, 2) ILMN_1689655 3122 Major histocompatibility

complex, class II, DR alphaDown-regulated genes HOXC13 ILMN_1759676 3229 Homeobox C13Down-regulated genes HTR3A ILMN_2371079 3359 5-Hydroxytryptamine

(serotonin) receptor 3A,ionotropic

Down-regulated genes IFI44 ILMN_1760062 10561 IFN-induced protein 44Down-regulated genes KRT8P44 ILMN_3182942 100129958 Keratin 8 pseudogene 44Down-regulated genes LAPTM5 ILMN_1772359 7805 Lysosomal protein

transmembrane 5Down-regulated genes LOC723805 1) ILMN_3293173, 2) ILMN_3205404 723805 IL-likeDown-regulated genes LOC90499 ILMN_3223843 90499 Uncharacterized LOC90499Down-regulated genes MCM7 1) ILMN_1663195, 2) ILMN_1704702 4176 Minichromosome maintenance

complex component 7Down-regulated genes METTL12 ILMN_3239525 751071 Methyltransferase like 12

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Table S6. Cont.

Signature nameGenesymbol Probe set name Gene ID Gene description

Down-regulated genes MGC27345 ILMN_1660412 157247 Uncharacterized proteinMGC27345

Down-regulated genes MT1IP ILMN_2136089 644314 Metallothionein 1I,pseudogene

Down-regulated genes MT2A ILMN_1686664 4502 Metallothionein 2ADown-regulated genes MYBPC2 ILMN_1799743 4606 Myosin binding protein

C, fast typeDown-regulated genes NQO1 ILMN_1720282 1728 NAD(P)H dehydrogenase,

quinone 1Down-regulated genes NXPH4 1) ILMN_2237211, 2) ILMN_1696333, 3) ILMN_1695893 11247 Neurexophilin 4Down-regulated genes OR2T11 ILMN_1760065 127077 Olfactory receptor, family 2,

subfamily T, member 11Down-regulated genes OSBPL9 ILMN_2313856 114883 Oxysterol binding

protein-like 9Down-regulated genes PAG1 1) ILMN_2055156, 2) ILMN_1736806, 3) ILMN_1673640 55824 Phosphoprotein associated

with glycosphingolipidmicrodomains 1

Down-regulated genes PCDHGC3 ILMN_1675428 5098 Protocadherin-γ subfamily C, 3Down-regulated genes PIM3 1) ILMN_1789781, 2) ILMN_1707748 415116 Pim-3 oncogeneDown-regulated genes PLD6 1) ILMN_3240586, 2) ILMN_1731518 201164 Phospholipase D family,

member 6Down-regulated genes POLR2J3 ILMN_1661516 548644 Polymerase (RNA) II (DNA

directed) polypeptide J3Down-regulated genes POLR3A ILMN_1681837 11128 Polymerase (RNA) III (DNA

directed) polypeptide A,155kDa

Down-regulated genes PTAFR ILMN_1746836 5724 Platelet-activating factorreceptor

Down-regulated genes RGS16 ILMN_1808226 6004 Regulator of G proteinsignaling 16

Down-regulated genes RGS8 ILMN_1808215 85397 Regulator of G proteinsignaling 8

Down-regulated genes RNF112 ILMN_1744676 7732 Ring finger protein 112Down-regulated genes S100A4 1) ILMN_1684306, 2) ILMN_1688780 6275 S100 calcium binding

protein A4Down-regulated genes SCARB1 ILMN_2183409 949 Scavenger receptor class B,

member 1Down-regulated genes SLC25A3 ILMN_1720703 5250 Solute carrier family 25

(mitochondrial carrier;phosphate carrier),member 3

Down-regulated genes SNORD12C ILMN_3249286 26765 Small nucleolar RNA, C/Dbox 12C

Down-regulated genes SNRPD3 ILMN_1794599 6634 Small nuclearribonucleoprotein D3polypeptide 18kDa

Down-regulated genes TAGLN2 ILMN_1691892 8407 Transgelin 2Down-regulated genes TMEM202 ILMN_2245523 338949 Transmembrane protein 202Down-regulated genes TMEM38A ILMN_1765584 79041 Transmembrane protein 38ADown-regulated genes TNF ILMN_1728106 7124 Tumor necrosis factorDown-regulated genes TPM3P9 ILMN_1761801 147804 Tropomyosin 3 pseudogene 9Down-regulated genes TSR1 ILMN_2092232 55720 TSR1, 20S rRNA accumulation,

homolog (Saccharomycescerevisiae)

Down-regulated genes UNC45A ILMN_1709860 55898 Unc-45 homolog A(Caenorhabditis elegans)

Down-regulated genes ZNF3 ILMN_2390739 7551 Zinc finger protein 3Down-regulated genes ZNF624 ILMN_1670122 57547 Zinc finger protein 624Down-regulated genes ZNF655 1) ILMN_2396292, 2) ILMN_1728528 79027 Zinc finger protein 655Down-regulated genes ZRANB2 ILMN_1662383 9406 Zinc finger, RAN-binding

domain containing 2Up-regulated genes ABTB1 1) ILMN_2367165, 2) ILMN_1802096 80325 Ankyrin repeat and BTB (POZ)

domain containing 1

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Table S6. Cont.

Signature nameGenesymbol Probe set name Gene ID Gene description

Up-regulated genes ADA ILMN_1803686 100 Adenosine deaminaseUp-regulated genes AFF3 ILMN_1775235 3899 AF4/FMR2 family, member 3Up-regulated genes BACH2 1) ILMN_2058468, 2) ILMN_1670695 60468 BTB and CNC homology 1, basic

leucine zipper transcriptionfactor 2

Up-regulated genes C15orf52 ILMN_1775330 388115 Chromosome 15 ORF 52Up-regulated genes C17orf58 1) ILMN_2398926, 2) ILMN_1700515, 3) ILMN_1712985 284018 Chromosome 17 ORF 58Up-regulated genes C7orf41 ILMN_1672605 222166 Chromosome 7 ORF 41Up-regulated genes CAB39L 1) ILMN_1783598, 2) ILMN_1660815 81617 Calcium binding protein 39-likeUp-regulated genes CARD10 ILMN_1743714 29775 Caspase recruitment domain

family, member 10Up-regulated genes CBLN3 ILMN_2053829 643866 Cerebellin 3 precursorUp-regulated genes CCDC41 ILMN_1799113 51134 Coiled-coil domain

containing 41Up-regulated genes CCNG2 1) ILMN_1747244, 2) ILMN_2228732 901 Cyclin G2Up-regulated genes CD40 1) ILMN_1779257, 2) ILMN_2367818 958 CD40 molecule, TNF receptor

superfamily member 5Up-regulated genes CDKN1B 1) ILMN_2196347, 2) ILMN_1722811 1027 Cyclin-dependent kinase

inhibitor 1B (p27, Kip1)Up-regulated genes CDKN2C ILMN_1656415 1031 Cyclin-dependent kinase

inhibitor 2C (p18, inhibitsCDK4)

Up-regulated genes CORO2A ILMN_1813746 7464 Coronin, actin bindingprotein, 2A

Up-regulated genes CTDSP2 ILMN_1692962 10106 CTD (carboxyl-terminaldomain, RNA polymerase II,polypeptide A) smallphosphatase 2

Up-regulated genes FAM53A ILMN_1658452 152877 Family with sequence similarity53, member A

Up-regulated genes FBXO32 ILMN_1703955 114907 F-box protein 32Up-regulated genes FCRL2 ILMN_1791329 79368 Fc receptor-like 2Up-regulated genes FCRL3 ILMN_1691693 115352 Fc receptor-like 3Up-regulated genes FZD7 ILMN_1804351 8324 Frizzled family receptor 7Up-regulated genes GCET2 ILMN_1667449 257144 Germinal center expressed

transcript 2Up-regulated genes HBP1 1) ILMN_1685415, 2) ILMN_2160764 26959 HMG-box transcription factor 1Up-regulated genes HCG27 ILMN_1746436 253018 HLA complex group 27

(nonprotein coding)Up-regulated genes HES6 ILMN_1694268 55502 Hairy and enhancer of split

6 (Drosophila)Up-regulated genes HIST1H2BD ILMN_1651496 3017 Histone cluster 1, H2bdUp-regulated genes HLA-DOB ILMN_1700428 3112 MHC, class II, DO-βUp-regulated genes HOMER2 ILMN_1671486 9455 Homer homolog 2 (Drosophila)Up-regulated genes HPCAL1 ILMN_1764850 3241 Hippocalcin-like 1Up-regulated genes ID3 ILMN_1732296 3399 Inhibitor of DNA binding 3,

dominant negativehelix–loop–helix protein

Up-regulated genes IFI16 ILMN_1710937 3428 IFN, γ-inducible protein 16Up-regulated genes IL4R ILMN_1652185 3566 IL-4 receptorUp-regulated genes IRAK2 ILMN_1745964 3656 IL-1 receptor-associated

kinase 2Up-regulated genes ISG20 ILMN_1659913 3669 IFN-stimulated exonuclease

gene 20kDaUp-regulated genes KIAA0125 1) ILMN_1707491, 2) ILMN_3187535 9834 KIAA0125Up-regulated genes KLHL24 ILMN_1678671 54800 Kelch-like 24 (Drosophila)Up-regulated genes KREMEN2 ILMN_2382290 79412 Kringle containing

transmembrane protein 2Up-regulated genes LAMP3 1) ILMN_2170813, 2) ILMN_2170814 27074 Lysosomal-associated

membrane protein 3Up-regulated genes LHFPL2 ILMN_1747744 10184 Lipoma HMGIC fusion

partner-like 2

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Table S6. Cont.

Signature nameGenesymbol Probe set name Gene ID Gene description

Up-regulated genes LINC00324 ILMN_1681252 284029 Long intergenic nonproteincoding RNA 324

Up-regulated genes LMTK3 ILMN_1668194 114783 Lemur tyrosine kinase 3Up-regulated genes LOC339352 ILMN_3289745 339352 Cytosolic thiouridylase subunit

1 homolog(Schizosaccharomycespombe) pseudogene

Up-regulated genes LOC730101 1) ILMN_3305735, 2) ILMN_3229424 730101 Uncharacterized LOC730101Up-regulated genes LTB 1) ILMN_2376205, 2) ILMN_2376204 4050 Lymphotoxin-β (TNF

superfamily, member 3)Up-regulated genes MAP1LC3A ILMN_1776188 84557 Microtubule-associated protein

1 light chain 3αUp-regulated genes METTL7A ILMN_1656285 25840 Methyltransferase-like 7AUp-regulated genes MIAT ILMN_1864900 440823 Myocardial infarction

associated transcript(nonprotein coding)

Up-regulated genes MID1IP1 1) ILMN_2165473, 2) ILMN_1668960 58526 MID1 interacting protein 1Up-regulated genes MST1 ILMN_1707464 4485 Macrophage stimulating 1

(hepatocyte growthfactor-like)

Up-regulated genes MST1P9 ILMN_2099259 11223 Macrophage stimulating 1(hepatocyte growthfactor-like) pseudogene 9

Up-regulated genes MYL2 1) ILMN_1688417, 2) ILMN_2113807 4633 Myosin, light chain 2, regulatory,cardiac, slow

Up-regulated genes N4BP2L1 1) ILMN_2344650, 2) ILMN_1799487 90634 NEDD4 binding protein 2-like 1Up-regulated genes NINJ1 ILMN_1815086 4814 Minjurin 1Up-regulated genes NLRP7 1) ILMN_1658632, 2) ILMN_1798063, 3) ILMN_1652366 199713 NLR family, pyrin domain

containing 7Up-regulated genes OAS1 1) ILMN_1672606, 2) ILMN_2410826, 3) ILMN_1675640 4938 2’-5′-oligoadenylate synthetase

1, 40/46kDaUp-regulated genes OPRL1 ILMN_2400922 4987 Opiate receptor-like 1Up-regulated genes PHYH ILMN_1773073 5264 Phytanoyl-CoA 2-hydroxylaseUp-regulated genes PIM2 ILMN_1748283 11040 Pim-2 oncogeneUp-regulated genes PLTP ILMN_1773389 5360 Phospholipid transfer proteinUp-regulated genes PNPLA8 1) ILMN_1680223, 2) ILMN_2180582 50640 Patatin-like phospholipase

domain containing 8Up-regulated genes PRR15L ILMN_1748970 79170 Proline rich 15-likeUp-regulated genes RAB26 ILMN_1790317 25837 RAB26, member RAS oncogene

familyUp-regulated genes RASAL1 ILMN_1793517 8437 RAS protein activator like 1

(GAP1 like)Up-regulated genes RGL4 ILMN_1663422 266747 Ral guanine nucleotide

dissociation stimulator-like 4Up-regulated genes RHBDD1 1) ILMN_1681543, 2) ILMN_2209766 84236 Rhomboid domain

containing 1Up-regulated genes SEC14L1 1) ILMN_1732575, 2) ILMN_2391912, 3) ILMN_2285802 6397 SEC14-like 1 (S. cerevisiae)Up-regulated genes SLC25A29 1) ILMN_2350801, 2) ILMN_1697544 123096 Solute carrier family 25

(mitochondrial carnitine/acylcarnitine carrier),member 29

Up-regulated genes SLC44A2 ILMN_1771987 57153 Solute carrier family 44,member 2

Up-regulated genes SLC44A3 ILMN_1658498 126969 Solute carrier family 44,member 3

Up-regulated genes SLC6A16 ILMN_1723287 28968 Solute carrier family 6,member 16

Up-regulated genes SNTA1 ILMN_1753241 6640 Syntrophin, α1Up-regulated genes SPOCK2 ILMN_1656287 9806 Sparc/osteonectin, cwcv and

kazal-like domainsproteoglycan (testican) 2

Up-regulated genes STMN3 ILMN_3244117 50861 Stathmin-like 3Up-regulated genes SUSD3 ILMN_1785570 203328 Sushi domain containing 3

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Table S6. Cont.

Signature nameGenesymbol Probe set name Gene ID Gene description

Up-regulated genes TCL1A ILMN_1788841 8115 T-cell leukemia/lymphoma 1AUp-regulated genes TMEM156 ILMN_2095660 80008 Transmembrane protein 156Up-regulated genes TRAM2 ILMN_1788783 9697 Translocation-associated

membrane protein 2Up-regulated genes TXNIP ILMN_1697448 10628 Thioredoxin interacting

proteinUp-regulated genes XPNPEP2 ILMN_1743357 7512 X-prolyl aminopeptidase

(aminopeptidase P) 2,membrane-bound

Up-regulated genes YPEL3 ILMN_1791147 83719 Yippee-like 3 (Drosophila)Up-regulated genes ZNF627 1) ILMN_1708787, 2) ILMN_2197519 199692 Zinc finger protein 627

Table S7. Gene expression signatures that are enriched with down- or up-regulated genes after PTEN induction

Signature name Category Defined genesEnrichment

scoreP value lowerthan (GSEA) FDR (GSEA)

Down-regulated signaturesMyc_overexpression_1.5x_up Signaling pathway 88 0.632 0.0010 0.0010HIF1α_2x_down Transcription factor target 41 0.595 0.0013 0.0088Tcell_cytokine_induced_IL2_IL7_IL15only Signaling pathway 24 0.563 0.0259 0.0470Myc_overexpression_2x_up Signaling pathway 36 0.560 0.0020 0.0183Notch_T-ALL_up_Palomero Signaling pathway 47 0.546 0.0021 0.0147HIF1α_1.5x_down Transcription factor target 215 0.545 0.0013 0.0012Myc_RNAi_OCILy3 Transcription factor target 54 0.539 0.0012 0.0084Notch_T-ALL_up_Sharma Signaling pathway 37 0.536 0.0109 0.0360Leucine_starve_down Cellular process 178 0.535 0.0010 0.0010Myeloid_Node1536 Cellular differentiation 19 0.507 0.1021 1.0000Tcell_cytokine_induced_prolif Signaling pathway 27 0.504 0.0457 0.0637Glutamine_starve_down Cellular process 313 0.501 0.0010 0.0010

Up-regulated signaturesBlood_Module-3.5_Undetermined Cellular differentiation 13 −0.626 0.0168 0.0474p53_up_Xray Transcription factor target 17 −0.620 0.0229 0.2123Blood_Module-1.3_B_cells Cellular differentiation 55 −0.591 0.0011 0.0023GC_B_cell_BLhigh_DLBCLlow Cellular differentiation 36 −0.583 0.0012 0.0211BCL6_repressed Transcription factor target 19 −0.554 0.0438 0.1555Myeloma_PR_subgroup_down Cancer differential 50 −0.553 0.0010 0.0411CNSonly_Node1460 Cellular differentiation 18 −0.542 0.0602 0.1571B_cell_up_anergy Cellular process 17 −0.542 0.0728 0.1464Thymic_DP_Tcell_gt_Thymic_progenitor_Tcell Cellular differentiation 65 −0.541 0.0012 0.0234NFkB_ChIPCHIP_Young_5factors Transcription factor target 15 −0.525 0.0881 0.1238GC_T_helper_up_Chtanova_and_Kim Cellular differentiation 19 −0.523 0.0986 0.1881Pan_B_U133plus Cellular differentiation 86 −0.514 0.0012 0.0217CLL_unmutated_gt_CLL_mutated Cancer differential 25 −0.509 0.0507 0.2735Muscle_Node1645 Cellular differentiation 24 −0.503 0.0503 0.1265Regulatory_Tcell_FOXP3+_4x_gt_CD4+Tcell Cellular differentiation 11 −0.501 0.2168 0.2046

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